THCCOOH and ETG in hair: analytical challenges and ... · Luca Morini. Department of Public Health,...

Luca Morini

Department of Public Health, Experimental and Forensic MedicineUniversity of PaviaVia Forlanini 12, 27100Pavia, Italy

THCCOOH and ETG in hair: analytical challenges and interpretation of results

in Forensic Toxicology.

Department of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

Metabolites in hair: why?Department of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

Advantages

Simple sample

collection

Easy storage

Simple sample preparation

Evaluation of a past use

Segmentalanalysis

Wide window of detection

Regional protocolDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

• New regional protocol for the determination of drugs of abuse and alcohol for the evaluation of drive license eligibility

• It has been developed and edited in 2014

• It regards all the procedures and the tests that should be performed on drivers in Lombardy

Italy – Lombardy - PaviaDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

Laboratories currently accredited for hair analysis in Lombardy


Pavia (1): Forensic Toxicology lab

Milan (4): Forensic Toxicology lab

2 HospitalsPrivate Lab

Varese (2): Health Local A gency

Private Lab

Como (1): Hospital

Como (1): Health Local Agency

Sondrio (1): Health Local Agency

Bergamo (1): Health Local Agency

Brescia (3):Forensic Toxicology lab

Health Local AgencyPrivate lab

Monza (2): Hospital

Private lab

Cremona (1): Health Local Agency

Regional Protocol: procedureDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

Hair analysis: Opiates Cocaine Cannabinoids Amfetamine MDMA Methadone Buprenorphine Ketamine Ethyl glucuronide (**)

Hair lenght for sample collection

a) Hair length ≥ 6 cm- analyis on the 6 cm proximal hair segment- analysis on two segments (0-3 cm; 3-6 cm)b) Hair lenght ≥3cm and <6 cm- analysis of the total amount of samplesc) Hair lenght < 3 cm- Postpone the sample collection- Collection of hair from other body area (*)d) Baldness- collection of hair from other body area (*)

Urine analysis Opiates Cocaine Cannabinoids Amfetamine MDMA Benzodiazepines Methadone Buprenorphine Ketamine

* Hair from other body area should be interprete with caution

** Pubic hair should not be used for EtG analysis. Hair from chest area should be preferred

Regional protocol: cutoffDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

Class Compound Cut-off (ng/mg)Opiates Morphine 0.2

6-acetylmorphine 0.2Codeine 0.2

Cocaine Cocaine 0.2Benzoylecgonine 0.05Cocaethylene 0.05Ecgonine methyl ester 0.05

Amfetamine Amfetamine 0.2Metamfetamine 0.2

MDMA MDMA 0.2MDA 0.2MDEA 0.2MBDB 0.2

Cannabinoids THC 0.05THCCOOH 0.001

Methadone Methadone 0.2EDDP 0.2

Buprenorphine Buprenorphine 0.05Norbuprenorphine 0.05

Ketamine Ketamine 0.5Ethyl glucuronide Ethyl glucuronide 0.03

THC analysis: sample preparation• Hair sample extracted in NaOH 5 M.• Liquid/liquid extraction with hexane-ethyl acetate mixture (9:1)

and 1 ml acetate buffer at pH 5 • Solvent soaked under nitrogen stream. Addition of 50µl

MSTFA, maintained at 75 °C for 15 minutes:• Chromatographic settings

– Gascromatography: HP 6890 Plus with HP 7683 Autosampler– Column: HP 5 Ultra 2, (5%-phenyl)-methylpolysiloxane (12 m x 0.2 mm i.d. x

0.33 μm)– Mass detector: HP 5973– Carrier gas: Helium (10.3 psi, 1 ml/min, constant flow)– Injection volume: 1 μl

Ramp: – Injector: pulsed splitless, 1 min, 25 psi , 250 °C– Column: 100 °C (2.5 min) 40 °C /min 180 °C 10 °C/min 290 °C (10 min)– Interface: 280 °C– Source: 230 °C


THCCOOH: sample preparation (L/L)• The remaining solution from THC analysis is acidified with

glacial acetic acid.• Liquid/liquid extraction with hexane-ethyl acetate mixture (9:1)

and 1 ml acetate buffer at pH 5 • Solvent soaked under nitrogen stream. Addition of 50µl PFPA

and 25µl HFIP and maintained at 75 °C for 1 h:• Solvent soaking followed by 50µ hexane addition• Chromatographic settings

– Capillary column HP-5MS (5%-phenyl)-methylpolysiloxane) (30 m x 0,25mm i.d., 0,25 μm).

– Carrier: He at 1,2 ml/min– NCI source at 150°C– Source Gas: Methane– Collision Gas: nitrogen– CE: 10 eV -5 eV– dwell time= 100 milliseconds


THCCOOH: sample preparation (SPE)• The remaining solution from THC analysis is acidified with

glacial acetic acid• Samples extractred thorugh SPE procedure using Bond Elut

Certify II cartridges

• Extraction procedure:– 2ml methanol– 2 ml sodium acetate buffer with 5% methanol– Sample loading– 2 ml methanol-water solution (1:1)– Elution with 2ml of hexane-ethyl acetate solution (75:25)

with 1% of glacial acetic acid.


THCCOOH: sample preparation (SPE)• The remaining solution from THC analysis is acidified with

glacial acetic acid• Samples extractred thorugh SPE procedure using UCT

cartridges

• Extraction procedure:– 1 x 3 mL CH3OH– 1 x 3 mL H2O– 1 x 1 mL 100 mm HCl– Load sample at 1-2 mL / minute– 1 x 3 mL DI H2O– 1 x 3 mL CH3CN /HCl (30:70)– Dry column (5 minutes at > 10 inches Hg)– 1 x 3 mL Hexanes/Ethyl acetate/Acetic Acid (49:49: 2)


Capillary coumn HP-5MS 5% phenil metil silox (30 m x 0,25mm i.d., 0,25 μm).

Carrier gas: He at 1,2 ml/min Soruce: NCI at 150°C Source Gas: Methane Collision Gas: Nitrogen CE: 10 eV -5 eV dwell time= 100 ms


Instrumentation

THCCOOH derivatizationDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

THCCOOH

THCCOOH-d3 (IS)

DERIVATIZATION PFPA HFIP

1h 60°C

THCCOOH-d3: m/z 623 m/z 495; (10 eV)

THCCOOH-d3: m/z 623 m/z 386; (5 eV)

THCCOOH: m/z 620 m/z 492; (10 eV)

THCCOOH: m/z 620 m/z 383; (5eV)

THCCOOH: chromatogramsDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

Real positive sample (0.6 pg/mg THCCOOH)

LOD= 0,02 pg/mgLOQ= 0,04 pg/mg

Calibration curveDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

SampleTHC

(ng/mg)

THCCOOH

(pg/mg)Sample

THC

(ng/mg)

THCCOOH

(pg/mg)

1 0.77 1.077 21 0.061 0.35

2 0.74 0.51 22 0.145 0.12

3 0.068 0.77 23 0.082 0.58

4 0.097 0.0495 24 0.071 0.123

5 0.06 0.23 25 0.137 0.89

6 0.24 1.53 26 0.198 Neg

7 0.56 0.4 27 0.925 1

8 0.28 23.84 28 0.122 Neg

9 0.07 2.27 29 0.225 Neg

10 0.15 Neg 30 0.07 Neg

11 0.052 0.25 31 0.086 Neg

12 0.155 0.67 32 0.955 1.87

13 0.169 1.8 33 0.06 0.117

14 0.099 0.72 34 0.068 Neg

15 0.091 0.086 35 0.254 0.113

16 0.08 Neg 36 0.07 Neg

17 0.083 0.34 37 0.079 Neg

18 0.107 0.6 38 0.82 1.5

19 0.29 0.39 39 0.13 Neg

20 0.073 0.176 40 0.12 0.1

THCCOOH: Real Samples

SampleTHC

(ng/mg)

THCCOOH

(pg/mg)Sample

THC

(ng/mg)

THCCOOH

(pg/mg)1 0.770 1.08 25 0.137 0.89

6 0.240 1.53 40 0.097 0.05

8 0.280 23.84 10 0.120 0.10

9 0.070 2.27 15 0.091 0.09

13 0.169 1.80 20 0.073 0.18

27 0.925 1.00 22 0.145 0.12

32 0.955 1.87 24 0.071 0.12

38 0.820 1.50 33 0.060 0.12

2 0.740 0.51 35 0.254 0.11

3 0.068 0.77 10 0.150 Neg

5 0.060 0.23 16 0.080 Neg

7 0.560 0.40 26 0.198 Neg

11 0.052 0.25 28 0.122 Neg

12 0.155 0.67 29 0.225 Neg

14 0.099 0.72 30 0.070 Neg

17 0.083 0.34 31 0.086 Neg

18 0.107 0.60 34 0.068 Neg

19 0.290 0.39 36 0.070 Neg

21 0.061 0.35 37 0.079 Neg

23 0.082 0.58 39 0.130 Neg

THCCOOH: Real Samples

THCCOOH: Real samples• Only 8 out 40 (20%) tested positive for either THC and

THCCOOH by using the proposed cut-off

• Decreasing the cut off to 0.2 pg/mg (the one suggested by the SoHT) we would assess a total of 21 cannabis consumers among the tested group

• The number of positive samples could be even more increased (75%) by using as cut-off the LOQ (0.04 pg/mg)


LiteratureDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

Questions still open for discussion

• The use of THCCOOH in hair is a reliable tool for discriminating cannabis users?

• What is the most reliable cutoff?

• Is it acceptable the use of a less sensitive method for THCCOOH detection in hair?


Ethyl glucuronide

• Direct ethanol metabolite

• Highly sensitive and specific markers for alcohol misuse diagnosis in the short, mid and long term, depending on the matrix evaluated.

UDP-glucuronosyltransferase

Ethanol β-D-ethylglucuronide (EtG)


LC-MS/MS: EtG ed EtS

• Direct Marker: high diagnostic sensitivity and specificty;

• Indicator of alcohol use/misuse in the short (blood/serum/plasma) mid (urine) and long (hair, nails) term;

• Determination of EtG in meconium is useful to evaluate intrauterine exposure to alcohol.


Regional Protocol: procedureDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

Hair analysis: Opiates Cocaine Benzoylecgonine Cannabinoids Amfetamine MDMA Methadone Buprenorphine Ketamine Ethyl glucuronide (**)

Hair lenght for sample collection

a) Hair length ≥ 6 cm- analyis on the 6 cm proximal hair segment- analysis on two segments (0-3 cm; 3-6 cm)b) Hair lenght ≥3cm and <6 cm- analysis of the total amount of samplesc) Hair lenght < 3 cm- Postpone the sample collection- Collection of hair from other body area (*)d) Baldness- collection of hair from other body area (*)

Urine analysis Opiates Cocaine Cannabinoids Amfetamine MDMA Benzodiazepines Methadone Buprenorphine Ketamine

* Hair from other body area should be interprete with caution

** Pubic hair should not be used for EtG analysis. Hair from chest area should be preferred

Regional protocolDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

Biomarker Teetotaller or occasional user

Grey zone Harmfulconsumption (≥ 60 g/day)

EtG ≤ 7.0 pg/mg 7.0 ≤ 30.0 pg/mg ≥ 30 pg/mg

CDT* ≤ 1.8 1.8 ≤ 2.2 ≥ 2.2

* Depending on the analytical procedure the cut-off could be different. The procedure should be stated

In case of a discordance between the two biomarker the EtG result should be preferred

Etg in hair: sample preparation• Hair (30 mg) washed with dichlormethane and methanol

• Addition of 1 ml water with EtG-D5

• Ultrasonication for two hours:

• SPE extraction on Strata-X-A Phenomenex:– 2 ml methanol; – 2 ml water– Sample loading– 2 ml water (2% of NH3)– 2 ml methanol– Elution with 2 ml methanol (5% formic acid)


EtG in hair: instrumental parameters• Solvent soaking under nitrogen stream. Addition of 75µl PFPA,

maintained at 50 °C for 1 h:• Solvent Soaked and remaining eluted in 50 µl hexane• Chromatographic parameters

– Capillary column HP-5MS 5% phenyl methyl silox (30 m x 0,25mm i.d., 0,25 μm).

– Carrier gas: He at 1,2 ml/min– Source NCI at 150°C– Source Gas: Methane– Collision Gas: Nitrogen– CE: 10 eV -5 eV– dwell time= 100 ms


EtG-d5: m/z 352 m/z 163; (5 eV)

EtG: m/z 347 m/z 163; (5 eV)

EtG: m/z 347 m/z 119; (5eV)

EtG: chromatogramsDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

Real positive sample (7 pg/mg)

LOD= 2 pg/mgLOQ= 3 pg/mg

Calibration curveDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

“The concentration of ethyl glucuronide in hair appears to correlate with EDI”Ethyl glucuronide in hair: Is it a reliable marker of chronic high levels of alcohol consumption? Politi L, Morini L, Leone F, Polettini A. Addiction. 2006 Oct;101(10):1408-12

“Variations in the EtG concentrations with respect to hair segments may provide an overview of the drinking history of patients. Moreover, EtG concentration in hair may help to estimate the daily alcohol intake”Segmental determination of ethyl glucuronide in hair: a pilot study.Appenzeller BM, Agirman R, Neuberg P, Yegles M, Wennig R. Forensic Sci Int. 2007 Dec 20;173(2-3):87-92. Epub 2007 Mar 6

“These findings suggest that measurements of EtG in hair may provide improved diagnostic information on alcohol abuse, due to a long retrospective time-window for detection and stability of EtG in hair in the decaying cadaver”Comparison of ethyl glucuronide in hair with phosphatidylethanol in whole blood as post-mortem markers of alcohol abuse. Bendroth P, Kronstrand R, Helander A, Greby J, Stephanson N, Krantz P. Forensic Sci Int. 2008 Mar 21;176(1):76-81. Epub 2007 Nov 19.

HEtG: literatureDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy

http://www.ncbi.nlm.nih.gov/pubmed/16968341



“Our results confirm further that HEtG is a sensitive and specific marker of chronic heavy drinking”Ethyl glucuronide in hair. A sensitive and specific marker of chronic heavy drinking. Morini L, Politi L, Polettini A. Addiction. 2009 Jun;104(6):915-20. Epub 2009 Apr 9

“Our results indicate that HEtG, as compared to CDT measured using different methods, is a selective marker of ethanol heavy chronic use providing considerably higher sensitivity”Comparison of ethyl glucuronide in hair with carbohydrate-deficient transferrin in serum as markers of chronic high levels of alcohol consumption. Morini L, Politi L, Acito S, Groppi A, Polettini A. Forensic Sci Int. 2009 Jul 1;188(1-3):140-3. Epub 2009 May 1

“The present assay appears convenient to carry out owing to the very small quantity of hair samples required to determine an effective heavy alcohol consumption ”Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography-tandem mass spectrometry following solid-phase extraction. Lamoureux F, Gaulier JM, Sauvage FL, Mercerolle M, Vallejo C, Lachâtre G. Anal Bioanal Chem. 2009 Aug;394(7):1895-901. Epub 2009 Jun 12.

HEtG: literatureDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy




“EtG analysis in hair is a useful tool for assessing fitness to drive in suspected drinking drivers; compared to CDT values it provides a direct and unequivocal marker for reliable abstinence monitoring over a period of several months, depending on the length of the hair”Abstinence monitoring of suspected drinking drivers: ethyl glucuronide in hairversus CDT.Liniger B, Nguyen A, Friedrich-Koch A, Yegles M. Traffic Inj Prev. 2010 Apr;11(2):123-6.

“It is recommended, that in context of driving ability diagnostics the EtG result is determinant”Comparison of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) concentrations in hair for testing abstinence. Albermann ME, Musshoff F, Madea B. Anal Bioanal Chem. 2011 Apr;400(1):175-81. Epub 2010 Dec 3.

“EtG can be used qualitatively to indicate alcohol consumption with a positive result providing strong evidence for an individual drinking within the past 3 months”Comparison of ethyl glucuronide in hair with self-reported alcohol consumption.Lees R, Kingston R, Williams TM, Henderson G, Lingford-Hughes A, Hickman M. Alcohol Alcohol. 2012 May-Jun;47(3):267-72. Epub 2012 Feb 14.

HEtG : literatureDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy




HEtG: cut-off e consensus

Consensus of the Society of Hair Testing on hair testing for chronic excessive alcohol consumption 2009.

Kintz P.Forensic Sci Int. 2010 Mar 20;196(1-3):2. Epub 2010 Jan 8.


• Highly sensitive and specific marker for excessive chronicalcohol consumption

• Cutoff: 30 pg/mg (since 2012 also the cutoff at 7.0 pg/mg)

• Consensus on EtG and FAEEs approved during the SoHTmeeting in Rome (2009) and revised every two years


HEtG: limitations• Hair treatments could hydrolized/eliminate EtG from the matrixEffect of bleaching on ethyl glucuronide in hair: an in vitro experiment.Morini L, Zucchella A, Polettini A, Politi L, Groppi A. Forensic Sci Int. 2010 May20;198(1-3):23-7. Epub 2009 Dec 3.

• EtG in pubic hair tends to overestimate EDIEthyl glucuronide determination: head hair versus non-head hair.Kerekes I, Yegles M, Grimm U, Wennig R. Alcohol Alcohol. 2009 Jan-Feb;44(1):62-6. Epub 2008 Nov 28

• EtG in axillary hair underestimates the concentration of EtG in head hair

A study of distribution of ethyl glucuronide in different keratin matrices.Pirro V, Di Corcia D, Pellegrino S, Vincenti M, Sciutteri B, Salomone A. Forensic Sci Int. 2011 Jul 15;210(1-3):271-7. Epub 2011 Apr 20





HEtG: limitations• In a recent study EtG in patients with severe decreased kidney

function is accumulated i hair in an higher ratio

Higher Levels of Hair Ethyl Glucuronide in Patients with Decreased KidneyFunction.Høiseth G, Morini L, Ganss R, Nordal K, Mørland J. Alcohol Clin Exp Res. 2012 Jun 14. doi: 10.1111/j.1530-0277.2012.01882.x. [Epub ahead of print]




Grazie per l’attenzione

Thanks for the attention

THCCOOH and ETG in hair: analytical challenges and ... · Luca Morini. Department of Public Health,...

Documents

Transcript of THCCOOH and ETG in hair: analytical challenges and ... · Luca Morini. Department of Public Health,...