FINAL DISSERATION DOCUMENT

Drugs of Abuse Testing in Fingernails

MSc Analytical and Forensic Science Bethan Jade Davies

12026506

Supervisor: Dr Richard Paul University of South Wales

September 2016

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Drugs of Abuse Testing in Fingernails Miss Bethan Jade Davies

Supervisor: Dr Richard Paul University of South Wales

September 2016 This Research Project was carried out in part fulfilment of the requirements for the award of Degree of Masters’ of Science

Analytical and Forensic Science

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Acknowledgements I would first like to thank everyone at Synergy Health Laboratories in Abergavenny for their endless help and support and for allowing this thesis possible. I would like to thank my University Supervisor Dr. Richard Paul at the University of South Wales for giving me the opportunity to carry out this thesis. Finally, I would like to dedicate and reflect my gratitude to my family and friends for providing me with the support and encouragement through-out my years of study and through-out writing this thesis. This accomplishment would not be possible without their support. Thank you.

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Contents

1.0 Introduction 1.1 Background of Tramadol 8 1.2 Pharmacokinetics of Tramadol 9 1.3 Background of Fingernails 9 1.4 Literature Review 10 1.5 Staten Island Ferry Disaster 13 1.6 Interference Study 14 2.0 Method, Materials and Reagents 2.1 Materials and Reagents 15 2.1.1 Equipment List 15 2.1.2 Critical Reagents and Consumables 15 2.1.3 General Reagents 16 2.2 Risk and COSHH Assessment 16

2.3 Calibration 17 2.3.1 Quality Control Samples 17 2.4 Procedure 18

2.4.1 Sample Washing 18 2.4.2 Incubation 18 2.4.3 LC/MS Preparation 19 2.4.4 LC/MS Acquisition 19 2.5 Validation 3.0 Results

3.1 Tramadol Results 23 3.2 Interference Study 25 4.0 Discussion 25 5.0 Conclusion 30 6.0 References 31 7.0 Appendix Appendix One Appendix Two Appendix Three

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Abstract

The aim of this research project is to determine whether it is possible to determine any traces of Tramadol in known positive fingernail samples. The purpose is to propose an alternative method for a non-invasive method of collection for the detection of drugs of abuse when the option to collect a hair sample is not possible. This method was carried out by through sample preparation, acid hydrolysis and reconstitution prior to being injection onto a Liquid Chromatogram coupled with a Mass Spectrometer (LC/MS) using positive fingernail samples collected by an anonymous donor. The method used was successful at discovering a concentration of Tramadol within the positive fingernail samples. The internal standard used for a deuterated version of Tramadol; Tramadol-D3. The LOD for this method calculated at 1.885ng/mg and the LOQ for this method is 5ng/mg. Statistical Analysis for the validation of the method was not possible to do a short calibration range but an Anova statistical test was carried out on the positive sample results to determine whether the null or alternative hypothesis would be accepted based upon a significant difference. The importance of this work is to demonstrate that there are multiple matrices that can be used for workplace drug testing. It is to express the importance of carrying out drug testing for the safety of individuals and the safety of other around.

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Tables and Figures

Table Page Table 1 Equipment List 15 Table 2 List of Reagents 16

Table 3 List of Drug Standards Used 16 Table 4 Risk Assessments 16

Table 5 Calibration Standards 17 Table 6 Quality Control Standards QC1 17 Table 7 Quality Control Standards QC2 17 Table 8 Quality Control Standards QC3 17

Table 9 LC/MS Transitions 20 Table 10 Summery Statistics for Tramadol Validation 20

Table 11 Summary of Analyte Concentration 24 Table 12 Anova Statistical Test 27

Figures Page Figure 1 Calibration Curve Tramadol 21

Figure 2 Inter-Day final Concentration for Tramadol 7.5ng/mg QC 21 Figure 3 Inter-Day final Concentration for Tramadol 75ng/mg QC 22 Figure 4 Inter-Day final Concentration for Tramadol 750ng/mg QC 22

Figure 5 Tramadol Result Peaks 23 Figure 6 Summary Table of Retention Times 25

Figure 7 Summary of Venlafaxine Concentration and Response 25 Figure 8 Summary of Tramadol Concentration and Response 26

Figure 9 Tramadol Nail Stability Test Results 30

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NOMCLATURE

Abbreviation Meaning MOR Morphine COD Codeine AC Acetyl Codeine

6-MAM 6-Monoacetylmorphine LOD Limit of Detection LOQ Limit of Quantification LC Liquid Chromatography MS Mass Spectrometry AP Acid Phosphate MA Methamphetamine

MDA Methelenedioxyamphetamine MDMA 3,4-Methylenedioxymethamphetamine

NKT Natural Killer T-Cells KET Ketamine EtG Ethyl Glucuronide

THCA Tetrahydrocannabinolic Acid BUP Buprenorphine

NBUP Norbuprenorphine HFBA Heptafluorobutyric Anhydride MDEA 3,4-Methylenedioxy-N-ethylamphetamine NKET Norketamine

BE Benzoylecgonine CE Cocaethylene

NCOC Norcocaine HCOD Hydrocodone HYM Hydromorphine QC Quality Control

UKAS United Kingdom Accreditation Service

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INTRODUCTION Screening for drugs of abuse has become very popular within work place testing arena. The purpose is to discourage employees from using any form of illicit drug, for personal safety and for the safety of other employees working with them. There have been many matrices used to determine whether an individual is abusing drugs with the most common being Urine, Blood and Hair. These biological matrices have been very successful in identifying numerous amounts of drugs of abuse ranging from cocaine, amphetamines and cannabis. The urine and blood sample collection can be an invasive method with strict guidelines, storage and the potential of being a biological hazard. Within the last decade there has been a demand for another matrix for testing which will be just as efficient and accurate for drug identification. Hair was brought into drug testing as it is a non-invasive method of collection and storage and transportation is much less of an issue in comparison to blood and urine. Medical conditions such as alopecia can prevent any hair being collected, therefore, individuals that cannot supply a hair sample. This opened up an opportunity to research into another matrix for drug identification such as Fingernails. The aim of this research project is to determine whether fingernails are capable of being used as a specimen to test for the abuse of drugs. Tramadol was chosen due to the recent addition to the Misuse of Drugs Act 1971 [1]. The overall objective to this project is to achieve confident, reproducible results for the detection of Tramadol within a fingernail. It is understood that this is the first project to determine the presence of Tramadol in finger nails.

1.0 BACKGROUND TO TRAMADOL Tramadol is a synthetic opiate, better known as an opioid [1]. It was first synthesised in 1962 by Grünenthal, GMbH in Germany to be used as an analgesic to treat moderate to severe pain that is taken orally in a tablet form. Tramadol stimulates brain receptors known as the P-opioid receptor which increases the brain serotonin levels therefore, mimicking the effects of general opiates. Tramadol has less side effects and a less addictive trait than other opiates. If an individual has taking an overdose of Tramadol there is a greater chance of seizures than any other opioid substance [2]. Tramadol has been used worldwide for decades but in recent years due to the opiate effect that Tramadol can cause, the well-known prescribed drugs is now being abused mainly by narcotic abusers. The main side effects known to be caused by Tramadol are drowsiness, tiredness and dizziness [1]. The Drug Enforcement Administration (DEA) in the United States of America has stated that Tramadol is mainly being abused in the States by narcotic addicts, health professionals and chronic pain patients [3]. Since June 2014, Tramadol has now been listed as a controlled substance under the United Kingdom Misuse of Drugs act 1971 as a Class C, Schedule 2 substance [1]. Tramadol is now only available by a prescription and not from an over the counter pharmacist. If an individual is caught in possession of Tramadol it can now result in a two years’ imprisonment sentence [1]. There has been a steady increase in deaths since 2010 that can be attributable to the administration of Tramadol and in 2014 the Home Office stated that there has been a 9% increase in deaths involving Tramadol which has now peaked at 240 deaths [2]. Even though the number of deaths associated with Tramadol has increased, the number of Tramadol prescription being distributed has remained stable therefore, indicating that the increase of deaths is associated with a misuse of Tramadol rather than an over prescription [5]. The number of cases where Tramadol has been abused is on the increase, causing a high demand for Tramadol identification within biological matrices. Tramadol has been successfully screened using a Gas Chromatography coupled with a Mass Spectrometer (GC-MS) and also Liquid Chromatography coupled with a Mass Spectrometer (LC-MS) within a wide range of

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matrices including blood, urine and hair. These screening and confirmation techniques have been very successful in identifying Tramadol dosages within a biological matrix.

1.1 PHARMACOKINETICS OF TRAMADOL

Tramadol is promptly and almost completely absorbed into the blood stream through oral administration and then it is rapidly distributed through-out the body [6]. Tramadol acts primarily upon two systems within the central nervous system. The pain transmission in the spinal cord is inhibited because Tramadol acts upon the norepinephrine and serotonin levels by supressing the reuptake into the central nervous system [6]. Tramadol is largely metabolised within the liver, on account of the fast hepatic metabolism. Tramadol only has a bioavailability of around 65-70% [6]. The main metabolites of Tramadol are; O-desmethyl-tramadol, N-desmethyl-tramadol [6]. These primary metabolites can be further metabolised into N,N-didesmethyltramadol, N,N,O-tridesmethyltramadol and N,O-desmethyltramadol [6]. The metabolites are formed because of the principle metabolic pathway which involves O- and N-demethylation catalysed by the cytochrome P450 and the isoenzymes 2D6 (CYP2D6), 2B6 (CYP2B6) and 3A4 (CYP3A4) [6]. During Phase II of metabolism, the O-desmethyl metabolites are coagulated with glucuronic acid and sulphur hydroxide prior to being excreted within the urine. Approximately 10-30% of the parent drug Tramadol is excreted unchanged within the urine [6]. The only metabolite that is pharmacologically active is O-desmethyltramadol [7]. O-desmethyltramadol is catalysed by the CYP2D6 isoenzyme and in comparison to Tramadol the O-desmethyl metabolite is more potent in the affinity of attraction to the P-opioid receptor. Tramadol is metabolised in the same process within every individual but, there is considerable variation between an individual’s response to a Tramadol dosage in respect to therapeutic and adverse effects [7].

1.2 BACKGROUND TO FINGERNAILS The structure of fingernails is very similar to that of hair. The keratin structure provides and maintains nails its structure and rigidity. There are no risks such as biological hazards associated with fingernails and storage is much less of a constraint. Fingernail testing has been around since 1965 where fingernails were originally used to detect arsenic poisoning [9]. There are many ways drugs can be incorporated within the fingernail as the drugs can fuse into germinal matrix following the nail bed [8]. Drugs of abuse can migrate and diffuse into the fingernails germinal matric and nail bed through sources of external contamination associated with sweat, sebum and salvia due to the absorbability and porousness of a fingernail [9]. The main diffusion passage into the fingernails is through the blood supply. Once the drug has been administered and integrated into the blood stream the drugs will diffuse across the cell membrane into the germinal matrix and into the nail bed of the fingernail [9]. The collection of fingernails is a non-invasive method. Collection is done through cutting the top section of the fingernail with stainless steel clippers without the requirement of a medical professional. In comparison to hair there is a greater chance of being able to collect samples from every individual. Unlike hair, Fingernails do not contain melanin removing the possibility of drugs binding to melanin and the known associated bias of this analytical variable. It is believed that some drug types have a greater affinity to certain types of melanin than others [9,10]. The main issue with using fingernails as a matrix is that the sample sizes are very small with an average growth being 3mm a month. This can vary as the growth rate is dependent on age, gender, season, diet exercise level and hereditary factors. Sample collection can be quite difficult but, on the other hand fingernails provide a large detection window with drugs being able to

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be accumulate within the fingernails until the blood supply is cut off and the sample is cut away [9, 10].

1.3 LITERATURE REVIEW There have been previous studies conducted within the last 5 years where Tramadol has been investigated within matrices for abuse of drugs. In 2014 a study was conducted by Patrizia Verri et al who investigated the determination of chronic Tramadol abuse within hair. The study used LC-MS/MS as its source of confirmation to detect Tramadol and its known metabolites O-desmethyltramadol (OMDT) and N-desmethyltramadol (NDMT). Hair samples were collected from 4 individuals that were known to be consuming Tramadol. The samples were collected from the posterior vertex of the scalp with the proximal and the distal ends of the hair identified and then stored at room temperature [3]. The hair samples were washed, cut into 2cm long segments and then pulverised using a Precellys 24 instrument. The samples were then subjected to a solid phase extraction as a form of clean-up [3]. Once the extensive sample preparation was complete the sample was injected onto the Agilent 1200 LC system coupled with a SCIEX API 4000 QTRAP mass analyser which was operating in positive ESI mode [3]. The individuals who supplied the hair samples were a mixture of male and female. The first case that provided a hair sample was a female in her 20’s undergoing therapy with amitriptyline and alprazolam [3]. The second and third case were male individuals undergoing treatment with methadone to treat a withdrawal of opioid consumption. The fourth case was a woman in her 30s who been previously treated with Tramadol under medical supervision because of abdominal pain. Once the abdominal pain had subsided, the patient admitted to still be consuming Tramadol and was increasing the dosage gradually. The results concluded that levels of Tramadol were detected in cases one, two and three within a range of 3.29 and 20.12ng/mg hair [3]. The concentrations of Tramadol within the hair of the fourth case was significantly higher at around 63.42-107.30 ng/mg of hair [3]. These finding are significant with a continuous use of Tramadol but the variability between the results could be a knock on effect from the variation in therapy the individuals are undertaking. Cases one, two and three were undergoing therapy for a separate drug issue that could potentially cause inhibition to the CYP2D6 enzyme which catalyses the formation of the O-desmethyltramadol causing a decrease in the concentration of the pharmacologically active metabolite [3]. Overall, the study concluded that it is possible to determine and quantify Tramadol within hair while using the concentration ratios of the parents and metabolite (OMDT/TRAM and (NDMT/TRAM) [3].). The hair samples may be helpful tools to monitor a historical consumption of an abuse of drugs whether it be a legal or illegal drug. A second study conducted in 2012 carried out by Milena M. Madry et al used the metabolite of parent drug concentrations in hair to determine a differentiation between Tramadol consumption, external contamination and passive exposure. This study began due to Tramadol being detected in a male’s hair sampled during an abstinence test prior to being re-instated his driving licence. The individual has denied consuming any Tramadol and claimed the presence of Tramadol in his hair is due to external contamination as he has been working in an industry that carries out the production of Tramadol [12]. The aim of the study was to determine whether it is possible to distinguish between Tramadol consumption and the external contamination of Tramadol through calculating the ratio between the parent analyte and the metabolites. The study began by taking a 20mg section of a hair sample from the individual who is trying to get their driving licence re-instated. The hair sections were then subjected to sample preparation and then injected onto an LC-MS/MS. The sample were injected

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onto a Dionex UltiMate 3000 HPLC system coupled with an Applied Biosystems 5500 Q Trap linear ion trap triple quadrupole mass spectrometer [12]. To determine whether it is possible that external contamination was a factor in the concentration of Tramadol within the hair from working at the Tramadol production company, proximal hair sections were taken from eight employees who are currently working at the production site [12]. The hair samples were analysed for concentrations of the parent drug Tramadol and the two major metabolites N-desmethyltramadol (NDMT) and O-desmethyltramadol (ODMT). In conjunction with the 8 hair samples taken from the employees, 75 hair samples were collected from known routine Tramadol consumers to provide a reference for concentration of Tramadol through oral consumption [12]. The departments within the Tramadol production company were segmented into 2 sections. The departments were segmented into the laboratory area of the company and the production section of the company. The 8 employees tested at the facility were tested based on what area of the company they worked. The production side of the company displayed concentrations of Tramadol ranging between 1780 and 21,460 pg/mg whereas at the laboratory area of the company the Tramadol concentration ranged between 130 and 380 pg/mg [12]. The concentration of NDMT was present in all 8 employees tested working at the facility. The concentration of NDMT discovered from the employees working in the production sector ranged from 90-2950 pg/mg and 13-17 pg/mg for individuals working in the laboratory sector [12]. The concentration of Tramadol within the 75 patient samples that were collected for reference ranged between 120 and 29,000 pg/mg of hair [12]. The NDMT and ODMT concentrations of the 75 patients ranged between 10 and 18,070 pg/mg of hair [12]. Once all the data was collected and interpreted, a ratio between the parent drug Tramadol and NDMT and ODMT was calculated to determine whether it is possible to distinguish between an administration of Tramadol and external contamination. The results concluded that the ratio of Tramadol and NDMT for the patients is above 0.037 and the ratio for Tramadol and ODMT for the patients is above 0.021 [12]. These values will provide evidence whether Tramadol was ingested or whether it was indeed caused by the external contamination from the working environment. The study as a collective concluded that it is possible to use the metabolite to parent drug ratio to determine whether an individual has ingested a drug or whether they have been subjected to external contamination [12]. The differentiation is mainly shown by the ODMT metabolite, even though the metabolite is produced by CYP2D6 which is also the isoenzyme that is involved in the breakdown of Tramadol, it is this ratio between the 75 patient’s hair samples collected and the 8 employees from the production facility that differed significantly.

There have been previous studies conducted with the detection of drugs of abuse within fingernails. There has been no previous study conducted where Tramadol has been determined in fingernails, all the previous studies have been carried out to determine other drugs of abuse. A study conducted by Min Shen et al who decided used fingernails as a specimen to quantify any presence of opiates using the LC-MS/MS method of confirmation. This study looked into the whether it is possible to discover and quantify 6-MAM, MOR, CO and AC within fingernails sample but also use hair as a source for comparison. The objective of this study was whether it is possible to determine the drugs and their metabolites in fingernails [13]. The study began by collecting 10 drug-free nail samples from 6 volunteers, all samples were cut and stored independently in a paper bag at room temperature [13]. The nail samples were subjected to thorough sample preparation, clean up and pulverisation using a freeze mill and liquid nitrogen [13]. Once the samples were completed pulverised the

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nails were reconstituted and liquid-liquid extraction was carried out using chloroform:2-propanol (9:1, v/v) for separation [13]. The results discovered that even though the structure of fingernails and hair are very similar, the drugs in hair can be influenced by the presence of melanin in the hair but further study will have to be carried out to prove this theory. The results for nails showed that out of 18 samples, 12 of them provided positive results for 6-MAM at a range of 0.10 to 1.37 ng/mg, MOR at a range of 0.58 to 3.16 ng/mg and COD at a range of LOQ to 0.27 ng/mg [13]. Heroin on the other hand was only present in three of the samples in conjunction with AC but heroin was found positive without any other drug or metabolite in only one of the nail samples [13]. The results for hair showed that 12 of the 18 samples provided resulted in revision to the cut off values recommended by the Society of Hair Testing (SOHT). The results ranged from 1.02 to 3.68 ng/mg for 6-MAM, 0.25 to 1.36 ng/mg for MOR and 0.42 to 14.07 ng/mg for COD [13]. The ratio of 6-MAM/MOR ranged from 1.79 to 6.00 which correlates to the recommended cut off values provided by the SOHT [13]. In comparison to the fingernails, AC was detected in eight of the hair sample and the concentration of AC ranged between LOQ and 0.91 ng/mg [13]. The study concluded that the determination of 6-MAM, MOR and COD in hair samples fell within the levels supplied by the Society of Hair Testing. Even though the levels of drugs detected were greater in hair than the levels detected in fingernails. Drugs were positively detected in fingernails therefore; it is possible to use fingernails as an alternative to hair samples in the future. A second study was conducted in 2010 by Jin Young Kim et al who used GC-MS to determine whether it is possible to quantify amphetamine-type stimulants, ketamine and their known metabolites using fingernails. The study investigated specifically at the discovery of AP, MA, MDA, MDMA, NKT and KET. The study began by collecting drug free fingernail samples from laboratory personnel. Positive fingernail samples were collected from known drug users by the Narcotics Departments at the District Prosecutors Office. There were a total of 7 positive fingernail samples obtained from individuals known to be administrating drugs of abuse for analysis. The fingernail samples were subjected to sample preparation followed by an aliquot of each sample being injected onto a GC-MS column. The GC-MS instrument used was an Agilent Technologies 5975 inert mass spectrometer coupled with a 6890N Gas Chromatographer [13]. The results displayed that out of the six drugs tested using the fingernail samples provided by the seven known drug abusers, MA was the drug that was most frequently detected as it was positively detected in six out of seven samples [14]. AP was detected in four out of the seven samples but as AP is the common metabolite of MA it was also detected in low levels of individuals who have been previously consuming MA [14]. MDMA, MDA, KET and NKT were all detected in a sample, but this was a results from a particular individual consuming a polydrug, but to prove this matter a further study would need to be carried out to determine a polydrug consumption pattern [14]. The study concluded that it was possible to determine each compound within the fingernail samples successfully while using the GC-MS. Fingernail testing can therefore be a very useful tool in discovering a chronic or historic abuse of drugs when it is not possible to obtain a hair sample. This method used was a fully validated method using a GC/MS separation technique for all six of the phenylalkylamine derivatives from the drug abuser’s fingernails through thorough sample preparation including alkaline hydrolysis, liquid- liquid extraction and HFB derivatisation [14].

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A more recent study conducted in 2015 published in the Journal of Analytical Toxicology by Irene Shu, looked at drug testing using fingernails as a matrix for 10,349 samples collected from high-risk cases over a time period of three years, between 2012 and 2014. The study investigated a vast array of drugs from amphetamines and the known metabolites, cocaine and opioids such as oxycodone and hydrocodone, morphine, codeine, methadone, cannabis, ethyl glucuronide (EtG) and barbiturates benzodiazepines, ketamine and finally Tramadol [14]. The study began by collecting 3mm clippings from all the nail samples that were submitted to the laboratory for analysis. These samples had been collected and delivered from drug courts, child advocacy, and drug treatment programmes etc. All the samples besides EtG, THCA, BUP and NBUP were subjected to screening prior to sample preparation and then for confirmation using LC-MS or GC-MS. The samples were separated due to the different sample preparation may be required dependent on what drug was being detected. The results showed that a total of 7,799 samples were analysed for amphetamines which included AMP, MET, MDMA, MDA and MDEA [15]. The major metabolites for both amphetamines and methamphetamines were present in 14.4% and 13.7% of the nail samples tested for that particular drug. The concentration for both amphetamines and methamphetamines in these samples ranged from 40 to 44,851 pg/mg [15]. The fingernails samples 0.3% showed a positive result for MDMA, 0.09% of the samples showed positive for MDA, and 0.05% positive for MDEA [15]. The study reported a finding of ketamine within two samples at a concentration of 3,772 and 12,632 pg/mg and NKET was discovered in one sample at a concentration of 201 pg/mg [15]. Cocaine, BE, NCOC and CE were also discovered in the fingernail samples out of a possible 7,787 samples tested with a concentration ranging from 20 to 265,063 pg/mg [15]. The highest concentration was shown by the major parent analyte for cocaine out of the overall nail samples collected that were tested for the particular drug group. Opioids were discovered within some of the nail samples for MOR, COD, 6-MAM, HCOD and HYM [15]. The concentration for all these opioids ranged at 40-118 and 229 pg/mg with the most abundant of the five opioids being HCOD [15]. THCA determination had to be differentiated between the external contamination of marijuana and the consumption of marijuana due to the potential of smoke becoming incorporated into fingernails through the porousness and permeability of a fingernail. The results displayed a concentration range between 0.04 and 262 pg/mg within the fingernails, EtG was discovered at concentrations of 20 to 3,754 from a possible 756 samples [15]. The overall study concluded that out all of the different drug groups tested in this research, all fingernails tested positive for at least one drug therefore, it can be assured fingernails can be a reliable matrix to use when testing for a chronic use of drugs whether they be legal or illegal substances. The sensitivity of the instrumentation allowed the determination of drugs of abuse and prescription drugs including their metabolites but, the study indicated that more research studies will need to be conducted to ensure that there is a better understanding for the significance and meaning of results when a fingernail sample is being tested.

1.3 STATEN ISLAND DISASTER The importance of work place testing cannot be stressed enough and on October 16th 2003, tragedy struck which may have been avoided if the correct work place testing procedure was in place. Pilot, assistant captain Richard J Smith was in charge of the Staten Island ferry which is used every day for commuters to cross the East River between New York City and Staten Island [16]. On October 16th 2003, the ferry boat crashed at high speed, hitting into the loading dock, causing wooden and concrete

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panels to smash into the side of ferry, carrying passengers through the passage of the vessel [16]. There were 10 casualties and dozens injured from the accident. The Pilot, Richard J Smith survived the crash who of which was accused of being slumped and unconscious over the controls of the ferry during the crossing, a co-worker stated [16]. It was suggested the Pilot attempted to commit suicide while in control of the ferry by slitting his left wrist. After the crash Richard Smith fled the scene, broke into his home as he has left his keys on the boat and locked himself into his bathroom to where he tried to commit suicide for a second time by shooting himself in the chest twice with a pellet gun [18]. He was emitted into hospital under critical condition to which he survived both suicide attempts. When questioned the pilot admitted to losing consciousness at the controls of the ferry after feeling lethargic and tired. These are some of the main side effects of Tramadol, to which later he admitted consuming Tramadol and Tylenol the morning of the accident. In August 2004, Pilot, Assistant Captain Richard J Smith pleaded guilty to manslaughter and was sentenced to 18 months’ imprisonment [17]. The New York Ferry Director Patrick Ryan also pleaded guilty to manslaughter due to the negligence and not providing adequate drug tests to staff members to ensure the safety of the commuters on the ferry. Mr Patrick Ryan was sentenced to a year and a day [18]. It is instances like these that reflect the true importance for work place testing and how this tragedy could have been easily avoided and the lives of the 10 casualties could have been spared through the use of a simple drug test. When individuals are taught about drugs of abuse, the instant thought are the drugs that are well known to be abused and are illegal such as Cocaine, Heroin, and Cannabis etc. This tragic event was caused by concealment of a legal, prescribed medication that caused impairment to an individual’s judgement. It is important to screen individuals not just for the well- known drugs of abuse but for prescribed drugs such as Tramadol.

1.4 INTERFERENCE STUDY Depression is a mental illness which is known around the globe and unfortunately it can affect 1 in 10 of individuals. Depression is a long lasting mood effect that can alter your everyday life and interactions. It is estimated that depression affects 2-6% of Children and Adolescents in a community and that depression is a leading cause of suicide among 10-19 year olds [19]. The purpose of an interference study is to verify that the compound discovered in this research project is actually the target analyte and not an interference or contamination. A drug known to be prescribed for the treatment of depression is Venlafaxine. Venlafaxine is very similar to Tramadol. Both compounds are very similar structurally in which both compounds include a methoxyphenyl, a N,N-desmethylamino and a hydroxycyclohexyl group [20]. Venlafaxine and Tramadol both inhibit the serotonin and norepinephrine reuptake in the brain and both compounds are enantioselectively metabolised by the same Cytochrome CYP450 isoenzyme CYP2D6 [20]. The serotonin system is known to control mood, emotion, sleep and appetite [19]. The main metabolites for Tramadol are; O-Desmethyltramadol and N-desmethyltramadol whereas, the main metabolite for venlafaxine is O-desmethylvenlafaxine as both compounds are metabolised in the same manner and both of the O-desmethyl metabolites for Venlafaxine and Tramadol are pharmacologically active [20].

Venlafaxine is prescribed for depression, anxiety, fears and social anxiety disorders in adults [21]. An overdose in Venlafaxine can result in hypertension, seizures, serotonin toxicity and cardiac condition abnormalities [21]. Venlafaxine, similar to Tramadol undergoes hepatic metabolism in the liver to form one major pharmacologically active metabolite O-desmethylvenlafaxine and two minor metabolites N-

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desmethylvenlafaxine and N,O-didesmethylvenlafaxine [21,23]. Venlafaxine is normally prescribed in tablet form and is administered orally, once ingested Venlafaxine has a bioavailability of 92% [22]. The main metabolites are formed in the same way as the metabolism of Tramadol in which the metabolism is catalysed by the CYP450 cytochrome isoenzymes: CYP2D6, CYP3A and CYP2C19 [22]. The (R)-enantiomers us the active enantiomer that inhibits both the noradrenaline and serotonin synaptic reuptake and the (S)-enantiomer inhibits the serotonin reuptake [23]. The aim of conducting the interference study is the verify that the compound discovered through the research project in Tramadol and not Venlafaxine as due to the high number of similarities Venlafaxine elutes from the LC column at a very similar retention time to Tramadol but exhibits slightly different characteristics on a mass spectrometer chromatogram.

2.0 METHODS, MATERIAL AND REAGENTS This chapter is a guide to the materials and reagents used through-out the method including a step by step guide on how the experiment was carried including the sample preparation and the LC/MS acquisition.

2.1 MATERIALS AND REAGENTS 2.1.1 EQUIPMENT LIST

Table 1 Equipment List Item Supplier

Measuring Flask 100 mL Fisher Scientific Volumetric Flask 10 mL Grade A Fisher Scientific

10PL - 100PL Variable Volume Pipette

Fisher Scientific

100PL - 1000PL Variable Volume Pipette

Fisher Scientific

Multi Stepper Pipette Fisher Scientific Fume Hood Fisher

Oxford A2605D Balance Lab 3 Techne Dri-Block DB-2A Fisher

Glass Pasteur Pipettes SSI Polypropylene tubes 70x12mm SSI

Polypropylene caps 12mm Lab 3 Turbo Vap LV Lab 3

Ultra-wave Sonicator Lab 3 Technologies Centrifuge Sigma 4K15 Lab 3 Technologies

Incubator Lab 3 Homogeniser (Fast Prep-24) MP Biomedical

Homogeniser Tubes MP Biomedical Tweezers

Fingernail Stainless Steel Clippers Single Use pierce-able sealing films Alphalabs EZP-100

96 Well Deep Plate for LC/MS Crawford Scientific LTD Cerex Processor Positive Pressure

Manifold SPE Crawford Scientific

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2.1.2 CRITICAL REAGENTS AND CONSUMABLES a. Column: Zorbax XDB 4.6x50mm 1.8PL Column (Agilent) b. Metal bead homogeniser tough tubes (MP Biomedical)

2.1.3 GENERAL REAGENTS

Table 2 List of General Reagents Reagent Grade Supplier

Deionised Water ELIX 35 Millipore Methanol HPLC Grade Sigma Aldrich

Acetonitrile HPLC Grade Sigma Aldrich Dichloromethane HPLC Grade Sigma Aldrich

Ammonium Acetate Analytical Grade Sigma Aldrich Formic Acid Analytical Grade Sigma Aldrich

Quantisal Buffer Manufacturer Immunanalysis Conc. Acetic Acid Analytical Grade Sigma Aldrich

Table 3 List of Drug Standards Used

Product Name Supplier Molecular Formula

Solvent Lot Number

Tramadol Hydrochloride

LGC C16H25NO2 HCL

Methanol 40527

O-Desmethyl Tramadol

Cerilliant C15H23NO2 HCL

Methanol FN08221402

N-Desmethyl Tramadol

Cerilliant C16H25NO2 HCL

Methanol FN10161403

Venlafaxine Hydrochloride

LGC C17H27NO2 HCL

Methanol 8787

O-Desmethyl Venlafaxine

LGC C16H25NO2 Powder 2398

N-Desmethyl Venlafaxine

LGC C16H25NO2 Power 2399

Tramadol-C13-D3 Hydrochloride

LGC C1513CH22D3NO2 HCL

Methanol 40525

*Certificate of Analysis can be found in Appendix Two 2.2 RISK AND COSHH ASSESSMENT

Table 4 Risk Assessments Process Appendix Number

Homogeniser Fast Prep-24 Appendix One Process Turbo-vap Sample Concentrator Appendix One

LCMS General Method Appendix One

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Table 5 COSHH Assessment Chemical Appendix Number Methanol Appendix One

Acetonitrile Appendix One Quantisal Buffer Appendix One Dichloromethane Appendix One

Concentrated Acetic Acid Appendix One Ammonium Acetate Appendix One

Concentrated Formic Acid Appendix One 2.3 CALIBRATION Table 5 Calibration Standards Calibration Standard

Target Concentration

(ng/mg)

Dilution Volume

(mL)

Volume Used (PL)

Final Concentration

(pg/mg)

Nails Concentration

pg/mg 1 0.0005 10 50 5 5 2 0.01 10 100 10 10 3 0.05 10 500 50 50 4 0.1 10 1000 100 100 5 0.5 10 500 500 500 6 1 10 1000 1000 1000

2.3.1 QUALITY CONTROL STANDARDS

*All quality control standards are internal (IQC) material therefore, ISO guide 34 compliant

Table 6 Quality Control Standards – QC1

Analyte Lot No. Stock Conc. Pg/mL

Intermediate Conc. Pg/mL

Target Conc. ng/mL

Final Conc. pg/mL

Volume Pipetted PL

Tramadol 40527 103.40 1.0 0.0075 7.5 75 N-Des

Tram FN10161403 108.93 1.0 0.0075 7.5 75

O-Des Tram

FN08221402 103.93 1.0 0.0075 7.5 75

Venlafaxine 8787 108.27 1.0 0.0075 7.5 75 N-Des Ven 2399 1000 1.0 0.0075 7.5 75 O-Des Ven 2398 1000 1.0 0.0075 7.5 75

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Table 7 Quality Control Standard – QC2 Analyte Lot No. Stock

Conc. Pg/mL


Target Conc. ng/mL

Final Conc. pg/mL

Volume Pipetted PL

Tramadol 40527 103.40 1.0 0.075 75 750 N-Des

Tram FN10161403 108.93 1.0 0.075 75 750

O-Des Tram

FN08221402 103.93 1.0 0.075 75 750

Venlafaxine 8787 108.27 1.0 0.075 75 750 N-Des Ven 2399 1000 1.0 0.075 75 750 O-Des Ven 2398 1000 1.0 0.075 75 750

Table 8 Quality Control Standard – QC3

Analyte Lot No. Stock Conc. Pg/mL


Target Conc. ng/mL

Final Conc. pg/mL

Volume Pipetted PL

Tramadol 40527 103.40 10.0 0.075 750 750 N-Des

Tram FN10161403 108.93 10.0 0.075 750 750

O-Des Tram

FN08221402 103.93 10.0 0.075 750 750

Venlafaxine 8787 108.27 10.0 0.075 750 750 N-Des Ven 2399 1000 10.0 0.075 750 750 O-Des Ven 2398 1000 10.0 0.075 750 750

2.4 PROCEDURE 2.4.1 SAMPLE WASHING

1. Remove fingernail from the packaging and using metal stainless steel fingernail clippers (Clippers were washed with methanol before use), cut the fingernails and weigh out approximately 10mg then place into a plastic tube.

2. All samples including the blank are washed in three stages. 3. Using a stepper with a 50-250PL tip on setting 2, add 1mL of deionised water to

each polypropylene tube. Cap the tubes and vortex for 5-10 minutes, let samples stand for 10 minutes.

4. Using a fresh glass pipette, remove all water from each individual tube and discard. 5. Using a stepper pipette with a 50-250PL tip on setting 2 add 1mL of methanol to

each nail sample, vortex for 15-20 seconds, remove the methanol and discard. 6. Place sample tubes into the fume cupboard and add 1mL of dichloromethane to

each sample, cap and vortex for 5-10 seconds. Allow samples to then stand for 10 minutes.

7. Remove the dichloromethane from the tube using a fresh glass pipette for each sample and discard.

8. Using a stepper pipette with a 50-250PL tip on setting 2 add 1mL of dichloromethane to each nail sample, vortex for 15-20 seconds and discard using a fresh glass pipette for each sample.

9. Using a stepper pipette with a 50-250PL tip on setting 2 add 1mL of dichloromethane to each nail sample, vortex for 15-20 seconds and leave the tubes to stand for 10 minutes.

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10. Pipette the dichloromethane out of the tube using a fresh glass pipette for each sample and discard.

11. Once the liquid has been removed from the fingernail samples allow the fingernail clipping to dry.

2.4.2 INCUBATION

1. Prepare eight fresh unused polypropylene tubes and label L1 to L6, Blank and QC.

2. Using a stepper pipette with a 50-250PL tip on setting 2 add 100PL of Tramadol internal standard into the polypropylene tube for each calibrator and wash.

3. Add 100PL of calibrators or QC into the designated tubes. The calibrators are then evaporated to dryness. Reconstitute with 250PL of 10% Quantisal buffer.

4. For the samples, blanks and QC’s the fingernail clippings need to be transferred into a homogeniser tube containing no liquid. Screw the lids onto the homogeniser tubes firmly and load onto the homogeniser so that the tubes are balanced and close the lid.

5. Turn the homogeniser on and press SET, RUN. The homogeniser will run for 20 seconds after which you press SET followed by the up arrow until program 2 is selected.

6. Press RUN to process the homogeniser for a 5-minute cycle. Press RUN again after five minutes and again after another 5 minutes. This process will have to be repeated between 8-10 times.

7. Once pulverisation is completed, remove the homogenisation tubes. Add 1mL of methanol into each tube, cap and mix well. Pour the entire contents (including the steel beads) into the extract tubes.

8. Add a further 1mL of methanol to each homogeniser tube, mix briefly and empty into the appropriate matching polypropylene tube. Add 20PL of acetic acid to each individual tube, cap and mix briefly on the vortex mixer.

9. Add 100PL of Tramadol internal standard into each tube containing the fingernail extract. This can be done using the stepper pipette with a 50-250PL tip on setting 2.

10. Incubate the sample, blank and QC’s for 2 hours at 60qC (Techne DriBlock) then sonicate for 15 minutes.

11. Cool the sample, blank and QC’s in the fridge for at least 5 minutes. Once cooled centrifuge at 1500rpm for 2 minutes and then remove the methanol and acetic acid into a fourth polypropylene tube.

12. Add 1mL of methanol using a 100-1000PL pipette to each tube containing sample. Cap and vortex briefly.

13. Centrifuge at 1500rpm for 2 minutes then transfer the methanol to the same the same tube from step 11 as for the primary extract.

2.4.3 LC/MS PREPARATION

1. Evaporate the sample tubes and wash tubes W1 and W2 to dryness using the Dri Block and Techne Sample Concentrator for 15 minutes. If some of the tubes are not evaporate completely to dryness allow an extra 5 minutes. The tubes must not be over dried.

2. Reconstitute each tube in 250PL 10% quantisal buffer in water using a stepper with a 50-250PL tip on setting 5. Cap tubes and vortex briefly.

3. Transfer all calibrators, samples and QCs into individual fresh wells on a 96 well deep plate using a 10-100PL variable volume pipette set at 100PL, using a fresh tip for each sample.

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4. Add 50PL of formic acid to each well, using a stepper with a 50-250PL tip on setting 1.

2.4.4 LC/MS ACQUISITION

The analysis was performed on an Agilent 1640 using the Nails Tramadol method. The method using a Zorbax XDB 4.6x50mm 1.8PL Column (Agilent) set up with a 50PL injection volume across a gradient ranging from 10% solvent B to 70% solvent B over a 4 minute injection coupled with a 1.5 minute equilibrium time to allow the LC pressure to return. Solvent A consisted of 50mM Ammonium acetate and Solvent B is HPLC Grade Acetonitrile. The LC flow rate was set to 0.500mL/min with a high pressure limit of 400.00 bar. The MS was powered in positive ESI mode under a dynamic MRM mode with a Delta EMV of 400. Gas Temperature running at 300oC and a gas flow of 13l/min, Nebuliser running at 50psi and Capillary running at 4000V.

Table 9 LC/MS Transitions Compound

Name ISTD Precursor

Ion Product

Ion Fragment

(V) CE (V)

Cell Acc (V)

Ret Time (Min)

Ret Window

N-Des Tram

No 250.2 65.1 35 89 4 3.6 2

N-Des Tram

No 250.2 51.1 35 100 4 3.6 2

N-Des Ven No 264.2 121 35 33 4 3.9 2 N-Des Ven No 264.2 78 35 85 4 3.9 2

O-Des Tram

No 250.2 77.1 40 77 4 2.9 2

O-Des Tram

No 250.2 50.9 40 100 4 2.9 2

O-Des Ven No 264.2 107 40 33 4 3.2 2 O-Des Ven No 264.2 77 40 77 4 3.2 2 Tramadol No 264.2 55.9 35 73 4 3.2 2 Tramadol No 264.2 50.9 35 100 4 3.2 2 Tramadol-

D3 Yes 268.3 58.2 100 12 7 3.2 2

Venlafaxine No 278.2 121 45 33 4 4 2 Venlafaxine No 278.2 78.1 45 85 4 4 2 2.5 VALIDATION

The validation and linearity for this method was tested using three different QC’s at known concentrations injected over three consecutive days. The concentration of each quality control standards can be found in section 2.3.1 of this report. The calibration curve is a combined standard, set over 6 calibration levels. The limit of detection (LOD) for this method is 1.885ng/mg and the limit of quantification (LOQ) is 5ng/mg.

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Figure 1: Calibration Curve Tramadol

Table 10: Summary Statistics for Tramadol Validation

Spiked Amount of Tramadol

7.5ng/mg 75ng/mg 750ng/mg Intra-Day Inter-Day Intra-Day Inter-Day Intra-Day Inter-Day

N 5.00 15.00 5.00 15.00 5.00 15.00 Mean 3.13 4.32 66.35 64.56 766.01 774.96

SD 0.49 1.62 12.09 6.52 12.67 736.1 %CV 15.54 37.53 18.22 10.09 1.65 3.95

95% Confidence 30.46 126.11 35.71 32.15 3.24 42.76

Figure 2 Inter-Day Final Concentration of 7.5ng/mg QC

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Figure 3 Inter-Day Final Concentrations of 75ng/mg QC

Figure 4 Inter-Day Final Concentration of 750ng/mg QC

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3.0 RESULTS 3.1 TRAMADOL RESULTS

The results gave a positive result for all six fingernail samples that were tested. The peaks shown below are indicative of tramadol being present in all 6 of the samples. These peaks show the retention time for Tramadol and the abundance at which the peak exists. Fingernail samples were collected from an anonymous donor who had been prescribed 50mg Tramadol tablets to take 4 times a day.

Figure 5 Tramadol Result Peaks

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Table 11 displays the concentrations as which each of the 6 analytes were discovered within each of the 6 samples tested.

Table 11 Summary of Analyte Concentration

Analyte Sample Concentration ng/ng 1 2 3 4 5 6

Tramadol 137415 133480 72110 90211 53699 27833 N-Desmethyl

Tramadol 9907 10417 4613 6273 3652 2248

O-Desmethyl Tramadol

32858 32223 16167 20926 12199 7387

Venlafaxine 65 15 9 18 24 23 N-Desmethyl Venlafaxine

40 32 11 16 22 12

O-Desmethyl Venlafaxine

34 719 14 437 258 2

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3.1 INTERFERENCE STUDY

The results of the interference study showed that using this method, it is possible to distinguish between Tramadol and Venlafaxine for the purpose of drugs testing using fingernails as a source of matrix. Using this method indicates that there is no source of interference associated with Venlafaxine when testing for Tramadol. The concentrations of Venlafaxine shown in table 11 reflect the very little or no substance within the fingernail samples that were tested. Figure 6 Summary Table of Retention Times

It is evident in Figure 6 that the retention times for Tramadol and Venlafaxine are significantly different. Tramadol is eluting at 3.281 whereas Venlafaxine is eluting at 3.728. This provides assurance that it is possible to distinguish between the two analytes during analysis even though both are structurally similar. Figure 7 Summary of Venlafaxine Concentration and Response

Figure 7 illustrates the final concentration of Venlafaxine within the positive samples. It is evident that the response of Venlafaxine within the positive samples are much less in comparison to the response of Venlafaxine within the calibration. This implies that there is little or no evidence that there is any trace of Venlafaxine within the 6 fingernail samples tested and there is no evidence of any kind of interference during this method.

4.0 DISCUSSION

Using the method described above, it was possible to quantify Tramadol within fingernail samples from an individual known to the prescribed Tramadol. The results showed Tramadol present at significant concentrations. It is noted that these concentrations are greater than the calibration range chosen for this project and because of this it is not possible to apply precision and accuracy analysis to the final concentrations of Tramadol present. In a further study it would be recommended to increase the calibration range to ensure that the high concentrations of Tramadol can be determined and statistical analysis can be applied. Studying multiple research articles as stated under the literature review chapter, the calibration ranges chosen were very low because due to levels detected within the literature. Additional variables

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that may have added to the high levels seen here include the amount of nail used in the analysis, in this case 10mg of nail sample was used which could account for the high concentration of Tramadol detected but, even if the nail samples were weighed out to 1mg, assuming a linear calibration curve the range used in this method would still not be sufficient. As a result, a larger calibration range would have to be used in a future study. Increasing the concentrations of the calibration standards would be an opportunity to investigate the linearity of the calibration curve at higher levels. This would be to determine whether the calibration curve is linear at the top end, or whether the curve plateaus, with this information it will be possible to determine whether the concentrations of Tramadol that were discovered in the fingernails are significant. The final concentrations of Tramadol reflected by the results in Table 11 are ranging between 27,800ng/mg and 137,415ng/mg in comparison to the calibration range of 5ng/mg and 1000ng/mg. In regards to a further study the concentration range would have to be increased, with the bottom calibration level beginning at approximately 1000ng/mg and continuing on a linear scale. Changing the calibration range would be beneficial as the 5ng/mg bottom calibration is working at the threshold of the instrument and method. Providing a higher concentration calibration range would be beneficial for statistical analysis and the precision and accuracy of the overall method. Figure 8 Summary of Tramadol Concentration and Response

Figure 8 illustrates the final concentrations of Tramadol including the response in comparison to the Tramadol-D3 internal standard used in the method. The response shown for the positive samples for Tramadol are significantly higher in comparison to the response associated with the calibration. This is valid because it is indicating that there is a great signal strength for Tramadol, implying that there is a high concentration of Tramadol present within the samples. Looking at the response associated with the Tramadol-D3 internal standard, it is clear that the value of the response within the samples decreases dramatically in comparison to the response associated with the calibration. This is due to loss of internal standard signal. A decrease in internal standard response isn’t necessary a bad thing but, there are reasons on why this could occur. In one form the internal standard response could be supressed through ion suppression. Ion suppression is caused by a presence of a less volatile substance that can alter the formation of the droplet, formed in the electrospray ionisation chamber [24]. Besides ion suppression the internal standard response may have been reduced during the sample preparation procedure. During this drying down period the samples may have become too dry reducing the level of internal standard. To rectify this, many previous studies have used a drying down method using a stream of nitrogen, as this method has been using a sample concentrator using air alone, drying down using nitrogen may be preferable.

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Figure 6 displays the retention time for each of the 6 individual analytes for this method. One of the key components for the project was to determine whether it is possible to distinguish Tramadol from Venlafaxine’s main metabolite N-Desmethyl Venlafaxine. Both analytes are structurally similar and produce the same fragmentation pathway. As displayed in figure 6, it was possible to distinguish between the two analytes. The method detected different transitions for both Tramadol and N-Desmethyl Venlafaxine as shown in table 9. The retention times for both analytes are different too, Tramadol is eluting at 3.281 and N-Desmethyl Tramadol is eluting at 3.600. Even though it is not possible to carry out a precision and accuracy statistical analysis of the results from the positive Tramadol samples. An Anova statistical analysis was carried out to represent the Null Hypothesis in relation to this method. The Null Hypothesis for this method is based upon that it is not possible to detect traces of Tramadol within fingernail samples when used as a biological matrix. The results of the Anova test are shown below;

Table 12 Anova Statistical Test SUMMARY

Groups Count Sum Average Variance Column 1 2 270895.6214 135447.8107 7743089.198 Column 2 2 162321.2431 81160.62155 163821509.4 Column 3 2 81533.3998 40766.6999 334511817.5

ANOVA

Source of Variation SS df MS F P-value F crit Between Groups 9028853702.93 2.00 4514426851.47 26.76 0.01 9.55 Within Groups 506076416.13 3.00 168692138.71

Total 9534930119.06 5.00

The results of the Anova display a p-value of 0.01, f-value of 26.76 and a f-critical value of 9.55. the f-value of 26.76 is greater that the f-critical value of 9.55, it statistically viable to reject the null hypothesis which states it’s not possible to detect traces of Tramadol within fingernail samples and accept the alternative hypothesis. The alternative hypothesis states that it is possible to detect traces of Tramadol when using fingernails as a matrix. The p-value at 0.01 level of significance demonstrates, that there is evidence of statistical significant and it is acceptable to reject the null hypothesis and accept the alternative hypothesis. Using an Anova test for the statistical analysis of the sample illustrates and verifies that it is possible to determine traces of Tramadol within fingernail samples. This test also allowed a statistical analysis when it was not possible to carry out the precision and accuracy because of the small calibration range. The recovery for the internal quality control samples was measured from blank fingernails being spiked with known concentrations of standard. This was done by taking blank fingernails and spiking with 7.5ng/mg, 75ng/mg and 750ng/mg. The blank fingernails were left to stand in a standard for a week prior to being tested. The

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IQCs were treated as if they were normal samples and were taken through the procedure from start to finish. The purpose of this recovery test is to investigate whether the nail matrix has any influence on the test, and whether the sample preparation has any influence on the overall method. The internal quality control samples were run 3 times to ensure there was enough data to calculate a recovery percentage for each individual concentration. The internal quality control standard at 7.5ng/mg provided a recovery of 96%,which is considered to be satisfactory. The concentration at 75ng/mg showed a percentage recovery at 118% and the top concentration at 750ng/mg had a percentage recovery at 79%. The overall recovery of fingernails is adequate in relation to the recovery percentages for each IQC concentration. In conjunction with the recovery percentage, coefficient of variance was also calculated to compare the recovery in relation to the variation between the three injections. The coefficient of variance calculated 7% for the 7.5ng/mg quality control standard indicating that there is very little variance within the recovery for the lowest standard. The middle IQC with a concentration of 75ng/mg, had a CV% of 18%, which is slightly higher than the lowest standard. The highest IQC at 750ng/mg showed the highest CV% and the greatest variation at 48%. This may be because the fingernail matrix had become saturated, reducing the uptake of the IQC and creating more variation between the recovery.

The calibration curve shows linearity across all 6 calibration points with an R2 value of 0.997. A 6-point calibration curve was used based upon a UKAS (United Kingdom Accreditation Service) recommendation, because if one calibration point is inadequate, there is the option of removal without obscuring linearity. The calibration curves for all 6 analytes can be found in appendix three, a calibration had to be analysed per analyte, as a combined calibration standard was used for each of the 6 standards.

The limit of detection for this method is 1.885ng/mg This value was calculated from injecting numerous blank samples, calculating the limit of blank and determining 3 standard deviations calculating the limit of detection. The limit of quantification for this method is 5ng/mg; this is based on the lowest detectable standard used within the method calibration. The Limit of Blank was calculated at 0.8055 from using the maximum of the relative response data as a result of injecting numerous blank samples.

The summary table for the Tramadol validation shown in Table 10 was determined by

injecting 3 quality control samples, 5 replicates over 3 consecutive days. This was carried out to determine the precision and accuracy of the overall method. Looking at the results displayed in the table, the most accurate quality control standard for this method is the highest quality control standard at 750ng/mg. A %CV of 1.65 for the inter-day data shows that there is very little variation between the data identifying that the standard deviation is equal to 1.65% of the average. The smaller the concentration of the quality control standard displays that the standard deviation and the percentage coefficient of variance increases. This indicates that there is greater variation at the lower levels of the calibration, this is because the concentration at 7.5ng/mg is reaching the limit of the method and working at the threshold of the instrument causing a greater margin of error. This margin of error can be reflected by the mean values shown in Table 10. The mean values portrayed at the 7.5ng/mg quality control standard is much lower at 3.13ng/mg and 4.32ng/mg than the actual concentration value. The accuracy and precision of this method is fluctuating between the different levels of the quality control samples depending on the concentration. The highest concentration 750ng/mg quality control standard is displaying the greatest level of accuracy and precision as there is not much variation between the data points and the

29

mean values are within an acceptable range. The lowest quality control standard has the greatest level of variance as 7.5ng/mg concentration as it is on the threshold of the instruments capability. To improve this, the method would have to be run on a more sensitive instrument that has a higher sensitivity to reach the very low concentrations.

Figures 2, 3 and 4 illustrate the final concentrations displayed inter-day for each of the

3 quality control samples. It is evident that the 7.5ng/mg QC concentration displays the most variation between days, with Day 3 being the most unpredictable. This can be caused from column degradation or solvent interference. The 75ng/mg QC concentration shows the least variation between the inter-day injections. The QC concentration at 750ng/mg shows very little variation between the inter-day injections besides Day 3 at injection 5, where there is no data obtained for this QC standard because of this, this data point was removed from the statistical analysis as an outlier. The reason for no data calculated may be due to the sample evaporating and drying out therefore, no injection occurred or there was a pipetting issue and there wasn’t enough sample volume being pipetted into the well causing insufficient sample being injected onto the column. Reproducibility graphs for each of the individual IQC’s can be found in appendix 3. These graphs illustrate the reproducibility of each IQC in comparison to standard deviation.

The measurement of uncertainty was calculated at 95% confidence level. The uncertainty of measurement takes into account the outliers associated with this method and expresses any doubt that can be reflected within the result. The data shown in Table 10, displays clearly that the best measurement of uncertainty is associated with the highest quality control standard at 750ng/mg. Value of 3.24 for the intra-day data indicates that were isn’t much variation and little doubt within the results. The most unreliable and most doubtful confidence level is associated with the inter-day results for the lowest concentration quality control standard at 7.5ng/mg. The reasoning for this, as stated previously is that this concentration is down at the bottom end and at the limit of the instruments capabilities therefore, any slight change within the results will cause a large variation and increase the margins of error. In regards to the other results, the measurement of uncertainty ranges between 30 and 40, indicating that there is some level of doubt especially at the bottom end of the quality control standard concentration. To rectify this, it may be an idea to alter the concentration of all quality control standards. Currently the quality control standards were made up based upon the concentration of the calibration standards. The first QC concentration was based upon half way concentration between calibration level 1 and 2. The second quality control at 75ng/mg was based upon mid concentration between calibration levels 3 and 4 and finally the concentration of the top QC was midway concentration between calibration level 5 and 6. In a future study it would be advisable to increase the quality control standard concentrations to the upper third of the overall method. As a small part of the research the live samples after the first injection were left to stand at room temperature for 24 hours for stability purposes. This stability test was done to determine whether there were any significant changes in the concentration of Tramadol within the fingernail samples when not stored in a refrigerator. 24 hours after the samples were injected on the LC/MS the quality control samples for one of the validation methods were re-injected. The purpose of the stability test is to ensure that if any issues or complications arose such as when the instrument had any down time or there is a problem with power, the samples that are ready to be analysed are not affected dramatically. The results are shown in the figure 9.

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Figure 9 Tramadol Nail Stability Test Results

The stability test that was carried out was unsuccessful. This is displayed in figure 9 as there is no data shown for any of the 6 analytes including calibration. It can be seen in the chromatograms that there are no visible peaks only background noise. There is also no peak for the internal standard. This may be due to evaporation from the room temperature at which the samples were left, causing the samples to dry out. Another source may be that after the first injection there was not enough sample left in the 96 deep well plate for another injection to occur as 50PL is quite a large injection volume. In a future study this is a topic to investigate further if the method was to be UKAS accredited. Investigation would be to determine whether it would be possible to reconstitute samples that have been left at room temperature and to carry out a comparison study with samples kept at room temperature or refrigerated.

5.0 CONCLUSION

From the evidence presented herein, it is possible to state that for the method used it is possible to determine traces of tramadol when using the fingernail matrix. Even though the calibration range was limited and below that required for the patient samples, the Anova statistical analysis verified that the null hypothesis can be rejected and the alternative hypothesis can be accepted. It was calculated that fingernail samples produce a good recovery percentage with a range from 79% - >100% therefore there is a significant amount of analyte that can be eluted from the samples. The high levels of Tramadol discovered within the 6 fingernail samples may have been caused by a continuous use of tramadol over a protracted time period, in this case 4 years. Fingernail samples are easy to handle, there is very limited concern with there used in terms of biological hazard and there is little chance that patients will not be able to produce at least some sample for testing. Therefore the use of this matrix could be viable within the workplace drug testing industry in the near future. This research concludes that there is another matrix that can be used for workplace drug testing. In future studies there would be an opportunity to expand on this topic and research into the detection of other forms of drugs of abuse, including prescription drugs. In the foreseeable future there could be position within the workplace drug testing arena for a method that could be used when a hair sample is not possible.

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6.0 REFERENCES 1Misuse of Drugs Act (1971) 2 Tramadol update review report agenda item 6.1 expert committee on drug dependence Thirty‐sixth meeting. [place unknown]: World Health Organization; 2014 Jun 16. 3Verri P, Rustichelli C, Palazzoli F, Vandelli D, Marchesi F, Ferrari A, Licata M. Tramadol chronic abuse: An evidence from hair analysis by LC tandem MS. Journal of Pharmaceutical and Biomedical Analysis. 2015 Jan; 102:450–8. 4Talk to Frank. [place unknown: publisher unknown]. Tramadol [cited 2016 Mar 28]. Available from: http://www.talktofrank.com/drug/tramadol. 5Statistics OFN. Deaths related to drug poisoning in England and wales: 2014 registrations. [place unknown]: Office for National Statistics; 2015 Sep 3. 6Ardakani YH, Rouini M-R. Pharmacokinetics of tramadol and its three main metabolites in healthy male and female volunteers. Biopharmaceutics & Drug Disposition. 2007;28(9):527–34. 7Bastami S, Haage P, Kronstrand R, Kugelberg FC, Zackrisson A-L, Uppugunduri S. Pharmacogenetic aspects of tramadol pharmacokinetics and pharmacodynamics after a single oral dose. Forensic Science International. 2014 May; 238:125–32. 8Meyer MR, Rosenborg S, Stenberg M, Beck O. First report on the pharmacokinetics of tramadol and O-desmethyltramadol in exhaled breath compared to plasma and oral fluid after a single oral dose. Biochemical Pharmacology. 2015 Dec;98(3):502–10. 9Cappelle D, Yegles M, Neels H, van Nuijs ALN, De Doncker M, Maudens K, Covaci A, Crunelle CL. Nail analysis for the detection of drugs of abuse and pharmaceuticals: A review. Forensic Toxicology. 2014 Dec 10;33(1):12–36. 10Kidwell DA, Lee EH, DeLauder SF. Evidence for bias in hair testing and procedures to correct bias. Forensic Science International. 2000 Jan;107(1-3):39–61. 11Yaemsiri S, Hou N, Slining M, He K. Growth rate of human fingernails and toenails in healthy American young adults. Journal of the European Academy of Dermatology and Venereology. 2010 Apr;24(4):420–3. 12Madry MM, Rust KY, Guglielmello R, Baumgartner MR, Kraemer T. Metabolite to parent drug concentration ratios in hair for the differentiation of tramadol intake from external contamination and passive exposure. Forensic Science International. 2012 Nov;223(1-3):330–4. 13Shen M, Chen H, Xiang P. Determination of opiates in human fingernail—Comparison to hair. Journal of Chromatography B. 2014 Sep;967:84–9. 14Kim JY, Shin SH, In MK. Determination of amphetamine-type stimulants, ketamine and metabolites in fingernails by gas chromatography–mass spectrometry. Forensic Science International. 2010 Jan;194(1-3):108–14. 15Shu I, Jones J, Jones M, Lewis D, Negrusz A. Detection of drugs in nails: Three year experience. Journal of Analytical Toxicology. 2015 Sep 16;39(8):624–8. 16Scott J, Rashbaum WK, Paul Von Zielbauer Contributed Reporting. THE FERRY CRASH: OVERVIEW; 10 die as Staten island ferry slams into pier. N.Y. / Region. 2003 Oct 16 [cited 2016 Apr 16]. Available from: http://www.nytimes.com/2003/10/16/nyregion/the-ferry-crash-overview-10-die-as-staten-island-ferry-slams- into-pier.html. 17 Whyte A. New York city officials scapegoat captain in Staten island ferry disaster - world socialist web site. 2003 Nov 5 [cited 2016 Apr 16]. Available from: https://www.wsws.org/en/articles/2003/11/ferr-n05.html. 18Rashbaum WK, Chan S. Pilot and supervisor sentenced in ’03 Staten island ferry crash. New York Region. 2006 [cited 2016 Apr 16]. Available from: http://www.nytimes.com/2006/01/10/nyregion/10ferry.html?_r=0.

http://www.talktofrank.com/drug/tramadol

http://www.nytimes.com/2003/10/16/nyregion/the-ferry-crash-overview-10-die-as-staten-island-ferry-slams-into-pier.html

http://www.nytimes.com/2003/10/16/nyregion/the-ferry-crash-overview-10-die-as-staten-island-ferry-slams-into-pier.html

https://www.wsws.org/en/articles/2003/11/ferr-n05.html

http://www.nytimes.com/2006/01/10/nyregion/10ferry.html?_r=0

32

19Whittington CJ, Kendall T, Fonagy P, Cottrell D, Cotgrove A, Boddington E. Selective serotonin reuptake inhibitors in childhood depression: Systematic review of published versus unpublished data. The Lancet. 2004 Apr;363(9418):1341–5. 20 Godoy ALPC, Rocha A, da Silva Souza C, Lanchote VL. Pharmacokinetics of venlafaxine enantiomers and their metabolites in psoriasis patients. The Journal of Clinical Pharmacology. 2015 Nov 11;56(5):567–75. 21 Liu Y, Jann M, Vandenberg C, Eap CB, Shamsi SA. Development of an enantioselective assay for simultaneous separation of venlafaxine and O-desmethylvenlafaxine by micellar electrokinetic chromatography-tandem mass spectrometry: Application to the analysis of drug–drug interaction. Journal of Chromatography A. 2015 Nov; 1420:119–28. 22Jornil J, Nielsen TS, Rosendal I, Ahlner J, Zackrisson AL, Boel LWT, Brock B. A poor metabolizer of both CYP2C19 and CYP2D6 identified by mechanistic pharmacokinetic simulation in a fatal drug poisoning case involving venlafaxine. Forensic Science International. 2013 Mar;226(1-3):e26–31. 23Markowitz JS, Patrick KS. Venlafaxine-tramadol similarities. Medical Hypotheses. 1998 Aug;51(2):167–8. 24 Annesley TM. Ion suppression in mass Spectrometry. Clinical Chemistry. 2003 Jul 1;49(7):1041–4.

33

Appendix One:

Health and Safety

Synergy Health Laboratory Services Document ID: 200.19007, December 2015 - Version 2 Controlled Document Page 1 of 1

HEALTH AND SAFETY RISK ASSESSMENT Location Food Testing Process Use of Homogeniser Fast Prep

24 1. Operation, task or area to be assessed:

Homogenising of tissue or hair samples 2. Potential hazardous conditions or unsafe practices:

Electrical, fast moving parts, pathology samples 3. Current risk control measures:

PAT testing, Lid locks when in use, PPE, Training provided in use, Risk assessments for samples being processed

Risk Assessment Likelihood of occurrence Number of people at risk Degree of Harm

5 = very likely 4 = probable 3 = possible 2 = unlikely 1 = no risk

2

5 = 50 plus 4 = 25-50 3 = 10-25

2 = 5-10 1 = Under 5

2

5 = fatal or multi fatal 4 = Major injury (RIDDOR)

3 = three day injury 2 = minor – first aid only

1 = No injury occurs

3

Risk Assessment ratings [multiply the scores] Likelihood of Occurrence

Number of people at risk

Degree of Harm

Total Degree of Risk

2 2 3 12 Low

Risk Rating: V/Low 0-9 Low 10-24 Medium 25-49 High 50-99 V/High 100+

4. Additional control measures required to reduce risk: N/A

Target Date: 5. Comments by Assessor:

Low risk if used correctly 6. Relevant standards, law and guidelines:

GLP Assessed by: Dr Philip Kindred

Prepared Date: 21/12/2015 Review Date: 21/12/2019

Synergy Health Laboratory Services Document ID: 200.80340, January 2015 - Version 2 Controlled Document Page 1 of 1

HEALTH AND SAFETY RISK ASSESSMENT Location Toxicology Process Turbovap sample concentrator 1. Operation, task or area to be assessed:

Drying down extracted samples 2. Potential hazardous conditions or unsafe practices:

Biohazardous materials, solvent exposure, hot surfaces 3. Current risk control measures:

Equipment designed to operate in these conditions, solvents vented through fume hood, staff vaccinated against BBV, samples extracted which removes pathogens



2

5 = 50 plus 4 = 25-50 3 = 10-25

2 = 5-10 1 = Under 5

1

5 = fatal or multi fatal 4 = Major injury (RIDDOR)

3 = three day injury 2 = minor – first aid only

1 = No injury occurs

2



Degree of Harm


2 1 2 4 V/Low


4. Additional control measures required to reduce risk: n.a


n.a 6. Relevant standards, law and guidelines:

Assessed by: Samantha Osmond


Synergy Health Laboratory Services Document ID: 200.12010, March 2016 - Version 3 Controlled Document Page 1 of 1

HEALTH AND SAFETY RISK ASSESSMENT Location Toxicology Confirmation Process LCMS Sample Processing 1. Operation, task or area to be assessed:

LCMS following sample preparation 2. Potential hazardous conditions or unsafe practices:

Flammable and toxic solvents, high temperatures and infection risks 3. Current risk control measures:

Staff trained to perform procedure, wear lab coats at all times and gloves when handling open samples or solvents. High temperature areas are clearly labelled, and solvents handled in a solvent cabinet. Solvents are stored in solvent bins and disposed of by a specialist company



3 5 = 50 plus

4 = 25-50 3 = 10-25

2 = 5-10 1 = Under 5

1 5 = fatal or multi fatal

4 = Major injury (RIDDOR) 3 = three day injury

2 = minor – first aid only 1 = No injury occurs

4



Degree of Harm


3 1 4 12 Low


4. Additional control measures required to reduce risk: None


Well controlled procedure 6. Relevant standards, law and guidelines:

Assessed by: Philip Kindred


Synergy Health Laboratory Services Document ID: 300.80510, April 2014 - Version 2 Controlled Document Page 1 of 1

COSHH ASSESSMENT RECORD SUBSTANCE

Cerilliant standards in methanol (1mL vials) MSDS Present

Yes

PROCESS/OR USE

Preparation of standards

MSDS Rev Number

RISKS TO HEALTH

Low

EH40 (MEL) If Applicable

8 hour TWA

15 minute STEL

CAN THE EXPOSURE BE ELIMINATED No

FIRST AID MEASURES Inhalation No Skin Contact Yes Ingestion Yes Eye Contact Yes

POSSIBLE EFFECTS FROM EXPOSURE Skin and eye contact can cause irritation, ingestion of significant quantities can lead to blindness

FIRE FIGHTING MEASURES (Tick Box)

SPILLAGE & ENVIRONMENTAL CONTROLS Absorb onto towelling or spillage kit and discard as clinical waste Wash small quantities down drains with plenty of water

PPE

(Tick Box)

CONTROL MEASURES

Store in spark free freezer Only trained staff may handle Small volumes handled

Assessment By: Richard Evers Date 24/4/2014 Review Date April 2016

2000ppm

250

X

X X X

Synergy Health Laboratory Services Document ID: 300.81400, January 2015 - Version 2 Controlled Document Page 1 of 1

COSHH ASSESSMENT RECORD SUBSTANCE Dichloromethane MSDS

Present

Yes

PROCESS OR USE

Laboratory solvent MSDS Rev Number

5

RISKS TO HEALTH

Irritating to respiratory tract, may cause cancer

EH40 (WEL)ppm If Applicable

8 hour TWA 100

15 minute STEL 300


FIRST AID MEASURES Inhalation Yes Move to fresh air Skin Contact Yes wash with plenty of water Ingestion Yes wash mouth with plenty of water Eye Contact Yes wash with plenty of water

POSSIBLE EFFECTS FROM EXPOSURE May cause weakness, shortage of breath and dizziness. May cause blurred vision with eye contact and abdominal pain with ingestion


SPILLAGE & ENVIRONMENTAL CONTROLS Do not empty down drains. Contact specialist removal company for disposal

PPE

(Tick Box)

CONTROL MEASURES

Staff training and PPE

Assessment By:

Samantha Osmond

Date 08/01/2015 Review Date 08/01/2017


COSHH ASSESSMENT RECORD DAC 92

SUBSTANCE

Acetic acid MSDS Present

YES

PROCESS/OR USE

Various uses

MSDS Rev Number

2

RISKS TO HEALTH

Caustic


8 hour TWA

15 minute STEL

CAN THE EXPOSURE BE ELIMINATED NO

FIRST AID MEASURES Inhalation Remove to fresh air. Skin Contact Wash with copious amounts of water. Remove contaminated clothing. Ingestion Wash out mouth with water. Eye Contact Flush with copious amounts of water.

POSSIBLE EFFECTS FROM EXPOSURE Respiratory and skin irritation.


SPILLAGE & ENVIRONMENTAL CONTROLS Try to prevent from entering drains Residues after analysis flushed with copious amounts of water.

PPE

(Tick Box)

CONTROL MEASURES

Stored in a cupboard. Prepare in fumehood


X X

X X X

Synergy Health Laboratory Services Document ID: 300.81800, May 2014 - Version 3 Controlled Document Page 1 of 1

COSHH ASSESSMENT RECORD

SUBSTANCE

Acetonitrile MSDS Present

YES

PROCESS/OR USE

Laboratory testing.

MSDS Rev Number

6.1

RISKS TO HEALTH


8 hour TWA

15 minute STEL


FIRST AID MEASURES Inhalation Remove to fresh air. Skin Contact Wash with copious amounts of water. Remove contaminated clothing. Ingestion Seek immediate medical attention. Eye Contact Flush with copious amounts of water.

POSSIBLE EFFECTS FROM EXPOSURE Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes.


SPILLAGE & ENVIRONMENTAL CONTROLS Try to prevent from entering drains

PPE

(Tick Box)

CONTROL MEASURES

Good laboratory practice. Self contained breathing apparatus to be worn in the event of a fire.

Assessment By: Richard Evers Date 27/05/14 Review Date May 2016

X

X X X X


COSHH ASSESSMENT RECORD SUBSTANCE

Cerilliant standards in acetonitrile (1mL vials) MSDS Present

Yes

PROCESS/OR USE

Preparation of standards

MSDS Rev Number

RISKS TO HEALTH

Low


8 hour TWA

15 minute STEL


FIRST AID MEASURES Inhalation No Skin Contact Yes Ingestion Yes Eye Contact Yes

POSSIBLE EFFECTS FROM EXPOSURE Skin and eye contact can cause irritation, ingestion of significant quantities can lead to blindness


SPILLAGE & ENVIRONMENTAL CONTROLS Absorb onto towelling or spillage kit and discard as clinical waste Wash small quantities down drains with plenty of water

PPE

(Tick Box)

CONTROL MEASURES

Store in spark free freezer Only trained staff may handle Small volumes handled


40 60

X

X X X

Synergy Health Laboratory Services Document ID: 300.8930, June 2014 - Version 2 Controlled Document Page 1 of 1


SUBSTANCE

Formic acid MSDS Present

YES

PROCESS/OR USE

Laboratory testing.

MSDS Rev Number

5.6

RISKS TO HEALTH


8 hour TWA

15 minute STEL


FIRST AID MEASURES Inhalation Remove to fresh air. Skin Contact Wash with copious amounts of water. Remove contaminated clothing. Ingestion Seek immediate medical attention. Do NOT induce vomiting. Eye Contact Flush with copious amounts of water.

POSSIBLE EFFECTS FROM EXPOSURE Causes severe skin burns and eye damage.


SPILLAGE & ENVIRONMENTAL CONTROLS Try to prevent from entering drains

PPE

(Tick Box)

CONTROL MEASURES

Good laboratory practice. Make up solutions using formic acid in fumehood Self contained breathing apparatus to be worn in the event of a fire.

Assessment By: Richard Evers Date 13/06/14 Review Date June 2016

X

X X X X

Synergy Health Laboratory Services Document ID: 300.81700, March 2016 - Version 4 Controlled Document Page 1 of 1


SUBSTANCE

Methonal MSDS Present

YES

PROCESS/OR USE

Various uses

MSDS Rev Number

6.3

RISKS TO HEALTH


8 hour TWA

15 minute STEL


FIRST AID MEASURES Inhalation Remove to fresh air and them lay down Skin Contact Wash with copious amounts of water. Ingestion Wash out mouth with water. Eye Contact Flush with copious amounts of water.

POSSIBLE EFFECTS FROM EXPOSURE Toxic by inhalation.


SPILLAGE & ENVIRONMENTAL CONTROLS Try to prevent from entering drains Dispose of in a waste solvent bottle. Containment level 2.

PPE

(Tick Box)

CONTROL MEASURES

Store in flammable solvent container. Used in a fume cabinet.

Assessment By: Philip Kindred Date 07/03/2016 Review Date March 2020

X X

X X X

Synergy Health Laboratory Services Document ID: 300.81300, July 2014 - Version 2 Controlled Document Page 1 of 1

COSHH ASSESSMENT RECORD SUBSTANCE Saliva Controls MSDS

Present

No

PROCESS OR USE

Used for screening and LCMSMS MSDS Rev Number

RISKS TO HEALTH

Potentially Infectious

EH40 (WEL)ppm If Applicable

8 hour TWA

15 minute STEL


FIRST AID MEASURES Inhalation No Skin Contact Yes Wash with copious amounts of water Ingestion Yes Wash out mouth with water Eye Contact Yes Flush with copious amounts of water

POSSIBLE EFFECTS FROM EXPOSURE


SPILLAGE & ENVIRONMENTAL CONTROLS Dispose of in clinical waste bin Containment level 1

PPE

(Tick Box)

CONTROL MEASURES

Store in Fridge

Assessment By: Richard Evers Date 14/07/14 Review Date July 2016

/9Safety data sheet

according to 1907/2006/EC, Article 31Printing date 02.10.2013 Revision: 02.10.2013Version number 2

37.0

1 Identification of the substance/mixture and of the company/undertaking

· Product identifier

· Trade name: Tramadol Hydrochloride 1.0 mg/ml in Methanol (as free base)· Reference number: LGCAMP0007.00-01· Relevant identified uses of the substance or mixture and uses advised against

No further relevant information available.· Application of the substance / the preparation Reference material for laboratory use only· Manufacturer/Supplier:

LGC Limited Tel :+44 (0) 20 8943 7000Queens Road Fax :+44 (0) 20 8943 2767Teddington eMail : [email protected] TW11 0LY Web : www.lgcstandards.comUNITED KINGDOM

· Further information obtainable from:Product safety departmenteMail : [email protected]

· Emergency telephone number: +44 (0) 20 8943 7000 (Monday - Friday : 8am - 5pm)

2 Hazards identification

· Classification of the substance or mixture· Classification according to Regulation (EC) No 1272/2008

GHS02 flame

Flam. Liq. 2 H225 Highly flammable liquid and vapour.

GHS06 skull and crossbones

Acute Tox. 3 H301 Toxic if swallowed.Acute Tox. 3 H311 Toxic in contact with skin.Acute Tox. 3 H331 Toxic if inhaled.

GHS08 health hazard

STOT SE 1 H370 Causes damage to organs.

· Classification according to Directive 67/548/EEC or Directive 1999/45/ECT+; Very toxic

R39/26/27: Very toxic: danger of very serious irreversible effects through inhalation and in contact withskin.

T; Toxic

R23/24/25: Toxic by inhalation, in contact with skin and if swallowed.

F; Highly flammable

R11: Highly flammable.· Information concerning particular hazards for human and environment:

The product has to be labelled due to the calculation procedure of the "General Classification guideline forpreparations of the EU" in the latest valid version.

(Contd. on page 2) GB

/9Safety data sheet


Trade name: Tramadol Hydrochloride 1.0 mg/ml in Methanol (as free base)

(Contd. from page 1)

37.0

· Classification system:The classification is according to the latest editions of the EU-lists, and extended by company and literaturedata.

· Label elements· Labelling according to Regulation (EC) No 1272/2008

The product is classified and labelled according to the CLP regulation.· Hazard pictograms

GHS02 GHS06 GHS08

· Signal word Danger

· Hazard-determining components of labelling:MethanolTramadol Hydrochloride

· Hazard statementsH225 Highly flammable liquid and vapour.H301+H311+H331 Toxic if swallowed, in contact with skin or if inhaled.H370 Causes damage to organs.

· Precautionary statementsP210 Keep away from heat/sparks/open flames/hot surfaces. - No smoking.P301+P310 IF SWALLOWED: Immediately call a POISON CENTER or doctor/physician.P303+P361+P353 IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin

with water/shower.P361 Remove/Take off immediately all contaminated clothing.P405 Store locked up.P501 Dispose of contents/container in accordance with local/regional/national/international

regulations.· Other hazards· Results of PBT and vPvB assessment· PBT: Not applicable.· vPvB: Not applicable.

3 Composition/information on ingredients· Chemical characterization: Mixtures· Description: Mixture: consisting of the following components.· Dangerous components:

CAS: 67-56-1EINECS: 200-659-6RTECS: PC1400000

Methanol T R23/24/25-39/23/24/25; F R11 Flam. Liq. 2, H225; Acute Tox. 3, H301; Acute Tox. 3, H311; Acute

Tox. 3, H331; STOT SE 1, H370

> 99%

CAS: 36282-47-0RTECS: -

Tramadol Hydrochloride T R25; Xi R38; N R51/53 Acute Tox. 3, H301; Aquatic Chronic 2, H411; Skin Irrit. 2, H315

< 1.0%

· Additional information: For the wording of the listed risk phrases refer to section 16. GB

(Contd. on page 3)

/9Safety data sheet




37.0

4 First aid measures

· Description of first aid measures· General information:

Immediately remove any clothing soiled by the product.Remove breathing equipment only after contaminated clothing has been completely removed.In case of irregular breathing or respiratory arrest provide artificial respiration.

· After inhalation:Supply fresh air or oxygen; call for doctor.In case of unconsciousness place patient in recovery position for transport.

· After skin contact: Immediately wash with water and soap and rinse thoroughly.· After eye contact: Rinse opened eye for several minutes under running water. Then consult a doctor.· After swallowing:

Rinse mouth. Do not induce vomiting.Call a doctor immediately.

· Information for doctor:· Most important symptoms and effects, both acute and delayed No further relevant information available.· Indication of any immediate medical attention and special treatment needed

No further relevant information available.

5 Firefighting measures

· Extinguishing media· Suitable extinguishing agents:

CO2, powder or water spray. Fight larger fires with water spray or alcohol resistant foam.· Special hazards arising from the substance or mixture

Formation of toxic gases is possible during heating or in case of fire.· Advice for firefighters· Protective equipment:

Mouth respiratory protective device.Wear self-contained respiratory protective device.

6 Accidental release measures

· Personal precautions, protective equipment and emergency proceduresWear protective equipment. Keep unprotected persons away.

· Environmental precautions:Dilute with plenty of water.Do not allow to enter sewers/ surface or ground water.

· Methods and material for containment and cleaning up:Absorb with liquid-binding material (sand, diatomite, acid binders, universal binders, sawdust).Dispose of contaminated material as waste according to item 13.Ensure adequate ventilation.

· Reference to other sectionsSee Section 7 for information on safe handling.See Section 8 for information on personal protection equipment.See Section 13 for disposal information.

7 Handling and storage

· Handling:· Precautions for safe handling

Ensure good ventilation/extraction at the workplace.(Contd. on page 4)

GB

/9Safety data sheet




37.0

Store in cool, dry place in tightly closed receptacles.Open and handle receptacle with care.Prevent formation of aerosols.

· Information about fire - and explosion protection:Keep ignition sources away - Do not smoke.Protect from heat.Protect against electrostatic charges.Keep respiratory protective device available.

· Conditions for safe storage, including any incompatibilities· Storage:· Requirements to be met by storerooms and receptacles:

Store in a cool location.Please refer to the manufacturer's certificate for specific storage and transport temperature conditions.Store only in the original receptacle.Keep container in a well-ventilated place. Keep away from sources of ignition and heat.

· Information about storage in one common storage facility: Store away from foodstuffs.· Further information about storage conditions:

Keep container tightly sealed.Store in cool, dry conditions in well sealed receptacles.Protect from heat and direct sunlight.

· Specific end use(s) No further relevant information available.

8 Exposure controls/personal protection

· Additional information about design of technical facilities: No further data; see item 7.

· Control parameters· Ingredients with limit values that require monitoring at the workplace:

67-56-1 MethanolWEL Short-term value: 333 mg/m³, 250 ppm

Long-term value: 266 mg/m³, 200 ppmSk

· Additional information: Lists used were valid at the time of SDS preparation.

· Exposure controls· Personal protective equipment:· General protective and hygienic measures:

Keep away from foodstuffs, beverages and feed.Immediately remove all soiled and contaminated clothingWash hands before breaks and at the end of work.Store protective clothing separately.Avoid contact with the eyes and skin.

· Respiratory protection:In case of brief exposure or low pollution use respiratory filter device. In case of intensive or longer exposureuse self-contained respiratory protective device.

· Protection of hands:

Protective gloves

The glove material has to be impermeable and resistant to the product/ the substance/ the preparation.Due to missing tests no recommendation to the glove material can be given for the product/ the preparation/the chemical mixture.


/9Safety data sheet




37.0

Selection of the glove material on consideration of the penetration times, rates of diffusion and thedegradation

· Material of glovesThe selection of the suitable gloves does not only depend on the material, but also on further marks of qualityand varies from manufacturer to manufacturer. As the product is a preparation of several substances, theresistance of the glove material can not be calculated in advance and has therefore to be checked prior to theapplication.

· Penetration time of glove materialThe exact break through time has to be found out by the manufacturer of the protective gloves and has to beobserved.

· Eye protection:

Tightly sealed goggles

9 Physical and chemical properties· Information on basic physical and chemical properties· General Information· Appearance:

Form: LiquidColour: Colourless

· Odour: Alcohol-like· Odour threshold: Not determined.

· pH-value: Not determined.

· Change in conditionMelting point/Melting range: -98 °CBoiling point/Boiling range: 65 °C

· Flash point: 12 °C

· Flammability (solid, gaseous): Not determined.

· Ignition temperature:Decomposition temperature: Not determined.

· Self-igniting: Product is not selfigniting.

· Danger of explosion: Product is not explosive. However, formation of explosive air/vapourmixtures is possible.

· Explosion limits:Lower: 6 Vol %Upper: 50 Vol %

· Vapour pressure: Not determined.

· Density at 20 °C: 0.8 g/cm³· Relative density Not determined.· Vapour density Not determined.· Evaporation rate Not determined.

· Solubility in / Miscibility withwater: Fully miscible.


/9Safety data sheet




37.0

· Partition coefficient (n-octanol/water): Not determined.

· Viscosity:Dynamic: Not determined.Kinematic: Not determined.

· Other information No further relevant information available.

10 Stability and reactivity· Reactivity Stable under normal conditions.· Chemical stability Stable under normal conditions.· Thermal decomposition / conditions to be avoided:

Formation of toxic gases is possible during heating or in case of fire.· Possibility of hazardous reactions No dangerous reactions known.· Conditions to avoid

Sources of ignition.Heat.

· Incompatible materials:Strong oxidizing agents.Strong acids.Reducing agents.Alkali metals.

· Hazardous decomposition products: Formation of toxic gases is possible during heating or in case of fire.

11 Toxicological information· Information on toxicological effects· Acute toxicity:· LD/LC50 values relevant for classification:

67-56-1 MethanolOral LD50 5628 mg/kg (rat)Dermal LD50 15800 mg/kg (rabbit)Inhalative LC50/4 h 83.9 mg/l (rat)

· Primary irritant effect:· on the skin: No irritating effect.· on the eye: No irritating effect.· Sensitization: No sensitizing effects known.· Additional toxicological information: Toxic

12 Ecological information· Toxicity· Aquatic toxicity:

67-56-1 MethanolLC50/96 h 24.000 mg/l (fish)

· Persistence and degradability No further relevant information available.· Behaviour in environmental systems:· Bioaccumulative potential No further relevant information available.· Mobility in soil No further relevant information available.


/9Safety data sheet




37.0

· Additional ecological information:· General notes:

Water hazard class 1 (German Regulation) (Self-assessment): slightly hazardous for waterDo not allow undiluted product to reach ground water, water course or sewage system.

· Results of PBT and vPvB assessment· PBT: Not applicable.· vPvB: Not applicable.· Other adverse effects No further relevant information available.

13 Disposal considerations· Waste treatment methods· Recommendation

Must not be disposed of together with household garbage. Do not allow product to reach sewage system.· European waste catalogue

Waste disposal key numbers from EWC have to be assigned depending on origin and processing.

· Uncleaned packaging:· Recommendation: Dispose of in accordance with national regulations.· Recommended cleansing agents: Water, if necessary together with cleansing agents.

14 Transport information· UN-Number· ADR, IMDG, IATA UN1230· ADR 1230 METHANOL, mixture· IMDG, IATA METHANOL, mixture

· Transport hazard class(es)· ADR, IMDG, IATA

· Class 3 Flammable liquids.· Label 3+6.1

· Packing group· ADR, IMDG, IATA II

· Environmental hazards:· Marine pollutant: No

· Special precautions for user Warning: Flammable liquids.· Danger code (Kemler): 336· EMS Number: F-E,S-D

· Transport in bulk according to Annex II ofMARPOL73/78 and the IBC Code Not applicable.

· Transport/Additional information:· ADR· Excepted quantities (EQ): E2· Limited quantities (LQ) 1L· Transport category 2


/9Safety data sheet




37.0

· Tunnel restriction code D/E

· UN "Model Regulation": UN1230, METHANOL, mixture, 3 (6.1), II

15 Regulatory information· Safety, health and environmental regulations/legislation specific for the substance or mixture· Philippines Inventory of Chemicals and Chemical Substances

67-56-1 Methanol

· Australian Inventory of Chemical SubstancesNone of the ingredients is listed.

· Standard for the Uniform Scheduling of Drugs and Poisons67-56-1 Methanol S5, S6

· Chemical safety assessment: A Chemical Safety Assessment has not been carried out.

16 Other informationThe information in this safety data sheet (SDS) has been prepared with due care and is true and accurate tothe best of our knowledge. The user must determine the suitability of the information for its particularpurpose, ensure compliance with existing laws and regulations, and be aware that other or additional safetyor performance considerations may arise when using, handling and/ or storing the material. The informationin this SDS does not purport to be all inclusive or a guarantee as to the properties of the material supplied,and should be used only as a guide. LGC makes no warranties or representations as to the accuracy andcompleteness of the information contained herein, shall not be held responsible for the suitability of thisinformation for the user's intended purposes or the consequences of such use, and shall not be liable for anydamage or loss, howsoever arising, direct or otherwise.

· Relevant phrasesH225 Highly flammable liquid and vapour.H301 Toxic if swallowed.H311 Toxic in contact with skin.H315 Causes skin irritation.H331 Toxic if inhaled.H370 Causes damage to organs.H411 Toxic to aquatic life with long lasting effects.

R11 Highly flammable.R23/24/25 Toxic by inhalation, in contact with skin and if swallowed.R25 Toxic if swallowed.R38 Irritating to skin.R39/23/24/25 Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if

swallowed.R51/53 Toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.

· Abbreviations and acronyms:RID: Règlement international concernant le transport des marchandises dangereuses par chemin de fer (Regulations Concerning theInternational Transport of Dangerous Goods by Rail)ICAO: International Civil Aviation OrganizationADR: Accord européen sur le transport des marchandises dangereuses par Route (European Agreement concerning the InternationalCarriage of Dangerous Goods by Road)IMDG: International Maritime Code for Dangerous GoodsIATA: International Air Transport AssociationGHS: Globally Harmonized System of Classification and Labelling of ChemicalsEINECS: European Inventory of Existing Commercial Chemical SubstancesELINCS: European List of Notified Chemical SubstancesCAS: Chemical Abstracts Service (division of the American Chemical Society)LC50: Lethal concentration, 50 percent


/9Safety data sheet




37.0

LD50: Lethal dose, 50 percent· Sources

Tables 3.1 and 3.2 from Annex 6 of EC 1272/2008, EC 1907/2006, EH40/2005 as amended 2011, Registry ofToxic Effects of Chemical Substances (RTECS), The Dictionary of Substances and their Effects, 1st Edition,IUCLID.

GB

/8Safety data sheet


36.0.36

1 Identification of the substance/mixture and of the company/undertaking

· Product identifier

· Trade name: Tramadol-13C-D3 Hydrochloride 0.1 mg/ml in Methanol (as free base)· Reference number: LGCAMP0007.90-02· Relevant identified uses of the substance or mixture and uses advised against

No further relevant information available.· Application of the substance / the preparation Reference material for laboratory use only· Manufacturer/Supplier:

LGC Limited Tel :+44 (0) 20 8943 7000Queens Road Fax :+44 (0) 20 8943 2767Teddington eMail : [email protected] TW11 0LY Web : www.lgcstandards.comUNITED KINGDOM

· Further information obtainable from:Product safety departmenteMail : [email protected]

· Emergency telephone number: +44 (0) 20 8943 7000 (Monday - Friday : 8am - 5pm)

2 Hazards identification

· Classification of the substance or mixture· Classification according to Regulation (EC) No 1272/2008

GHS02 flame

Flam. Liq. 2 H225 Highly flammable liquid and vapour.

GHS06 skull and crossbones

Acute Tox. 3 H301 Toxic if swallowed.Acute Tox. 3 H311 Toxic in contact with skin.Acute Tox. 3 H331 Toxic if inhaled.

GHS08 health hazard

STOT SE 1 H370 Causes damage to organs.

· Classification according to Directive 67/548/EEC or Directive 1999/45/ECT; Toxic

R23/24/25-39/23/24/25: Toxic by inhalation, in contact with skin and if swallowed. Toxic: danger of veryserious irreversible effects through inhalation, in contact with skin and ifswallowed.

F; Highly flammable

R11: Highly flammable.· Information concerning particular hazards for human and environment:

The product has to be labelled due to the calculation procedure of the "General Classification guideline forpreparations of the EU" in the latest valid version.

· Classification system:The classification is according to the latest editions of the EU-lists, and extended by company and literaturedata.


/8Safety data sheet


Trade name: Tramadol-13C-D3 Hydrochloride 0.1 mg/ml in Methanol (as free base)


36.0.36

· Label elements· Labelling according to Regulation (EC) No 1272/2008

The product is classified and labelled according to the CLP regulation.· Hazard pictograms

GHS02 GHS06 GHS08

· Signal word Danger

· Hazard-determining components of labelling:MethanolTramadol-13C-D3 Hydrochloride

· Hazard statementsH225 Highly flammable liquid and vapour.H301+H311+H331 Toxic if swallowed, in contact with skin or if inhaled.H370 Causes damage to organs.

· Precautionary statementsP210 Keep away from heat/sparks/open flames/hot surfaces. - No smoking.P301+P310 IF SWALLOWED: Immediately call a POISON CENTER or doctor/physician.P303+P361+P353 IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin

with water/shower.P361 Remove/Take off immediately all contaminated clothing.P405 Store locked up.P501 Dispose of contents/container in accordance with local/regional/national/international

regulations.· Other hazards· Results of PBT and vPvB assessment· PBT: Not applicable.· vPvB: Not applicable.

3 Composition/information on ingredients· Chemical characterization: Mixtures· Description: Mixture: consisting of the following components.· Dangerous components:

CAS: 67-56-1EINECS: 200-659-6RTECS: PC1400000

Methanol T R23/24/25-39/23/24/25; F R11 Flam. Liq. 2, H225; Acute Tox. 3, H301; Acute Tox. 3, H311; Acute

Tox. 3, H331; STOT SE 1, H370

>99%

RTECS: - Tramadol-13C-D3 Hydrochloride T R25; Xi R38; N R51/53 Acute Tox. 3, H301; Aquatic Chronic 2, H411; Skin Irrit. 2, H315

< 0.1%

· Additional information: For the wording of the listed risk phrases refer to section 16.

4 First aid measures· Description of first aid measures· General information:

Immediately remove any clothing soiled by the product.Remove breathing equipment only after contaminated clothing has been completely removed.


/8Safety data sheet




36.0.36

In case of irregular breathing or respiratory arrest provide artificial respiration.· After inhalation:

Supply fresh air or oxygen; call for doctor.In case of unconsciousness place patient in recovery position for transport.

· After skin contact: Immediately wash with water and soap and rinse thoroughly.· After eye contact: Rinse opened eye for several minutes under running water. Then consult a doctor.· After swallowing:

Rinse mouth. Do not induce vomiting.Call a doctor immediately.

· Information for doctor:· Most important symptoms and effects, both acute and delayed No further relevant information available.· Indication of any immediate medical attention and special treatment needed

No further relevant information available.

5 Firefighting measures

· Extinguishing media· Suitable extinguishing agents:

CO2, powder or water spray. Fight larger fires with water spray or alcohol resistant foam.· Special hazards arising from the substance or mixture

Formation of toxic gases is possible during heating or in case of fire.· Advice for firefighters· Protective equipment:

Mouth respiratory protective device.Wear self-contained respiratory protective device.

6 Accidental release measures

· Personal precautions, protective equipment and emergency proceduresWear protective equipment. Keep unprotected persons away.

· Environmental precautions:Dilute with plenty of water.Do not allow to enter sewers/ surface or ground water.

· Methods and material for containment and cleaning up:Absorb with liquid-binding material (sand, diatomite, acid binders, universal binders, sawdust).Dispose of contaminated material as waste according to item 13.Ensure adequate ventilation.

· Reference to other sectionsSee Section 7 for information on safe handling.See Section 8 for information on personal protection equipment.See Section 13 for disposal information.

7 Handling and storage

· Handling:· Precautions for safe handling

Ensure good ventilation/extraction at the workplace.Store in cool, dry place in tightly closed receptacles.Open and handle receptacle with care.Prevent formation of aerosols.

· Information about fire - and explosion protection:Keep ignition sources away - Do not smoke.Protect against electrostatic charges.


/8Safety data sheet




36.0.36

Keep respiratory protective device available.

· Conditions for safe storage, including any incompatibilities· Storage:· Requirements to be met by storerooms and receptacles:

Store in a cool location.Please refer to the manufacturer's certificate for specific storage and transport temperature conditions.Store only in the original receptacle.Keep container in a well-ventilated place. Keep away from sources of ignition and heat.

· Information about storage in one common storage facility: Store away from foodstuffs.· Further information about storage conditions:

Keep container tightly sealed.Store in cool, dry conditions in well sealed receptacles.

· Specific end use(s) No further relevant information available.

8 Exposure controls/personal protection

· Additional information about design of technical facilities: No further data; see item 7.

· Control parameters· Ingredients with limit values that require monitoring at the workplace:

67-56-1 MethanolWEL Short-term value: 333 mg/m³, 250 ppm

Long-term value: 266 mg/m³, 200 ppmSk

· Additional information: Lists used were valid at the time of SDS preparation.

· Exposure controls· Personal protective equipment:· General protective and hygienic measures:

Keep away from foodstuffs, beverages and feed.Immediately remove all soiled and contaminated clothingWash hands before breaks and at the end of work.Store protective clothing separately.Avoid contact with the eyes and skin.

· Respiratory protection:In case of brief exposure or low pollution use respiratory filter device. In case of intensive or longer exposureuse self-contained respiratory protective device.

· Protection of hands:

Protective gloves

The glove material has to be impermeable and resistant to the product/ the substance/ the preparation.Due to missing tests no recommendation to the glove material can be given for the product/ the preparation/the chemical mixture.Selection of the glove material on consideration of the penetration times, rates of diffusion and thedegradation

· Material of glovesThe selection of the suitable gloves does not only depend on the material, but also on further marks of qualityand varies from manufacturer to manufacturer. As the product is a preparation of several substances, theresistance of the glove material can not be calculated in advance and has therefore to be checked prior to theapplication.


/8Safety data sheet




36.0.36

· Penetration time of glove materialThe exact break through time has to be found out by the manufacturer of the protective gloves and has to beobserved.

· Eye protection:

Tightly sealed goggles

9 Physical and chemical properties· Information on basic physical and chemical properties· General Information· Appearance:

Form: LiquidColour: Colourless

· Odour: Alcohol-like· Odour threshold: Not determined.

· pH-value: Not determined.

· Change in conditionMelting point/Melting range: -98 °CBoiling point/Boiling range: 65 °C

· Flash point: 12 °C

· Flammability (solid, gaseous): Not determined.

· Ignition temperature:Decomposition temperature: Not determined.

· Self-igniting: Product is not selfigniting.

· Danger of explosion: Product is not explosive. However, formation of explosive air/vapourmixtures is possible.

· Explosion limits:Lower: 6 Vol %Upper: 50 Vol %

· Vapour pressure: Not determined.

· Density at 20 °C: 0.8 g/cm³· Relative density Not determined.· Vapour density Not determined.· Evaporation rate Not determined.

· Solubility in / Miscibility withwater: Fully miscible.

· Partition coefficient (n-octanol/water): Not determined.

· Viscosity:Dynamic: Not determined.Kinematic: Not determined.

· Other information No further relevant information available. GB

(Contd. on page 6)

/8Safety data sheet




36.0.36

10 Stability and reactivity

· Reactivity Stable under normal conditions.· Chemical stability Stable under normal conditions.· Thermal decomposition / conditions to be avoided:

Formation of toxic gases is possible during heating or in case of fire.· Possibility of hazardous reactions No dangerous reactions known.· Conditions to avoid

Sources of ignition.Heat.

· Incompatible materials:Strong oxidizing agents.Strong acids.Reducing agents.Alkali metals.

· Hazardous decomposition products: Formation of toxic gases is possible during heating or in case of fire.

11 Toxicological information

· Information on toxicological effects· Acute toxicity:· LD/LC50 values relevant for classification:

67-56-1 MethanolOral LD50 5628 mg/kg (rat)Dermal LD50 15800 mg/kg (rabbit)Inhalative LC50/4 h 83.9 mg/l (rat)

· Primary irritant effect:· on the skin: No irritating effect.· on the eye: No irritating effect.· Sensitization: No sensitizing effects known.· Additional toxicological information:

The product shows the following dangers according to the calculation method of the General EUClassification Guidelines for Preparations as issued in the latest version:Toxic

12 Ecological information

· Toxicity· Aquatic toxicity:

67-56-1 MethanolLC50/96 h 24.000 mg/l (fish)

· Persistence and degradability No further relevant information available.· Behaviour in environmental systems:· Bioaccumulative potential No further relevant information available.· Mobility in soil No further relevant information available.· Additional ecological information:· General notes:

Water hazard class 1 (German Regulation) (Self-assessment): slightly hazardous for waterDo not allow undiluted product to reach ground water, water course or sewage system.

· Results of PBT and vPvB assessment· PBT: Not applicable.


/8Safety data sheet




36.0.36

· vPvB: Not applicable.· Other adverse effects No further relevant information available.

13 Disposal considerations· Waste treatment methods· Recommendation

Must not be disposed of together with household garbage. Do not allow product to reach sewage system.· European waste catalogue

Waste disposal key numbers from EWC have to be assigned depending on origin and processing.

· Uncleaned packaging:· Recommendation: Dispose of in accordance with national regulations.· Recommended cleansing agents: Water, if necessary together with cleansing agents.

14 Transport information· UN-Number· ADR, IMDG, IATA UN1230· ADR 1230 METHANOL, mixture· IMDG, IATA METHANOL, mixture

· Transport hazard class(es)· ADR, IMDG, IATA

· Class 3 Flammable liquids.· Label 3+6.1

· Packing group· ADR, IMDG, IATA II

· Environmental hazards:· Marine pollutant: No

· Special precautions for user Warning: Flammable liquids.· Danger code (Kemler): 336· EMS Number: F-E,S-D

· Transport in bulk according to Annex II ofMARPOL73/78 and the IBC Code Not applicable.

· Transport/Additional information:· ADR· Limited quantities (LQ) 1L· Transport category 2 · Tunnel restriction code D/E

· UN "Model Regulation": UN1230, METHANOL, mixture, 3 (6.1), II GB

(Contd. on page 8)

/8Safety data sheet




36.0.36

15 Regulatory information· Chemical safety assessment: A Chemical Safety Assessment has not been carried out.

16 Other informationThe information in this safety data sheet (SDS) has been prepared with due care and is true and accurate tothe best of our knowledge. The user must determine the suitability of the information for its particularpurpose, ensure compliance with existing laws and regulations, and be aware that other or additional safetyor performance considerations may arise when using, handling and/ or storing the material. The informationin this SDS does not purport to be all inclusive or a guarantee as to the properties of the material supplied,and should be used only as a guide. LGC makes no warranties or representations as to the accuracy andcompleteness of the information contained herein, shall not be held responsible for the suitability of thisinformation for the user's intended purposes or the consequences of such use, and shall not be liable for anydamage or loss, howsoever arising, direct or otherwise.

· Relevant phrasesH225 Highly flammable liquid and vapour.H301 Toxic if swallowed.H311 Toxic in contact with skin.H315 Causes skin irritation.H331 Toxic if inhaled.H370 Causes damage to organs.H411 Toxic to aquatic life with long lasting effects.

R11 Highly flammable.R23/24/25 Toxic by inhalation, in contact with skin and if swallowed.R25 Toxic if swallowed.R38 Irritating to skin.R39/23/24/25 Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if

swallowed.R51/53 Toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.

· Abbreviations and acronyms:RID: Règlement international concernant le transport des marchandises dangereuses par chemin de fer (Regulations Concerning theInternational Transport of Dangerous Goods by Rail)ICAO: International Civil Aviation OrganizationADR: Accord européen sur le transport des marchandises dangereuses par Route (European Agreement concerning the InternationalCarriage of Dangerous Goods by Road)IMDG: International Maritime Code for Dangerous GoodsIATA: International Air Transport AssociationGHS: Globally Harmonized System of Classification and Labelling of ChemicalsLC50: Lethal concentration, 50 percentLD50: Lethal dose, 50 percent

· SourcesTables 3.1 and 3.2 from Annex 6 of EC 1272/2008, EC 1907/2006, EH40/2005 as amended 2011, Registry ofToxic Effects of Chemical Substances (RTECS), The Dictionary of Substances and their Effects, 1st Edition,IUCLID.

GB

Sigma - A1542 Page 1 of 6

SIGMA-ALDRICH sigma-aldrich.com SAFETY DATA SHEET

according to Regulation (EC) No. 1907/2006 Version 5.2 Revision Date 08.03.2014

Print Date 24.04.2014 SECTION 1: Identification of the substance/mixture and of the company/undertaking 1.1 Product identifiers

Product name : Ammonium acetate

Product Number : A1542 Brand : Sigma REACH No. : A registration number is not available for this substance as the substance

or its uses are exempted from registration, the annual tonnage does not require a registration or the registration is envisaged for a later registration deadline.

CAS-No. : 631-61-8

1.2 Relevant identified uses of the substance or mixture and uses advised against Identified uses : Laboratory chemicals, Manufacture of substances

1.3 Details of the supplier of the safety data sheet Company : Sigma-Aldrich Company Ltd.

The Old Brickyard NEW ROAD, GILLINGHAM Dorset SP8 4XT UNITED KINGDOM

Telephone : +44 (0)1747 833000 Fax : +44 (0)1747 833313 E-mail address : [email protected]

1.4 Emergency telephone number Emergency Phone # : +44 (0)1747 833100

SECTION 2: Hazards identification 2.1 Classification of the substance or mixture

Not a hazardous substance or mixture according to Regulation (EC) No. 1272/2008. This substance is not classified as dangerous according to Directive 67/548/EEC.

2.2 Label elements The product does not need to be labelled in accordance with EC directives or respective national laws.

2.3 Other hazards - none SECTION 3: Composition/information on ingredients 3.1 Substances

Formula : C2H7NO2 Molecular Weight : 77.08 g/mol CAS-No. : 631-61-8 EC-No. : 211-162-9 No components need to be disclosed according to the applicable regulations.


SECTION 4: First aid measures 4.1 Description of first aid measures

If inhaled If breathed in, move person into fresh air. If not breathing, give artificial respiration.

In case of skin contact Wash off with soap and plenty of water.

In case of eye contact Flush eyes with water as a precaution.

If swallowed Never give anything by mouth to an unconscious person. Rinse mouth with water.

4.2 Most important symptoms and effects, both acute and delayed The most important known symptoms and effects are described in the labelling (see section 2.2) and/or in section 11

4.3 Indication of any immediate medical attention and special treatment needed no data available

SECTION 5: Firefighting measures 5.1 Extinguishing media

Suitable extinguishing media Use water spray, alcohol-resistant foam, dry chemical or carbon dioxide.

5.2 Special hazards arising from the substance or mixture Carbon oxides, nitrogen oxides (NOx)

5.3 Advice for firefighters Wear self contained breathing apparatus for fire fighting if necessary.

5.4 Further information no data available

SECTION 6: Accidental release measures 6.1 Personal precautions, protective equipment and emergency procedures

Avoid dust formation. Avoid breathing vapours, mist or gas. For personal protection see section 8.

6.2 Environmental precautions Do not let product enter drains.

6.3 Methods and materials for containment and cleaning up Sweep up and shovel. Keep in suitable, closed containers for disposal.

6.4 Reference to other sections For disposal see section 13.

SECTION 7: Handling and storage 7.1 Precautions for safe handling

Provide appropriate exhaust ventilation at places where dust is formed. For precautions see section 2.2.

7.2 Conditions for safe storage, including any incompatibilities Store in cool place. Keep container tightly closed in a dry and well-ventilated place.

Recommended storage temperature: 2 - 8 °C

Hygroscopic.


7.3 Specific end use(s) Apart from the uses mentioned in section 1.2 no other specific uses are stipulated

SECTION 8: Exposure controls/personal protection 8.1 Control parameters

Components with workplace control parameters Contains no substances with occupational exposure limit values.

8.2 Exposure controls Appropriate engineering controls General industrial hygiene practice.

Personal protective equipment Eye/face protection Use equipment for eye protection tested and approved under appropriate government standards such as NIOSH (US) or EN 166(EU).

Skin protection Handle with gloves. Gloves must be inspected prior to use. Use proper glove removal technique (without touching glove's outer surface) to avoid skin contact with this product. Dispose of contaminated gloves after use in accordance with applicable laws and good laboratory practices. Wash and dry hands. The selected protective gloves have to satisfy the specifications of EU Directive 89/686/EEC and the standard EN 374 derived from it. Full contact Material: Nitrile rubber Minimum layer thickness: 0.11 mm Break through time: 480 min Material tested:Dermatril® (KCL 740 / Aldrich Z677272, Size M) Splash contact Material: Nitrile rubber Minimum layer thickness: 0.11 mm Break through time: 480 min Material tested:Dermatril® (KCL 740 / Aldrich Z677272, Size M) data source: KCL GmbH, D-36124 Eichenzell, phone +49 (0)6659 87300, e-mail [email protected], test method: EN374 If used in solution, or mixed with other substances, and under conditions which differ from EN 374, contact the supplier of the CE approved gloves. This recommendation is advisory only and must be evaluated by an industrial hygienist and safety officer familiar with the specific situation of anticipated use by our customers. It should not be construed as offering an approval for any specific use scenario. Body Protection Choose body protection in relation to its type, to the concentration and amount of dangerous substances, and to the specific work-place., The type of protective equipment must be selected according to the concentration and amount of the dangerous substance at the specific workplace.

Respiratory protection Respiratory protection is not required. Where protection from nuisance levels of dusts are desired, use type N95 (US) or type P1 (EN 143) dust masks. Use respirators and components tested and approved under appropriate government standards such as NIOSH (US) or CEN (EU).

Control of environmental exposure Do not let product enter drains.


SECTION 9: Physical and chemical properties 9.1 Information on basic physical and chemical properties

a) Appearance Form: solid Colour: white

b) Odour no data available

c) Odour Threshold no data available

d) pH 6.5 - 7.5 at 77.1 g/l at 25 °C

e) Melting point/freezing point

Melting point/range: 110 - 112 °C

f) Initial boiling point and boiling range

no data available

g) Flash point no data available

h) Evapouration rate no data available

i) Flammability (solid, gas) no data available

j) Upper/lower flammability or explosive limits

no data available

k) Vapour pressure no data available

l) Vapour density no data available

m) Relative density no data available

n) Water solubility 77.1 g/l at 20 °C - completely soluble

o) Partition coefficient: n-octanol/water

no data available

p) Auto-ignition temperature

no data available

q) Decomposition temperature

no data available

r) Viscosity no data available

s) Explosive properties no data available

t) Oxidizing properties no data available

9.2 Other safety information no data available

SECTION 10: Stability and reactivity 10.1 Reactivity

no data available

10.2 Chemical stability Stable under recommended storage conditions.

10.3 Possibility of hazardous reactions no data available

10.4 Conditions to avoid Exposure to moisture.

10.5 Incompatible materials Strong oxidizing agents, Strong acids


10.6 Hazardous decomposition products Other decomposition products - no data available In the event of fire: see section 5

SECTION 11: Toxicological information 11.1 Information on toxicological effects

Acute toxicity no data available

LD50 Intraperitoneal - mouse - 736 mg/kg Skin corrosion/irritation no data available

Serious eye damage/eye irritation no data available

Respiratory or skin sensitisation no data available

Germ cell mutagenicity no data available Carcinogenicity IARC: No component of this product present at levels greater than or equal to 0.1% is identified as

probable, possible or confirmed human carcinogen by IARC.

Reproductive toxicity no data available

Specific target organ toxicity - single exposure no data available

Specific target organ toxicity - repeated exposure no data available

Aspiration hazard no data available

Additional Information RTECS: AF3675000 Gastrointestinal disturbance, To the best of our knowledge, the chemical, physical, and toxicological properties have not been thoroughly investigated.

SECTION 12: Ecological information 12.1 Toxicity

Toxicity to fish LC50 - Cyprinus carpio (Carp) - 56 mg/l - 48 h

12.2 Persistence and degradability Biodegradability Result: - Readily biodegradable.

12.3 Bioaccumulative potential no data available

12.4 Mobility in soil no data available

12.5 Results of PBT and vPvB assessment PBT/vPvB assessment not available as chemical safety assessment not required/not conducted

12.6 Other adverse effects no data available


SECTION 13: Disposal considerations 13.1 Waste treatment methods

Product Offer surplus and non-recyclable solutions to a licensed disposal company.

Contaminated packaging Dispose of as unused product.

SECTION 14: Transport information 14.1 UN number

ADR/RID: - IMDG: - IATA: -

14.2 UN proper shipping name ADR/RID: Not dangerous goods IMDG: Not dangerous goods IATA: Not dangerous goods

14.3 Transport hazard class(es) ADR/RID: - IMDG: - IATA: -

14.4 Packaging group ADR/RID: - IMDG: - IATA: -

14.5 Environmental hazards ADR/RID: no IMDG Marine pollutant: no IATA: no

14.6 Special precautions for user no data available

SECTION 15: Regulatory information

This safety datasheet complies with the requirements of Regulation (EC) No. 1907/2006.

15.1 Safety, health and environmental regulations/legislation specific for the substance or mixture no data available

15.2 Chemical Safety Assessment For this product a chemical safety assessment was not carried out

SECTION 16: Other information

Further information Copyright 2014 Sigma-Aldrich Co. LLC. License granted to make unlimited paper copies for internal use only. The above information is believed to be correct but does not purport to be all inclusive and shall be used only as a guide. The information in this document is based on the present state of our knowledge and is applicable to the product with regard to appropriate safety precautions. It does not represent any guarantee of the properties of the product. Sigma-Aldrich Corporation and its Affiliates shall not be held liable for any damage resulting from handling or from contact with the above product. See www.sigma-aldrich.com and/or the reverse side of invoice or packing slip for additional terms and conditions of sale.

34

Appendix Two:

Certificate of Analysis

LGC GmbH, Im Biotechnologiepark, TGZ II, D-14943 Luckenwalde, Germany

8 pages © 2012 LGC limited. All rights reserved. LGC Standards is part of the LGC Group. LoGiCal is a registered trademark of LGC Standards GmbH


Reference Material - Primary Standard Product Name: Tramadol Hydrochloride 1.0 mg/ml in Methanol (as free base)

Catalogue Number: LGCAMP0007.00-01

Lot Number: 40527

CAS Number: 36282-47-0

Molecular Formula: C16H25NO2 HCl

Molecular Weight: 299.84

Solvent: Methanol

Volume per Ampoule: Not less than 1 ml 1

Long-term Storage: 2 to 8 °C, dark

Expiry Date: February-2016

Intended Use: The primary aim of this material is for identification, calibration and quantification.

Component Concentration (“free base”) Uncertainty see product name 1.000 mg/ml 2 U = 0.006 mg/ml 3 Uncertainty of the concentration is expressed as an expanded uncertainty in accordance with ISO 17025 and Guide 34 at the about 95 % level of confidence using a coverage factor of k = 2 and has been calculated by statistical analysis of our production system and incorporates uncertainty of the purity, material density and balance and weighing technique.

Concentration based on material weighings and material purity factor (assay of the neat material).

The solution’s concentration and homogeneity are verified by independent method. LGC certifies that this standard meets the specification stated in this certificate and warrants this product to meet the stated acceptance criteria through the retest date when stored unopened as recommended. Product should be used shortly after

opening to avoid concentration changes due to evaporation. Warranty does not apply to ampoules stored after opening. Release Date: Luckenwalde, February 2014 Signed: Dr. Sabine Schröder Unit for Reference Materials 1 Ampoules are overfilled to ensure a minimum 1 ml volume fill. We advise laboratories to use measured volumes of this standard solution before diluting to the desired concentration. 2 The value is based on the results of analytical techniques, which calibration and verification was carried out with standards traceable to SI-units. The value is expressed on a "free base" basis. The concentration with its uncertainty is valid in the range between 19 °C and 25 °C.

The identity is verified by data from international scientific literature. Gravimetrically prepared using qualified balances calibrated annually by accredited calibration service. Calibration verification performed daily prior to use utilizing weights traceable to SI via other mass standards.

3 The uncertainty “U” is the expanded uncertainty estimated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM). It is corresponding to a level of confidence of about 95 %. Standard uncertainties are indicated with “u”.

LGCAMP0007.00-01 Lot Number 40527

LGC GmbH, Im Biotechnologiepark, TGZ II, D-14943 Luckenwalde, Germany page 2/8

© 2012 LGC limited. All rights reserved. LGC Standards is part of the LGC Group. LoGiCal is a registered trademark of LGC Standards GmbH

Additional information

Concentration (“as is”) 1.138 mg/ml

Verification of Concentration and Homogeneity

Lot Number

Verified Concentration (mg/ml) Result Acceptance Criteria

% RSD - Homogeneity Result Acceptance Criteria

40527 1.001 ± 3 % 0.820 � 3 % Concentration verified by HPLC Solution Standard Assay Parameters External Calibration (100 % amount)

Analysis Method HPLC

Column: Hypersil Gold (C18), 5 µm, 150 x 4.6 mm Number of Measurements: 6

Injector: Auto; 1 µl; 1.0 mg/ml in Methanol

Flow: 1.0 ml/min, 40 °C Detector: 215 nm

Conditions: mob. Phase A: Water + 0.1 % H3PO4, mob. Phase B: Acetonitrile + 0.1 % H3PO4

0-6 min A/B 80/20, 6-9 min A/B to 20/80, 9-11 min A/B to 80/20, 11-20 min A/B 80/20 (v/v)

Neat Material Data

Product Name: Tramadol Hydrochloride




Compound Lot: 1392

Test Method Result

Melting Point (°C)* SOP 06-010 181 °C

1H-NMR Spectrum* SOP 06-053 conform / complies to structure

IR Spectrum* SOP 06-036 conform / complies to structure

Mass Spectrum (ESI)* SOP 06-022 conform / complies to structure

Assay by carbon titration (free base)* Elementary Analysis 87.82 %

The expanded uncertainty according to the assay is U = 0.56 % (about 95 % level of confidence using a coverage factor of k = 2).

Assay by carbon titration (“as is”) 99.98 %

*: Validated method performed by ISO/IEC 17025 accredited testing lab

The assay of the neat material is verified by the 100 % method using HPLC, corrected with water

(KFT) and residual solvents.




I. Identity

The identity of the reference substance (neat material) was established by the following analyses.

Ia. 1H-NMR Spectrum

Conditions: 400 MHz, CDCl3

The structure is confirmed with the signals of the spectrum and their interpretation.




Ib. Mass Spectrum

Method: EI, 70eV, detector temperature: 280 °C

m/z fragments (M = free base)

263 [ M ]

188 [ C13H16O ]

107 [ C7H7O ]

58 [ C3H8N ]

The signals of the mass spectrum and their interpretation are consistent with the structural formula.




Ic. IR Spectrum

Method: attenuated total reflection fourier transform infrared (ATR-FTIR) spectroscopy

The signals of the IR spectrum and their interpretation are consistent with the structural formula.

Id. Melting Point

181 °C

Ie. UV Spectrum

Method: HPLC (DAD-detection)




II. Assay by Elementary Analysis (Carbon Titration – neat material)

Method: percentage carbon found in relation to percentage carbon as calculated for molecular formula

Results:

Arithmetic mean (n=3) 99.98 % (mass fraction)

Uncertainty U 0.64 %

III. Purity

IIIa. High Performance Liquid Chromatography (HPLC)

The purity of the reference substance (neat material) was analysed by high performance liquid

chromatography (HPLC).

Minutes

0 5 10 15 20 25 30 35

mA

U

0

50

100

150

200

250

mA

U

0

50

100

150

200

250

5,1

7

Surveyor PDA 1-215nmRetention Time




Area Percent Report - Sorted by Signal

Pk # Retention Time Area Area % 1 5.17 2690920 100.00

Totals 2690920 100.00

For the calculation the system peaks were ignored. The content of the analyte was determined as ratio

of the peak area of the analyte and the cumulative areas of the purities, added up to 100 %.

HPLC Conditions:

Column: Conditions: Detector: Injector:

RP 60 Select B 1.0 ml/min, 40 °C DAD Auto

5 µm, 125 x 4 mm 0 – 6 min Water/Acetonitrile 80/20

6 – 15 min Water/Acetonitrile to 70/30

15 – 20 min Water/Acetonitrile 70/30


25 – 35 min Water/Acetonitrile 80/20 (v/v);

0.1 % H3PO4

215 nm 3 µl; 0.164 mg/ml in

Water/Acetonitrile 50/50 (v/v)

Results:

Arithmetic mean (n=6) 100 %

IIIb. Water Content

Method: coulometric Karl Fischer titration

Results:


IIIc. Residual Solvents

Method: 1H-NMR

No significant amounts of residual solvents were detected (< 0.05 %).




IV. Stability and Homogeneity

Accelerated stability studies indicate no significant instability. The given validity period is based on this

data. This is backed up by historical data over the range of several years for the neat substance.

Homogeneity assured by validated process of preparation (incl. ampoulation), verified by homogeneity

testing (HPLC).

V. Further Information

General

For laboratory use only. Not suitable for human or animal consumption.

This material conforms to the characteristics of a primary standard as described within ISO Guide 30

(Terms and definitions used in connection with reference materials).

The certified values quoted in this certificate are LGC’s best estimate of the true values within the

stated uncertainties and based on the techniques described in this certificate.

Handling of the RM

Before usage of the RM, it should be allowed to warm to room temperature. The concentration with its

uncertainty is guaranteed in the range between 19 °C and 25 °C. The uncertainty accounts for the

temperature-dependent density in this range.

Quality Control Assessment

The product quality is controlled by regularly performed quality control tests (retests).

D-023FN10161403

Revision 00Page 1 of 7

Product of USA

Catalog Number: D-023Lot: FN10161403Expiration:Description: N-Desmethyl-cis-tramadol HCl in Methanol.

Nominal concentration is adjusted for HCl content.Packaging: Solution in 2 mL amber USP Type I glass ampoule

containing not less than 1 mL of certified solution.Storage: Store unopened in freezer (-10 °C to -25 °C).Shipping: Ambient. See Stability Section.Intended Use: This Certified Reference Material is suitable for the in vitro identification, calibration, and quantification of the

analyte(s) in analytical and R&D applications. Not suitable for human or animal consumption.Instructions for Use: Users should quantitatively transfer desired volume using established good laboratory practices to

spike into matrix or to dilute to the desired concentration. Each ampoule is intended for one-time use.Safety: Danger. See Safety Data Sheet

i Expiration date has been established through real time stability studies.i Ampoules are overfilled to ensure a minimum 1 mL volume can be transferred when using a 1 mL Class A volumetric pipette.i For quantitative applications, the minimum sample size for intended use is 1 µL.

Analyte

Certified Reference Material - Certificate of Analysis

N-Desmethyl-cis-tramadol, Primary Standard

October 2019

Certified Concentration Value

HO

CH2NCH3H

H

CH3O

HCl

Cerilliant Corporation 811 Paloma Drive, Suite A, Round Rock, TX 78665 800-848-7837 / 512-238-9974

yN-Desmethyl-cis-tramadol

i

i

i

i Additional certification information available upon request.

Metrological Traceabilityi

i

i Fill volume is gravimetrically verified throughout the dispensing process using qualified and calibrated balances.i Concentration is verified against an independently prepared calibration solution gravimetrically prepared.i

Darron Ellsworth, Quality Assurance Manager Date

Uncertainty of the concentration is expressed as an expanded uncertainty in accordance with ISO 17025 and Guide 34 at the approximate 95% confidence interval using a coverage factor of k = 2 and has been calculated by statistical analysis of our production system and incorporates uncertainty of the mass balance purity factor, material density, balance, and weighing technique. This standard is prepared gravimetrically and mass results are reported on the conventional basis for weighing in air. Nominal concentration is calculated based on: the actual measured mass; Mass Balance Purity Factor of the analyte(s); measured mass of the solution; and the density of the pure diluent at 20 °C.

January 13, 2015

Cerilliant certifies that this standard meets the specifications stated in this certificate and warrants this product to meet the statedacceptance criteria through the expiration/retest date when stored unopened as recommended. Product should be used shortly afteropening to avoid concentration changes due to evaporation. Warranty does not apply to ampoules stored after opening.

1.000 ± 0.005 mg/mL

This standard has been gravimetrically prepared using balances that have been fully qualified and calibrated to ISO 17025 requirements. All calibrations utilize NIST traceable weights which are calibrated externally by a qualified ISO 17025 accredited calibration laboratory to NIST standards. Qualification of each balance includes thassignment of a minimum weighing by a qualified and ISO 17025 accredited calibration vendor taking into consideration the balance and installed environmental conditions to ensure compliance with USP tolerances of NMT 0.1% relative error. Balance calibration adjustments are performed weekly utilizing the balance's internal adjustment mechanism. Calibration verifications are performed pre-use. Weigh tapes from the calibration verification are included in the production batch record for this standard. Production data package available upon request.

Each raw material utilized has been identified and thoroughly characterized through the use of multiple analytical techniques. Spectral data is provided on subsequent pages of this COA. The density and material Mass Balance Purity Factor is traceable to the SI and higher order reference standards through mass measurement and instrument qualification and calibrations.

This standard has been prepared and certified under the ISO Guide 34, ISO/IEC 17025, ISO 9001 and ISO 13485 standards. This standard meets the requirements of a Certified Reference Material and a Primary Standard as defined by ISO and is traceable to the SI and higher order standards through an unbroken chain of comparisons.

Concentration is corrected for chromatographic purity, residual water, residual solvents and residual inorganics. Nominal concentration is adjusted for HCl content. No adjustment required before use.


D-023FN10161403


Product of USA

Solution Standard Verification

Standard Solution Assay Parameters Calibration CurveAnalysis Method: UV/Vis Calibration Curve: Linear RegressionWavelength: 275 nm Number of Points: 4Slit Width: 1.0 nm Linearity (r) :Response: 0.5 s

Standard Solution Lot Number

New Lot FN10161403Previous Lot FN03201401

i Concentration is verified through multiple analyses and is calculated as the average of multiple analyses compared to an independently prepared calibration solution.i Within-sample and between-sample homogeneity of the New Lot is ensured through rigorous production process controls statistically analyzed to evaluate risk and

verified by analysis. Multiple samples pulled from across the lot using a random stratified sampling plan were analyzed to verify homogeneity. % RSD results shownabove for the New Lot demonstrate ampoule-to-ampoule homogeneity

Analyte Certification - Mass Balance Purity Factor

Solution standard verification demonstrates confirmation that the specified requirements for the Primary Standard have been fulfilled and validated under ISO 13485.

1.000

Each analyte is thoroughly identified and characterized using an orthogonal approach A mass balance purity factor is assigned incorporating

Concentration accuracy and within- and between-bottle homogeneity are analytically verified against an independently prepared calibration solution and to the prior lot.

1.0100.71.005 ± 3% ≤ 3%

Verified Concentration (mg/mL) %RSD - HomogeneityActual Results Acceptance Criteria Actual Results Acceptance Criteria

± 3%


i The primary chromatographic purity is calculated as the average of two independently performed analyses utilizing two different methods. Acceptance criteria requires the purity values to be within 0.5% of each other.

i The primary chromatographic purity value is used to calculate the Mass Balance Purity Factor.i A secondary chromatographic purity method is utilized as a control.i Mass Balance Purity Factor = [(100 - wt% residual solvent - wt% residual water - wt% residual inorganics) x Chromatographic Purity/100]. i Mass Balance Purity Factor does not include adjustment for chiral and/or isotopic purity.

1 Validated analytical method

Each analyte is thoroughly identified and characterized using an orthogonal approach. A mass balance purity factor is assigned incorporating chromatographic purity and residual impurities. The mass balance purity factor is utilized to calculate the weighing adjustment necessary to ensure accuracy of the solution standard concentration.

Material Characterization SummaryResultsMethodAnalytical Test

Material Lot NumberCAS Chemical Molecular Molecular Salt

Number Formula Weight (salt) Weight (base) Adjustment

N-Desmethyl-cis-tramadol HCl FN041009-03 1018989-94-0 C15H23NO2·HCl 285.81 249.35 1.146

98.3%

SP10-0135

Primary Chromatographic Purity by HPLC/UV Analysis

96.55%

1.69%

SP10-0101

AM1087 1 0.38%

<0.2%

Consistent with Structure

Mass Balance Purity Factor

Secondary Chromatographic Purity by GC/FID Analysis

Inorganic Content by Microash Analysis

Residual Solvent Analysis by GC/FID Headspace

AM1346 1Residual Water Analysis by Karl Fischer Coulometry

Identity by LC/MS AnalysisUSP <761>, SP10-0116Identity by 1H-NMR Analysis

Consistent with StructureSP10-0107

98.6%SP10-0102


D-023FN10161403


Product of USA

Spectral and Physical Data

HPLC/UVColumn: Betasil Phenyl, 5 µ, 150 x 4.6 mmMobile Phase: Acetonitrile::20 mM Ammonium acetate, pH 3

(30::70)Flow Rate: 1.0 mL/minWavelength: 275 nm

Data File Name: RMD-070 P LC 5 2013-09-12 19-42-26\042-0301.D Instrument: LC#5Sample Name: FN041009-03Acquired:

1 4.40 1.17 0.17

September 12, 2013

Peak # Ret Time Area Height Area %

10.49 2.07 0.12 0.13

5 8.73 1.05 0.07 0.076 9.47 6.68 0.37 0.42

0.072 4.89 1570.96 144.64 98.813 5.93 3.73 0.33 0.234 6.65 4.21 0.39 0.27

7


Residual Solvent Analysis by GC/FID Headspace Column:Temp Program:Carrier Gas: HeliumFlow Rate: 2.0 mL/minDetector Heater Temp: 250°CInjector: Headspace SamplerHS Oven Temp: 60°CVial Equilibration: 10 minutes

Data File Name: GC 9 2013-09-17 18-59-09\012F0301.D Instrument: GC#9Sample Name: FN041009-03Acquired:

Peak Compound

1 Acetonitrile2 Hexane3 Unknown #14 NMPTotal

BQL- Below Quantitation Limit

1.54 BQLNA NA

0.38

44.43 BQL114.51 0.38

DB-ALC1 30 m x 0.53 mm, 3 µm film thickness40°C (12 min) to 220°C at 40°C/min (5.5 min)

September 17, 2013

Area Weight %


D-023FN10161403


Product of USA

Spectral and Physical Data (cont.)

GC/FIDColumn: DB-5ms, 30 m x 0.53 mm ID, 1.5 µm film thicknessTemp Program: 40°C to 140°C at 40°C/min

140°C to 280°C at 5°C/min hold 5 minutesInjector Temp: Cool-on-ColumnDetector Temp: 325°C

Data File Name: RMD-070 GC 10 2013-09-18 22-17-33\052B0201.DInstrument: GC#10Sample Name: FN041009-03Acquired: September 18, 2013

2 11.61 6.82 1.52 0.03 22 18.90 0.90 0.241 9.91 1.01 0.24 0.01 21 18.63 41.49 9.14

Peak # Ret Time Area Height Area % Peak # Ret Time Area Height Area %

0.210.00


0.0120 18.34 84.38 19.26 0.42 40 32.31 1.29 0.21

0.0119 18.13 29.60 7.77 0.15 39 32.21 1.17 0.19 0.0118 17.95 19719.30 4052.46 98.26 38 31.97 1.87 0.22

0.0117 17.58 0.67 0.16 0.00 37 31.39 3.39 0.51 0.0216 17.38 7.11 1.55 0.04 36 30.48 2.30 0.38

0.0215 17.02 0.91 0.24 0.00 35 30.33 4.20 0.55 0.0214 16.50 46.84 9.23 0.23 34 30.04 3.14 0.53

0.0113 16.34 4.53 1.05 0.02 33 28.44 0.63 0.13 0.0012 15.98 33.58 7.53 0.17 32 27.36 1.09 0.18

0.0211 15.72 1.76 0.32 0.01 31 27.25 0.55 0.11 0.0010 15.35 2.14 0.42 0.01 30 25.55 4.75 0.84

0.009 15.17 1.25 0.24 0.01 29 24.42 2.09 0.31 0.018 14.97 17.50 3.66 0.09 28 24.22 0.67 0.10

0.007 13.76 24.21 4.57 0.12 27 24.01 1.31 0.16 0.016 13.43 0.85 0.19 0.00 26 23.19 0.57 0.11

0.015 13.17 1.76 0.39 0.01 25 21.43 1.34 0.19 0.014 12.94 7.13 1.60 0.04 24 20.28 1.45 0.303 12.75 0.66 0.14 0.00 23 20.05 3.28 0.60 0.02


D-023FN10161403


Product of USA


1H NMR Instrument: JEOL ECS 400Solvent: DMSO-D6

Cerilliant Corporation 811 Paloma Drive, Suite A, Round Rock, TX 78665 800-848-7837 / 512-238-9974Cerilliant Corporation 811 Paloma Drive, Suite A, Round Rock, TX 78665 800-848-7837 / 512-238-9974

D-023FN10161403


Product of USA


LC/MS

Column: Acquity UPLC BEH C18, 1.7 µm, 2.1 x 50 mm Flow Rate: 0.4 mL/minMobile Phase: A:: 0.1% Formic Acid in Water Scan Range: 100-1200 amu

B:: Acetonitrile Ionization: Electrospray, Positive IonGradient: Data File Name: W10021304

Instrument: Waters XEVO G2 QTOFSample Name: FN041009-03Acquired:7.1 90 10 October 02, 2013

8.0 90 10

Time (min) %A %B0.0 90 106.0 50 50

Cone Voltage 20N-Desmethyl-cis-tramadol HClRMD-070 FN041009-03

%

100W10021314 502 (1.829) Cm (502:520) 1: TOF MS ES+

2.00e7250.1808

Theoretical [M + H]+: 250.1807Found [M + H]+: 250.1808


m/z100 200 300 400 500 600 700 800 900 1000 1100

%

0

251.1841


D-023FN10161403


Product of USA

Stability

Mean Kinetic Temperature (MKT) Time Period/Result

4°CRoom Temperature 21°C

Short term stability studies have been performed under accelerated conditions for a period of up to four weeks. Short term data isutilized to predict long term stability and to support transport conditions and normal laboratory use. Real-time stability studiesare performed at the recommended storage conditions over the life of the product.

Short Term Stability: A summary of accelerated stability findings for this product is listed below.

Storage Condition

Long Term Stability: Long term stability has been assessed for Freezer storage (-10 °C to -25 °C) conditions. Stability of a minimum of 60 months has been established through real-time stability studies.

Short Term Storage: Stability data supports short term storage for up to 12 months at Refrigerate conditions.

Transport/Shipping: Stability studies support the transport of this product at ambient conditions.

RefrigeratorFreezer -15°C

40°C 40°C

No decrease in purity was noted after four weeks.


COA Revision History

00Revision No. Date

January 13, 2015Reason for Revision

Initial version


T-035FN08221402


Product of USA

Catalog Number: T-035Lot: FN08221402Expiration:Description: O-Desmethyl-cis-tramadol HCl in Methanol.

Nominal concentration is adjusted for HCl content.Packaging: Solution in 2 mL amber USP Type I glass ampoule

containing not less than 1 mL of certified solution.Storage: Store unopened in freezer (-10 °C to -25 °C).Shipping: Ambient. See Stability Section.Intended Use: This Certified Reference Material is suitable for the in vitro identification, calibration, and quantification of the

analyte(s) in analytical and R&D applications. Not suitable for human or animal consumption.Instructions for Use: Users should quantitatively transfer desired volume using established good laboratory practices to

spike into matrix or to dilute to the desired concentration. Each ampoule is intended for one-time use.Safety: Danger. See Safety Data Sheet

i Expiration date has been established through real time stability studies.i Ampoules are overfilled to ensure a minimum 1 mL volume can be transferred when using a 1 mL Class A volumetric pipette.i For quantitative applications, the minimum sample size for intended use is 1 µL.

Certified Reference Material - Certificate of AnalysisO-Desmethyl-cis-tramadol, Primary Standard

October 2019

OHOH

NCH3

CH3

ClH


AnalyteO-Desmethyl-cis-tramadol

i

i

i Concentration is corrected for chromatographic purity, residual water, residual solvents and residual inorganics. Nominal concentration is adjusted for HCl content. No adjustment required before use.

i Additional certification information available upon request.

Metrological Traceabilityi

i

i Fill volume is gravimetrically verified throughout the dispensing process using qualified and calibrated balances.i Concentration is verified against an independently prepared calibration solution gravimetrically prepared.i

Darron Ellsworth, Quality Assurance Manager Date

Cerilliant certifies that this standard meets the specifications stated in this certificate and warrants this product to meet the statedacceptance criteria through the expiration/retest date when stored unopened as recommended. Product should be used shortly after opening to avoid concentration changes due to evaporation. Warranty does not apply to ampoules stored after opening.

1.000 ± 0.005 mg/mLUncertainty of the concentration is expressed as an expanded uncertainty in accordance with ISO 17025 and Guide 34 at the approximate 95% confidence interval using a coverage factor of k = 2 and has been calculated by statistical analysis of our production system and incorporates uncertainty of the mass balance purity factor, material density, balance, and weighing technique.

This standard is prepared gravimetrically and mass results are reported on the conventional basis for weighing in air. Nominal concentration is calculated based on: the actual measured mass; Mass Balance Purity Factor of the analyte(s); measured mass of the solution; and the density of the pure diluent at 20 °C.

Each raw material utilized has been identified and thoroughly characterized through the use of multiple analytical techniques. Spectral data is provided on subsequent pages of this COA. The density and material Mass Balance Purity Factor is traceable to the SI and higher order reference standards through mass measurement and instrument qualification and calibrations.

Certified Concentration Value

This standard has been prepared and certified under the ISO Guide 34, ISO/IEC 17025, ISO 9001 and ISO 13485 standards. This standard meets the requirements of a Certified Reference Material and a Primary Standard as defined by ISO and is traceable to the SI and higher order standards through an unbroken chain of comparisons.

December 09, 2014

This standard has been gravimetrically prepared using balances that have been fully qualified and calibrated to ISO 17025 requirements. All calibrations utilize NIST traceable weights which are calibrated externally by a qualified ISO 17025 accredited calibration laboratory to NIST standards. Qualification of each balance includes the assignment of a minimum weighing by a qualified and ISO 17025 accredited calibration vendor taking into consideration the balance and installed environmental conditions to ensure compliance with USP tolerances of NMT 0.1% relative uncertainty. Balance calibration adjustments are performed weekly utilizing the balance's internal adjustment mechanism. Calibration verifications are performed pre-use. Weigh tapes from the calibration verification are included in the production batch record for this standard. Production data package available upon request.

OHOH

NCH3

CH3

ClH


T-035FN08221402


Product of USA

Solution Standard Verification

Standard Solution Assay Parameters Calibration Curve

Analysis Method: UV/Vis Calibration Curve: Linear RegressionWavelength: 274 nm Number of Points: 4Slit Width: 1.0 nm Linearity (r) :Response: 0.5 s

Standard Solution Lot Number

New Lot FN08221402Previous Lot FN111111-01

i Concentration is verified through multiple analyses and is calculated as the average of multiple analyses compared to an independently prepared calibration solution.i Within-sample and between-sample homogeneity of the New Lot is ensured through rigorous production process controls statistically analyzed to evaluate risk and

verified by analysis. Multiple samples pulled from across the lot using a random stratified sampling plan were analyzed to verify homogeneity. % RSD results shownabove for the New Lot demonstrate ampoule-to-ampoule homogeneity.

Analyte Certification - Mass Balance Purity Factor

Solution standard verification demonstrates confirmation that the specified requirements for the Primary Standard have been fulfilled and validated under ISO 13485.

Concentration accuracy and within- and between-bottle homogeneity are analytically verified against an independently prepared calibration solution and to the prior lot.

Verified Concentration (mg/mL) %RSD - HomogeneityActual Results

1.000

1.006 ± 3%

Acceptance Criteria Actual Results Acceptance Criteria

0.996 ± 3% 0.5 ≤ 3%


y f y

Lot Number

FN011712-01

i The primary chromatographic purity is calculated as the average of two independently performed analyses utilizing two different methods. Acceptance criteria requires the purity values to be within 0.5% of each other.

i The primary chromatographic purity value is used to calculate the Mass Balance Purity Factor.i A secondary chromatographic purity method is utilized as a control.i Mass Balance Purity Factor = [(100 - wt% residual solvent - wt% residual water - wt% residual inorganics) x Chromatographic Purity/100]. i Mass Balance Purity Factor does not include adjustment for chiral and/or isotopic purity.

1 Validated analytical method

99.20%

0.72%

SP10-0101

AM1087 1 0.01%

< 0.2%


Mass Balance Purity Factor

SP10-0105

Results

Identity by GC/MS Analysis

USP <761>, SP10-0116

Material Characterization Summary

Each analyte is thoroughly identified and characterized using an orthogonal approach. A mass balance purity factor is assigned incorporating chromatographic purity and residual impurities. The mass balance purity factor and salt adjustment are utilized to calculate the weighing adjustment necessary to ensure accuracy of the solution standard concentration.

ChemicalFormula

Primary Chromatographic Purity by HPLC/UV Analysis

Inorganic Content by Microash Analysis

Residual Solvent Analysis by GC/FID Headspace

AM1346 1

1.146

Residual Water Analysis by Karl Fischer Coulometry

NA 285.81 249.35

Weight (base)Molecular

NumberCAS

C15H23NO2·HClO-Desmethyl-cis-tramadol HCl

Material AdjustmentSalt

99.9%

Method

SP10-0135

99.9%

Secondary Chromatographic Purity by GC/FID Analysis

Identity by 1H-NMR Analysis

Analytical Test


SP10-0102

Weight (salt)Molecular


T-035FN08221402


Product of USA

Spectral and Physical Data

HPLC/UVColumn: Prodigy ODS3, 5µm, 4.6 x 250 mmMobile Phase: A:: Acetonitrile

B:: 0.1% Phosphoric Acid in WaterGradient:

Flow Rate: 1.0 mL/minWavelength: 215 nm

Data File Name: RMT-143 P LC 4 2014-08-29 14-46-03\012-0201.DInstrument: LC#4Sample Name: FN011712-01Acquired:

Peak #

123 0.35 0.08 0.01

5 9525.0 5 95

August 29, 2014

Ret Time Area Height

Time A B0.0 5 95

18.0 75 2518.1

Area %

2.72 0.76 0.16 0.027.78 4041.48 757.91 99.929.48

45

GC/FIDColumn: DB-5ms, 30 m x 0.53 mm ID, 1.5 µm film thicknessTemp Program: 40°C to 180°C at 40°C/min

180°C to 280°C at 5°C/min, hold 12 minInjector Temp: Cool-on-ColumnDetector Temp: 325°C

Data File Name: S:\GC\GC6\2012\0212\F0209204.DInstrument: GC#6Sample Name: FN011712-01Acquired:

13 21.62 1.83 0.30 0.02

11 12.57 9321.54 2052.19 99.8812 19.80 1.73 0.36 0.02

9 11.43 0.88 0.22 0.0110 11.78 0.21 0.05 0.00

7 10.64 0.36 0.08 0.008 10.99 0.50 0.12 0.01

5 9.96 3.45 0.93 0.046 10.41 0.28 0.07 0.00

3 8.37 0.49 0.11 0.014 8.55 0.24 0.06 0.00

1 5.68 0.67 0.17 0.012 6.61 0.23 0.08 0.00

February 09, 2012

Peak # Ret Time Area Height Area %

9.69 0.35 0.09 0.019.86 1.65 0.35 0.04

m5 10 15 20 25 30

pA

0

500

1000

1500

2000

FID1 A, (0212\F0209204.D)

5.6

82 6

.611

8.3

73 8

.549

9.9

58 1

0.41

2 1

0.63

6 1

0.98

7 1

1.43

3 1

1.78

0 1

2.56

7

19.

804

21.

618


T-035FN08221402


Product of USA


Residual Solvent Analysis by GC/FID Headspace Column:Temp Program:Carrier Gas: HeliumFlow Rate: 2.0 mL/minDetector Heater Temp: 250°CInjector: Headspace SamplerHS Oven Temp: 60°CVial Equilibration: 10 minutes

Data File Name: C:\CHEM\32\1\DATA\GC90212\D0202218.DInstrument: GC#9Sample Name: FN011712-01Acquired:

Peak Compound

1 Acetone2 NMPTotal

DB-ALC1 30 m x 0.53 mm, 3 µm film thickness40°C (12 min) to 220°C at 40°C/min (5.5 min)

February 02, 2012

Area Weight %

4.40 0.01NA NA

0.01

min2.5 5 7.5 10 12.5 15 17.5 20

pA

20

40

60

80

100

FID1 A, (GC90212\D0202218.D)

9.2

58 -

Ace

tone


1H NMR Instrument: JEOL ECS 400Solvent: DMSO-D6

min2.5 5 7.5 10 12.5 15 17.5 20

pA

20

40

60

80

100

FID1 A, (GC90212\D0202218.D)

9.2

58 -

Ace

tone


T-035FN08221402


Product of USA


GC/MS


T-035FN08221402


Product of USA

Stability

Time Period/Result

Short term stability studies have been performed under accelerated conditions for periods up to one week. Short term data isutilized to predict long term stability and to support transport conditions and normal laboratory use. Real-time stability studiesare performed at the recommended storage conditions over the life of the product.

Storage Condition Mean Kinetic Temperature (MKT)

Short Term Stability: A summary of accelerated stability findings for this product is listed below.

Freezer

Long Term Stability: Long term stability has been assessed for Freezer storage (-10 °C to -25 °C) conditions. Stability of a minimum of 60 months has been established through real-time stability studies.

Short Term Storage: Stability data supports short term storage up to 12 months at Refrigerate conditions.

Refrigerator 4°CRoom Temperature 21°C

40°C 40°CTransport/Shipping : Stability data supports transport of this product at ambient conditions.

-15°C

No decrease in purity was noted after one week.


COA Revision History

Reason for RevisionInitial version

Revision No.00

DateDecember 09, 2014





Reference Material - Primary Standard Product Name: Venlafaxine Hydrochloride 1.0 mg/ml in Methanol (as free base)


Lot Number: 8787




Solvent: Methanol



O

N

OH

x HCl

Expiry Date: August-2016





opening to avoid concentration changes due to evaporation. Warranty does not apply to ampoules stored after opening. Release Date: Luckenwalde, April 2012 Signed: Dr. Sabine Schröder Unit for Reference Materials 1 Ampoules are overfilled to ensure a minimum 1 ml volume fill. We advise laboratories to use measured volumes of this standard solution before diluting to the desired concentration. 2 The value is based on the results of analytical techniques, which calibration and verification was carried out with standards traceable to SI-units. The value is expressed on a "free base" basis. The concentration with its uncertainty is valid in the range between 19 °C and 25 °C.









Lot Number



8787 1.004 ± 3 % 0.670 � 3 % Concentration verified by HPLC Solution Standard Assay Parameters External Calibration (100 % amount)


Column: 60 RP Select B, 5 µm, 125 x 4 mm Number of Measurements: 6





Neat Material Data

Product Name: Venlafaxine Hydrochloride




Compound Lot: 393.00.09.02

Test Method Result







Assay by carbon titration (“as is”) 100.10 %

*: Validated method performed by ISO/IEC 17025 accredited testing lab






I. Identity


Ia. 1H-NMR Spectrum

Conditions: 400 MHz, DMSO-d6





Ib. Mass Spectrum

Method: 4.5 kV ESI; vaporization temperature: 200 °C, direct inlet


277.97 [ M + H ]

260.00 [ MH – H2O ]

214.99 [ M – H2O – N(CH3)2 ]

120.98 [ CH3OC7H6 ]





Ic. IR Spectrum



Id. Melting Point

211 °C

Ie. UV Spectrum


nm200 220 240 260 280 300 320 340

mAU

0

200

400

600

800

1000

1200

225nm

275nm






Results:



III. Purity




Minutes

0 2 4 6 8 10 12 14 16 18 20

mA

U

0

100

200

300

mA

U

0

100

200

300

2.9

0 15

.30

Surveyor PDA-225nm

Retention Time


Pk # Retention Time Area Area % 1 2.90 2685674 99.85 2 15.30 4023 0.15

Totals 2689697 100.00






HPLC Conditions:



5 µm, 125 x 4 mm Water/Acetonitrile 70/30 (v/v);

0.1 % H3PO4

225 nm 5 µl; 0.0608 mg/ml in


Results:

Arithmetic mean (n=6) 99.85 %

IIIb. Water Content


Results:



Method: 1H-NMR

No significant amounts of residual solvents were detected (< 0.05 %).





testing (HPLC).





General






Handling of the RM






Revision Date Reason for Revision

00 April 2012 Release of the Lot – initial version

01 August 2012 Copyright added

02 September 2014 Expiry Date adapted

4 pages

Catalogue Number: LGCFOR0393.04

Lot Number: 2399

Long Term Storage: 2 to 8 °C, dark

Appearance: white solid

Melting Point: 165 °C

Assay ‘as is’: 98.9 %

Reference Substance

N-Desmethylvenlafaxine Hydrochloride

O

HN

OH

x HCl

Molecular Formula: C16H25NO2 HCl Molecular Weight: 299.84 CAS Number: [ 93413-90-2 ]

Date of shipment: 2012-04-16

This certificate is valid for two years from the date of shipment provided the substance is stored under the recommended conditions.

N-Desmethylvenlafaxine Hydrochloride lot number 2399 page 2/4

I. Identity

The identity of the reference substance was established by 1H-NMR spectroscopy. The structure is

confirmed with the signals of the spectrum and their interpretation.


II. Purity

The purity of the reference substance was analysed by high performance liquid chromatography (HPLC).

HPLC Conditions:



5 µm, 125 x 4 mm 0-9 min Water/Acetonitrile 80/20

9-15 min Water/Acetonitrile to 65/35

15-20 min Water/Acetonitrile 65/35

20-25 min Water/Acetonitrile to 80/20

25-35 min Water/Acetonitrile 80/20 (v/v);

0.1 % H3PO4

225 nm 3 µl; 0.0637 mg/ml in



Minutes

0 5 10 15 20 25 30 35

mA

U

0

20

40

60

80

100

120

mA

U

0

20

40

60

80

100

120

1.6

82

.29

2.9

9

6.9

2

8.0

0

9.4

8

10

.59

10

.89

12

.69

14

.87

16

.12

16

.80

18

.13

Surveyor PDA 6-225nm

Retention T ime


Pk # Retention Time Area Area % 1 1.68 307 0.02 2 2.29 135 0.01 3 2.99 1418 0.08 4 6.92 706 0.04 5 8.00 1777812 99.07 6 9.48 6155 0.34 7 10.59 894 0.05 8 10.89 1595 0.09 9 12.69 1167 0.07 10 14.87 719 0.04 11 16.12 1550 0.09 12 16.80 1365 0.08 13 18.13 760 0.04

Totals 1794583 100.00

For the calculation the system peaks were ignored. The content of the analyte was determined as ratio of

the peak area of the analyte and the cumulative areas of the purities, added up to 100 %.

Results:

Average 99.04 %

Number of results n=6

Standard deviation 0.04 %


III. Water Content

Method: Karl Fischer titration

Results:

Average 0.18 %


IV. Residual Solvents

Method: 1H-NMR

No residual solvents were detected.

V. Final Result

Total impurities (HPLC) 0.96 %

Water content 0.18 %

Residual solvents 0.00 %

Assay (100 % method) 98.86 %

The assay is assessed to be 98.9 % ‘as is’

The assay 'as is' is equivalent to the assay based of the not anhydrous and not dried substance

respectively.

Release Date: 2010-06-02 LGC GmbH

Dr. Sabine Schröder

Product Release

5 pages

Catalogue Number: LGCFOR0393.01

Lot Number: 2398

Long Term Storage: 2 to 8 °C, dark

Appearance: white solid

Melting Point: 223 °C

Assay ‘as is’: 98.8 %

Reference Substance

O-Desmethylvenlafaxine

HO

N

OH

Molecular Formula: C16H25NO2 Molecular Weight: 263.38 CAS Number: [ 93413-62-8 ]

Date of shipment: 2013-06-11

This certificate is valid for two years from the date of shipment provided the substance is stored under the recommended conditions.

O-Desmethylvenlafaxine lot number 2398 page 2/5

I. Identity

The identity of the reference substance was established by following analyses.

Ia. 1H-NMR Spectrum




Ib. IR Spectrum

Method: Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) Spectroscopy


II. Purity

The purity of the reference substance was analysed by high performance liquid chromatography (HPLC).

HPLC Conditions:


ODS-AQ 1.0 ml/min, 40 °C DAD Auto

5 µm, 250 x 4.6 mm 0 – 10 min Water/Acetonitrile 85/15





0.1 % H3PO4

225 nm 4 µl; 0.120 mg/ml in



Minutes

0 5 10 15 20 25 30 35

mA

U

0

50

100

150

200

250

300

mA

U

0

50

100

150

200

250

300

11

.01 1

4.8

9

19

.38

20

.43

Surveyor PDA 3-225nm

Retention T ime


Pk # Retention Time Area Area % 1 11.01 3999491 99.11 2 14.89 16525 0.41 3 19.38 18885 0.47 4 20.43 603 0.01

Totals 4035504 100.00

For the calculation the system peaks were ignored. The content of the analyte was determined as ratio of

the peak area of the analyte and the cumulative areas of the purities, added up to 100 %.

Results:

Average 99.11 %


Standard deviation 0.01 %

III. Water Content

Method: Karl Fischer titration

Results:

Average 0.34 %



IV. Residual Solvents

Method: 1H-NMR

No residual solvents were detected.

V. Final Result

Total impurities (HPLC) 0.89 %

Water content 0.34 %

Residual solvents 0.00 %

Assay (100 % method) 98.77 %

The assay is assessed to be 98.8 % ‘as is’

The assay 'as is' is equivalent to the assay based of the not anhydrous and not dried substance

respectively.

Release Date: 2010-06-01 LGC GmbH

Dr. Sabine Schröder

Product Release




Reference Material - Primary Standard Product Name: Tramadol-13C-D3 Hydrochloride 0.1 mg/ml in Methanol (as free base)


Lot Number: 40525

CAS Number: unlisted

Molecular Formula: C1513CH22D3NO2 HCl


Solvent: Methanol



Expiry Date: December-2015





opening to avoid concentration changes due to evaporation. Warranty does not apply to ampoules stored after opening. Release Date: Luckenwalde, January 2014 Signed: Dr. Sabine Schröder Unit for Reference Materials 1 Ampoules are overfilled to ensure a minimum 1 ml volume fill. We advise laboratories to use measured volumes of this standard solution before diluting to the desired concentration. 2 The value is based on the results of analytical techniques, which calibration and verification was carried out with standards traceable to SI-units. The value is expressed on a "free base" basis. The concentration with its uncertainty is valid in the range between 19 °C and 25 °C.









Lot Number



40525 0.1020 ± 3 % 1.493 � 3 % Concentration verified by HPLC

Solution Standard Assay Parameters External Calibration (100 % amount)


Column: 60 RP Select B, 5 µm, 125 x 4 mm Number of Measurements: 6





Neat Material Data

Product Name: Tramadol-13C-D3 Hydrochloride

CAS Number: unlisted

Molecular Formula: C1513CH22D3NO2 HCl


Compound Lot: 40524

Test Method Result





Isotopic Purity by HRMS 0.30 % D2, 99.70 % D3



Assay by carbon titration (“as is”)* 99.71 %

*: Validated method performed by ISO/IEC 17025 accredited testing lab. Purity factor does not include adjustment for chiral and/or isotopic purity.






I. Identity


Ia. 1H-NMR Spectrum

Conditions: 400 MHz, CDCl3





Ib. Mass Spectrum

Method: 4.5 kV ESI; vaporization temperature: 200 °C, direct inlet


268.02 [ MH ]

250.01 [ MH – H2O ]





Ic. IR Spectrum



Id. Melting Point

181 °C

Ie. UV Spectrum







Results:



III. Purity




Minutes

0 5 10 15 20 25 30 35

mA

U

0

50

100

150

200

250

mA

U

0

50

100

150

200

250

4.1

1

11

.59

12

.43

Surveyor PDA 4-215nmRetention Time





Pk # Retention Time Area Area % 1 4.11 2723128 99.60 2 11.59 2574 0.09 3 12.43 8351 0.31

Totals 2734053 100.00



HPLC Conditions:



5 µm, 125 x 4 mm 0 – 6 min Water/Acetonitrile 80/20





0.1 % H3PO4

215 nm 3 µl; 0.188 mg/ml in


Results:

Arithmetic mean (n=3) 99.60 %

IIIb. Water Content


Results:



Method: 1H-NMR

Result: 0.26 % tert-Butyl methyl ether








testing (HPLC).


General






Handling of the RM






35

Appendix Three: Extra Material

Tramadol Hydrochloride Calibration Data Calibration Curve

Retention Time for Each Calibration Point

N-Desmethyl Tramadol Calibration Data Calibration Curve


O-Desmethyl Tramadol Calibration Data Calibration Curve


Venlafaxine Hydrochloride Calibration Data Calibration Curve


N-Desmethyl Venlafaxine Calibration Data Calibration Curve


O-Desmethyl Venlafaxine Calibration Data Calibration Curve


Reproducibility Charts for Quality Control Concentrations

School of Applied Science, University of South Wales

1

Information Sheet Study Title Drugs of Abuse Testing in Fingernails Invitation paragraph I would like to invite you to take part in a research study. Before you decide you need to understand why the research is being done. Please take time to read the following information carefully. Ask questions if anything you read is not clear or would like more information. Take time to decide or not to take part. For the past decade hair testing for drugs of abuse has become increasingly popular but there may be cases where people cannot provide a hair sample for medical reasons there is a need for a new, non-invasive matrix to be used. Fingernails are very similar to hair; they can incorporate drugs within the nail bed for a significant amount of time. The collection is non invasive and can easily be transported. Since 2014, Tramadol has become a controlled substance and can only be used with a prescription. Therefore, for this project I have decided to research whether fingernails can be used for the testing of drugs of abuse and whether it is possible to detect Tramadol. What is the purpose of the study? Purpose of this study is to determine whether it is possible to use fingernails to detect drugs of abuse. This research study is going to try and detect Tramadol specifically as from 2014 Tramadol became a control substance as part of the Misuse of Drugs Act 1971 and the level of individuals abusing Tramadol is rising. Why have I been invited? I have chosen you as a participant as it has come to my knowledge that you have been prescribed a particular dose of Tramadol on a regular basis. Do I have to take part? It is up to you whether you decide to take part. We will describe the study and go through the information sheet that I will give to you. We will ask you to sign a consent form to show that you have agreed to take part in the project. You are free to withdraw at any point, without giving a reason. What will happen to me if I take part?

x All I require is fingernail clipping samples. x Information regarding the dosage of Tramadol that is prescribed i.e. what dosage

you are currently taking? How often do you have to take Tramadol? What will I have to do? Using clippers, remove as much as the as the white area of fingernai l possible and place into a seal proof bag and write down the current dosage of Tramadol that you have been using from prescription.


2

What are the possible benefits of taking part? We cannot promise that the study will benefit you personally, but the information we get from the study will help determine whether individuals are abusing drugs and whether it is possible to use a different sample method that is not invasive in comparison to drawing blood and a urine test. What if there is a problem? If you have any concerns about the study in any way, please do not be afraid to contact myself on my mobile or by email, I will do my best to answer your questions. Will my taking part in the study be kept confidential? This is an anonymous donation and there will be no record of Name, Address, Telephone number. If there are any samples left over and not used during the analysis, these samples will be destroyed indefinitely. All information which is collected during the course of this research will be kept strictly confidential. What will happen if I don’t carry on with the study? If you decide to withdraw from the study all the information and data collected from you, to date will be destroyed and removed from the research project. What will happen to the results of the research study? As this is part of a Masters Degree Dissertation the project results will be shared with the University of South Wales which will include the Project Supervisor and an External Examiner for marking. The results will be read by me, possibly staff members at Synergy Health and my university supervisor. If you have any queries regarding who will have access to the results from the research project please do not hesitate to ask. Who is organising or sponsoring the research? The research study is being collaborated with a drugs testing company Synergy Health PLC who are based in Abergavenny. It is at the site where I will be conducting my research. Other than this, the research project is being fulfilled as part of a Masters Dissertation for the University of South Wales. Further information and contact details: If you have any questions please do not hesitate to ask, you may contact myself by telephone or email. Mobile: 07583653270 Home Email: [email protected] University Email: [email protected]

mailto:[email protected]

mailto:[email protected]


1

CONSENT FORM Project Title: Drugs of Abuse Testing in Fingernails

I agree to take part in the above School of Applied Science (University of South Wales)

research project. I have had the project explained to me, and I have read the Participant

Information Sheet. I understand that agreeing to take part means that I am willing to:

x Provide samples of Fingernails

x Provide information regarding Tramadol dosage

Data Protection This information will be held and processed for the following purpose(s): Quantification of Tramadol detectable in Fingernails I understand that any information I provide is confidential, and that no information that could lead to the identification of any individual will be disclosed in any reports on the

project, or to any other party. No identifiable personal data will be published.

OR The identifiable data will be shared with Synergy Health Laboratories and the University of

South Wales. This organisation has made a written agreement with the University to abide by the Data Protection Principles.

OR

I understand that no forenames and surnames will be collected to protect my identity from being made public.

I agree that Bethan Davies can process this information about me. I understand that this

information will be used only for the purpose(s) set out in this statement and my consent is conditional on the University complying with its duties and obligations under the Data

Protection Act 1998.

FINAL DISSERATION DOCUMENT

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