Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 184–189
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Determination of XLR-11 and its metabolites in hair by liquid chromatography–tandem mass spectrometry Meejung Park ∗ , Seonghoon Yeon, Jaesin Lee, Sangwhan In Drug and Forensic Toxicology Division, National Forensic Service, 10 Ipchunro, Wonju , Kangwon-do 220-170, Republic of Korea
a r t i c l e
i n f o
Article history: Received 24 February 2015 Received in revised form 22 May 2015 Accepted 24 May 2015 Available online 27 May 2015 Keywords: Hair analysis XLR-11 Synthetic cannabinoids Metabolites
a b s t r a c t 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 disposition of synthetic cannabinoids including cyclopropylindoles (UR-144 and XLR-11) and their metabolites in hair. XLR-11 has been widely abused in South Korea recently. Identification of metabolites in hair can be an important proof of synthetic cannabinoids use because it can exclude the possibility of passive smoke exposure. In this study, we described a quantitative analytical method of XLR-11 and its metabolites (UR-144, UR-144 N-5-hydroxypentyl metabolite, UR-144 N-4-hydroxypentyl metabolite, UR-144 N-pentanoic acid metabolite and XLR-11 N4-hydroxypentyl metabolite) in hair by liquid chromatography with ESI-MS/MS. The target analytes were extracted with methanol from washed and cut hair samples and the extracts were evaporated, filtered and analyzed by LC–MS/MS with electrospray ion source in positive-ionization mode. JWH-018-d9 and JWH-018 N-5-hydroxypentyl metabolite-d5 were used as internal standards. Chromatographic separation was completed within 15 min. No interferences were detected in 10 blank hair samples. In intraand inter-assay precision and accuracy study, CV (%) and bias (%) were below 12. The limit of detection (LOD) was 0.1 ∼ 2 pg/mg and the limit of quantification (LOQ) was 0.2–2 pg/mg, respectively. The validation results proved that the method was selective, accurate and precise with acceptable linearity within calibration range. No significant variation was observed by different sources of matrices. This method was applied to hair samples from 14 individual suspects of XLR-11 use. In this result, XLR-11, UR-144, UR144 N-5-hydroxypentyl metabolite and UR-144 N-pentanoic acid metabolite, XLR-11 N-4-hydroxypentyl metabolite were detected. The concentration of XLR-11 as a parent drug was much higher than other metabolites. UR-144 N-5-hydroxy metabolite and UR-144 N-pentanoic acid were detected mainly in the authentic hair samples from suspected of XLR-11 use. UR-144 N-4- hydroxypentyl metabolite was not detected in all cases. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Hair analysis has been regarded as an alternative method to urine analysis in forensic and criminal cases. Hair has unique advantages such as provision of long history of drug use, relatively noninvasiveness, samples’ good stability and track of time of intake by segmental analysis [1]. Since synthetic cannabinoids emerged globally as the new psychoactive substances (NPS) in 2008, a wide range of their analogs have been abused. Most of the classical synthetic cannabinoids such as JWH-018 or JWH-073 have CB1 receptor affinity and psychotropic effects higher than 9 -
∗ Corresponding author. Tel.: +82 339025451; fax: +82 339025932. E-mail address: [email protected] (M. Park). http://dx.doi.org/10.1016/j.jpba.2015.05.022 0731-7085/© 2015 Elsevier B.V. All rights reserved.
tetrahydrocannabinol (THC) [2,3]. which is the active ingredient in cannabis. Some of the CB2 selective agonists such as UR-144 have been found in herbal highs intended for recreational use [4]. The recent increased appearance of analogues with a halogen substituent including AM-2201, XLR-11, and MAM-2201 has been noted [5]. These fluorine modifications increase CB1 receptor affinity of synthetic cannabinoids [6]. A new ‘temporary scheduling system’, added to the Act on the Control of Narcotics, entered into force in South Korea. Under the Act UR-144 and XLR-11 were added to temporary scheduled NPS for 3 years in June 2014 in South Korea. Many studies have been reported on the determination and identification of synthetic cannabinoids in urine samples [7–10], and several researches on the determination of JWH-018, JWH-073, AM-2201, JWH-122 and MAM-2201 in hair samples were reported [11,12]. However, no study on the distribution of XLR-11 and
M. Park et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 184–189
185
Fig. 1. Chemical structures of XLR-11 and its metabolites.
UR-144 in hair samples has yet been conducted. XLR-11 and UR144 have the 2,2,3,3-tetramethylcyclopropyl (TMCP) ring. Recently, some researches were published regarding possible metabolites of XLR-11 in human hepatocytes and urines [13,14]. Fig. 1 shows the chemical structures and proposed metabolites of XLR-11 and UR144. The purpose of this study was to establish and validate an analytical method for simultaneous detection of XLR-11 and some of its metabolites in hair. This method was applied to investigate the distribution of XLR-11 and its metabolites in authentic human hair samples. 2. Materials and methods 2.1. Chemicals and reagents XLR-11, UR-144, UR-144 N-4-hydroxypentyl metabolite (UR144 N-4-OH M), UR-144 N-5-hydroxypentyl metabolite (UR-144 N-5-OH M), UR-144 N-pentanoic acid metabolite (UR-144 N-COOH M) and XLR-11 N-4-hydroxypentyl metabolite (XLR-11 N-4-OH M), JWH-018-d9 , JWH-018 N-5-hydroxypentyl metabolite-d5 (JWH018 N-5-OH M-d5 ) were purchased from Cayman Chemical (Ann Arbor, MI, USA). All solvents were high-performance liquid chromatography (HPLC) grade. Ammonium formate and formic acid were obtained from Fluka (St. Louis, MO, USA). HPLC-grade methanol and acetonitrile were purchased from J.T. Baker (MT, USA). Deionized water was produced by use of an Elga Purelab Option-Q ultra-pure water system (Lane End UK).
2.2. Sample preparation 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). XLR-11 and its metabolites in the prepared hair were extracted by use of HPLC-grade methanol at 38 ◦ C, with gentle magnetic stirring, for at least 18 h. JWH-018-d9 and JWH018 N-5-hydroxypentyl metabolite-d5 (50 L, 200 pg/ml internal standard mixed solution) were used as internal standards for the parent drugs (XLR-11 and UR-144) 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 L of a 1:1 (v/v) mixture of methanol and mobile phase component A, and 5 L was injected for LC–MS/MS analysis. 2.3. LC–MS/MS analysis The LC–MS/MS analysis was performed on an Agilent 1290 infinity UHPLC system consisting of pump, autosampler, on-line degasser, and column compartment (Agilent Technologies, CA, USA) and an AB SCIEX QTRAP® 5500 MS/MS (AB SCIEX, MA, USA). The chromatographic separation was performed in a Zorbax Eclipse plus C18 (RRHD 2.1 × 100 mm, 1.8 m, Agilent Technologies, CA,
Table 1 MRM transitions, retention times and conditions of each analyte and internal standards. Compound
Precursor ion (m/z)
Product ion (m/z)
Retention time (min)
Declustering potential (V)
Collision energy (V)
Collision cell exit potential (V)
XLR11 UR-144
328
6.6
363
UR-144 N-4-OH M
351
UR-144 N-5-OH M
312
UR-144 N-COOH M
328
XLR-11 N-4-OH M
342
JWH-018-d3
351
JWH-018 N-5-OH M-d5
363
125 55 155 127 155 127 125 144 125 55 125 55 155 127 155 127
111 161 140 140 75 75 120 120 116 116 121 161 75 140 140 105
25 65 30 65 33 65 28 43 25 57 27 65 33 65 33 65
10 8 12 12 14 14 16 15 14 14 14 8 14 10 12 12
5.5 9.7 10.1 6.7 6.2 6.0 4.2
Quantifier ions are underlined. The values of entrance potential for all analyte are same same as 10 V.
186
M. Park et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 184–189
Fig. 2. Representative chromatograms of XLR-11 and its metabolites in drug-free (A) and authentic hair samples (B).
USA, fitted with 0.3 m in-line filter) which was maintained at 40 ◦ C. The LC–MS/MS conditions were same with our previous report on the analysis of naphthoylindole-based synthetic cannabinoids and their metabolites [11]. The individual duration time was 15 min. The temperature of autosampler was maintained at 10 ◦ C. The MS system was operated using ESI in positive mode. The individual MRM transitions, retention times and other experimental parameters are shown in Table 1. Data were processed using Analyst 1.6 software. 2.4. Validation of methods The following parameters were evaluated: selectivity, matrix effect, recovery, process efficiency, linearity, limit of detection
(LOD), limit of quantification (LOQ), precision, accuracy, and processed sample stability. The analytical method was validated using spiked drug-free human hair pooled from five different volunteers, except for the evaluation of selectivity, matrix effect, recovery, and process efficiency, as described in previous studies [12,15]. Working standard solutions of 10 ng/ml, 1 ng/ml and 100 pg/ml for XLR-11, UR-144, UR-144 N-4-OH M, UR-144 N-5-OH M, UR144 N-COOH M and XLR-11 N-4-OH M were made by dilution. All primary and working solutions were stored at –20 ◦ C. To investigate the linearity, five sets of calibrators (2, 5, 10, 20, 50, 100 and 200 pg/10 mg hair) were analyzed and the regression coefficients were calculated using 1/x weighting factor. LOD and LOQ were determined by analyzing blank hair matrix fortified with known drug concentrations. Each concentration was measured in
M. Park et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 184–189
187
Table 2 Matrix effect, recovery and process efficiency in human hair (n = 5). Compound
Conc. (pg/10mg hair)
5 20 5 20 5 20 5 20 5 20 5 20 10 10
XLR-11 UR-144 UR-144 N-4-OH M UR-144 N-5-OH M UR-144 N-COOH M XLR-11 N-4-OH M JWH-018-d3 JWH-018 N-5-OH M-d5
Matrix effect (%)
Recovery (%)
Process efficiency (%)
Mean
CV
Mean
CV
Mean
CV
80 38 31 25 85 73 80 75 70 78 73 68 55 67
14 12 14 15 13 7 8 10 11 4 12 10 8 9
84 103 105 94 66 98 64 58 105 94 103 87 93 97
10 12 10 7 7 9 7 8 11 8 5 12 9 6
67 37 33 22 55 72 56 70 70 73 66 59 85 97
11 10 10 10 12 10 9 10 9 8 11 8 9 7
CV: coefficient of variation. Table 3 Precision and accuracy in human hair. Compound
XLR-11 UR-144 UR-144 N-4-OH M UR-144 N-5-OH M UR-144 N-COOH M XLR-11 N-4-OH M
Conc. (pg/10mg hair)
5 20 5 20 5 20 5 20 5 20 5 20
Precision (CV, %)
Accuracy (bias, %)
Intra- assay (n = 5)
Inter-assay (n = 20)
Intra- assay (n = 5)
Inter-assay (n = 20)
3.1 7.9 6.1 6.2 4.5 7.5 10.1 11.1 10.2 6.5 7.6 5.5
11.7 8.3 8.6 9.8 6.6 9.5 9.8 7.1 10.4 10.1 9.3 6.4
4.8 −7.4 −7.0 −4.9 −3.5 −6.2 −7.2 −5.9 −7.8 −10.3 −1.9 −6.5
−2.6 −6.6 −8.6 −5.2 −7.6 −8.5 −5.6 −8.0 −9.4 −9.2 −3.4 −8.9
CV: coefficient of variation
five replicates. LOD was defined as the lowest concentration giving a response of three times the average baseline noise defined from five unfortified samples. LOQ was defined as the lowest observed concentration for 10 replicates with less than ±20% coefficient of variation (CV) for precision and less than ±20% for bias. To assess the selectivity of the method, ten different sources of blank hair were analyzed to check the absence of responses interfering with the signals of 6 target drugs and 2 internal standards. The matrix effect, recovery, and process efficiency were determined by comparing the analysis of five neat standards (set 1), extracts of five different sources of blank hair spiked with analytes after extraction (set 2), and extracts of the same five different sources of blank hair spiked with analytes before extraction (set 3) at 5 pg/10 mg hair and 20 pg/10 mg hair of XLR-11 and its metabolites (matrix effect, a percentage of the response of set 2 samples in relation to those of set 1 samples; recovery, a percentage of the response of set 3 samples in relation to that of set 2 samples; process efficiency, a percentage of the response of set 3 samples in relation to set 1 samples). Method precision and accuracy were examined by analyzing drug-free hair samples spiked with low (5 pg/10 mg hair) and high (20 pg/10 mg hair) concentrations of XLR-11 and its metabolites, respectively. The five sets of each sample were analyzed on four different days. Intra-assay and inter-assay precision and accuracy were estimated as CV and bias, respectively. The processed sample stability was examined at 2.5 h intervals over 20 h.
2.5. Quantitative analysis of human authentic hair samples Hair samples from 14 individuals suspected of XLR-11 use were provided by the law enforcement agency. All hair samples were prepared and analyzed as described above. 3. Results and discussion A quantitative LC–MS/MS method for simultaneous detection of XLR-11 and its metabolites in hair has been developed and fully validated. This method was sensitive and selective for the detection and quantification of TMCP cannabinoids (UR-144 and XLR-11) and showed satisfactory validation results and was successfully applied in the analysis of authentic human hair samples. We used simple agitation-based extraction in methanol solution for 18 h. Chromatographic separation was completed within 15 min. Fig. 2 shows representative chromatograms of XLR-11 and its metabolites obtained from drug-free (A) and authentic hair samples (B). No endogenous substances interfered with analysis of XLR-11 and its metabolites. The LOD and the LOQ were 0.1 pg/10 mg and 0.2 pg/mg hair for XLR-11, UR-144, UR-144 N-4-OH M, UR-144 N-5-OH M and UR-144 N-COOH M, respectively. The LOD and the LOQ were 2 pg/10 mg hair for XLR-11 N-4-OH M. Calibration curves for these compounds were prepared. Their correlation coefficients (R) were larger than 0.99 for all analytes. The results of matrix effect, recovery and process efficiency of XLR-11 and its metabolites in human
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Table 4 Concentrations of XLR-11 and its metabolites in authentic human hair samples (pg/mg). Case No.
Gender
Hair color
XLR-11
UR-144
UR-144 N-4-OH M
UR-144 N-5-OH M
UR-144 N-COOH M
XLR-11 N-4-OH M
1 2 3 4 5 6 7 8 9 10 11 12 13 14
M M M M M M F M M F M M M M
Black Black Black Black Black Black Black Black Black Black Blonde Black Black Black
1105.0 4250.0 1200.0 5350.0 335.3 1046.7 142.7 813.3 17.8 176.0 12.6 37.9 0.8 4.4
0.6 0.4 0.8 0.6 1.5 0.5 ND 1.6 ND ND ND 0.4 ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND
0.4 0.3 0.6 0.2 18.8 39.7 6.7 0.2
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Determination of XLR-11 and its metabolites in hair by liquid chromatography–tandem mass spectrometry Meejung Park ∗ , Seonghoon Yeon, Jaesin Lee, Sangwhan In Drug and Forensic Toxicology Division, National Forensic Service, 10 Ipchunro, Wonju , Kangwon-do 220-170, Republic of Korea
a r t i c l e
i n f o
Article history: Received 24 February 2015 Received in revised form 22 May 2015 Accepted 24 May 2015 Available online 27 May 2015 Keywords: Hair analysis XLR-11 Synthetic cannabinoids Metabolites
a b s t r a c t 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 disposition of synthetic cannabinoids including cyclopropylindoles (UR-144 and XLR-11) and their metabolites in hair. XLR-11 has been widely abused in South Korea recently. Identification of metabolites in hair can be an important proof of synthetic cannabinoids use because it can exclude the possibility of passive smoke exposure. In this study, we described a quantitative analytical method of XLR-11 and its metabolites (UR-144, UR-144 N-5-hydroxypentyl metabolite, UR-144 N-4-hydroxypentyl metabolite, UR-144 N-pentanoic acid metabolite and XLR-11 N4-hydroxypentyl metabolite) in hair by liquid chromatography with ESI-MS/MS. The target analytes were extracted with methanol from washed and cut hair samples and the extracts were evaporated, filtered and analyzed by LC–MS/MS with electrospray ion source in positive-ionization mode. JWH-018-d9 and JWH-018 N-5-hydroxypentyl metabolite-d5 were used as internal standards. Chromatographic separation was completed within 15 min. No interferences were detected in 10 blank hair samples. In intraand inter-assay precision and accuracy study, CV (%) and bias (%) were below 12. The limit of detection (LOD) was 0.1 ∼ 2 pg/mg and the limit of quantification (LOQ) was 0.2–2 pg/mg, respectively. The validation results proved that the method was selective, accurate and precise with acceptable linearity within calibration range. No significant variation was observed by different sources of matrices. This method was applied to hair samples from 14 individual suspects of XLR-11 use. In this result, XLR-11, UR-144, UR144 N-5-hydroxypentyl metabolite and UR-144 N-pentanoic acid metabolite, XLR-11 N-4-hydroxypentyl metabolite were detected. The concentration of XLR-11 as a parent drug was much higher than other metabolites. UR-144 N-5-hydroxy metabolite and UR-144 N-pentanoic acid were detected mainly in the authentic hair samples from suspected of XLR-11 use. UR-144 N-4- hydroxypentyl metabolite was not detected in all cases. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Hair analysis has been regarded as an alternative method to urine analysis in forensic and criminal cases. Hair has unique advantages such as provision of long history of drug use, relatively noninvasiveness, samples’ good stability and track of time of intake by segmental analysis [1]. Since synthetic cannabinoids emerged globally as the new psychoactive substances (NPS) in 2008, a wide range of their analogs have been abused. Most of the classical synthetic cannabinoids such as JWH-018 or JWH-073 have CB1 receptor affinity and psychotropic effects higher than 9 -
∗ Corresponding author. Tel.: +82 339025451; fax: +82 339025932. E-mail address: [email protected] (M. Park). http://dx.doi.org/10.1016/j.jpba.2015.05.022 0731-7085/© 2015 Elsevier B.V. All rights reserved.
tetrahydrocannabinol (THC) [2,3]. which is the active ingredient in cannabis. Some of the CB2 selective agonists such as UR-144 have been found in herbal highs intended for recreational use [4]. The recent increased appearance of analogues with a halogen substituent including AM-2201, XLR-11, and MAM-2201 has been noted [5]. These fluorine modifications increase CB1 receptor affinity of synthetic cannabinoids [6]. A new ‘temporary scheduling system’, added to the Act on the Control of Narcotics, entered into force in South Korea. Under the Act UR-144 and XLR-11 were added to temporary scheduled NPS for 3 years in June 2014 in South Korea. Many studies have been reported on the determination and identification of synthetic cannabinoids in urine samples [7–10], and several researches on the determination of JWH-018, JWH-073, AM-2201, JWH-122 and MAM-2201 in hair samples were reported [11,12]. However, no study on the distribution of XLR-11 and
M. Park et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 184–189
185
Fig. 1. Chemical structures of XLR-11 and its metabolites.
UR-144 in hair samples has yet been conducted. XLR-11 and UR144 have the 2,2,3,3-tetramethylcyclopropyl (TMCP) ring. Recently, some researches were published regarding possible metabolites of XLR-11 in human hepatocytes and urines [13,14]. Fig. 1 shows the chemical structures and proposed metabolites of XLR-11 and UR144. The purpose of this study was to establish and validate an analytical method for simultaneous detection of XLR-11 and some of its metabolites in hair. This method was applied to investigate the distribution of XLR-11 and its metabolites in authentic human hair samples. 2. Materials and methods 2.1. Chemicals and reagents XLR-11, UR-144, UR-144 N-4-hydroxypentyl metabolite (UR144 N-4-OH M), UR-144 N-5-hydroxypentyl metabolite (UR-144 N-5-OH M), UR-144 N-pentanoic acid metabolite (UR-144 N-COOH M) and XLR-11 N-4-hydroxypentyl metabolite (XLR-11 N-4-OH M), JWH-018-d9 , JWH-018 N-5-hydroxypentyl metabolite-d5 (JWH018 N-5-OH M-d5 ) were purchased from Cayman Chemical (Ann Arbor, MI, USA). All solvents were high-performance liquid chromatography (HPLC) grade. Ammonium formate and formic acid were obtained from Fluka (St. Louis, MO, USA). HPLC-grade methanol and acetonitrile were purchased from J.T. Baker (MT, USA). Deionized water was produced by use of an Elga Purelab Option-Q ultra-pure water system (Lane End UK).
2.2. Sample preparation 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). XLR-11 and its metabolites in the prepared hair were extracted by use of HPLC-grade methanol at 38 ◦ C, with gentle magnetic stirring, for at least 18 h. JWH-018-d9 and JWH018 N-5-hydroxypentyl metabolite-d5 (50 L, 200 pg/ml internal standard mixed solution) were used as internal standards for the parent drugs (XLR-11 and UR-144) 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 L of a 1:1 (v/v) mixture of methanol and mobile phase component A, and 5 L was injected for LC–MS/MS analysis. 2.3. LC–MS/MS analysis The LC–MS/MS analysis was performed on an Agilent 1290 infinity UHPLC system consisting of pump, autosampler, on-line degasser, and column compartment (Agilent Technologies, CA, USA) and an AB SCIEX QTRAP® 5500 MS/MS (AB SCIEX, MA, USA). The chromatographic separation was performed in a Zorbax Eclipse plus C18 (RRHD 2.1 × 100 mm, 1.8 m, Agilent Technologies, CA,
Table 1 MRM transitions, retention times and conditions of each analyte and internal standards. Compound
Precursor ion (m/z)
Product ion (m/z)
Retention time (min)
Declustering potential (V)
Collision energy (V)
Collision cell exit potential (V)
XLR11 UR-144
328
6.6
363
UR-144 N-4-OH M
351
UR-144 N-5-OH M
312
UR-144 N-COOH M
328
XLR-11 N-4-OH M
342
JWH-018-d3
351
JWH-018 N-5-OH M-d5
363
125 55 155 127 155 127 125 144 125 55 125 55 155 127 155 127
111 161 140 140 75 75 120 120 116 116 121 161 75 140 140 105
25 65 30 65 33 65 28 43 25 57 27 65 33 65 33 65
10 8 12 12 14 14 16 15 14 14 14 8 14 10 12 12
5.5 9.7 10.1 6.7 6.2 6.0 4.2
Quantifier ions are underlined. The values of entrance potential for all analyte are same same as 10 V.
186
M. Park et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 184–189
Fig. 2. Representative chromatograms of XLR-11 and its metabolites in drug-free (A) and authentic hair samples (B).
USA, fitted with 0.3 m in-line filter) which was maintained at 40 ◦ C. The LC–MS/MS conditions were same with our previous report on the analysis of naphthoylindole-based synthetic cannabinoids and their metabolites [11]. The individual duration time was 15 min. The temperature of autosampler was maintained at 10 ◦ C. The MS system was operated using ESI in positive mode. The individual MRM transitions, retention times and other experimental parameters are shown in Table 1. Data were processed using Analyst 1.6 software. 2.4. Validation of methods The following parameters were evaluated: selectivity, matrix effect, recovery, process efficiency, linearity, limit of detection
(LOD), limit of quantification (LOQ), precision, accuracy, and processed sample stability. The analytical method was validated using spiked drug-free human hair pooled from five different volunteers, except for the evaluation of selectivity, matrix effect, recovery, and process efficiency, as described in previous studies [12,15]. Working standard solutions of 10 ng/ml, 1 ng/ml and 100 pg/ml for XLR-11, UR-144, UR-144 N-4-OH M, UR-144 N-5-OH M, UR144 N-COOH M and XLR-11 N-4-OH M were made by dilution. All primary and working solutions were stored at –20 ◦ C. To investigate the linearity, five sets of calibrators (2, 5, 10, 20, 50, 100 and 200 pg/10 mg hair) were analyzed and the regression coefficients were calculated using 1/x weighting factor. LOD and LOQ were determined by analyzing blank hair matrix fortified with known drug concentrations. Each concentration was measured in
M. Park et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 184–189
187
Table 2 Matrix effect, recovery and process efficiency in human hair (n = 5). Compound
Conc. (pg/10mg hair)
5 20 5 20 5 20 5 20 5 20 5 20 10 10
XLR-11 UR-144 UR-144 N-4-OH M UR-144 N-5-OH M UR-144 N-COOH M XLR-11 N-4-OH M JWH-018-d3 JWH-018 N-5-OH M-d5
Matrix effect (%)
Recovery (%)
Process efficiency (%)
Mean
CV
Mean
CV
Mean
CV
80 38 31 25 85 73 80 75 70 78 73 68 55 67
14 12 14 15 13 7 8 10 11 4 12 10 8 9
84 103 105 94 66 98 64 58 105 94 103 87 93 97
10 12 10 7 7 9 7 8 11 8 5 12 9 6
67 37 33 22 55 72 56 70 70 73 66 59 85 97
11 10 10 10 12 10 9 10 9 8 11 8 9 7
CV: coefficient of variation. Table 3 Precision and accuracy in human hair. Compound
XLR-11 UR-144 UR-144 N-4-OH M UR-144 N-5-OH M UR-144 N-COOH M XLR-11 N-4-OH M
Conc. (pg/10mg hair)
5 20 5 20 5 20 5 20 5 20 5 20
Precision (CV, %)
Accuracy (bias, %)
Intra- assay (n = 5)
Inter-assay (n = 20)
Intra- assay (n = 5)
Inter-assay (n = 20)
3.1 7.9 6.1 6.2 4.5 7.5 10.1 11.1 10.2 6.5 7.6 5.5
11.7 8.3 8.6 9.8 6.6 9.5 9.8 7.1 10.4 10.1 9.3 6.4
4.8 −7.4 −7.0 −4.9 −3.5 −6.2 −7.2 −5.9 −7.8 −10.3 −1.9 −6.5
−2.6 −6.6 −8.6 −5.2 −7.6 −8.5 −5.6 −8.0 −9.4 −9.2 −3.4 −8.9
CV: coefficient of variation
five replicates. LOD was defined as the lowest concentration giving a response of three times the average baseline noise defined from five unfortified samples. LOQ was defined as the lowest observed concentration for 10 replicates with less than ±20% coefficient of variation (CV) for precision and less than ±20% for bias. To assess the selectivity of the method, ten different sources of blank hair were analyzed to check the absence of responses interfering with the signals of 6 target drugs and 2 internal standards. The matrix effect, recovery, and process efficiency were determined by comparing the analysis of five neat standards (set 1), extracts of five different sources of blank hair spiked with analytes after extraction (set 2), and extracts of the same five different sources of blank hair spiked with analytes before extraction (set 3) at 5 pg/10 mg hair and 20 pg/10 mg hair of XLR-11 and its metabolites (matrix effect, a percentage of the response of set 2 samples in relation to those of set 1 samples; recovery, a percentage of the response of set 3 samples in relation to that of set 2 samples; process efficiency, a percentage of the response of set 3 samples in relation to set 1 samples). Method precision and accuracy were examined by analyzing drug-free hair samples spiked with low (5 pg/10 mg hair) and high (20 pg/10 mg hair) concentrations of XLR-11 and its metabolites, respectively. The five sets of each sample were analyzed on four different days. Intra-assay and inter-assay precision and accuracy were estimated as CV and bias, respectively. The processed sample stability was examined at 2.5 h intervals over 20 h.
2.5. Quantitative analysis of human authentic hair samples Hair samples from 14 individuals suspected of XLR-11 use were provided by the law enforcement agency. All hair samples were prepared and analyzed as described above. 3. Results and discussion A quantitative LC–MS/MS method for simultaneous detection of XLR-11 and its metabolites in hair has been developed and fully validated. This method was sensitive and selective for the detection and quantification of TMCP cannabinoids (UR-144 and XLR-11) and showed satisfactory validation results and was successfully applied in the analysis of authentic human hair samples. We used simple agitation-based extraction in methanol solution for 18 h. Chromatographic separation was completed within 15 min. Fig. 2 shows representative chromatograms of XLR-11 and its metabolites obtained from drug-free (A) and authentic hair samples (B). No endogenous substances interfered with analysis of XLR-11 and its metabolites. The LOD and the LOQ were 0.1 pg/10 mg and 0.2 pg/mg hair for XLR-11, UR-144, UR-144 N-4-OH M, UR-144 N-5-OH M and UR-144 N-COOH M, respectively. The LOD and the LOQ were 2 pg/10 mg hair for XLR-11 N-4-OH M. Calibration curves for these compounds were prepared. Their correlation coefficients (R) were larger than 0.99 for all analytes. The results of matrix effect, recovery and process efficiency of XLR-11 and its metabolites in human
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Table 4 Concentrations of XLR-11 and its metabolites in authentic human hair samples (pg/mg). Case No.
Gender
Hair color
XLR-11
UR-144
UR-144 N-4-OH M
UR-144 N-5-OH M
UR-144 N-COOH M
XLR-11 N-4-OH M
1 2 3 4 5 6 7 8 9 10 11 12 13 14
M M M M M M F M M F M M M M
Black Black Black Black Black Black Black Black Black Black Blonde Black Black Black
1105.0 4250.0 1200.0 5350.0 335.3 1046.7 142.7 813.3 17.8 176.0 12.6 37.9 0.8 4.4
0.6 0.4 0.8 0.6 1.5 0.5 ND 1.6 ND ND ND 0.4 ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND
0.4 0.3 0.6 0.2 18.8 39.7 6.7 0.2
