Anal Bioanal Chem DOI 10.1007/s00216-013-7423-y
RESEARCH PAPER
Deposition of JWH-018, JWH-073 and their metabolites in hair and effect of hair pigmentation Jihyun Kim & Sanghwan In & Yuran Park & Meejung Park & Eunmi Kim & Sooyeun Lee
Received: 11 June 2013 / Revised: 7 October 2013 / Accepted: 7 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Analysis of drugs in hair is often used as a routine method to obtain detailed information about drug ingestion. However, few studies have been conducted on deposition of synthetic cannabinoids and metabolites in hair. The first purpose of this study was to establish and validate an analytical method for detection of JWH-018, JWH-073, and their metabolites in hair, by use of UHPLC–MS–MS, for forensic application. The second purpose was to investigate the distribution of synthetic cannabinoids metabolites in hair and the effect of hair pigmentation, by use of an animal model. For this, JWH-073 was chosen as a representative synthetic cannabinoid. Finally, the developed method was applied to hair samples from 18 individuals suspected of synthetic cannabinoids use. JWH-018, JWH-073, and their metabolites were extracted from hair with methanol. The extract was then filtered and analyzed by UHPLC–MS–MS with an electrospray ion source in positive-ionization mode. Validation proved the method was selective, sensitive, accurate, and precise, with acceptable linearity within the calibration ranges. No significant variations were observed when different sources of both human and rat hair were used. The animal study demonstrated that JWH-073 N-COOH M was the major metabolite of JWH-073 in rat hair, and hair pigmentation did not have a significant effect on incorporation of JWH-073 and its metabolites into hair. In the analysis of 18 authentic hair samples, only JWH-018, JWH-018 N-5-OH M, and JWH-073 were detected, with wide variation in concentrations. J. Kim : S. In : Y. Park : M. Park : E. Kim Narcotic Analysis Division, National Forensic Service, 139 Jiyangno, Yangcheon-gu, Seoul 158-707, Republic of Korea S. Lee (*) College of Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-gu, Daegu 704-701, Republic of Korea e-mail: [email protected]
Keywords Synthetic cannabinoids . JWH-018 . JWH-073 . Drug abuse . Hair analysis . UHPLC–MS–MS
Introduction JWH-018 (naphthalene-1-yl-(1-pentyl-1H -indol)-3yl)methanone) is believed to be the most widely distributed synthetic cannabinoid in Korea, based on analysis of seized drugs in our laboratory (National Forensic Service, Seoul, Korea). JWH-073 (naphthalene-1-yl-(1-butylindol-3yl)methanone) is often present as a minor component of JWH-018. In previous studies [1, 2], JWH-018 and JWH073 were shown to have psychoactive effects similar to those of strong agonists of cannabinoid type 1 (CB1) receptor, for example Δ9-tetrahydrocannabinol (THC). It was also reported that ingestion of synthetic cannabinoids can cause anxiety, psychosis, seizures, tachycardia, nausea, etc. [3–5]. Recently, many studies have been conducted to clarify the metabolism of JWH-018 and JWH-073 [6–8], and quantitative analytical methods for urine samples have been developed for forensic and clinical applications [9, 10]. Because both JWH-018 and JWH-073 are rapidly metabolized, these parent drugs are not detected in urine [8, 10]. Many different metabolites are formed by hydroxylation, carboxylation, and/or dealkylation, and these have been identified in urine samples from drug users [6–10] and rats [8]. In forensic and clinical laboratories, analysis of drugs in hair is often used to prove ingestion of drugs of abuse, because hair enables longer surveillance with less invasion of privacy than urine analysis. It has been proposed that drugs are incorporated into hair by binding to such intracellular components as melanins, lipids, and proteins. Analysis of hair is commonly used to verify history of drug use by sectional analysis, to provide supplementary information on drug use to facilitate interpretation of urine drug analysis, and to
J. Kim et al.
determine gestational drug exposure [11, 12]. Recently, five synthetic cannabinoids (JWH-018, JWH-073, JWH-200, JWH-250, and HU-210) and THC, cannabidiol, and cannabinol were analyzed in 175 hair samples from cannabis users by validated ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS– MS). In this analysis, JWH-018, JWH-073 and/or JWH-250 were detected in 14 samples [13]. Hutter et al. developed and validated a method for simultaneous detection of 22 synthetic cannabinoids in hair, and applied the method to authentic hair samples from chronic synthetic cannabinoid consumers [14]. In both studies, only the parent drugs were included in the list of analytes, but this does not exclude the possibility of external contamination, nor does it provide conclusive evidence of active drug consumption. Accordingly, detection of the THC metabolite 11-nor-Δ9-tetrahydrocannabinol-9carboxylic acid (THCCOOH), in a hair sample is an important evidence of cannabis use [15–17]. However, no study on the distribution of metabolites of synthetic cannabinoids in hair and the effect of pigmentation on the deposition of synthetic cannabinoids and metabolites in hair has yet been conducted. Therefore, the first purpose of this study was to establish and validate an analytical method for simultaneous detection of JWH-018 and JWH-073 and some of their metabolites in hair, by use of UHPLC–MS–MS, for forensic application. The second purpose was to investigate the incorporation of metabolites of synthetic cannabinoids into hair, and the effect of pigmentation on the deposition of synthetic cannabinoids and their metabolites in hair, by use of an animal model. For this latter purpose, JWH-073 was chosen as representative synthetic cannabinoid. Finally, the developed method was applied to 18 hair samples from individuals suspected of use of synthetic cannabinoids.
and acetonitrile were purchased from J.T. Baker (MT, USA). Ammonium formate and formic acid were supplied by Sigma–Aldrich (MO, USA). Deionized water was produced by use of an Elga Purelab Option-Q ultra-pure water system (Lane End, UK). Animals Five male lean Zucker rats (Orient Bio, Seoul, Korea) were allowed at least one-week acclimation in a laboratory animal facility before experiments. The rats were provided with tap water and a commercial diet ad libitum. The facility was maintained at a temperature of 24±2 °C, a relative humidity of 50±20 %, and with a 12-h light–dark cycle. Human or rat hair sample preparation
Material and methods
Before extraction, possible contaminants on the surface of hair strands were eliminated by washing twice with 2 mL methanol then twice with 2 mL distilled water and again twice with 2 mL methanol, by use of reservoirs installed in a manifold. The hair samples were through-flow-dried in each reservoir at room temperature, cut finely into 1–2 mm pieces with scissors and weighed accurately (ca 10 mg for human hair; ca 20 mg for rat hair). JWH-018, JWH-073, and their metabolites in the prepared hair were extracted by use of HPLC-grade methanol at 38 °C, with gentle magnetic stirring, for at least 16 h. JWH-018-d 9 and JWH-018 N -(5hydroxypentyl) metabolite-d5 (50 μL, 100 pg mL−1 internal standard mixed solution) were used as internal standards for the parent drugs (JWH-018 and JWH-073) and metabolites, respectively. The methanolic extracts were collected in glass tubes and evaporated to dryness under nitrogen gas at 45 °C. The residue was dissolved in 100 μL of a 1:1 (v/v) mixture of methanol and mobile phase component A, and 5 μL was injected for UHPLC–MS–MS analysis.
Chemicals and reagents
UHPLC–MS–MS analysis
JWH-018, JWH-018 N -(4-hydroxypentyl) metabolite (JWH018 N-4-OH M), JWH-018 N-(5-hydroxypentyl) metabolite (JWH-018 N-5-OH M), JWH-018 N -pentanoic acid metabolite (JWH-018 N-COOH M), JWH-073, JWH-073 N -(3-hydroxybutyl) metabolite (JWH-073 N-3-OH M), JWH-073 N -(4-hydroxybutyl) metabolite (JWH-073 N-4OH M), JWH-073 N -butanoic acid metabolite (JWH-073 NCOOH M), JWH-018-d9, and JWH-018 N -(5-hydroxypentyl) metabolite-d5 (JWH-018 N-5-OH M-d5) were obtained from Cayman Chemical (MI, USA). JWH-073 (purity 99 %) samples were provided by the law enforcement agencies and assayed by the Narcotic Analysis Division of the National Forensic Service. Highperformance liquid chromatography (HPLC)-grade methanol
UHPLC–MS–MS analysis was conducted with an Agilent 1290 infinity UHPLC (CA, USA) and an AB Sciex Qtrap 5500 MS/MS (MA, USA). An Eclipse plus C18 (RRHD 2.1 mm × 100 mm, 1.8 μm, Agilent Technologies, CA, USA) with a 1290 in-line filter was used; the column oven temperature was 40 °C. The mobile phase was a gradient prepared from 2 mmol L−1 aqueous ammonium formate containing 0.2 % (v /v ) formic acid (component A) and 2 mmol L−1 ammonium formate in acetonitrile containing 0.2 % (v/v) formic acid (component B). The initial mobile phase, 20 % B, was rapidly increased to 50 % B in 1 min, then maintained at this composition for 3 min, followed by an increase, in 1 min, to 90 %, which was maintained for 1.5 min. Finally, the initial conditions were restored for
Deposition of JWH-018, JWH-073, and their metabolites in hair
4.5 min. The flow rate was 500 μL min−1, and the total run time was 11 min. The MS system was operated with an electrospray ion source in the positive-ionization mode. The optimum conditions for ionization were found to be: ion source voltage, 5500 V; turbo gas temperature, 600 °C ; curtain gas, 30 psi; collision gas, medium; gas 1 (nebulizing gas), 50 psi; and gas 2 (heater gas), 55 psi. Two multiple reaction monitoring (MRM) transitions were chosen for each analyte. The individual MRM transitions, retention times, and other experimental conditions are summarized in Table 1. Data were processed by use of Analyst 1.6 software. Validation study The analytical method was validated, as described elsewhere [18, 19], by use of spiked drug-free human hair provided voluntarily by the laboratory staff. Selectivity, matrix effect, recovery, process efficiency, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and stability were evaluated. To assess the selectivity of method, ten different sources of drug-free hair were analyzed to confirm the absence of interference with the signals from Table 1 MRM transitions, retention times, and other conditions for MS analysis of JWH-018, JWH-073, their metabolites, and internal standards Compound
Precursor Product RT DP EP CE CXP ion (m/z) ion (m/z) (min) (V) (V) (V) (V)
JWH-018
342
JWH-018 N-4-OH M
358
JWH-018 N-5-OH M
358
JWH-018 N-COOH M
372
JWH-073
328
JWH-073 N-3-OH M
344
JWH-073 N-4-OH M
344
JWH-073 N-COOH M
358
JWH-018-d9
351
JWH-018 363 N-5-OH M -d5
155 127 155 127 155 127 155 127 155 127 155 127 127 101 155 127 155 127 155 127
6.2 4.3 4.2 3.9 5.9 4.1 3.6 3.5 6.2 4.2
140 105 140 100 190 140 140 100 145 120 140 90 90
10 10 10 10 10 10 10 10 10 10 10 10 10
31 59 31 65 27 63 29 65 31 53 31 67 59
12 12 10 14 12 12 14 10 12 10 12 12 10
86 10 140 10 55 10
99 31 63
12 12 16
140 105 190 140
33 65 30 65
14 10 12 12
10 10 10 10
Quantifier ions are in bold RT, retention time; DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential
each analyte and the internal standard. Matrix effect, recovery, and process efficiency were determined by comparing five neat standards, five extracts obtained from drug-free hair from five different sources spiked with the analytes after extraction, and five extracts obtained from drug-free hair from five different sources spiked with the analytes before extraction; the hair was spiked at low (1 pg/10 mg hair) and high (100 pg/ 10 mg hair) concentrations [19]. For determination of other validation data, combined drugfree hair from five different sources was used. To demonstrate linearity, five sets of calibrators (1, 2, 5, 10, 20, 50, and 100 pg/10 mg hair) were prepared and analyzed, and regression coefficients were calculated from the results by using 1/x as a weighting factor. For the LOD and LOQ, drug-free hair samples spiked with each analyte at concentrations below 1 pg/10 mg hair were evaluated. The analyte concentration at which the signal-to-noise ratio was greater than 3 was chosen as the LOD. The analyte concentration for which the coefficient of variation (CV) was less than 20 % for precision and less than ±20 % for bias was selected as the LOQ. Method precision and accuracy were determined by analyzing drug-free hair samples spiked with the low and high concentrations of each analyte. Five sets of each sample were analyzed on five different days. Repeatability, between-day precision, and intermediate precision (as CV) were estimated by use of one-way ANOVA with the grouping variable “day” at the respective concentrations. Accuracy (as bias) was calculated as the percentage difference between the grand mean of all 25 measurements and the respective nominal concentration, for each concentration. In-process stability under samplepreparation conditions was monitored 2, 4, 6, 12, and 24 h after spiking hair with the analytes. The stability at each time was calculated by comparison with the initial concentrations of analytes. Processed sample stability was examined at 2.5-h intervals over 24 h. For analysis of rat hair, matrix effect, recovery, and process efficiency were also investigated at low (1 pg/20 mg hair) and high (100 pg/20 mg hair) concentrations, as described above, by using pigmented and nonpigmented drug-free hair from five different lean Zucker rats. Animal study All procedures had been approved by the Animal Care and Use Committee at the National Forensic Service. When the rats were six weeks old, they were weighed and their pigmented and nonpigmented hair was shaved from the dorsal region by use of an electric shaver before drug administration. After three days, JWH-073 suspension in 2 % Tween-80 was administered intraperitoneally at 10 mg kg−1 to rats once per day for twenty weekdays for four weeks. Five weeks after initial treatment, the newly grown pigmented and nonpigmented hair was shaved
J. Kim et al.
(A) JWH-018
JWH-018 N-4-OH M
JWH-018 N-5-OH M
JWH-018 N-COOH M
JWH-073
JWH-073 N-3-OH M
JWH-073 N-4-OH M
JWH-073 N-COOH M
JWH-018
JWH-018 N-4-OH M
JWH-018 N-5-OH M
JWH-018 N-COOH M
JWH-073
JWH-073 N-3-OH M
JWH-073 N-4-OH M
JWH-073 N-COOH M
JWH-018
JWH-018 N-4-OH M
JWH-018 N-5-OH M
JWH-018 N-COOH M
JWH-073
JWH-073 N-3-OH M
JWH-073 N-4-OH M
JWH-073 N-COOH M
(B)
(C)
Deposition of JWH-018, JWH-073, and their metabolites in hair
Fig. 1
Representative chromatograms of JWH-018, JWH-073, and their metabolites obtained from drug-free (a ), fortified (LOQ, b ), and authentic (case 2, segment 1, c) hair samples
and collected. Each shaved sample was washed, finely cut, weighed (ca 20 mg) and analyzed as described above. Analysis of authentic human hair samples Hair samples from 18 individuals suspected of synthetic cannabinoids use were provided by the law enforcement agency. Hair samples were divided into 3-cm lengths on request. Hair samples were analyzed up to 12 cm of hair length. The samples were prepared and analyzed as described above.
Results and discussion In a previous study of the metabolism of JWH-018 and JWH073, monohydroxylation was assumed to be a common pathway [8]. According to other studies, JWH-018 N-5-OH M and JWH-018 N-COOH M were the main metabolites detected in urine after JWH-018 ingestion whereas JWH073 N-COOH M was the primary metabolite identified after consumption of JWH-073 [20, 21]. Therefore, an analytical method was established to concurrently analyze JWH-018, JWH-073, and their most abundant monohydroxylated and carboxylated metabolites in hair. Previous studies on detection of synthetic cannabinoids in hair had only included the parent drugs in the analysis [13, 14]. In this study both parent drugs and metabolites were investigated in hair samples. In the initial stages of this study, negative ionization was tried for analysis of acidic metabolites; however, the sensitivity was no better than that of the positive ionization. Analysis of JWH-018 N-4-OH M and JWH-018 N-5-OH M was not straightforward, because of their similar chemical structures, which could prevent chromatographic separation, and identical MRM transitions. In previous studies, either separation of those compounds was not tried or the former compound was not included in the list of analytes [20–22]. In this study, the resolution between JWH-018 N-4-OH M and JWH-018 N-5-OH M was 1.2, which was considered minimally sufficient for quantitative analysis. Separation of the two compounds on chromatograms (Fig. 1b) was similar to that achieved by Chimalakonda et al. [23], who separated the compounds under isocratic mobile phase conditions. Figure 1 shows representative chromatograms of JWH018, JWH-073, and their metabolites obtained from drugfree, fortified, and authentic hair samples. No endogenous substances interfered with analysis of JWH-018, JWH-073, or the metabolites. JWH-018, JWH-018 N-5-OH M and
JWH-073 were clearly detected in chromatograms (Fig. 1c) obtained from the first hair segment from an individual suspected of synthetic cannabinoids use (case 2 in Table 7). Results from determination of matrix effect, recovery, and process efficiency for low and high concentrations of each analyte and internal standard in human hair are summarized in Table 2. The matrix effect ranged from 20 % (JWH-073) to 112 % (JWH-073 N-4-OH M) at low analyte concentrationsand from 22 % (JWH-073) to 89 % (JWH-073 N-4-OH M) at high concentrations. For most of the analytes, different levels of ion suppression were observed at both concentrations; this is a general phenomenon in electrospray ionization, although slight ion enhancement (112 %) was observed for JWH-073 N-4-OH M at high concentrations. However, CV values for the five hair samples from different sources were never >15 %, which meant no significant variation was caused by different matrices. Recovery was higher than 78 %, which was acceptable. Process efficiency, which is the combined outcome of matrix effect and recovery, ranged from 17– 106 %. The matrix effect, recovery, and process efficiency results for JWH-018-d9 and JWH-018 N-5-OH M-d5 were similar in pattern to those for JWH-018 and JWH-018 N-5OH M, respectively. Table 2 Matrix effect, recovery, and process efficiency at low and high concentrations of each analyte and internal standard in human hair (n =5 each) Compound
Concentration Matrix (pg/10 mg hair) effect (%)
Recovery (%)
Process efficiency (%)
Mean CV Mean CV Mean CV JWH-018 JWH-018 N-4-OH M
1 100 1 100
JWH-018 N-5-OH M
1 100 JWH-018 1 N-COOH M 100 JWH-073 1 100 JWH-073 1 N-3-OH M 100 JWH-073 1 N-4-OH M 100 JWH-073 N1 COOH M 100 JWH-018-d9 5 JWH-018 N-5- 5 OH M-d5 CV, coefficient of variation
59 56 89 85
4 6 3 4
78 94 93 95
3 6 3 4
47 47 83 81
3 5 3 4
83 88 92 79 20 22 85 67 112 89 92 85
4 4 8 8 4 4 8 7 11 5 7 3
100 98 101 97 83 96 97 101 94 98 95 94
8 2 9 4 6 2 5 3 18 2 15 4
83 86 93 77 17 21 82 67 106 87 87 80
8 2 9 4 6 2 5 3 18 2 15 4
53 78
15 6
79 95
9 4
42 74
9 4
J. Kim et al. Table 3 LOD, LOQ, and linearity for analysis of human hair Compound
LOD (pg/10 mg hair)
LOQ (pg/10 mg hair)
Calibration curve (n =5) a
JWH-018 JWH-018 N-4-OH M JWH-018 N-5-OH M JWH-018 N-COOH M JWH-073 JWH-073 N-3-OH M JWH-073 N-4-OH M JWH-073 N-COOH M
0.5 0.5 0.5 0.5 0.5 0.5 1 0.5
1 1 1 1 1 1 1 1
The LOD was 0.5 pg/10 mg hair for every compound except JWH-073 N-4-OH M, for which the LOD was 1 pg/ 10 mg hair. The LOQ was 1 pg/10 mg hair for every analyte. The regression coefficients (R) of the calibration curves were higher than 0.997 for every analyte (Table 3). The repeatability, between-day precision, and intermediate precision were acceptable (i.e., below 20 % CV at low concentration and below 15 % CV at high concentration). Accuracy was also within acceptance limits (below ±20 % bias at low concentration and below ±15 % CV at high concentration) (Table 4). The in-process stability results were within the acceptance criteria (±10 %), which meant that all analytes in hair are considered stable in the extraction solvent under the conditions tested (Table 5).
Table 4 Precision and accuracy of analysis of human hair
Regression coefficient (R, mean, n =5)
Mean
SD
Mean
SD
0.040 0.147 0.236 0.188 0.059 0.108 0.197 0.180
0.005 0.011 0.033 0.020 0.006 0.013 0.025 0.047
0.009 0.022 0.006 0.060 0.019 0.080 0.025 0.134
0.024 0.038 0.051 0.096 0.019 0.152 0.039 0.115
0.999 0.999 0.998 0.997 0.999 0.999 0.999 0.997
Linear regression analysis of peak areas of analytes or internal standards plotted against injection time over 24 h confirmed that processed sample stability was also satisfactory, i.e., negative slopes were not obtained (results not shown). The LOD and LOQ for rat hair were the same as those for human hair (e.g. 1 pg/20 mg hair for LOQ). Matrix effect, recovery, and process efficiency results for pigmented and nonpigmented rat hair are shown in Table 6. The matrix effect values for pigmented hair ranged from 68 % (JWH-073) to 113 % (JWH-073 4-OH M) and from 34 % (JWH-018) to 88 % (JWH-073 4-OH M) at low and high concentrations, respectively. CV values were no higher than 18 %. The matrix effect for nonpigmented hair ranged from 80 % (JWH-073) to
Compound
Concentration (pg/10 mg hair)
Repeatability (CV, %)
Between-day precision (CV, %)
Intermediate precision (CV, %)
Accuracy (bias, %)
JWH-018
1 100 1 100 1 100
5.9 8.4 9.2 6.3 8.8 5.8
1.0 5.6 5.7 6.3 8.5 6.2
6.0 10.0 10.8 8.9 12.2 8.5
7.3 −1.0 3.4 1.2 2.6 1.8
1 100 1 100 1 100 1 100 1 100
9.8 6.6 8.3 9.2 9.5 6.0 8.9 6.2 11.9 7.5
4.2 10.0 7.9 9.8 8.2 7.0 10.0 6.6 0.7 3.4
10.6 12.0 11.4 13.5 12.6 9.2 13.4 9.1 12.0 8.3
0.5 0.2 3.6 −1.5 -.3.5 2.6 −1.9 −1.0 0.7 5.5
JWH-018 N4-OH M JWH-018 N5-OH M JWH-018 NCOOH M JWH-073 JWH-073 N3-OH M JWH-073 N4-OH M JWH-073 NCOOH M CV, coefficient of variation
b
Deposition of JWH-018, JWH-073, and their metabolites in hair Table 5 In-process stability under the sample-preparation conditions used for human hair
Compound
Concentration (pg/10 mg hair)
Extraction time (h) 2
JWH-018 JWH-018 N-4-OH M JWH-018 N-5-OH M JWH-018 N-COOH M JWH-073 JWH-073 N-3-OH M JWH-073 N-4-OH M Results are presented as mean percentage of initial concentration of analytes (n =3)
JWH-073 N-COOH M
4
6
12
24
1
105
96
102
100
100
100 1 100 1 100 1 100 1 100 1 100 1 100 1 100
92 101 105 98 99 95 95 95 101 92 104 92 100 102 100
91 101 102 102 103 97 97 95 105 96 104 96 105 99 102
90 100 100 99 102 95 103 95 104 95 101 95 104 100 101
90 105 102 101 102 94 97 94 104 96 103 100 101 102 100
92 100 104 100 103 97 98 94 105 97 106 95 106 101 104
118 % (JWH-018 5-OH M) and 60 % (JWH-018) to 101 % (JWH-018 4-OH M) at low and high concentrations, respectively. The CV values were below 15 % for each
analyte, except for JWH-018 4-OH M (18 %) and JWH-018 5-OH M (16 %) at low concentrations in pigmented hair; these were not regarded as significant variations.
Table 6 Matrix effect, recovery, and process efficiency at low and high concentrations for each analyte and internal standard in rat hair (n =5 each) Compound
JWH-018 JWH-018 N-4-OH M JWH-018 N-5-OH M JWH-018 N-COOH M JWH-073 JWH-073 N-3-OH M JWH-073 N-4-OH M JWH-073 N-COOH M JWH-018-d9 JWH-018 N-5-OH M-d5 CV, coefficient of variation
Concentration Pigmented hair (pg/20 mg rat hair) Matrix effect Recovery (%) (%)
Nonpigmented hair Process efficiency (%)
Matrix effect (%)
Recovery (%)
Process efficiency (%)
Mean
CV
Mean CV Mean
CV
Mean
CV
Mean CV Mean
CV
1
80
4
106
14
74
16
92
12
88
8
72
14
100 1 100 1 100 1 100 1 100 1 100 1 100 1 100
34 92 67 99 74 95 83 68 55 92 73 113 88 98 83
5 18 8 16 11 7 8 10 11 15 11 10 5 9 4
109 132 131 131 140 107 133 86 105 127 136 104 136 104 109
6 11 11 9 7 11 7 10 13 9 7 4 7 10 6
55 121 102 129 99 102 99 58 58 117 99 117 98 102 91
4 10 5 9 5 11 5 7 7 8 5 5 5 10 5
60 112 101 118 98 101 96 80 67 104 99 114 96 98 90
4 12 5 7 6 16 4 5 4 2 5 12 6 8 3
100 102 100 113 100 100 100 91 100 114 100 106 100 104 100
7 7 5 3 6 12 4 7 6 10 5 12 7 3 3
34 110 86 120 82 97 81 68 42 107 79 111 91 92 81
14 12 14 9 14 13 7 5 10 9 14 13 9 3 7
5 5
76 115
11 4
138 132
12 6
125 58
5 5
124 83
11 6
91 106
10 4
62 118
7 5
J. Kim et al.
Concentration (pg/mg)
120
Pigmented Nonpigmented
100 80 60 40 20 0 JWH-073
JWH-073 N-3- JWH-073 NOH M COOH M
Fig. 2 Comparison of amounts JWH-073 and its metabolites in pigmented and nonpigmented rat hair (n =3 for JWH-073 N-COOH M in nonpigmented hair; n =4 for JWH-073 N-3-OH M in nonpigmented hair; n =5 for others)
To investigate the distribution of synthetic cannabinoids and metabolites in hair and the effect of hair pigmentation, the JWH-073 suspension was fed to five lean Zucker rats with both pigmented and nonpigmented hair. Although wide variations of quantitative results were obtained, depending on individual differences, JWH-073 and its two metabolites, JWH-073 N-3-OH M and JWH-073 N-COOH M, were detected in all pigmented and nonpigmented hair samples, whereas JWH-073 N-4-OH M was detected below the LOQ or was not detectable. Quantitative results for JWH-073, JWH-073 N-3-OH M, and JWH-073 N-COOH M in pigmented and nonpigmented hair are compared in Fig. 2. Some results below the LOQ were not included; n =3 for JWH-073 N-COOH M in nonpigmented hair, n = 4 for JWH-073 N-3-OH M in nonpigmented hair, and n =5 for other occasions. The concentration of JWH-073 in both
Table 7 Quantitative analysis for JWH-018, JWH-018 N-5-OH M, JWH-073, and THCCOOH in hair samples from individuals suspected of synthetic cannabinoids use Case no.
Gender
Age
Hair colora
1
Male
24
Black
2
Female
26
Black
Hair length (cm)
JWH-018 (pg mg−1)
JWH-018 N-5-OH M (pg mg−1)
JWH-073 (pg mg−1)
THCCOOHb (pg mg−1) NA
0-3c
88
ND
3
3-6c
117
ND
ND
NA
6-9c
56
ND
ND
NA
0-3c 3-6c
237 524
8 11
11 20
0.06
6-9c
688
12
20
9-12c
753
12
22
11 14
9 5
3
Male
27
Blonde
0-3c 3-6c
RESEARCH PAPER
Deposition of JWH-018, JWH-073 and their metabolites in hair and effect of hair pigmentation Jihyun Kim & Sanghwan In & Yuran Park & Meejung Park & Eunmi Kim & Sooyeun Lee
Received: 11 June 2013 / Revised: 7 October 2013 / Accepted: 7 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Analysis of drugs in hair is often used as a routine method to obtain detailed information about drug ingestion. However, few studies have been conducted on deposition of synthetic cannabinoids and metabolites in hair. The first purpose of this study was to establish and validate an analytical method for detection of JWH-018, JWH-073, and their metabolites in hair, by use of UHPLC–MS–MS, for forensic application. The second purpose was to investigate the distribution of synthetic cannabinoids metabolites in hair and the effect of hair pigmentation, by use of an animal model. For this, JWH-073 was chosen as a representative synthetic cannabinoid. Finally, the developed method was applied to hair samples from 18 individuals suspected of synthetic cannabinoids use. JWH-018, JWH-073, and their metabolites were extracted from hair with methanol. The extract was then filtered and analyzed by UHPLC–MS–MS with an electrospray ion source in positive-ionization mode. Validation proved the method was selective, sensitive, accurate, and precise, with acceptable linearity within the calibration ranges. No significant variations were observed when different sources of both human and rat hair were used. The animal study demonstrated that JWH-073 N-COOH M was the major metabolite of JWH-073 in rat hair, and hair pigmentation did not have a significant effect on incorporation of JWH-073 and its metabolites into hair. In the analysis of 18 authentic hair samples, only JWH-018, JWH-018 N-5-OH M, and JWH-073 were detected, with wide variation in concentrations. J. Kim : S. In : Y. Park : M. Park : E. Kim Narcotic Analysis Division, National Forensic Service, 139 Jiyangno, Yangcheon-gu, Seoul 158-707, Republic of Korea S. Lee (*) College of Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-gu, Daegu 704-701, Republic of Korea e-mail: [email protected]
Keywords Synthetic cannabinoids . JWH-018 . JWH-073 . Drug abuse . Hair analysis . UHPLC–MS–MS
Introduction JWH-018 (naphthalene-1-yl-(1-pentyl-1H -indol)-3yl)methanone) is believed to be the most widely distributed synthetic cannabinoid in Korea, based on analysis of seized drugs in our laboratory (National Forensic Service, Seoul, Korea). JWH-073 (naphthalene-1-yl-(1-butylindol-3yl)methanone) is often present as a minor component of JWH-018. In previous studies [1, 2], JWH-018 and JWH073 were shown to have psychoactive effects similar to those of strong agonists of cannabinoid type 1 (CB1) receptor, for example Δ9-tetrahydrocannabinol (THC). It was also reported that ingestion of synthetic cannabinoids can cause anxiety, psychosis, seizures, tachycardia, nausea, etc. [3–5]. Recently, many studies have been conducted to clarify the metabolism of JWH-018 and JWH-073 [6–8], and quantitative analytical methods for urine samples have been developed for forensic and clinical applications [9, 10]. Because both JWH-018 and JWH-073 are rapidly metabolized, these parent drugs are not detected in urine [8, 10]. Many different metabolites are formed by hydroxylation, carboxylation, and/or dealkylation, and these have been identified in urine samples from drug users [6–10] and rats [8]. In forensic and clinical laboratories, analysis of drugs in hair is often used to prove ingestion of drugs of abuse, because hair enables longer surveillance with less invasion of privacy than urine analysis. It has been proposed that drugs are incorporated into hair by binding to such intracellular components as melanins, lipids, and proteins. Analysis of hair is commonly used to verify history of drug use by sectional analysis, to provide supplementary information on drug use to facilitate interpretation of urine drug analysis, and to
J. Kim et al.
determine gestational drug exposure [11, 12]. Recently, five synthetic cannabinoids (JWH-018, JWH-073, JWH-200, JWH-250, and HU-210) and THC, cannabidiol, and cannabinol were analyzed in 175 hair samples from cannabis users by validated ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS– MS). In this analysis, JWH-018, JWH-073 and/or JWH-250 were detected in 14 samples [13]. Hutter et al. developed and validated a method for simultaneous detection of 22 synthetic cannabinoids in hair, and applied the method to authentic hair samples from chronic synthetic cannabinoid consumers [14]. In both studies, only the parent drugs were included in the list of analytes, but this does not exclude the possibility of external contamination, nor does it provide conclusive evidence of active drug consumption. Accordingly, detection of the THC metabolite 11-nor-Δ9-tetrahydrocannabinol-9carboxylic acid (THCCOOH), in a hair sample is an important evidence of cannabis use [15–17]. However, no study on the distribution of metabolites of synthetic cannabinoids in hair and the effect of pigmentation on the deposition of synthetic cannabinoids and metabolites in hair has yet been conducted. Therefore, the first purpose of this study was to establish and validate an analytical method for simultaneous detection of JWH-018 and JWH-073 and some of their metabolites in hair, by use of UHPLC–MS–MS, for forensic application. The second purpose was to investigate the incorporation of metabolites of synthetic cannabinoids into hair, and the effect of pigmentation on the deposition of synthetic cannabinoids and their metabolites in hair, by use of an animal model. For this latter purpose, JWH-073 was chosen as representative synthetic cannabinoid. Finally, the developed method was applied to 18 hair samples from individuals suspected of use of synthetic cannabinoids.
and acetonitrile were purchased from J.T. Baker (MT, USA). Ammonium formate and formic acid were supplied by Sigma–Aldrich (MO, USA). Deionized water was produced by use of an Elga Purelab Option-Q ultra-pure water system (Lane End, UK). Animals Five male lean Zucker rats (Orient Bio, Seoul, Korea) were allowed at least one-week acclimation in a laboratory animal facility before experiments. The rats were provided with tap water and a commercial diet ad libitum. The facility was maintained at a temperature of 24±2 °C, a relative humidity of 50±20 %, and with a 12-h light–dark cycle. Human or rat hair sample preparation
Material and methods
Before extraction, possible contaminants on the surface of hair strands were eliminated by washing twice with 2 mL methanol then twice with 2 mL distilled water and again twice with 2 mL methanol, by use of reservoirs installed in a manifold. The hair samples were through-flow-dried in each reservoir at room temperature, cut finely into 1–2 mm pieces with scissors and weighed accurately (ca 10 mg for human hair; ca 20 mg for rat hair). JWH-018, JWH-073, and their metabolites in the prepared hair were extracted by use of HPLC-grade methanol at 38 °C, with gentle magnetic stirring, for at least 16 h. JWH-018-d 9 and JWH-018 N -(5hydroxypentyl) metabolite-d5 (50 μL, 100 pg mL−1 internal standard mixed solution) were used as internal standards for the parent drugs (JWH-018 and JWH-073) and metabolites, respectively. The methanolic extracts were collected in glass tubes and evaporated to dryness under nitrogen gas at 45 °C. The residue was dissolved in 100 μL of a 1:1 (v/v) mixture of methanol and mobile phase component A, and 5 μL was injected for UHPLC–MS–MS analysis.
Chemicals and reagents
UHPLC–MS–MS analysis
JWH-018, JWH-018 N -(4-hydroxypentyl) metabolite (JWH018 N-4-OH M), JWH-018 N-(5-hydroxypentyl) metabolite (JWH-018 N-5-OH M), JWH-018 N -pentanoic acid metabolite (JWH-018 N-COOH M), JWH-073, JWH-073 N -(3-hydroxybutyl) metabolite (JWH-073 N-3-OH M), JWH-073 N -(4-hydroxybutyl) metabolite (JWH-073 N-4OH M), JWH-073 N -butanoic acid metabolite (JWH-073 NCOOH M), JWH-018-d9, and JWH-018 N -(5-hydroxypentyl) metabolite-d5 (JWH-018 N-5-OH M-d5) were obtained from Cayman Chemical (MI, USA). JWH-073 (purity 99 %) samples were provided by the law enforcement agencies and assayed by the Narcotic Analysis Division of the National Forensic Service. Highperformance liquid chromatography (HPLC)-grade methanol
UHPLC–MS–MS analysis was conducted with an Agilent 1290 infinity UHPLC (CA, USA) and an AB Sciex Qtrap 5500 MS/MS (MA, USA). An Eclipse plus C18 (RRHD 2.1 mm × 100 mm, 1.8 μm, Agilent Technologies, CA, USA) with a 1290 in-line filter was used; the column oven temperature was 40 °C. The mobile phase was a gradient prepared from 2 mmol L−1 aqueous ammonium formate containing 0.2 % (v /v ) formic acid (component A) and 2 mmol L−1 ammonium formate in acetonitrile containing 0.2 % (v/v) formic acid (component B). The initial mobile phase, 20 % B, was rapidly increased to 50 % B in 1 min, then maintained at this composition for 3 min, followed by an increase, in 1 min, to 90 %, which was maintained for 1.5 min. Finally, the initial conditions were restored for
Deposition of JWH-018, JWH-073, and their metabolites in hair
4.5 min. The flow rate was 500 μL min−1, and the total run time was 11 min. The MS system was operated with an electrospray ion source in the positive-ionization mode. The optimum conditions for ionization were found to be: ion source voltage, 5500 V; turbo gas temperature, 600 °C ; curtain gas, 30 psi; collision gas, medium; gas 1 (nebulizing gas), 50 psi; and gas 2 (heater gas), 55 psi. Two multiple reaction monitoring (MRM) transitions were chosen for each analyte. The individual MRM transitions, retention times, and other experimental conditions are summarized in Table 1. Data were processed by use of Analyst 1.6 software. Validation study The analytical method was validated, as described elsewhere [18, 19], by use of spiked drug-free human hair provided voluntarily by the laboratory staff. Selectivity, matrix effect, recovery, process efficiency, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and stability were evaluated. To assess the selectivity of method, ten different sources of drug-free hair were analyzed to confirm the absence of interference with the signals from Table 1 MRM transitions, retention times, and other conditions for MS analysis of JWH-018, JWH-073, their metabolites, and internal standards Compound
Precursor Product RT DP EP CE CXP ion (m/z) ion (m/z) (min) (V) (V) (V) (V)
JWH-018
342
JWH-018 N-4-OH M
358
JWH-018 N-5-OH M
358
JWH-018 N-COOH M
372
JWH-073
328
JWH-073 N-3-OH M
344
JWH-073 N-4-OH M
344
JWH-073 N-COOH M
358
JWH-018-d9
351
JWH-018 363 N-5-OH M -d5
155 127 155 127 155 127 155 127 155 127 155 127 127 101 155 127 155 127 155 127
6.2 4.3 4.2 3.9 5.9 4.1 3.6 3.5 6.2 4.2
140 105 140 100 190 140 140 100 145 120 140 90 90
10 10 10 10 10 10 10 10 10 10 10 10 10
31 59 31 65 27 63 29 65 31 53 31 67 59
12 12 10 14 12 12 14 10 12 10 12 12 10
86 10 140 10 55 10
99 31 63
12 12 16
140 105 190 140
33 65 30 65
14 10 12 12
10 10 10 10
Quantifier ions are in bold RT, retention time; DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential
each analyte and the internal standard. Matrix effect, recovery, and process efficiency were determined by comparing five neat standards, five extracts obtained from drug-free hair from five different sources spiked with the analytes after extraction, and five extracts obtained from drug-free hair from five different sources spiked with the analytes before extraction; the hair was spiked at low (1 pg/10 mg hair) and high (100 pg/ 10 mg hair) concentrations [19]. For determination of other validation data, combined drugfree hair from five different sources was used. To demonstrate linearity, five sets of calibrators (1, 2, 5, 10, 20, 50, and 100 pg/10 mg hair) were prepared and analyzed, and regression coefficients were calculated from the results by using 1/x as a weighting factor. For the LOD and LOQ, drug-free hair samples spiked with each analyte at concentrations below 1 pg/10 mg hair were evaluated. The analyte concentration at which the signal-to-noise ratio was greater than 3 was chosen as the LOD. The analyte concentration for which the coefficient of variation (CV) was less than 20 % for precision and less than ±20 % for bias was selected as the LOQ. Method precision and accuracy were determined by analyzing drug-free hair samples spiked with the low and high concentrations of each analyte. Five sets of each sample were analyzed on five different days. Repeatability, between-day precision, and intermediate precision (as CV) were estimated by use of one-way ANOVA with the grouping variable “day” at the respective concentrations. Accuracy (as bias) was calculated as the percentage difference between the grand mean of all 25 measurements and the respective nominal concentration, for each concentration. In-process stability under samplepreparation conditions was monitored 2, 4, 6, 12, and 24 h after spiking hair with the analytes. The stability at each time was calculated by comparison with the initial concentrations of analytes. Processed sample stability was examined at 2.5-h intervals over 24 h. For analysis of rat hair, matrix effect, recovery, and process efficiency were also investigated at low (1 pg/20 mg hair) and high (100 pg/20 mg hair) concentrations, as described above, by using pigmented and nonpigmented drug-free hair from five different lean Zucker rats. Animal study All procedures had been approved by the Animal Care and Use Committee at the National Forensic Service. When the rats were six weeks old, they were weighed and their pigmented and nonpigmented hair was shaved from the dorsal region by use of an electric shaver before drug administration. After three days, JWH-073 suspension in 2 % Tween-80 was administered intraperitoneally at 10 mg kg−1 to rats once per day for twenty weekdays for four weeks. Five weeks after initial treatment, the newly grown pigmented and nonpigmented hair was shaved
J. Kim et al.
(A) JWH-018
JWH-018 N-4-OH M
JWH-018 N-5-OH M
JWH-018 N-COOH M
JWH-073
JWH-073 N-3-OH M
JWH-073 N-4-OH M
JWH-073 N-COOH M
JWH-018
JWH-018 N-4-OH M
JWH-018 N-5-OH M
JWH-018 N-COOH M
JWH-073
JWH-073 N-3-OH M
JWH-073 N-4-OH M
JWH-073 N-COOH M
JWH-018
JWH-018 N-4-OH M
JWH-018 N-5-OH M
JWH-018 N-COOH M
JWH-073
JWH-073 N-3-OH M
JWH-073 N-4-OH M
JWH-073 N-COOH M
(B)
(C)
Deposition of JWH-018, JWH-073, and their metabolites in hair
Fig. 1
Representative chromatograms of JWH-018, JWH-073, and their metabolites obtained from drug-free (a ), fortified (LOQ, b ), and authentic (case 2, segment 1, c) hair samples
and collected. Each shaved sample was washed, finely cut, weighed (ca 20 mg) and analyzed as described above. Analysis of authentic human hair samples Hair samples from 18 individuals suspected of synthetic cannabinoids use were provided by the law enforcement agency. Hair samples were divided into 3-cm lengths on request. Hair samples were analyzed up to 12 cm of hair length. The samples were prepared and analyzed as described above.
Results and discussion In a previous study of the metabolism of JWH-018 and JWH073, monohydroxylation was assumed to be a common pathway [8]. According to other studies, JWH-018 N-5-OH M and JWH-018 N-COOH M were the main metabolites detected in urine after JWH-018 ingestion whereas JWH073 N-COOH M was the primary metabolite identified after consumption of JWH-073 [20, 21]. Therefore, an analytical method was established to concurrently analyze JWH-018, JWH-073, and their most abundant monohydroxylated and carboxylated metabolites in hair. Previous studies on detection of synthetic cannabinoids in hair had only included the parent drugs in the analysis [13, 14]. In this study both parent drugs and metabolites were investigated in hair samples. In the initial stages of this study, negative ionization was tried for analysis of acidic metabolites; however, the sensitivity was no better than that of the positive ionization. Analysis of JWH-018 N-4-OH M and JWH-018 N-5-OH M was not straightforward, because of their similar chemical structures, which could prevent chromatographic separation, and identical MRM transitions. In previous studies, either separation of those compounds was not tried or the former compound was not included in the list of analytes [20–22]. In this study, the resolution between JWH-018 N-4-OH M and JWH-018 N-5-OH M was 1.2, which was considered minimally sufficient for quantitative analysis. Separation of the two compounds on chromatograms (Fig. 1b) was similar to that achieved by Chimalakonda et al. [23], who separated the compounds under isocratic mobile phase conditions. Figure 1 shows representative chromatograms of JWH018, JWH-073, and their metabolites obtained from drugfree, fortified, and authentic hair samples. No endogenous substances interfered with analysis of JWH-018, JWH-073, or the metabolites. JWH-018, JWH-018 N-5-OH M and
JWH-073 were clearly detected in chromatograms (Fig. 1c) obtained from the first hair segment from an individual suspected of synthetic cannabinoids use (case 2 in Table 7). Results from determination of matrix effect, recovery, and process efficiency for low and high concentrations of each analyte and internal standard in human hair are summarized in Table 2. The matrix effect ranged from 20 % (JWH-073) to 112 % (JWH-073 N-4-OH M) at low analyte concentrationsand from 22 % (JWH-073) to 89 % (JWH-073 N-4-OH M) at high concentrations. For most of the analytes, different levels of ion suppression were observed at both concentrations; this is a general phenomenon in electrospray ionization, although slight ion enhancement (112 %) was observed for JWH-073 N-4-OH M at high concentrations. However, CV values for the five hair samples from different sources were never >15 %, which meant no significant variation was caused by different matrices. Recovery was higher than 78 %, which was acceptable. Process efficiency, which is the combined outcome of matrix effect and recovery, ranged from 17– 106 %. The matrix effect, recovery, and process efficiency results for JWH-018-d9 and JWH-018 N-5-OH M-d5 were similar in pattern to those for JWH-018 and JWH-018 N-5OH M, respectively. Table 2 Matrix effect, recovery, and process efficiency at low and high concentrations of each analyte and internal standard in human hair (n =5 each) Compound
Concentration Matrix (pg/10 mg hair) effect (%)
Recovery (%)
Process efficiency (%)
Mean CV Mean CV Mean CV JWH-018 JWH-018 N-4-OH M
1 100 1 100
JWH-018 N-5-OH M
1 100 JWH-018 1 N-COOH M 100 JWH-073 1 100 JWH-073 1 N-3-OH M 100 JWH-073 1 N-4-OH M 100 JWH-073 N1 COOH M 100 JWH-018-d9 5 JWH-018 N-5- 5 OH M-d5 CV, coefficient of variation
59 56 89 85
4 6 3 4
78 94 93 95
3 6 3 4
47 47 83 81
3 5 3 4
83 88 92 79 20 22 85 67 112 89 92 85
4 4 8 8 4 4 8 7 11 5 7 3
100 98 101 97 83 96 97 101 94 98 95 94
8 2 9 4 6 2 5 3 18 2 15 4
83 86 93 77 17 21 82 67 106 87 87 80
8 2 9 4 6 2 5 3 18 2 15 4
53 78
15 6
79 95
9 4
42 74
9 4
J. Kim et al. Table 3 LOD, LOQ, and linearity for analysis of human hair Compound
LOD (pg/10 mg hair)
LOQ (pg/10 mg hair)
Calibration curve (n =5) a
JWH-018 JWH-018 N-4-OH M JWH-018 N-5-OH M JWH-018 N-COOH M JWH-073 JWH-073 N-3-OH M JWH-073 N-4-OH M JWH-073 N-COOH M
0.5 0.5 0.5 0.5 0.5 0.5 1 0.5
1 1 1 1 1 1 1 1
The LOD was 0.5 pg/10 mg hair for every compound except JWH-073 N-4-OH M, for which the LOD was 1 pg/ 10 mg hair. The LOQ was 1 pg/10 mg hair for every analyte. The regression coefficients (R) of the calibration curves were higher than 0.997 for every analyte (Table 3). The repeatability, between-day precision, and intermediate precision were acceptable (i.e., below 20 % CV at low concentration and below 15 % CV at high concentration). Accuracy was also within acceptance limits (below ±20 % bias at low concentration and below ±15 % CV at high concentration) (Table 4). The in-process stability results were within the acceptance criteria (±10 %), which meant that all analytes in hair are considered stable in the extraction solvent under the conditions tested (Table 5).
Table 4 Precision and accuracy of analysis of human hair
Regression coefficient (R, mean, n =5)
Mean
SD
Mean
SD
0.040 0.147 0.236 0.188 0.059 0.108 0.197 0.180
0.005 0.011 0.033 0.020 0.006 0.013 0.025 0.047
0.009 0.022 0.006 0.060 0.019 0.080 0.025 0.134
0.024 0.038 0.051 0.096 0.019 0.152 0.039 0.115
0.999 0.999 0.998 0.997 0.999 0.999 0.999 0.997
Linear regression analysis of peak areas of analytes or internal standards plotted against injection time over 24 h confirmed that processed sample stability was also satisfactory, i.e., negative slopes were not obtained (results not shown). The LOD and LOQ for rat hair were the same as those for human hair (e.g. 1 pg/20 mg hair for LOQ). Matrix effect, recovery, and process efficiency results for pigmented and nonpigmented rat hair are shown in Table 6. The matrix effect values for pigmented hair ranged from 68 % (JWH-073) to 113 % (JWH-073 4-OH M) and from 34 % (JWH-018) to 88 % (JWH-073 4-OH M) at low and high concentrations, respectively. CV values were no higher than 18 %. The matrix effect for nonpigmented hair ranged from 80 % (JWH-073) to
Compound
Concentration (pg/10 mg hair)
Repeatability (CV, %)
Between-day precision (CV, %)
Intermediate precision (CV, %)
Accuracy (bias, %)
JWH-018
1 100 1 100 1 100
5.9 8.4 9.2 6.3 8.8 5.8
1.0 5.6 5.7 6.3 8.5 6.2
6.0 10.0 10.8 8.9 12.2 8.5
7.3 −1.0 3.4 1.2 2.6 1.8
1 100 1 100 1 100 1 100 1 100
9.8 6.6 8.3 9.2 9.5 6.0 8.9 6.2 11.9 7.5
4.2 10.0 7.9 9.8 8.2 7.0 10.0 6.6 0.7 3.4
10.6 12.0 11.4 13.5 12.6 9.2 13.4 9.1 12.0 8.3
0.5 0.2 3.6 −1.5 -.3.5 2.6 −1.9 −1.0 0.7 5.5
JWH-018 N4-OH M JWH-018 N5-OH M JWH-018 NCOOH M JWH-073 JWH-073 N3-OH M JWH-073 N4-OH M JWH-073 NCOOH M CV, coefficient of variation
b
Deposition of JWH-018, JWH-073, and their metabolites in hair Table 5 In-process stability under the sample-preparation conditions used for human hair
Compound
Concentration (pg/10 mg hair)
Extraction time (h) 2
JWH-018 JWH-018 N-4-OH M JWH-018 N-5-OH M JWH-018 N-COOH M JWH-073 JWH-073 N-3-OH M JWH-073 N-4-OH M Results are presented as mean percentage of initial concentration of analytes (n =3)
JWH-073 N-COOH M
4
6
12
24
1
105
96
102
100
100
100 1 100 1 100 1 100 1 100 1 100 1 100 1 100
92 101 105 98 99 95 95 95 101 92 104 92 100 102 100
91 101 102 102 103 97 97 95 105 96 104 96 105 99 102
90 100 100 99 102 95 103 95 104 95 101 95 104 100 101
90 105 102 101 102 94 97 94 104 96 103 100 101 102 100
92 100 104 100 103 97 98 94 105 97 106 95 106 101 104
118 % (JWH-018 5-OH M) and 60 % (JWH-018) to 101 % (JWH-018 4-OH M) at low and high concentrations, respectively. The CV values were below 15 % for each
analyte, except for JWH-018 4-OH M (18 %) and JWH-018 5-OH M (16 %) at low concentrations in pigmented hair; these were not regarded as significant variations.
Table 6 Matrix effect, recovery, and process efficiency at low and high concentrations for each analyte and internal standard in rat hair (n =5 each) Compound
JWH-018 JWH-018 N-4-OH M JWH-018 N-5-OH M JWH-018 N-COOH M JWH-073 JWH-073 N-3-OH M JWH-073 N-4-OH M JWH-073 N-COOH M JWH-018-d9 JWH-018 N-5-OH M-d5 CV, coefficient of variation
Concentration Pigmented hair (pg/20 mg rat hair) Matrix effect Recovery (%) (%)
Nonpigmented hair Process efficiency (%)
Matrix effect (%)
Recovery (%)
Process efficiency (%)
Mean
CV
Mean CV Mean
CV
Mean
CV
Mean CV Mean
CV
1
80
4
106
14
74
16
92
12
88
8
72
14
100 1 100 1 100 1 100 1 100 1 100 1 100 1 100
34 92 67 99 74 95 83 68 55 92 73 113 88 98 83
5 18 8 16 11 7 8 10 11 15 11 10 5 9 4
109 132 131 131 140 107 133 86 105 127 136 104 136 104 109
6 11 11 9 7 11 7 10 13 9 7 4 7 10 6
55 121 102 129 99 102 99 58 58 117 99 117 98 102 91
4 10 5 9 5 11 5 7 7 8 5 5 5 10 5
60 112 101 118 98 101 96 80 67 104 99 114 96 98 90
4 12 5 7 6 16 4 5 4 2 5 12 6 8 3
100 102 100 113 100 100 100 91 100 114 100 106 100 104 100
7 7 5 3 6 12 4 7 6 10 5 12 7 3 3
34 110 86 120 82 97 81 68 42 107 79 111 91 92 81
14 12 14 9 14 13 7 5 10 9 14 13 9 3 7
5 5
76 115
11 4
138 132
12 6
125 58
5 5
124 83
11 6
91 106
10 4
62 118
7 5
J. Kim et al.
Concentration (pg/mg)
120
Pigmented Nonpigmented
100 80 60 40 20 0 JWH-073
JWH-073 N-3- JWH-073 NOH M COOH M
Fig. 2 Comparison of amounts JWH-073 and its metabolites in pigmented and nonpigmented rat hair (n =3 for JWH-073 N-COOH M in nonpigmented hair; n =4 for JWH-073 N-3-OH M in nonpigmented hair; n =5 for others)
To investigate the distribution of synthetic cannabinoids and metabolites in hair and the effect of hair pigmentation, the JWH-073 suspension was fed to five lean Zucker rats with both pigmented and nonpigmented hair. Although wide variations of quantitative results were obtained, depending on individual differences, JWH-073 and its two metabolites, JWH-073 N-3-OH M and JWH-073 N-COOH M, were detected in all pigmented and nonpigmented hair samples, whereas JWH-073 N-4-OH M was detected below the LOQ or was not detectable. Quantitative results for JWH-073, JWH-073 N-3-OH M, and JWH-073 N-COOH M in pigmented and nonpigmented hair are compared in Fig. 2. Some results below the LOQ were not included; n =3 for JWH-073 N-COOH M in nonpigmented hair, n = 4 for JWH-073 N-3-OH M in nonpigmented hair, and n =5 for other occasions. The concentration of JWH-073 in both
Table 7 Quantitative analysis for JWH-018, JWH-018 N-5-OH M, JWH-073, and THCCOOH in hair samples from individuals suspected of synthetic cannabinoids use Case no.
Gender
Age
Hair colora
1
Male
24
Black
2
Female
26
Black
Hair length (cm)
JWH-018 (pg mg−1)
JWH-018 N-5-OH M (pg mg−1)
JWH-073 (pg mg−1)
THCCOOHb (pg mg−1) NA
0-3c
88
ND
3
3-6c
117
ND
ND
NA
6-9c
56
ND
ND
NA
0-3c 3-6c
237 524
8 11
11 20
0.06
6-9c
688
12
20
9-12c
753
12
22
11 14
9 5
3
Male
27
Blonde
0-3c 3-6c
