Validation of an Enzyme-Linked Immunosorbent Assay Screening

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An ELISA and a liquid chromatography–tandem mass spectrometry(LC–MS–MS) confirmation method were developed and validatedfor the identification and quantitation of ketamine and its majormetabolite norketamine in urine samples. The Neogen® ketaminemicroplate ELISA was optimized with respect to sample andenzyme conjugate volumes and the sample preincubation timebefore addition of the enzyme conjugate. The ELISA kit wasvalidated to include an assessment of the dose-response curve,intra- and interday precision, limit of detection (LOD), andcross-reactivity. The sensitivity and specificity were calculatedby comparison to the results from the validated LC–MS–MSconfirmation method. An LC–MS–MS method was developedand validated with respect to LOD, lower limit of quantitation(LLOQ), linearity, recovery, intra- and interday precision,and matrix effects. The ELISA dose-response curve was atypical S-shaped binding curve, with a linear portion of thegraph observed between 25 and 500 ng/mL for ketamine. Thecross-reactivity of 200 ng/mL norketamine to ketamine was2.1%, and no cross-reactivity was detected with 13 commondrugs tested at 10,000 ng/mL. The ELISA LOD was calculatedto be 5 ng/mL. Both intra- (n = 10) and interday (n = 50)precisions were below 5.0% at 25 ng/mL. The LOD for ketamineand norketamine was calculated statistically to be 0.6 ng/mL.The LLOQ values were also calculated statistically and were1.9 ng/mL and 2.1 ng/mL for ketamine and norketamine,respectively. The test linearity was 0–1200 ng/mL with correlationcoefficient (R2) > 0.99 for both analytes. Recoveries at 50,500, and 1000 ng/mL range from 97.9% to 113.3%. Intra-(n = 5) and interday (n = 25) precisions between extractsfor ketamine and norketamine were excellent (< 10%). Matrixeffects analysis showed an average ion suppression of 5.7%for ketamine and an average ion enhancement of 13.0%for norketamine for urine samples collected from six individuals.

A comparison of ELISA and LC–MS–MS results demonstrated asensitivity, specificity, and efficiency of 100%. These resultsindicated that a cutoff value of 25 ng/mL ketamine in the ELISAscreen is particularly suitable and reliable for urine testing in aforensic toxicology setting. Furthermore, both ketamine andnorketamine were detected in all 34 urine samples collectedfrom individuals socializing in pubs by the Royal MalaysianPolice. Ketamine concentrations detected by LC–MS–MS rangedfrom 22 to 31,670 ng/mL, and norketamine concentrationsranged from 25 to 10,990 ng/mL. The concentrations ofketamine and norketamine detected in the samples are mostikely indicative of ketamine abuse.

Introduction

Ketamine was first synthesized in 1962 by Calvin Stevens atthe Parke Davis Laboratory in Detroit, Michigan. It was firstmarketed under the tradename Ketalar™ in 1970 followingFood and Drug Administration (FDA) approval for human useas a short-acting general anesthetic drug that was also usedfor animals (1,2). Ketamine produces narcotic effects similarto phencyclidine (PCP) and hallucinogenic effects similar tolysergic acid diethylamide (LSD), making it popular with userswho sought a dissociative experience (3). The recreational useof ketamine as a rave, party, and nightclub drug has widened,thus increasing public concerns about the potential hazardsof this drug (2,4). Ketamine abuse was reported in the UnitedKingdom and Europe in the 1990s (5). In the United Kingdom,ketamine was classified as a Class C drug under the Misuse ofDrugs Act 1971 and therefore carries up to 2 years imprison-ment and/or an unlimited fine for possession and up to 14years imprisonment and/or an unlimited fine for possession

Validation of an Enzyme-Linked Immunosorbent AssayScreening Method and a Liquid Chromatography–Tandem Mass Spectrometry Confirmation Method forthe Identification and Quantification of Ketamine andNorketamine in Urine Samples from Malaysia

Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.

Journal of Analytical Toxicology, Vol. 33, July/August 2009

Norlida Harun*, Robert A. Anderson, and Eleanor I. MillerForensic Medicine and Science, Division of Cancer Sciences and Molecular Pathology, Faculty of Medicine,University of Glasgow, Scotland, G12 8QQ United Kingdom

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to supply or supplying (6). Its misuse later spread to Asiancountries, in particular, China, Malaysia, Taiwan, and Singa-pore (1). In the United States, ketamine has been classified asa club drug by the National Institute on Drug Abuse (NIDA)(7). The rapid growth of ketamine misuse worldwide has led tothe development of various methods such as gas chromatog-raphy–mass spectrometry (GC–MS) (8–16), liquid chro-matography (LC)–MS (17–20), a combination of GC–MS andLC–MS (22), and emzyme-linked immunosorbent assay(ELISA) (10,15,20).

Ketamine undergoes N-demethylation by liver microsomalcytochrome P450 enzymes CYP 3A4, CYP 2B6, and CYP 2C9 toform its primary metabolite, norketamine (22). Norketamineundergoes dehydrogenation to form dehydronorketamine,which is then conjugated with glucuronic acid before excre-tion in urine. The basic ketamine metabolic pathway is shownin Figure 1 (23).

Urine continues to be a widely used specimen for the anal-ysis of drugs of abuse in some situations, such as workplacetesting, for a number of reasons including the non-invasivemethod of collection, simple sample pre-treatment and anal-ysis, the large volume of sample that can be obtained for anal-ysis, the wide drug detection window compared to blood, andthe presence of the parent drug and metabolites in high con-centrations compared to other matrices (24). Ketamine andnorketamine can be detected in urine up to 5 and 6 days, re-spectively, after administration, and dehydronorketamine canbe detected in urine for up to 10 days (25).

Currently, ELISA is popular within the forensic toxicologycommunity (26) and is, at the time of writing, the only im-munoassay system available for the rapid, qualitative screen-ing of ketamine and its metabolites (20). The test is fast, sim-ple, can be automated to screen a large number of samplessimultaneously, requires a small sample volume, and can beapplied in the analysis of a range of biological matrices.

LC–MS–MS has become a useful tool in the analysis of drugsof abuse in urine. In this study, electrospray ionization (ESI),a soft ionization technique in LC–MS–MS analysis, was appliedin the selected reaction monitoring (SRM) mode to obtain bet-ter sensitivity and satisfy identification criteria requirements(24).

Although 177.91 kg ketamine was seized by the MalaysianRoyal Police in 2007 (27), ketamine was not included in rou-tine investigations when drug abuse suspects were tested bythe National Agency of Drug Abuse in Malaysia. Consequently,there are no data currently available on the extent of ketamineabuse in Malaysia. The aim of this current study was to de-velop and validate an ELISA screening method and anLC–MS–MS confirmation method to be used in tandem for therespective identification and quantitation of ketamine and nor-ketamine in urine samples. This method would be very usefulin a workplace testing or forensic toxicology setting. Prelimi-nary data collected from this study provide information on theconcentrations of ketamine and norketamine typically foundin urine samples collected from individuals frequenting pubsand clubs in Malaysia.

Materials and Methods

Reagents, standards, and specimensKetamine ELISA kits (product number: 109419) were pur-

chased from Neogen (Lexington, KY). The kits contained 96antibody coated microplate wells, wash buffer (phosphatebuffer saline solution containing a surfactant, pH 7), ketamineenzyme conjugate labeled with horseradish peroxidase (HRP),3,3',5,5'-tetramethylbenzidine (TMB) substrate solution withhydrogen peroxide (H2O2), a red stop solution containing 1 NH2SO4, and negative (0 ng/mL) and positive controls (2000ng/mL) prepared in synthetic urine. Phosphate buffer saline(PBS) pH 7 was purchased from Immunalysis® (Pomona, CA)and contained bovine serum albumin and non-azide preserva-tives.

Ketamine, norketamine, ketamine-d4, norketamine-d4,and the drug standards used to test the cross-reactivityof the ELISA kit, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-methylamphetamine (MDMA), 3,4-methylenedioxyethylam-phetamine (MDEA), cocaine, benzoylecgonine, diazepam,nordiazepam, morphine, methadone, 6-monoacetylmorphine(6-MAM), and phencyclidine (PCP), were obtained from

Promochem (Teddington, U.K.). TiletamineHCl powder was purchased from Sigma-Aldrich (Dorset, U.K.). Ammonium acetateand ammonium formate were purchasedfrom Fluka (Buchs, Switzerland). HPLC-grade formic acid, methanol, and acetonitrilewere from BDH (Poole, U.K.). β-Glu-curonidase Type HP-2 from Helix pomatia(100 U/μL) was obtained from Sigma-Aldrich.Synergi Hydro RP column (150-mm length,2.0-mm i.d., 4-µm particle size) waspurchased from Phenomenex (Torrance, CA)along with a guard column (4.0 mm ×2.0 mm, 5 µm) with the same packing asthe main column. World Wide MonitoringClean Screen® columns (ZSDAU 020) usedfor drug extraction in the LC–MS–MS method


Figure 1. Basic ketamine metabolic pathway (23).

cyclohexanone glucuronide derivatives

hydroxylation and conjugation

norketamine dehydronorketamine

dehydrogenation

ketamine

N-demethylation

* the chiral point

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were purchased from United Chemical Technologies (Bristol,PA).

Thirty-four urine samples were obtained from the NarcoticDepartment of the Royal Malaysian Police. All urine specimens(10 mL volume in each container) were freeze-dried at –54°Cby the Drug Laboratory, Pathology Department, KualaLumpur General Hospital in Malaysia using a Cole Palmer 1 LBenchtop Freeze Dry System. The lyophilized samples weresent by courier to the forensic toxicology laboratory at the Uni-versity of Glasgow. Each of the samples was reconstituted with10 mL deionized water prior to analysis by ELISA andLC–MS–MS. These samples were previously screened using aqualitative GC–MS method with a cutoff of 350 ng/mL at theDrug Laboratory, Pathology Department, Malaysia. TheseGC–MS results were compared to the ELISA and LC–MS–MSresults obtained in this study (Table I). Ten negative urine con-trols were obtained from volunteers among the Glasgow lab-oratory personnel.

Microplate well ELISAOptimization of ELISA procedure

Combinations of different sample volumes(20 and 40 µL), enzyme conjugate volumes(90 and 180 µL) and sample pre-incubationtimes before addition of enzyme conjugate (0,30, and 60 min) were tested.

A Miniprep 75 automatic pipettor pur-chased from Tecan (San Jose, CA) was usedto dilute samples and pipette all calibrators,quality control samples and case samplesinto the microplate wells. Twenty microlitersof diluted calibrator, quality control, or casesample (diluted 1:10 online with PBS) wasloaded into the microplate wells in duplicate.After pipetting the samples, 180 µL of dilutedenzyme conjugate (diluted 1:180, v/v, accord-ing to kit manufacturer instructions) wasadded into each microplate well. The mi-croplate was then left in the dark at roomtemperature for 45 min. Then the wells werewashed manually five times with 300 µL washbuffer to remove any unbound sample orresidual conjugate reagent remaining inthe wells. K-Blue substrate (150 µL) wasthen added to the microplate wells and leftto incubate in the dark at room temperaturefor a further 30 min. The reaction wasstopped by adding 50 µL stop solution (1 NH2SO4) to each well, turning the contents yel-low. The plate was read at a wavelength 450nm using a Sunrise Remote EIA reader byTecan (Grödlg, Austria).

Method validation for ELISADose-response curve. A dose-response

curve was generated for urine spiked at con-centrations of 0, 10, 25, 50, 100, 500, 1000,2000, 4000, and 8000 ng/mL ketamine. The

test was performed in duplicate and the data were expressed asmean of the B/B0 (%) readings where B is the absorbance read-ing of the bound calibrator and B0 is the absorbance value ofthe blank calibrator. The ketamine concentration range usedin the dose-response curve spanned the range previously re-ported in the literature for ketamine concentrations detectedin urine (2,8,10,12,14,15,17).

Limit of detection (LOD). Ketamine was spiked at concen-trations of 0, 0.5, 1, 2, 4, 5, 10, 25, and 50 ng/mL to establisha calibration curve. Ten negative urine samples were usedin the calculation. The LOD absorbance value was calculatedas the concentration having a signal-to-noise ratio of 3:1.This absorbance value was matched to the absorbance valueproduced by one of the ketamine-spiked calibrators on thegraph.

Intra- and interday precision. The intraday precision ofB/B0 (%) values was determined by spiking 10 × 1 mL


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Table I. GC–MS, ELISA, and LC–MS–MS Urine Sample Results

GC–MS Qualitative ELISA LC–MS–MS LC–MS–MS Ratio ofSample Screening at Concentration Ketamine* Norketamine* Norketamine/Number Cutoff 350 ng/mL (ng/mL) (ng/mL) (ng/mL) Ketamine

1 Positive > 125 2830 6960 2.52 Positive > 125 880 3970 4.53 Positive > 125 2670 6800 2.54 Positive > 125 1200 1230 1.05 Positive > 125 5340 4070 0.86 Positive > 125 1210 6960 5.87 Positive > 125 4570 420 0.18 Positive > 125 920 6400 7.09 Positive > 125 150 1830 12.2

10 Positive > 125 200 10,990 55.011 Positive > 125 790 5250 6.612 Positive 32 31 690 22.213 Positive > 125 11,070 8810 0.814 Positive 37.4 36 1540 42.815 Positive 25.8 22 1080 49.116 Positive 29.6 27 1250 46.217 Positive > 125 8460 220 0.0318 Positive > 125 130 8980 69.119 Positive > 125 1150 10,320 9.020 Positive > 125 560 8650 15.421 Positive > 125 130 10,680 82.222 Positive > 125 790 3270 4.123 Positive > 125 33 740 22.424 Positive > 125 120 1020 8.525 Positive > 125 130 250 1.926 Positive > 125 190 200 1.127 Positive > 125 17,260 1040 0.0628 Positive > 125 2610 3250 1.229 Positive > 125 1810 2200 1.230 Positive > 125 2360 3800 1.631 Positive > 125 8150 1000 0.132 Positive > 125 220 1000 4.533 Positive > 125 31,670 25 0.00134 Positive > 125 4560 5620 1.2

* High concentrations are those equal to or higher than the high control, 1000 ng/mL.

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drug-free urine with 25 ng ketamine (cutoff value). Eachspiked sample was analyzed in duplicate. The test was carriedout on the same plate and on the same day. For interdayprecision, the intraday test was carried out on five differentplates on five different days. The mean B/B0 (%) was calculatedfor n = 50.

Cross-reactivity study. Ketamine was spiked into blankurine to produce concentrations of 2, 4, 5, 10, 20, and 50ng/mL and norketamine was spiked into other blank urinesamples to produce concentrations of 25, 50, 75, 150, and 200ng/mL. A blank urine sample was also prepared (B0). The cross-reactivity of norketamine to ketamine was calculated at 200ng/mL by comparing the ketamine and norketamine calibra-tion curves plotted in Microsoft Excel®. Thirteen commonlyabused drugs and tiletamine (an anesthesic with a similarchemical structure to ketamine) were individually tested inseparate microplate wells in duplicate at a concentration of10,000 ng/mL to test the kit cross-reactivity to these com-pounds.

Sensitivity and specificity. The ELISA test sensitivity andspecificity were calculated using Eq. 1 and 2.

Sensitivity = (TP × 100)/(TP + FN) Eq. 1

Specificity = (TN × 100)/(TN + FP) Eq. 2

To define the equation parameters, a true-positive result(TP) produced both positive screening and confirmation re-sults, a false-positive result (FP) produced a positive screeningand negative confirmation result, a true-negative result (TN)produced a negative result for screening and confirmation, anda false-negative result (FN) was negative for screening but pos-itive in the confirmation test.

Procedure for sample analysis using ELISAUrine (100 µL) was pipetted into disposable borosilicate glass

culture tubes (75 × 12 mm). The samples were vortex mixedthen diluted 1:10 online with PBS pH 7 (900 µL). This proce-dure was performed to minimize potential urine matrix effects.Each ELISA run consisted of a set of calibrators includingblank urine and three blank urine samples spiked to produce25, 50, and 125 ng/mL ketamine. The ELISA administrationcutoff was set at 25 ng/mL based on the LOD calculation. In ad-dition, this concentration formed part of the linear portion ofthe S-shaped dose-response curve. Negative and positive con-trols (0 and 2000 ng/mL) were provided by the manufacturerto verify the performance of the ELISA test. These were dilutedin the same manner as the calibrators and samples, vortexmixed, and distributed at the beginning and end of the plate tomonitor assay performance.

LC–MS–MS analysisSample hydrolysis

Standards were prepared by spiking 1 mL of blank urinewith 50, 100, 200, 400, 800, and 1200 ng ketamine and norke-tamine standards. One blank urine with no standard or inter-nal standard, and one blank urine containing 100 ng internalstandards were also prepared. One milliliter of 1 M sodium ac-etate buffer (pH 5.0) was added to each tube along with 40 µLof β-glucuronidase crude enzyme solution (Helix pomatia) wasadded to each tube. The tubes were capped and placed in anoven at 60°C. After 3 h, the tubes were removed from the ovenand left at room temperature to cool. After cooling, 3 mL phos-phate buffer (0.1 M, pH 5.0) was added to each tube, and themixture was vortex mixed. The pH was adjusted to pH 5.0using 1 M potassium hydroxide. All tubes were centrifuged at2500 rpm for 10 min prior to loading on solid-phase extrac-tion (SPE) columns.

SPEWorld Wide Monitoring (ZSDAU020) SPE cartridges were

first conditioned with 3.0 mL methanol, followed by 3.0 mLdistilled water, and 1.0 mL phosphate buffer (0.1 M, pH 5.0)without the application of a vacuum. Then the urine sampleswere loaded onto the SPE cartridges. The cartridges werewashed sequentially with 3 mL phosphate buffer (0.1 M, pH5.0) and 1.0 mL acetic acid (1.0 M, pH 5.0) in an attempt to re-move potential interferences present in the in urine matrix.The columns were dried thoroughly under full vacuum for 5min. Two milliliters of methanol/aqueous ammonium hydrox-ide (98:2, v/v) was used to elute the analytes and deuterated

internal standards. The SPE eluant was evap-orated to dryness under nitrogen gas, using aheating block set at 40°C. Finally, the residueswere reconstituted with 150 μL mobile phaseand vortex mixed.

LC–MS–MS set upA Surveyor HPLC system (Thermo Finni-

gan, San Jose, CA) linked to an LCQ Deca XPPlus ion trap MS (Thermo Finnigan) was usedfor detection. The equipment also came with a

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Table II. Gradient Program of the Mobile Phase

3 mM AmmoniumTime Formate + 0.001% Acetonitrile Flow Rate(min) Formic Acid (%A) (%B) (µL/min)

0 95 5 25013 74 26 25022 20 80 25026 5 95 25030 95 5 25036 95 5 250

Table III. The LC–MS–MS Optimized Parameters for Ketamine andNorketamine

Sheath Auxiliary Capillary Collision Precursor ProductCompound Gas (AU) Gas (AU) Temperature (°C) Energy (%) Ions Ions

Ketamine 25 20 210 26 238 220*, 207Norketamine 25 15 250 25 224 207*, 206

* Quantitation ions.

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Surveyor Autosampler (AS 3000) and a data processing system,Xcalibur 1.3. LC analysis was carried out using a mobile phasegradient program combining 3 mM ammonium formate buffer+ 0.001% formic acid (pH 3) and acetonitrile at a flow rateof 0.25 mL/min as shown in Table II. A 20-µL sample was in-jected onto the Synergi Hydro RP LC column using partialloop mode.

The mass spectral data acquired for both analytes andtheir deuterated internal standards are based on electrospray(ESI) positive ion mode [M + H]+. The probe voltage usedwas 4.5 kV. The capillary temperature, sheath and auxiliarygas flow rates, and collision energies were optimized duringtuning for each analyte. The optimized parameters are givenin Table III. Internal standard data was collected by selectedion monitoring (SIM) for identification of the parent ionsof ketamine-d4 (m/z 242) and norketamine-d4 (m/z 228)and analyte data was collected by selected reaction monitor-ing (SRM) over the mass range m/z 60–250. SRM was usedwhere one parent ion and two product ions were identifiedfor a compound and this fulfilled the European Union re-quirement for identification and confirmation of illicit drugs(24). The precursor ions for ketamine and norketamine wereat m/z 238 and m/z 224, respectively. The precursor and prod-uct ions are shown in Table III. The quantitation ion was themajor product ion produced on precursor fragmentation. Theratios of quantitation ion to internal standard and qualitativeions to quantitation ion were calculated. For positive sampleidentification, the ratio of quantitation ion to internal stan-dard was either greater than or within ± 20% of the ratio forthe lowest calibration standard. The qualitative ion to quanti-tation ion ratio was also calculated, and the qualitative ion ra-tios were acceptable if they were within ± 20% of the corre-sponding calibrator or control.

Method validationFor the LC–MS–MS method, the parame-

ters investigated as part of the validation pro-cedure were limit of detection (LOD), lowerlimit of quantification (LLOQ), linearity, re-covery, intra- and interday precision, accuracy,and urine matrix effects.

Linearity and determination of LODs andLLOQs. Urine calibration standards were pre-pared by spiking the appropriate amount ofthe ketamine and norketamine stock solutions(1 and 10 ng/µL) into test tubes containingdrug-free urine to provide final concentrationsof ketamine and norketamine of 0, 25, 50, 100,200, 400, 800, and 1200 ng/mL. 100 ng ofdeuterated IS was added to each tube with ex-ception of the blank. Two replicate analyseswere performed for each concentration toevaluate linearity for statistical purposes. Theslope and standard error of the calibrationcurves were calculated and the peak-area ra-tios of analyte to deuterated internal standardwere used for sample quantification throughthese calibration curves.

LODs were calculated statistically using Eq. 3 and 4, whereyB is the intercept, sB is the standard error of the regressionline, and m is the slope (28,29).

yLOD = yB + 3sB Eq. 3

LOD = (yLOD – yB)/m Eq. 4

LLOQs were calculated using a similar method, but 10times the standard error of the regression line was used(Eq. 5 and 6).

yLOQ = yB + 10sB Eq. 5

LOQ = (yLOQ – yB)/m Eq. 6

Precision and accuracy. The intraday precision was deter-mined by spiking five replicates of 50, 500, and 1000 ng/mLketamine and norketamine standards in blank urine andanalysing them on the same day. The interday precisionwas the same experiment as intraday only carried out onfive different days to produce (n = 25) results. The accuracieswere determined by comparing the mean calculated concen-tration of the five spiked urine samples with the targetconcentration.

Matrix effect assessment. This study was conducted to assessthe potential interference caused by the urine matrix duringanalysis. Three replicates of 1 mL blank urine from six indi-viduals were run through the SPE procedure together withthree replicates of 2 mL SPE loading buffer (0.1 M phosphatebuffer, pH 5.0). Each of the SPE eluants was spiked with 50,500, and 1000 ng/mL of ketamine and norketamine and 100ng of internal standard after the extraction. The % matrix ef-fect was calculated according to Eq. 7, where a and b are de-fined as the peak-area ratio of analyte to internal standard in

Table IV. Experimental Conditions for Method Optimization

Experimental Condition:Urine Diluted 1:10 (v/v) with PBS

Volume of Volume of Samplesample enzyme pre-incubation Average(µL) conjugate (µL) time (min) B/B0 (%) S.D. % R.S.D.

20* 90 0 84.16 3.09 4.9030 84.38 1.37 1.6360 83.95 1.63 1.94

180* 0* 74.07 3.09 4.9030 74.58 1.73 2.3260 74.42 0.94 1.26

40 0 83.52 3.12 4.5030 82.93 1.09 1.2960 80.85 1.65 2.04

180 0 73.94 2.22 2.7130 72.14 3.12 5.0560 71.52 1.75 2.38

* Parameters used for method validation.

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SPE loading solution without urine matrix and human urinematrix, respectively.

Percentage of matrix effect = b/a × 100% Eq. 7

A value of less than 100% indicates analyte ion suppressionwhereas a value of more than 100% indicates analyte ion en-hancement. Both effects are attributed to the urine matrix effect.

Recovery study. Ketamine and norketamine at 50, 500, and1000 ng/mL were spiked into 1 mL of blank urine (n = 3)and extracted using the SPE procedure. Two unextracted stan-dards were also prepared at each concentration without inter-nal standards and were kept in the refrigerator throughoutthe extraction. Ketamine-d4 and norketamine-d4 internal stan-dards (100 ng) were added to each tube before the sampleswere evaporated under nitrogen at 40°C. The % recoverywas determined by comparing peak-area ratios obtained fromextracted ketamine and norketamine versus the correspond-ing peak-area ratios of the same concentration of unextractedstandards.

Results

ELISA resultsMethod development

Sample pre-incubation time and sample volume were not

significant factors and different times and volumes demon-strated similar % B/B0 results (± 13%). The most importantvariable was clearly the volume of enzyme conjugate used,which showed that a larger volume (180 µL as recommendedby the kit manufacturers) was better than a smaller volume(90 µL), producing a decrease in B/B0 (%) response of 10%(Table IV). An assay cutoff of 25 ng/mL was chosen because itproduced an average B/B0 (%) of 75%. If a calibrator with ahigher B/B0 (%) value was used, the number of false positiveswould be increased. In this study, no sample pre-incubationtime, a smaller sample volume (20 µL) and a larger volume ofenzyme conjugate (180 µL) were optimum and hence chosenfor method validation This finding agrees with a study for op-timization of an ELISA method for cocaine in hair done byLachhenmier et al. (30).

Method validationThe ELISA validation procedure in this study was based on

ELISA validation procedures published previously in the liter-ature for the analysis of drugs of abuse within the forensic tox-icology field (31–33). The ELISA kit manufacturers determineda plate sensitivity of 8 ng/mL ketamine in buffer compared to10 ng/mL ketamine (1.25-fold lower sensitivity) in neat urine.Therefore, the blank, calibrators, and quality control sampleswere prepared in blank urine for this study in order to take theurine matrix effect into account.

Dose-response curve. The B/B0 (%) values were calculatedwhere B is the absorbance value of the bound calibrator and B0is the absorbance value of the blank calibrator. Both the x andy-axes were converted into log-scales and the results, as ex-pected, demonstrated the inverse relationship between con-centration and absorbance. The higher the ketamine concen-tration, the lower the B/B0 % due to the lower quantity ofenzyme conjugate that binds to the antibody sites on the mi-croplate wells compared to the analyte. The graph also indi-cated that the ketamine assay S-shaped binding curve had alinear portion between 25 and 500 ng/mL ketamine, which lev-eled off after 2000 ng/mL ketamine. (Figure 2). Based on thedose-response curve in which 25 ng/mL ketamine was the low-est concentration on the linear portion of the binding curve,this concentration was selected for the assay cutoff, which is inagreement with manufacturer’s recommendations. Also, by se-lecting a range of calibrators related to the linear portion of

Figure 2. Ketamine ELISA dose-response curve in urine (0.5–8000ng/mL).

Figure 3. The cross-reactivity of norketamine to ketamine in the NeogenELISA kit.

Table V. LC–MS–MS Parameters for Ketamine,Norketamine, Ketamine-d4, and Norketamine-d4 in theESI Positive Mode

Precursor Ion Product Ions Collision EnergyCompound (m/z) (m/z) (%)

Ketamine 238 220*, 207 26Norketamine 224 207*, 206 26Ketamine-d4 242 224*, 211 25Norketamine-d4 228 211*, 183 25

* Quantitation ions (100% relative abundance compared to 10% relativeabundance of precursor ion).

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this graph, a semi-quantitative estimation of sample concen-tration was obtained. The absorbance response for the mainmetabolite norketamine stated in the manufacturer’s kit in-sert and also in the cross-reactivity conducted for this study,were very low (4.6% and 2.1%, respectively) and was thereforenot a significant factor in the preparation of the dose-responsecurve.

Assay precision and LOD. The test demonstrated excellentintra- and interday precision results. The %R.S.D. for the intraday precision for 10 blankurine samples spiked with ketamine at 25ng/mL was 2.5%, and the % R.S.D. for the in-terday precision for 10 samples spiked at thesame concentration and analyzed on five dif-ferent days (n = 50) was 4.8%.

This study determined the LOD to be ap-proximately 5.0 ng/mL ketamine which waslower than the LOD reported by the manufac-turer in the kit insert. This LOD value was notused as the cutoff value of this test to mini-mize the number of false-positive (FP) results.An administrative cutoff of 25 ng/mL ke-tamine which produced a B/B0 (%) value of ap-proximately 75% was selected and usedthroughout the study in an attempt to make abetter distinction between positive and nega-tive results.

Cross-reactivity studies. Referring to themanufacturer’s insert, norketamine is the onlyrelated drug that cross-reacts with the ke-tamine assay. The manufacturer found that aconcentration of 196.7 ng/mL ketamine cor-responded to a norketamine cross-reactivity of4.6% towards the ketamine assay. The mi-croplate is directed towards ketamine andcross-reacts 100% with it. As shown in Figure3, 200 ng/mL norketamine cross-reacted toketamine at 4.2 ng/mL. The cross-reactivitywas calculated relative to ketamine calibrationcurve using Microsoft Excel, and the value wasfound to be 2.1%. The common drugs ofabuse, which were tested at a concentration of10,000 ng/mL, including amphetamine,methamphetamine, MDA, MDMA, MDEA, co-caine, benzoylecgonine, diazepam, nor-diazepam, morphine, methadone, 6-MAM,PCP, and structurally similar anesthetic, tile-tamine, did not produce absorbance values inthe assay equal to or less than the assay cutofflevel 25 ng/mL and therefore were noted asbeing non cross-reactive with this ELISA assayat this concentration.

Case samples results: sensitivity and speci-ficity of the ELISA assay. Forty-four samples(34 positive and 10 negative) were analyzedusing the ketamine ELISA kit. The assay cut-off 25 ng/mL was chosen based on the dose-response curve. If samples produced B/B0 (%)

absorbance greater than the cutoff concentration, the resultswere reported as negative. The assay is semi-quantitative andsamples with B/B0 (%) between the cutoff concentration (25ng/mL) and the highest calibrator (125 ng/mL) were reportedas values between these concentrations (i.e., reportedas 25–125 ng/mL). Some samples produced B/B0 % valueswhich were less than the highest calibrator and these were re-ported as > 125 ng/mL. Ten samples screened as negative, and

Figure 4. Chromatograms of blank urine (A), extracted 5 ng/mL ketamine and norketamine stan-dard (B), and positive ketamine and norketamine case sample (C).

Positive case sample containing ketamine at 17,260 ng/mL and norketamine at 1040 ng/mL

Extracted 5 ng/mL ketamine and norketamine standards

Blank urine extract

C

B

A

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34 samples screened as positive. Using 25 ng/mL ketamine asthe cutoff and based on the comparison of the ELISA and theLC–MS–MS results (Table I), the sensitivity for the test was100% and the specificity was 100% according to Eq. 1 and 2.ELISA is a semi-quantitative assay and the sensitivity andspecificity were calculated based solely on a positive or nega-tive results and did not take ELISA concentrations into ac-count. For example, norketamine was present in sample 15 at1080 ng/mL with a cross-reactivity of 2.1%, which translatesinto 23 ng/mL plus the 22 ng/mL ketamine in the sample, it isnot surprising that the sample screened at that level. A simi-lar result was obtained for sample 23 because of the cross-re-activity of norketamine.

LC–MS–MS resultsThe optimum tuning parameters, precursor and product

quantitation and qualification ions are shown in Table V. Theseions were used for method validation.

The product ion chromatograms and spectra for blank urine

without analyte or deuterated internal standards, 5 ng/mL ke-tamine and norketamine standard plus deuterated internalstandard and a positive case sample for ketamine and norke-tamine plus deuterated internal standard are shown in Figure4. The chromatograms show, from top to bottom, precursorions for ketamine and norketamine, two product ions and adeuterated internal standard precursor ion.

Linearity, LOD, and LLOQThe correlation coefficients (R2) of the calibration curves

were greater than 0.99 for both ketamine and norketamine(n = 3). The linearity of the LC–ESI-MS–MS method was eval-uated within the range 0–1200 ng/mL, using eight pointsacross the curve. The average slopes, intercepts, and R2 valuesare summarized in Table VI and Figure 5.

The LODs for ketamine and norketamine were both approx-imately 0.6 ng/mL as calculated statistically according to Eq.3 and 4. The LLOQ values were determined as 1.9 ng/mL forketamine and 2.1 ng/mL for norketamine.

Intra- and interday precision and accuracyThe intraday precision and accuracy were evaluated by ana-

lyzing five replicates of three spiked samples in the same day.The precision between extracts was calculated as the % relativestandard deviation (% R.S.D.). Interday precision and accuracywere determined by analyzing five spiked samples on five dif-ferent days (n = 25). The % R.S.D.s for the intra- and interdayprecision are shown in Table VII. The % accuracy was deter-mined by comparing the mean calculated concentration of thespiked urine samples with the target concentration. The intra-and interday accuracies for all the samples ranged from 96.6%to 105.2%. Therefore, the precision and accuracy values wereexcellent and were well within the recommendations issued bythe Society of Forensic Toxicologists of ± 20% (34).

Matrix effect analysisMost researchers include the assessment of matrix effects

in their LC–MS–MS method development to ensure thatthe chromatographic separation developed using differentmatrices is not affected or that the separation shows minimalor acceptable effects, at least when doing quantitative analysis(35). The matrix effects caused by the interferences in

the urine were acceptable for both ketamineand the metabolite nor-ketamine. Matrixeffects analysis for ketamine showed ionsuppression of 8.6% for 50 ng/mL, 4.7%for 500 ng/mL, and 4.0% for 1000 ng/mL.Norketamine showed ion enhancement of19.7% for 50 ng/mL, 6.3% for 500 ng/mL,and 12.9% for 1000 ng/mL. A summary of theobserved matrix effects is given in Table VIII.

Recovery studiesKetamine and norketamine recoveries in

human urine samples for low (50 ng/mL),medium (500 ng/mL), and high (1000 ng/mL)concentrations are presented in Table IX. Therecoveries for both analytes at all three con-

Figure 5. Calibration curves for ketamine and norketamine.

Table VI. Analytical Characteristics of LC–ESI-MS–MSMethod for Ketamine and Norketamine in Urine

AverageLinear Number of Coefficient (R2)Range Calibration Linear Correlation

Compound (ng/mL) Points Equation (n = 3)

Ketamine 0–1200 8 y = 0082x + 0.08 0.9995Norketamine 0–1200 8 y = 0.0004x + 0.011 0.9979

Table VII. Intraday and Interday Imprecision and Accuracy of the SpikedSamples of Ketamine and Norketamine in Urine

SpikedIntraday Imprecision (n = 5) Interday Imprecision (n = 25)

Concentration Accuracy R.S.D. Accuracy R.S.D.Analyte (ng/mL) Mean ± S.D. (%) (%) Mean ± S.D. (%) (%)

Ketamine 50.0 49.0 ± 0.8 98.0 1.7 49.8 ± 2.5 99.6 5.0500.0 499.0 ± 6.2 99.8 1.3 497.9 ± 3.9 99.6 0.8

1000.0 997.1 ± 7.4 99.7 0.7 996.1 ± 5.0 99.6 2.2

Norketamine 50.0 47.4 ± 3.3 94.8 7.0 48.0 ± 1.7 95.9 3.6500.0 497.4 ± 4.5 99.5 0.9 498.1 ± 2.6 99.6 0.5

1000.0 997.1 ± 0.5 99.7 0.5 995.2 ± 2.7 99.5 0.3

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centrations were > 97%, with excellent % RSD values < 8%.

Application of LC–MS–MS method to urine samplesThirty-four urine samples obtained from the Royal

Malaysian Police were tested using a calibration curve with ke-tamine and norketamine concentrations ranging from 0 to1200 ng/mL. Case samples which contained ketamine and nor-ketamine concentrations greater than 1200 ng/mL were di-luted 10 times or in some cases 100 times with phosphatebuffer (0.1 M, pH 5.0) and reanalyzed so that the diluted con-centration fell within the set calibration concentration range.A urine specimen was considered positive by LC– MS–MS ifketamine or norketamine was detected at a concentrationhigher than the method LLOQ of 2 ng/mL.

For the LC–MS–MS analysis, a concentration equal to ormore than the high control (1000 ng/mL) is defined as a highconcentration. All 34 samples were confirmed positive byLC–MS–MS with most of them containing high concentrationsof ketamine and norketamine. The data showed no consistentratio of norketamine to ketamine. The average ratio was 14.2and the ratio median was 4.3. The ratio of norketamine to ke-tamine ranged from 0.001 to 82.2 with norketamine beinghigher than ketamine in 27 out of 34 samples. The results arelisted in Table I.

Discussion

Immunoassay tests for drugs of abuse screening require tobe rapid and efficient because of the large number of samplestypically encountered in forensic toxicology laboratories. It isalso extremely important that these screening tests are highlysensitive and specific. In this study the ELISA test was quickand simple to perform (3 h for analysis). The optimized testconditions required a small sample volume of diluted urine(20 µL diluted urine) with no sample pre-incubation time in-cluded.

The manufacturer’s kit insert claimed that the ELISA testsensitivity was better in buffer compared to urine with a B/B0% approximately 20% lower in buffer than in urine. In thisstudy the comparison was tested using spiked ketamine at 25ng/mL in buffer (n = 3) and urine (n = 3); with a blank bufferand a blank urine included and the results were found to besimilar to the manufacturer’s findings. This was in agreementwith Huang et al. (20) who found that, when using the Neogenkit for urine samples, the lower concentration ketamine stan-dards generated higher B/B0 (%) values than the same stan-dard concentration prepared in buffer. Conversely, the authorsalso observed that for higher concentration ketamine stan-dards, the B/B0 (%) was lower than for same standard concen-tration prepared in buffer.

The test was also very specific to ketamine and showeda low cross-reactivity to norketamine and no cross-reactivityto 13 commonly tested drugs of abuse at a concentrationof 10,000 ng/mL. In the case of sample 23, in which theketamine concentration determined by ELISA was 33 ng/mLand the norketamine concentration was 740 ng/mL, the

large norketamine concentration resulted in a relatively largecross-reactivity to produce an ELISA response greater than125 ng/mL. Huang et al. (20) found that the Neogen KetamineELISA kit showed some cross-reactivity (0.3%) to the otherketamine metabolite, dehydronorketamine (DHNK), andthat this decreased with increasing concentrations of DHNK(20).

The ketamine ELISA assay was shown to be a sensitivetest (LOD 5.0 ng/mL), in agreement with the manufacturer’sfindings. The LOD was not used as the cutoff value, howeverto minimize the number of false-positive results. Ketamineis a weakly basic drug (pKa 7.5) and is metabolized into glu-curonide conjugates during phase II metabolism before excre-tion in urine. Because of low cross-reactivity of norketamineand dehydronorketamine, the use of a 25 ng/mL ketaminecut off could result in some case samples with higher concen-trations of norketamine or dehydronorketamine or theirglucuronide producing a negative screening result if theketamine concentration in the sample is low. The cutoff valueas determined for this assay was shown to be fit for purposeand had no norketamine cross-reactivity issues at the levelsthat were detected for these particular samples. However, thismay pose a problem in a clinical setting involving low thera-peutic concentrations of ketamine. One possible solution inthis scenario would be to include a hydrolysis step in theELISA protocol in order to release conjugated ketamine andnorketamine.

Wieber et al. (36) found that a very small percentageof unchanged ketamine (2.3%), norketamine (1.6%), and

Table VIII. Matrix effects for Ketamine and Norketaminein Urine (n = 6 individuals)

Spiked Concentration Matrix Effect (%)Analyte (ng/mL) [R.S.D.]

Ketamine 50 91.4 [2.3]500 95.3 [10.4]

1000 96.0 [5.1]

Norketamine 50 80.3[13.2]500 95.7 [11.9]

1000 87.1[10.7]

Table IX. Recoveries for Ketamine and Norketamine inHuman Urine Samples

SpikedConcentration % Recovery

Analyte (ng/mL) (n = 5) S.D. % R.S.D.

Ketamine 50 113.4 0.1 5.1500 107.6 0.6 3.6

1000 98.3 1.8 7.1

Norketamine 50 102.1 0.1 1.1500 104.3 0.5 5.6

1000 97.9 0.3 3.8

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dehydronorketamine (16.2%) are eliminated in urine, whereas80% is present as the glucuronide conjugates of hydroxylatedmetabolites of ketamine. The aim of this study was to detecttotal and free ketamine and norketamine in urine samples. Al-though LC–MS–MS analysis could en-compass the detection ofglucuronide conjugates (37), there were no ketamine or nor-ketamine glucuronides standards available in the commercialmarket at the time of conducting this study.

The LC–MS–MS method detected free ketamine and norke-tamine in the urine samples and, because the sample concen-trations were unknown at the time of testing, it was thoughtbest to enhance ketamine and norketamine sensitivity bycleaving the glucuronides. A previously published study byKronstrand et al. (37) determined that direct LC–MS–MS anal-ysis of buprenorphine and norbuprenorphine glucuronideswithout hydrolysis was only suitable for a screening methodand this could be compared to the ELISA method in this cur-rent study. Their study found that inclusion of a hydrolysis stepand SPE clean up increased the sensitivity of the LC–MS–MSmethod 20-fold (from 20 to 1 µg/L) for both analytes (37).

With this procedure, the LODs for ketamine and norke-tamine obtained were low, (both approximately 0.6 ng/mL),and the LLOQ values were 1.9 and 2.1 ng/mL, respectively.SPE was used to extract ketamine and norketamine. WorldWide Monitoring Clean Screen® columns (ZSDAU 020)columns have been used in many studies for detection of drugsof abuse with good recoveries (38,39). The SPE methodselected operated by a mixed-mode cationic exchange mecha-nism based on the sorbent composition of C8 chains andbenzene sulfonic acid (BSA) residues. Therefore, ketamineand its metabolite norketamine are most likely retained onthe column via both hydrophobic and ionic interactions re-sulting in high recoveries for both analytes. The SPE stepalso helped in obtaining low LODs and reducing the matrixeffects as shown for the chromatogram of the blank urinesample in Figure 3 (35).

Both ketamine and norketamine were detected byLC–MS–MS in all of the 34 urine specimens tested fromthe Royal Malaysian Police. However, the concentrationsof ketamine and norketamine in each sample vary widelywith no consistency of ratios between ketamine and norke-tamine. This could be caused of a number of factors suchas dose and route of administration of ketamine, the timeinterval between administration and collection of the urinesample, the subject’s rate of metabolism, weight and health;thus the duration of the drug in the body and severity of effectsvaries from one person to another (40). The concentrationrange of ketamine in the urine samples was between 22and 31,670 ng/mL, and the concentration of norketaminewas between 25 and 10,990 ng/mL. Wieber et al. (36) reportedthat the half-lives of ketamine and norketamine in urinewere 3.37 ± 0.14 h and 4.21 ± 0.35 h, respectively, and thatboth analytes were undetectable after 22 h (approximately1 day). Therefore those samples which have ketamine concen-trations greater than norketamine in this study were probablycollected soon after use. The high concentrations of ketamineand norketamine in most samples may suggest regular intakeof ketamine by those users (20).

In this study, a comparison of ELISA and LC–MS–MS resultshas been made for ketamine and norketamine detection inurine. The linear range of Neogen ELISA kit for ketaminewas evaluated as 25–500 ng/mL Another paper which usedan ELISA cutoff of 100 ng/mL ketamine as well as a GC–MSmethod with a cutoff of ≤ 2.6 ng/mL demonstrated poorcorrelation between methods (less than 30%) in 43 samples(21). In another study (15), the data demonstrated 90.9%sensitivity and 98.9% specificity with 1% false-positive resultswhen an ELISA cutoff concentration of 10 ng/mL ketaminewas used with a GC–MS cutoff value of 15 ng/mL ketamine.In this study, the use of an ELISA cutoff concentration of25 ng/mL ketamine and an LC–MS–MS cutoff concentrationof 2 ng/mL yielded 100% sensitivity and specificity on com-parison of the two methods. The combination of an ELISAscreening test and an LC–MS–MS confirmation analysis pro-duced an acceptable, highly sensitive, specific and efficient sys-tem for the determination of ketamine and norketamine inurine samples.

Conclusions

A simple, rapid, and efficient ELISA test for ketamine hasbeen optimized and validated for the analysis of ketamine andnorketamine in urine samples. The Neogen ELISA kit is ex-tremely sensitive and specific for ketamine screening at a cut-off concentration of 25 ng/mL coupled with an LC–MS–MScutoff of 2 ng/mL. The ELISA test cross-reacts with ketamineby 100% and demonstrated some cross-reactivity to its mainmetabolite norketamine. The kit demonstrated excellent pre-cision for ketamine.

An LC–MS–MS confirmation method was validated for thequantitation of ketamine and norketamine in human urine.The method demonstrated excellent linearity, LOD, LLOQ, ac-curacy, and precision with acceptable matrix interference ef-fects. The screening efficiency of ELISA has been validated andevaluated together with this LC–MS–MS confirmation methodusing 34 urine specimens collected from suspected drug userswere tested and found to be positive. The combination of testsdemonstrated excellent efficiency; sensitivity and specificity,with no false positive and false negative results for this partic-ular set of samples.

Therefore, a combination of ELISA and LC–MS–MS can beused reliably as components of a two-approach routine teststrategy (screening and confirmation) for the determinationof ketamine in urine specimens. This study also found that ke-tamine and norketamine are present in all specimens with 47%ketamine and 79% norketamine present at high concentra-tions (> 1000 ng/mL) and highlighted that ketamine is beingabused in Malaysia.

Acknowledgments

This study was supported by a scholarship from the Depart-

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ment of Public Service and the Ministry of Health, Malaysia forthe corresponding author’s postgraduate study in forensic tox-icology at the University of Glasgow, Scotland. The authorsalso gratefully thank the Narcotic Department within the RoyalMalaysian Police for the provision of urine samples.

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Manuscript received January 14, 2009;revision received April 14, 2009.

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