Forensic Science International 194 (2010) 108–114

Determination of amphetamine-type stimulants, ketamine and metabolites infingernails by gas chromatography–mass spectrometry

Jin Young Kim *, Soon Ho Shin, Moon Kyo In

Drug Analysis Laboratory, Forensic Science Division, Supreme Prosecutors’ Office, 706 Banporo, Seocho-gu, Seoul 137-730, Republic of Korea

A R T I C L E I N F O

Article history:

Received 9 September 2009

Received in revised form 14 October 2009

Accepted 17 October 2009

Available online 18 November 2009

Keywords:

Amphetamine-type stimulants

Ketamine

GC–MS

Nail analysis

A B S T R A C T

A gas chromatography–mass spectrometry (GC–MS) method was developed and validated for the

simultaneous qualification and quantification of methamphetamine (MA), amphetamine (AP), 3,4-

methylenedioxy-N-methylamphetamine (MDMA), 3,4-methylenedioxy-N-amphetamine (MDA), keta-

mine (KET) and norketamine (NKT) in fingernails. Fingernail samples (20 mg) were washed with distilled

water and methanol, digested with 1.0 M sodium hydroxide at 95 8C for 30 min, and then extracted with

ethyl acetate. Extract solutions were evaporated to dryness, derivatized using heptafluorobutyric

anhydride (HFBA) at 60 8C for 30 min, and analyzed by GC–MS. The linear ranges were 0.1–20.0 ng/mg

for AP, MDMA and NKT, 0.2–20.0 ng/mg for MA and MDA, and 0.4–20.0 ng/mg for KET, with the

coefficients of determination (r2 � 0.9989). The intra- and inter-day precisions were within 7.1% and

10.6%, respectively. The intra- and inter-day accuracies were �10.9% to 0.8% and �4.3% to 4.5%,

respectively. The limits of detections (LODs) and the limits of quantifications (LOQs) for each analyte

were lower than 0.094 ng/mg and 0.314 ng/mg, respectively. The recoveries were in the range of 72.3–

94.9%. The average fingernail growth rates of two subjects for three years and six subjects for two months

were 3.12 mm/month and 3.16 mm/month, respectively. The method proved to be suitable also for the

simultaneous detection and quantification of MA, MDMA, KET and their metabolites in fingernails.

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1. Introduction

Amphetamine-type stimulants (ATSs) such as amphetamine(AP), methamphetamine (MA), 3,4-methylenedioxy-N-ampheta-mine (MDA), and 3,4-methylenedioxy-N-methylamphetamine(MDMA), are abused drugs that have stimulant and hallucinogenicproperties [1–3]. As these synthetic drugs are simple to produce,inexpensive to buy, and usually have a long-lasting effect, theirabuse became widespread universally and has become a majorpublic health issue in many East Asian countries [4]. MA is a potentand highly addictive psychostimulant which is the most abusedillegal drug and ranked as the primary drug of concern in Korea [5].Unlike other drugs, the abuse of MA and MDMA increaseddramatically in the late 1990s [6]. Ecstasy (MDMA) and itsderivatives are central nervous stimulants including euphoria andalertness. Ecstasy is usually not pure MDMA and used incombination with other substances such as ketamine (KET),MDA, and caffeine [7,8]. KET is a short-acting but powerfulgeneral anaesthetic which depresses the nervous system. Itundergoes extensive hepatic metabolism, primarily via N-demethylation to norketamine (NKT) [9]. In recent years, KET

* Corresponding author. Tel.: +82 2 535 4173; fax: +82 2 535 4175.

E-mail address: paxus@spo.go.kr (J.Y. Kim).

0379-0738/$ – see front matter � 2009 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.forsciint.2009.10.023

abuse is increasing in East Asia and Korea [10,11]. The increasingabuse of these illicit drugs requires rapid, sensitive analyticalmethods for analysis in biological samples.

The matrices commonly used for drug testing are urine, blood,hair, oral fluid, sweat, and nails in forensic chemistry andtoxicology. Drug testing in keratinized matrices of hair and nailshas gradually been gaining attention because of its advantagesover conventional urine or blood analysis [12,13]. The collection ofthese samples is simple and noninvasive. Furthermore, as theparent drug and its metabolite are incorporated inside the matrix,they are stable for long periods of time and difficult to alter. It hasbeen shown that the axial distribution of a drug in the nail platecould provide a relatively long-term window depending on thelength of nail [14]. The rate of fingernail growth has been reportedto be approximately about 0.1 mm/day, 3 mm/month and38.1 mm/year [15–17]. To date, ATSs, cannabinoids, opiates,cocaine, phencyclidine, benzodiazepines and methadone areamong the drugs that have been detected in nails. These studieshave been based on several instrumental methods includingimmunoassay [18,19], high performance liquid chromatography(HPLC) [19], gas chromatography–mass spectrometry (GC–MS)[18,20–25], and liquid chromatography–mass spectrometry (LC–MS) [26]. Due to its specificity and sensitivity, the GC–MS methodin the selected-ion monitoring (SIM) mode has been routinelyemployed for the detection of abused drugs in nails.

Table 1Measurement of mean fingernail growth rate of two subjects for three years (mm/

month).

Subject 1 Subject 2 Representative value

Thumb (right) 2.92 2.98 2.95

Thumb (left) 2.96 2.93 2.94

Forefinger (right) 2.97 2.95 2.96

Forefinger (left) 2.97 2.98 2.98

Middle finger (right) 3.16 3.28 3.22

Middle finger (left) 3.29 3.22 3.25

Third finger (right) 3.43 3.43 3.43

Third finger (left) 3.36 3.37 3.36

Little finger (right) 3.10 – 3.10

Little finger (left) 2.98 – 2.98

Standard deviation 0.19 0.21 0.19

Mean value 3.11 3.14 3.12

J.Y. Kim et al. / Forensic Science International 194 (2010) 108–114 109

Although GC–MS is known to be a sensitive technique for ATSsanalysis, a derivatization step is sometimes required because ofsimilar fragmentation patterns for target analytes and, conse-quently, poor diagnostic ions in the mass spectrum [27]. Therefore,it is important to choose the proper derivatization technique forovercoming these problems.

In this study, we are focused on the development and validationof a sensitive method for simultaneous determination of MA,MDMA, KET and their major metabolites. To increase detectionsensitivity, the efficiency of derivatization was investigated withthree different acylation reagents. The method was also evaluatedfor its feasibility and applicability to fingernail samples obtainedfrom drug abusers.

2. Material and methods

2.1. Reagents and materials

The reference compounds AP, MA, MDA, MDMA, NKT and KET were purchased

from Cerilliant (Austin, TX, USA) at a concentration of 1000 mg/mL in methanol, and

methanolic solutions of the deuterated internal standards, AP-d8, MA-d11, MDA-d5,

MDMA-d5, NKT-d4 and KET-d4 at 100 mg/mL were also purchased from Cerilliant.

Heptafluorobutyric anhydride (HFBA), pentafluoropropionic anhydride (PFPA) and

trifluoroacetic anhydride (TFAA) were supplied from Acros Organics (Geel,

Belgium). HPLC-grade methanol and ethyl acetate were supplied from J.T. Baker

(Phillipsburg, NJ, USA). The water was purified using a Direct-Q water purification

system (Millipore, Bedford, MA, USA).

Working standard solutions (0.1 mg/mL, 1.0 mg/mL, 10.0 mg/mL) of AP, MA,

MDA, MDMA, NKT, and KET, and a combined standard solution of the internal

standards (0.5 mg/mL) AP-d8, MA-d11, MDA-d5, MDMA-d5, NKT-d4, and (1.0 mg/mL)

KET-d4, were prepared in methanol. All of these solutions were stored at �20 8C in

the dark until used.

2.2. Fingernail specimens

Drug-free fingernails obtained from eight laboratory personnel were used to

prepare the matrix for the control and calibration samples and to measure the

growth rate of fingernail. Fingernail samples of drug abusers were obtained from

the Narcotics Departments at the District Prosecutors’ Offices. The samples were

obtained by cutting the excess overhang of the nail plate. A total of seven samples

were collected from drug abusers including positive samples tested for drug use

during a screening test of urine or hair samples by GC–MS. The length of the nail

clippings was measured and special treatments such as manicuring and artificial

nail tips were noted. The study was approved by the Ethics Committee of Forensic

Sciences Division at Supreme Prosecutors’ Office, and written informed consent was

obtained from each participant before fingernail sampling.

2.3. Sample preparation

Fingernail sample was first washed with water (5 mL) and subsequently washed

twice with methanol (5 mL). It was then air-dried, weighed 20 mg of sample, and

transferred to a test tube (12 � 100 mm) containing 50 mL of the combined internal

standard solution (0.5 mg/mL for AP-d8, MA-d11, MDA-d5, MDMA-d5, NKT-d4, and

1.0 mg/mL for KET-d4).

Nail sample was hydrolyzed by incubation in 1 mL of 1.0 M sodium hydroxide at

95 8C for 30 min. After cooling, each sample was extracted with 3 mL of ethyl acetate

for 10 min. Sample was centrifuged at 3500 rpm for 5 min, then the organic layer was

transferred to a new test tube. Subsequently, extract solution was concentrated to

dryness under a nitrogen stream at 40 8C and 30 kPa. It was then dried in a vacuum

desiccator over P2O5–KOH for at least 10 min. Heptafluorobutyryl (HFB) derivatives of

Table 2Measurement of mean fingernail growth rate of six subjects for two months (mm/mon

Subject 3 Subject 4 Subject 5

Thumb (right) 3.33 2.62 3.36

Thumb (left) 3.01 2.33 3.45

Forefinger (right) 3.52 2.63 3.56

Forefinger (left) 2.93 2.61 3.64

Middle finger (right) 3.81 2.66 3.43

Middle finger (left) 3.22 2.48 3.82

Third finger (right) 3.68 2.49 3.60

Third finger (left) 3.20 2.42 4.38

Little finger (right) 3.34 2.20 3.45

Little finger (left) 2.85 2.12 3.54

Standard deviation 0.32 0.19 0.30

Mean value 3.29 2.46 3.62

AP, MA, MDA, MDMA, NKT and KET were formed by reaction of the sample with 50 mL

ethyl acetate and 50 mL HFBA in a dry heating block at 60 8C for 30 min, followed by

drying under a nitrogen stream. The residue was reconstituted with 50 mL of ethyl

acetate. An aliquot (1 mL) of sample solution was injected into the GC–MS.

2.4. GC–MS analysis

GC–MS analyses were performed with an Agilent Technologies 5975 inert mass

spectrometer (Santa Clara, CA, USA) equipped with a 6890N GC and 7683B

automatic liquid sampler. Data acquisition and analysis were performed using

standard software supplied by the manufacturer (Agilent Tech., MSD Chemstation

D.02.00). Separation was achieved with a capillary column (DB-5MS,

30 m � 0.25 mm i.d., 0.25 mm, J&W Scientific, Folsom, CA, USA) with helium as

the carrier gas at a flow rate of 1.1 mL/min. The GC temperature program was as

follows: initial temperature was 90 8C for 3.0 min, increased to 170 8C at a rate of

15 8C/min, held for 2.0 min, increased to 210 8C at a rate of 25 8C/min, held for

1.5 min, then increased to 230 8C at a rate of 20 8C/min, held for 0.5 min, finally

increased to 300 8C at a rate of 40 8C/min, and held for 0.3 min. Splitless injection

mode was used with a purge-on time of 0.1 min at flow rate of 16.5 mL/min. The

injector and the transfer line temperatures were 260 8C and 280 8C, respectively. The

mass spectrometer was operated under positive ion EI conditions (70 eV) with

selected-ion monitoring for quantification. Quantifier and qualifier ions were

monitored in respective groups for each compound that changed with elution time.

2.5. Validation of analytical method

The method was validated and tested according to protocol before the

application to real samples [28,29]. Selectivity, matrix effect, linearity, limits of

detection (LOD) and quantification (LOQ), precision and accuracy, and recovery

were assayed for six phenylalkylamine derivatives in fingernail.

To evaluate selectivity, drug-free nail samples were extracted and analyzed for

peaks interfering with the detection of the analytes or the internal standards. In

addition, potential interference from phenylalkylamine derivatives was investi-

gated by spiking 20 mg of drug-free fingernail with 20 ng of the aforementioned

substances through the entire procedure.

The potential for carryover was evaluated by injecting the highest point of the

calibration curve, followed by solvent blank, and measuring the area of peaks

present at the analyte retention times. For routine analysis of fingernail samples,

ethyl acetate blanks were run between each pair of samples.

Calibration curves were constructed over the LOQ for all the analytes. Linear

regression analysis was performed on the peak area ratios of analyte to internal

standard versus analyte concentrations. The LODs and LOQs for each analyte were

th).

Subject 6 Subject 7 Subject 8 Mean value

4.10 3.41 3.65 3.41

3.75 3.29 2.96 3.13

3.71 2.95 2.98 3.22

3.21 3.20 2.66 3.04

3.93 3.43 2.75 3.33

2.96 3.38 2.87 3.12

3.88 3.10 2.96 3.29

3.14 3.35 3.01 3.25

3.40 2.85 2.70 2.99

2.85 2.84 2.51 2.78

0.44 0.23 0.31 0.19

3.49 3.18 2.90 3.16

Fig. 1. Evaluation of acylating derivatization reagents for the analytes with heptafluorobutyric anhydride (HFBA), pentafluoropropionic anhydride (PFPA) and trifluoroacetic

anhydride (TFAA).

J.Y. Kim et al. / Forensic Science International 194 (2010) 108–114110

estimated in accordance with the baseline noise from drug-free fingernail extracts.

The baseline noise was evaluated by recording the detector response over a period

of about 10 times the peak width. The LOD was obtained as the concentration of a

sample that provided a peak with a height three times the baseline noise level and

the LOQ was calculated as 10 times the baseline noise level.

Seven replicates at the four different quality control (QC) sample concentrations

(0.4 ng/mg, 2.0 ng/mg, 7.5 ng/mg and 15.0 ng/mg) were added to drug-free

fingernail samples and extracted as above for the determination of intra- and

inter-day precision and accuracy. The inter-day precision and accuracy were

determined for four independent experimental days. To determine the precision,

relative standard deviation (%RSD) was calculated for the replicate measurements.

Expressed accuracy (%bias) as the relative error of the calculated concentrations is

calculated by the degree of agreement between the measured and nominal

concentrations of the fortified samples.

Analytical recoveries were calculated by comparing the peak areas obtained

when calibration samples were analyzed by adding the reference compounds in the

extract from drug-free samples prior to and after the extraction procedure. The

recoveries were assessed by QC samples using seven replicates for each QC sample

concentration (0.4 ng/mg, 2.0 ng/mg, 7.5 ng/mg and 15.0 ng/mg).

3. Results and discussion

3.1. Fingernail growth rate

The rate of fingernail growth has been reported to beapproximately about 3 mm a month [16]. Actual growth rate isdependent upon age, gender, season, exercise level, diet, andhereditary factors [30]. Fingernails require about three to sixmonths to regrow completely [24].

Fingernail growth rate was measured using a vernier calipers(Mitutoyo, Kawasaki, Japan) based on specimens collected fromeight laboratory staffs. Individual fingernail growth rate data andmean values are detailed in Tables 1 and 2. The mean fingernailgrowth rates of two subjects for three years were 3.11 mm/monthand 3.14 mm/month (mean 3.12) while those of six subjects fortwo months ranged from 2.46 mm/month to 3.62 mm/month(mean � SD, 3.16 � 0.19). These growth rate data could be used toconfirm the long-term administration of illicit drugs in fingernails assegmental hair analysis was used to verify both their previous drug

Table 3Seasonal patterns in fingernail growth rate of two subjects for three years (mm/

month).

Spring Summer Autumn Winter

Subject 1

Range 3.01–3.43 2.83–3.27 2.81–3.18 2.79–3.25

Standard deviation 0.16 0.15 0.14 0.16

Mean value 3.24 3.05 2.96 3.09

Subject 2

Range 2.88–3.58 3.07–3.57 2.92–3.44 2.75–3.50

Standard deviation 0.25 0.20 0.20 0.27

Mean value 3.30 3.34 3.11 2.99

history and their recent enforced abstinence [31]. Table 3 shows theseasonal patterns in fingernail growth rate of two subjects for threeyears. There was no significant difference in fingernail growthbetween seasons within two subjects. The resulting data wereinsufficient to assess the regular trend for more rapid growth insummer and slower in winter [32,33].

3.2. GC–MS analysis

Derivatization in gas chromatograph (GC) or GC–MS improvesoverall chromatographic selectivity and non-tailing peak shapes,

Fig. 2. Comparison of the mean derivatization efficiencies at different temperatures.

Fig. 3. EI mass spectra for trimethylsilyl (TMS) and heptafluorobutyryl (HFB) derivatives of the analytes.

J.Y. Kim et al. / Forensic Science International 194 (2010) 108–114 111

Fig. 4. Merged selected ion chromatograms for heptafluorobutyryl (HFB)

derivatives of the analytes and internal standards, including (a) drug-free

fingernail, (b) drug-fortified fingernail at 1.0 ng/mg of each analyte, and (c)

polydrug-user fingernail samples.

J.Y. Kim et al. / Forensic Science International 194 (2010) 108–114112

producing new compounds with altered polarity and volatility andforming distinctive mass spectral fragment ions. The acylatingreagents such as TFAA, PFPA and HFBA are used for chemicalderivatization of amphetamine-type stimulants. Derivatizationwith HFBA gave better selectivity and recovery for all the analytescompared to TFAA and PFPA (Fig. 1). The optimum conditions forderivatization were a reaction temperature of 60 8C and a reactiontime of 30 min (Fig. 2), which provided the best overallchromatographic selectivity and derivatization yields on theanalytes.

Fig. 3 shows the EI mass spectra and mass fragmentationpatterns, as recorded with the quadrupole mass spectrometer, ofthe TMS (trimethylsilyl) and HFB derivatives of the analytes. TMSderivatives are prepared by reaction with MSTFA (N-methyl-N-trimethylsilyltrifluoroacetamide). The m/z 73, corresponding tothe TMS group, is prominent in nearly all TMS mass spectra. TMSderivative mass spectra are characterized by abundant [C9H17]+ ionfor AP and MDA, and [C6H16NSi]+ ion for MA and MDMA. Incomparison to TMS derivatization, the HFB derivatives were morepreferable because they provide more specific mass spectrometricinformation. Under the conditions used for analysis (nominalelectron energy 70 eV), molecular ions of MDA and MDMA wereobserved except for AP and MA, in which the molecular ion was oflow relative intensity and not the base peak ion. The spectra of AP–HFB and MA–HFB were characterized by base peaks at m/z 240 and254 corresponding to [M–C7H7]+ ion and intense peak [C9H10]+ ionat m/z 118. The base peaks in the spectra of MDA–HFB and MDMA–HFB were correspond to [M–C6H5F7NO]+ ion (m/z 135) and [M–C8H7O2]+ ion (m/z 254) of each. [C10H10O2]+ ion at m/z 162 is a peakcharacteristic of the HFB derivatives of MDA and MDMA.Characteristic ions (qualification and quantification ions) of HFBderivatives of the analytes including NKT and KET are described inTable 4.

3.3. Method validation

The selectivity of the method was assessed by analyzing blank,drug-fortified, and polydrug-user fingernail samples. Representa-tive chromatograms obtained from GC–MS SIM mode are shown inFig. 4. All analytes were well separated with good peak shapes.Fig. 4 showed no interfering peaks from endogenous substances orco-extracted compounds.

Evaluation of the potential carryover was carried out byinjecting the highest extracted calibrator into the GC–MS, followedby an ethyl acetate blank. Result showed that there was nocarryover detected. Subsequently, ethyl acetate blanks were usedthroughout the sample sequence to assure that no sample-to-sample contamination occurred due to sample overloading.

Table 4Retention times, molecular weights, and ions monitored for GC–MS analysis for HFB

derivatives.

Compound Retention

time (min)

Molecular

weight

Ions monitored (m/z)

Quantifier

ions

Qualifier

ions

AP-d8–HFB 7.83 339 243 – –

AP–HFB 7.87 331 240 118 91

MA-d11–HFB 8.88 356 260 – –

MA–HFB 8.92 345 254 118 210

MDA-d5–HFB 11.46 380 167 – –

MDA–HFB 11.49 375 162 135 375

MDMA-d5–HFB 12.46 394 258 – –

MDMA–HFB 12.49 389 254 162 210

NKT-d4–HFB 13.02 424 388 – –

NKT–HFB 13.05 420 384 356 340

KET-d4–HFB 14.51 438 374 – –

KET–HFB 14.53 434 370 210 362

Seven-point calibration curves for each analyte were estab-lished with three replicates at each concentration. The linearranges were 0.1–20.0 ng/mg for AP, MDMA and NKT, 0.2–20.0 ng/mg for MA and MDA, and 0.4–20.0 ng/mg for KET, withgood correlation coefficients (r2 � 0.9989). LODs for eachcompound were lower than 0.094 ng/mg, based on the con-centration of analyte corresponding to a signal plus 3 standarddeviations (SD) from the mean of seven replicates of drug-freefingernail. LOQs, defined as the concentration of analyte giving asignal equivalent to above 10 SD of blank signal, were between0.050 ng/mg and 0.314 ng/mg for each analyte (Table 5).Analytical recovery, accuracy, and precision experiments wereperformed at four concentrations (0.4 ng/mg, 2.0 ng/mg, 7.5 ng/mg and 15.0 ng/mg), covering the calibration range. The intra-day (n = 3) and inter-day (n = 4) accuracy (%bias) and precision(%RSD) were assessed by analyzing seven QC samples spikedwith the analytes at four different concentrations (0.4 ng/mg,2.0 ng/mg, 7.5 ng/mg and 15.0 ng/mg). The intra- and inter-dayprecisions were within 7.1% and 10.6%, respectively. The intra-and inter-day accuracies were �10.9% to 0.8% and �4.3% to 4.5%,respectively. Analytical recoveries at four concentration levels(0.4 ng/mg, 2.0 ng/mg, 7.5 ng/mg and 15.0 ng/mg) in sevenreplicates were 72.3–94.9% (Table 6). Considering the complex-ity of the biological matrix, we regard these results assatisfactory.

Table 5Calibration curve results, LOD, and LOQ for analytes.

Analyte Concentration range (ng/mg) Slope y-intercept Linearitya (r2) LODb (ng/mg) LOQc (ng/mg)

AP 0.1–20.0 0.4261 0.0034 0.9997 0.019 0.063

MA 0.2–20.0 0.4371 0.0347 0.9995 0.044 0.147

MDA 0.2–20.0 0.7349 �0.0298 0.9994 0.043 0.143

MDMA 0.1–20.0 0.4200 �0.0001 0.9997 0.016 0.053

NKT 0.1–20.0 0.2232 0.0066 0.9996 0.015 0.050

KET 0.4–20.0 0.1258 0.0217 0.9989 0.094 0.314

a Linearity is described by the correlation coefficient for the calibration curve.b Limit of detection (LOD).c Limit of quantification (LOQ) were based on the concentration corresponding to a signal plus 3 and 10 standard deviations from the mean of seven replicates of drug-free

fingernail.

Table 6Results of method validation.

Analyte Nominal concentration (ng/mg) Recovery (% mean� SDa) Intra-day (n = 3) Inter-day (n = 4)

Precisionb

(%RSD)

Accuracyc

(%bias)

Precision

(%RSD)

Accuracy

(%bias)

AP 0.4 82.1�2.8 4.2 �5.8 1.7 0.4

2.0 79.4�0.3 1.6 �1.1 0.9 �0.6

7.5 76.4�2.7 0.6 �0.8 1.0 �2.3

15.0 80.6�0.8 1.3 �0.3 0.7 �1.0

MA 0.4 78.8�2.1 4.5 �10.9 10.6 �4.3

2.0 74.8�0.4 5.6 �3.2 0.9 0.8

7.5 72.3�2.6 1.2 0.1 0.6 �1.8

15.0 75.9�1.6 1.7 �1.3 1.1 �1.6

MDA 0.4 89.4�1.6 4.8 �3.3 5.7 4.5

2.0 89.0�0.7 2.1 �1.2 1.4 �0.4

7.5 88.1�0.6 1.3 �0.7 1.3 �1.9

15.0 91.8�0.5 0.9 �0.1 1.5 �1.3

MDMA 0.4 91.1�0.9 2.9 �5.8 2.9 �1.2

2.0 92.4�0.7 1.6 �1.6 0.8 �1.1

7.5 89.6�1.2 1.2 0.4 1.0 �2.2

15.0 93.3�0.5 1.1 0.1 0.5 �1.4

NKT 0.4 87.9�2.8 2.6 �9.0 3.9 �1.8

2.0 87.4�0.1 1.6 0.2 0.8 0.6

7.5 85.8�1.0 3.5 �1.5 1.3 �1.7

15.0 87.4�1.4 0.7 �0.2 0.9 �1.5

KET 2.0 89.4�1.2 7.1 0.7 4.0 0.5

7.5 92.9�1.8 2.3 0.6 3.6 �0.9

15.0 94.9�1.6 2.4 0.8 3.7 1.1

a Standard deviation.b Expressed as the coefficient of variance of the peak area ratios of analyte/internal standard.c Calculated as [(mean calculated concentration�nominal concentration)/nominal concentration]�100.

Table 7Analytes concentration in positive nail samples.

Analyte Analyzed samples Positive samples Range (ng/mg)

MA 7 6 (1)a 0.23–2.09

AP 7 4 (1)a <0.063b

MDMA 7 1 (1)a 0.46

MDA 7 1 (1)a <0.143b

NKT 7 1 (1)a <0.050b

KET 7 1 (1)b <0.314b

a The number of poly-consumption case (MA + AP + MDMA + MDA + NKT + KET) is

indicated in parenthesis.b Below limit of quantification.

J.Y. Kim et al. / Forensic Science International 194 (2010) 108–114 113

3.4. Application to real samples from drug abusers

The applicability of the method was examined using realfingernail samples from suspected illicit drug abusers. A total ofseven fingernail samples obtained by drug abusers were analyzedfor MA, AP, MDMA, MDA, NKT and KET. MA was the mostfrequently detected compound and was detected in six samples. APwas detected in four samples, which is a metabolite of MA, and that

could be found at low levels in fingernail of MA drug abusers[24,34]. MDMA, MDA, KET and NKT were detected together in onlyone sample, and their use was related with polydrug consumptionin combination with MA, MDMA and ketamine. But it may notaccurately reflect the polydrug use patterns due to the lack of realuse cases (Table 7).

4. Conclusions

A rapid, sensitive GC–MS method was developed and validatedfor measuring AP, MA, MDA, MDMA, NKT and KET in fingernails.The method includes alkaline hydrolysis, liquid–liquid extractionstep and HFB derivatization of analytes. The collection of fingernailsamples is simple and noninvasive in comparison of urine andblood. The axial distribution of a drug in the nail plate would besimilar to the way drugs are distributed in hair [24]. Fingernailscould be especially useful alternative for retrospective investiga-tion of chronic and past drug consumption when it is impossible toobtain hair specimen. This method was successfully applied for thedetermination of six phenylalkylamine derivatives in fingernailsamples from drug abusers.

J.Y. Kim et al. / Forensic Science International 194 (2010) 108–114114

Acknowledgements

This study was supported in part by a grant (M10640010000-06N4001-00100) from the National R&D program of Ministry ofEducation, Science and Technology (MEST) and National ResearchFoundation of Korea (NRF).

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