Journal of Analytical Toxicology 2015;39:280 –286 doi:10.1093/jat/bkv013 Advance Access publication March 3, 2015
Article
Detection Times of Carboxylic Acid Metabolites of the Synthetic Cannabinoids JWH-018 and JWH-073 in Human Urine Solfrid Hegstad1*, Andreas A. Westin1 and Olav Spigset1,2 1
Department of Clinical Pharmacology, St Olav University Hospital, Trondheim, Norway, and 2Department of Laboratory Medicine, Children’s and Women’s Health, Norwegian University of Science and Technology, Trondheim, Norway
*Author to whom correspondence should be addressed. Email:[email protected]
Over the past years, use of synthetic cannabinoids has become increasingly popular. To draw the right conclusions regarding new intake of these substances in situations of repeated urinary drug testing, knowledge of their elimination rate in urine is essential. We report data from consecutive urine specimens from five subjects after ingestion of synthetic cannabinoids. Urinary concentrations of the carboxylic acid metabolites JWH-018-COOH and JWH-073-COOH were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) with a limit of quantification of 0.1 ng/mL. In these subjects, specimens remained positive over a period of 20–43 (mean 27) days for JWH-018-COOH and over a period of 11–25 (mean 19) days for JWH-073-COOH. Detection times were shorter for subjects that appeared to have ingested only one, or a few, doses prior to urine collection in the study. Creatinine-normalized concentrations (CN-concentrations) slowly declined throughout the follow-up period in all subjects, suggesting that no new intake had taken place during this period. Mean elimination half-lives in urine were 14.0 (range 4.4 – 23.8) days for CN-JWH-018-COOH and 9.3 (range 3.6 –16.8) days for CN-JWH-073-COOH. These data show that urine specimens could be positive for JWH-018-COOH for more than 6 weeks and JWH-073-COOH for more than 3 weeks after ingestion. However, such long detection periods require a low limit of quantification.
Introduction Testing for drugs-of-abuse in urine is requested in various situations, including health care, workplace, military and criminal justice settings. Test results may be used to distinguish between a previous and a recent drug intake based upon changes in the (creatinine-normalized) drug concentration between two positive specimens from the same individual. However, urinary detection times depend both on the pharmacological properties of the drug, the sensitivity of the drug test and the subjects’ frequency of use. For some drugs, such as cannabis and some benzodiazepines, excretion can be prolonged, especially following chronic use (1, 2). Synthetic cannabinoids are a new and commonly abused class of designer drugs (3). They are often marketed as herbal blends or incense, with brand names such as ‘K2’ and ‘Spice’. Nicknames such as ‘legal highs’ and ‘herbal highs’ promote perceived safety, but in reality their pharmacological properties are largely unknown (3). The synthetic cannabinoid products are easily available on the Internet, are usually smoked like marijuana, and induce similar intoxicating effects (4). Despite international efforts to control these products, surveys and poison control data indicate that they are still readily available (3). The increasing number of available synthetic cannabinoid products and
metabolites pose a great challenge on analytical laboratories to have updated methods for the drug detection. JWH-018 and JWH-073 were among the first synthetic cannabinoids available on the market (5). In Norway, JWH-018 and AM-2201 were the most frequently synthetic cannabinoids detected in blood samples from drivers suspected of impaired driving during a 7 weeks’ period in 2011 –2012 (6). Several studies on the characterization of the metabolites of JWH-018 and JWH-073 in urine have been performed (7 – 17). These studies describe carboxylation and hydroxylation with subsequent glucuronidation as the metabolic pathways. In a recent study, metabolites of JWH-018 and JWH-073 were detected in urine for 2 –3 days with peak concentrations of 3 –16.5 h after smoking a single dose (18). Moreover, based on their experience with other samples at that laboratory (18), the authors suggest that these metabolites remain detectable in urine for about 2 – 3 weeks after ending a chronic use. Except for that report, we are not aware of any other published data on urinary detection times after intake of synthetic cannabinoids. The aim of this study is to present data on the concentration range and time span for detection of the metabolites JWH-018 N-pentanoic acid (JWH-018-COOH) and JWH-073 N-butanoic acid (JWH-073-COOH) in urine collected from the same individuals. The implications these findings may have for the interpretation related to new drug ingestion or not, are also discussed. Material and methods Chemicals and reagents The synthetic cannabinoid metabolites 5-(3-(1-naphthoyl)1H-indol-1-yl) pentanoic acid (JWH-018-COOH) and 4-(3(1-naphthoyl)-1H-indol-1-yl) butanoic acid (JWH-073-COOH), as well as the internal standard JWH-018-COOH-d4, were purchased from Chiron (Trondheim, Norway). The enzyme b-glucuronidase (Type 2 HP-2 from Helix pomatia, 100 000 units/mL) was obtained from Sigma-Aldrich (St Louis, MO). LC–MS grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany) and formic acid 98% from VWR (Leuven, Belgium). 96-well sample collection plates (1 mL) and polypropylene cap mat round wells for 96-well plates were purchased from Waters (Milford, MA). Sample preparation Calibrator, QC sample or urine sample (100 mL) was mixed with 20 mL internal standard (JWH-018-COOH-d4; 3 ng/mL), 60 mL 0.2 M ammonium acetate buffer ( pH 4.8) and 20 mL b-glucuronidase (25 000 U/mL), and incubated at 658C for 1 h, thereby hydrolyzing glucuronide conjugated analytes. An aliquot of 50 mL was diluted with 200 mL methanol/water (60/40, v/v), and centrifuged at 1800 g (Rotanta 460, Hettich Lab Technology,
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Tuttlingen, Germany) for 5 min. All dilution steps were done in 96-well plates using a Tecan pipetting robot. Instruments An Acquity UPLC I-Class FTN system (Waters) was used for separation, applying an Acquity HSS-T3 column (2.1 100 mm, 1.8 mm) maintained at 508C. The mobile phase consisted of 0.1% formic acid in water (A) and 100% acetonitrile (B). The system was run with a linear gradient from 40% B to 98% B for 2.5 min. The flow rate was 0.6 mL/min and the injection volume was 5 mL. Mass detection was performed by positive ion mode electrospray MS-MS with a Xevo TQ-S tandem-quadrupole mass spectrometer (Waters). The capillary voltage was set to 2.0 kV, the source block temperature was 1208C, and the desolvation gas nitrogen was heated to 6508C and delivered at a flow rate of 1000 L/h. The m/z 372.1 .155.0 (cone voltage: 60 V, collision energy: 25 eV) and m/z 372.1 .127.0 (cone voltage: 60 V, collision energy: 55 eV) transitions were monitored for JWH-018-COOH and the m/z 358.1 .155.0 (cone voltage: 30 V, collision energy: 23 eV) and m/z 358.1 .127.0 (cone voltage: 30 V, collision energy: 43 eV) transitions were monitored for JWH-073-COOH. For the internal standard JWH-018-COOH-d4 the m/z 376.1 .155.0 (cone voltage: 60 V, collision energy: 25 eV) transition was monitored. System operation and data acquisition were controlled using the Mass Lynx 4.1 software (Waters). A representative chromatogram is presented in Figure 1. Method validation The six-point calibration curves (three replicates of each standard) were based on peak-area ratios of the analyte relative to the internal standard using a weighted (1/x) linear line, which
excluded the origin. The correlation coefficient was above 0.999 with concentrations of 0.10, 0.30, 1.0, 3.0, 6.0 and 10.0 ng/mL. The limit of quantification (LOQ) was determined with a signal to noise ratio .10 at the lowest calibrator concentration (0.10 ng/mL). LOQ samples of 0.10 ng/mL were run in one replicate on 10 different days and the coefficients of variation (CV) and the bias were in the range 6.2 – 9.3 and 21.9 to 1.9%, respectively. Within-assay CVs were estimated by analysis of 10 separate replicates of quality control (QC) samples at three concentrations (0.50, 2.5 and 8.0 ng/mL) in a single assay, and were in the range 2.8 – 4.3%. Between-assay CVs were determined by analysis of aliquots of each QC concentration at 10 different days, one replicate in each assay and were in the range 2.2 – 6.7%. Bias was in the range 24.4 to þ1.0%. Matrix effects (ME) were evaluated by the method by Matuszewski et al. (19). ME in percent was calculated as ME% ¼ (Peak intensitymatrix/Peak intensitymethanol/water) 100. Relative ME (CV%) expresses the precision of peak intensity in matrix. ME% corrected with the internal standard (IS) was calculated as ¼ [(Peak intensitymatrix/Peak IS intensitymatrix)/(Peak intensitymethanol/water/Peak IS intensitymethanol/water)] 100. Relative ME corrected with the IS (CV %) expresses the precision of Peak intensity/Peak IS intensity in matrix. Six replicates of urine from six different individuals were analyzed at two concentrations level (0.50 and 8.0 ng/mL). ME % ranged from 70 to 90% and CVs from 7.7 to 17.5%. When corrected with internal standard the ME % were from 94 to 104% and CVs from 2.1 to 5.8%. Specificity of the methods was investigated by selecting substances with almost the same MHþ and MH– ions (+2 atomic mass units) as the analytes. The substances tested were amisulpride (MW 369.5), prochlorperazine (MW 373.9), thioridazine (MW 370.6) and JWH-203-COOH (MW 369.8). Potential endogenous
Figure 1. MRM-chromatograms of JWH-073-COOH (upper part) and JWH-018-COOH (lower part) with quantitative and qualitative transitions. Left panel represents the lowest quality control sample with concentrations of 0.5 ng/mL for both compounds. Right panel is an authentic sample with concentrations of 0.73 ng/mL for JWH-073-COOH and 1.03 ng/mL for JWH-018-COOH.
Detection Times of Carboxylic Acid Metabolites 281
interferences were assessed by analyzing 10 urine specimens from different individuals. No interferences were noted. The QC samples were found to be stable in urine for 7 days at 48C and for 6 weeks at 2208C, respectively. Diluted QC samples were found to be stable in the autosampler for 4 days at 48C. Subjects and specimens Every year our laboratory receives several hundred urinary samples for the analysis of synthetic cannabinoids, including JWH-018 and JWH-073. The results from these analyses are stored in a large database. After approval from the Regional Ethics Committee, we retrieved data from subjects with serial samples positive for JWH-018-COOH and JWH-073-COOH from our database. Five subjects in the database were of particular interest because there were numerous samples obtained during a limited period of time. All samples were sent from the same drug rehabilitation clinic. According to the staff at this clinic, they had over a short period of time experienced several episodes where suspected drug use among inpatients had been undetectable by regular urinary drug screening tests. The suspected inpatients resided in a closed ward, and all had their belongings searched prior to unit entry. However, they all had access to a common living room, and were only intermittently monitored during nighttime. Moreover, inpatients were allowed to leave the ward for short periods of time, such as for jogging, and could after special application get permission to leave for longer periods, from hours to days. Thus, drug use during hospitalization could not be ruled out, and the clinic staff decided to monitor the suspected inpatients with repeated urine samplings at least once or twice weekly, and had sent the specimens to our laboratory for additional testing. All urinary samples had been obtained under close surveillance. Calculations In order to control for differences in hydration and urine output, creatinine-normalized urinary concentrations (CN-concentrations) of JWH-018-COOH and JWH-073-COOH were determined by dividing the drug concentration (in ng/mL) by the creatinine concentration (in mg/dL). Then the result was multiplied by 100 in order to report the results in nanograms of drug per milligram of creatinine excreted (ng/mg). Creatinine was analyzed photometrically after complex formation with picric acid in an alkaline solution by a routine method (Creatinine Jaffe´ Gen.2 (CRJ2 U)) on a Cobas Intergra 400þ multianalyzer (Roche Diagnostics, Basel, Switzerland). Elimination half-lives (t1/2) of JWH-018-COOH and JWH-073COOH in urine were calculated from the CN-concentrations by the pharmacokinetic program package Kinetica, version 5.0 (Thermo Scientific, Waltham, MA, USA). By using a mixed loglinear model, the parameter estimate describing the decrease of the log-concentrations (lz) was calculated using the best-fit logregression line of the samples representing the elimination phase. The elimination half-life was calculated as ln 2/lz.
Results The subjects included were three males and two females, with a mean age of 28.6 (range 20– 40) years. Except for synthetic cannabinoids, no illicit drugs were detected (20). Concentrations of 282 Hegstad et al.
JWH-018-COOH and JWH-073-COOH in the collected urinary samples from each subject are presented in Table I. Specimens were positive over a period of 20 – 43 (mean 27.4) days for JWH-018-COOH and over a period of 11 – 25 (mean 19.4) days for JWH-073-COOH (Tables I and II). In addition to the positive samples, three of the five subjects had negative specimens within the sequence of sampling. Subject B had negative samples after 31 and 37 days, subject C had a negative sample after 19 days and subject D had a negative sample after 8 days (Table I). In all these samples, creatinine concentrations were low (in the range of 39 – 65 mg/dL), indicating intake of a large volume of fluid prior to sampling. Excluding samples with low creatinine levels, JWH-018-COOH was found alone in the last positive sample in four subjects (A, B, C, E), whereas in the fifth subject (D), both JWH-018-COOH and JWH-073-COOH were found in the last positive sample. CN-concentrations and corresponding regression lines representing lz are presented in Table I and Figure 2. Calculated urinary elimination half-lives were 4.4 – 23.8 (mean 14.0) days for CN-JWH-018-COOH and 3.6 – 16.8 (mean 9.3) days for CN-JWH-073-COOH (Table II). There were no previous samples available from subjects A and B before the first positive sample was obtained. In contrast, in subjects C, D and E, samples had been obtained earlier during their same stay at the clinic; 1, 4 and 3 days before the index sample, respectively. They were all negative (Table II). Thus, in these subjects it was verified that the drug use had taken place over no more than one to a few days. Discussion The principal finding in the present study is that urine specimens could be positive for JWH-018-COOH for more than 6 weeks and JWH-073-COOH for more than 3 weeks after ingestion of synthetic cannabinoids. Even when the duration of use prior to the index sampling was documented to be no more than 4 days, making tissue accumulation less likely, metabolites were detected for about 3 weeks. We consider it being a strength of this study that we could follow the excretion of JWH-018 and JWH-073 metabolites for a prolonged period of time with frequent urinary samplings. The index sample from all subjects dated from the same day, except for two subjects who were not tested that day, but were tested 2 days later. This indicates that the drug ingestion took place at approximately the same time, and possibly inside the institution. Thus, it cannot be ruled out that additional intakes had taken place after the baseline samples were obtained, despite the ward surveillance. However, the steadily falling slopes of the CN-concentrations (Figure 2) clearly indicate residual urinary excretion and no new ingestion during the follow-up period. This study has some weaknesses, mostly caused by its naturalistic design. There were only five subjects included, and we had no information regarding dose and type of herbal mixture consumed. Moreover, only one metabolite of each compound was analyzed. As various synthetic cannabinoids can produce overlapping metabolites, it might be necessary to analyze more than one metabolite of each compound to determine the intake of a specific substance with certainty (7). For example, in a recent study, two subjects consuming JWH-018 were found to excrete JWH-073 metabolites as well as JWH-018 metabolites (8). On
Table I. Concentrations of Creatinine, JWH-018-COOH and JWH-071-COOH in Individual Consecutive Urinary Samples From five Subjects (A –E) who had Ingested JWH-018 and JWH-073 Subject
Variable
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Sample 11
Sample 12
A
Day Creatinine JWH-018-COOH JWH-073-COOH CN-JWH-018-COOH CN-JWH-073-COOH Day Creatinine JWH-018-COOH JWH-073-COOH CN-JWH-018-COOH CN-JWH-073-COOH Day Creatinine JWH-018-COOH JWH-073-COOH CN-JWH-018-COOH CN-JWH-073-COOH Day Creatinine JWH-018-COOH JWH-073-COOH CN-JWH-018-COOH CN-JWH-073-COOH Day Creatinine JWH-018-COOH JWH-073-COOH CN-JWH-018-COOH CN-JWH-073-COOH
0 67 1.35 1.67 2.02 2.50 0 135 4.49 2.18 3.34 1.62 0 48 0.57 0.56 1.20 1.18 0 60 0.81 0.82 1.35 1.37 0 269 1.24 0.68 0.46 0.25
6 117 0.90 0.63 0.77 0.54 3 119 0.41 0.33 0.35 0.28 0.5 37 0.41 0.21 1.13 0.58 2 91 0.21 0.11 0.23 0.12 2 180 0.46 0.41 0.26 0.23
12 81 0.26 0.17 0.32 0.21 4 258 0.68 0.46 0.26 0.18 6 193 1.03 0.73 0.53 0.38 7 115 0.18 0.10 0.16 0.09 7 251 0.24 0.18 0.10 0.07
15 93 0.15 0.11 0.16 0.12 9 242 0.50 0.26 0.21 0.11 11 181 0.20 0.18 0.11 0.10 8 51 ,LOQ ,LOQ – – 16 300 0.22 0.21 0.07 0.07
19 71 0.14 0.12 0.20 0.17 11 157 0.25 0.13 0.16 0.08 19 39 ,LOQ ,LOQ – – 14 181 0.24 0.20 0.13 0.11 24 325 0.13 ,LOQ 0.04 –
22 213 0.27 0.23 0.13 0.11 17 355 0.42 0.27 0.12 0.08 20 154 0.10 ,LOQ 0.07 – 21 213 0.22 0.10 0.10 0.05 30 154 ,LOQ ,LOQ – –
25 290 0.27 0.23 0.09 0.08 23 214 0.25 0.16 0.12 0.07 25 42 ,LOQ ,LOQ – – 23 129 ,LOQ ,LOQ – – – – – – – –
29 143 0.10 ,LOQ 0.07 – 24 436 0.39 0.20 0.09 0.05 – – – – – – – – – – – – – – – – – –
36 83 ,LOQ ,LOQ – – 31 65 ,LOQ ,LOQ – – – – – – – – – – – – – – – – – – – –
– – – – – – 37 48 ,LOQ ,LOQ – – – – – – – – – – – – – – – – – – – –
– – – – – – 43 213 0.14 ,LOQ
– – – – – – 52 192 ,LOQ ,LOQ – – – – – – – – – – – – – – – – – – – –
B
C
D
E
– – – – – – – – – – – – – – – – – – –
Day zero is defined as the day when the first positive sample was obtained. Creatinine ¼ Urinary creatinine concentration (mg/dL). JWH-018-COOH ¼ Urinary JWH-018-COOH concentration (ng/mL). JWH-073-COOH ¼ Urinary JWH-073-COOH concentration (ng/mL). CN-JWH-018-COOH: Creatinine-normalized urinary JWH-018-COOH concentration (ng/mg). CN-JWH-073-COOH: Creatinine-normalized urinary JWH-073-COOH concentration (ng/mg). ,LOQ ¼ below limit of quantification.
Table II. Detection Times and Elimination Half-lives of JWH-018-COOH and JWH-073-COOH in Urine Based upon Data from five Subjects (A –E) Subject
Time since last negative sample
JWH-018-COOH
JWH-073-COOH
Time to last positive sample (days)
Elimination half-life (days)
Time to last positive sample (days)
Elimination half-life (days)
A B C D E Mean + SD
NA NA 1 day 4 days 3 days –
29 43 20 21 24 27 + 8
8.6 19.8 4.4 23.8 13.6 14.0 + 7.9
25 24 11 21 16 19 + 5
7.8 10.3 3.6 16.8 8.1 9.3 + 4.9
NA, not applicable (no previous sampling); SD, standard deviation.
the basis of this finding, the authors hypothesize that JWH-018 could be demethylated to JWH-073 in humans, although they cannot exclude that the subjects had had previous, non-reported intakes of JWH-073. Interestingly, the metabolite pattern in urine was the same in these two subjects as in the third subject who consumed a mixture of JWH-018 and JWH-073. Unfortunately, no ratios between these metabolites are presented in that study. In another study, a somewhat higher concentration of JWH-018-COOH than of JWH-073-COOH was found after a single smoke of a blend known to contain a mixture of JWH-018 and JWH-073 (18). In the present study, concentrations of JWH-018-COOH were, with the exception of two single samples, always higher than
those of JWH-073-COOH. The mean ratio was 1.47, with a range from 0.81 to 2.20. This range is very close to the urinary ratios (mean 1.35, range 0.81 – 2.16) than can be calculated based upon the concentrations found in five subjects who were accused for possession of herbal products shown to contain both JWH-018 and JWH-073 (15). In other subjects in that study, JWH-018-COOH/ JWH-073-COOH ratios can be calculated to 20 –40. We consider that these data indicate that those with the highest ratios had ingested JWH-018 only, whereas those with a ratio closer to 1 most likely had ingested a mix. It is well known also from other studies that JWH-018 and JWH-073 are often mixed in herbal products (8, 17, 21). Another indication of what has been ingested can be found from the urinary Detection Times of Carboxylic Acid Metabolites 283
Figure 2. Creatinine-normalized concentrations (CN-concentrations) of JWH-018-COOH () and JWH-073-COOH (W) in individual consecutive urinary samples from five subjects (A– E) who had ingested synthetic cannabinoids. The dashed and dotted lines show the regression lines for CN-018-COOH and CN-JWH-073-COOH, respectively.
elimination curves in our subjects, where the half-lives were shorter for CN-JWH-073-COOH than for CN-JWH-018-COOH (Figure 2, Table II). If JWH-073 were produced from JWH-018, 284 Hegstad et al.
only, the half-lives for CN-JWH-073-COOH could not have been shorter than for CN-JWH-018-COOH, as the production rate of JWH-073 from JWH-018 would then be the rate-limiting step.
Thus, JWH-073 most likely stems from another source. Although we cannot with certainty conclude whether the subjects in our study had ingested JWH-018 only or a mixture of JWH-018 and JWH-073, we therefore consider the latter being more likely. Biotransformation of the synthetic cannabinoid AM-2201 produces metabolites identical to those of JWH-018, including JWH-018-COOH (7). Therefore, hydroxylated metabolites specific to AM-2201 have been used to differentiate between intake of AM-2201 and JWH-018 (7). We included AM-2201 –5-hydroxy in our method (data not shown), but this metabolite was not detected in any of the samples. Consequently, there was no evidence that the blend consumed by the subjects in the present study contained AM-2201. An LOQ as low as 0.1 ng/mL is essential in order to be able to follow the excretion over weeks instead of days. For example, with a limit of 0.5 ng/mL, analytes would have been detected in about 1 week in subjects A and B, and in the first sample only for the other three subjects. One of the authors of a previous study (18) smoked a blend known to contain a mixture of JWH-018 and JWH-073. With an LOQ of 0.1 ng/mL, they were able to monitor the urinary excretion for 65 h, and suggest a detection window of 2 –3 days following a single intake. As this time frame was shorter than for subject C in our study (who also may have had a single intake) we suggest that the doses consumed by our subjects were higher than the one smoked by the drug-naive volunteer in the study by Jager et al. (18). Jager et al. also report that their experience with routine samples analyzed at their laboratory suggests that metabolites remain detectable for 2 – 3 weeks after cessation of use. That time frame is shorter than the detection times found in our study. Due to the prolonged urinary excretion of metabolites of JWH-018 and JWH-073, it could be a challenge to determine whether serial positive samples represent residual excretion from a previous intake, or a new intake. To clarify this issue, calculations of CN-concentrations are essential. During residual excretion, the elimination curves of CN-JWH-018-COOH and CN-JWH-073-COOH should follow a steadily declining slope without any spikes, as illustrated in Figure 2. As the patterns of urinary metabolite excretion are close to those seen after intake of conventional cannabis products, it seems logical to suggest that the disposition of JWH-018 and JWH-073 follow a twocompartment model, with an early distribution phase followed by a prolonged period of redistribution and metabolism, in the same way as D9-tetrahydrocannabinol (THC) (22, 23). Consequently, the rate-limiting step in the elimination process of JWH-018 and JWH-073 could be redistribution from tissue depots back into circulation. Related to this assumption, it is worth noting that subject A and subject B who had the longest metabolite elimination times of the subjects included in our study, lacked negative samples taken prior to the first positive specimen in the series (Table II). Thus, it is not unlikely that these two subjects were chronic users, and that their slow metabolite elimination was caused by prior tissue accumulation during chronic use. Interestingly, they were also both women. As a consequence of the prolonged elimination phase, a positive sample after a negative sample does not necessarily represent a new intake. Variations in hydration and urine output (indirectly measured by creatinine concentrations in urine) may cause the concentrations of JWH-018-COOH and/or JWH-073-COOH to fluctuate above and below the LOQ for the
analytical method. In our study, subject B had two negative specimens (on Day 31 and Day 37) during the course of sampling, whereas subject C and subject D had one negative specimen each (on Day 19 and Day 8, respectively) (Table I). In these samples the creatinine concentrations were low, varying from 39 to 65 mg/dL. Thus, when the urine is as dilute as in these cases, a negative sample may well be falsely negative. Without calculated CN-concentrations of the analytes, the subjects risk being wrongly accused of new drug intake. For cannabis, various algorithms have been suggested to aid the interpretation of serial positive findings in urine after single and chronic use (2, 24 –26). Taking into consideration the similarities in pharmacokinetics, it seems reasonable that similar algorithms could be applied when differentiating new synthetic cannabinoid use from residual excretion. However, far more background information, e.g., related to the amount and the exact type of synthetic cannabinoid ingested, is needed before reliable algorithms can be produced for these substances. In conclusion, the present study indicates that urinary specimens could be positive for JWH-018-COOH for more than 6 weeks and JWH-073-COOH for more than 3 weeks after ingestion of synthetic cannabinoids, based upon a method with an LOQ as low as 0.1 ng/mL. Even when the duration of use was no more than 4 days prior to the index sampling, making tissue accumulation less likely, metabolites were detected for about 3 weeks. References 1. Lennesta˚l, R., Lakso, H.A., Nilsson, M., Mjo¨rndal, T. (2008) Urine monitoring of diazepam abuse- new intake or not? Journal of Analytical Toxicology, 32, 402–407. 2. Schwilke, E.W., Gullberg, R.G., Darwin, W.D., Chiang, C.N., Cadet, J.L., Gorelick, D.A. et al. (2011) Differentiating new cannabis use from residual urinary cannabinoid excretion in chronic, daily cannabis users. Addiction, 106, 499– 506. 3. Gunderson, E.W. (2013) Synthetic cannabinoids: a new frontier of designer drugs. Annals of Internal Medicine, 159, 563– 564. 4. Brents, L.K., Prather, P.L. (2014) The K2/Spice phenomenon: emergence, identification, legislation and metabolic characterization of synthetic cannabinoids in herbal incense products. Drug Metabolism Reviews, 46, 72 –85. 5. Fattore, L., Fratta, W. (2011) Beyond THC: The new generation of cannabinoid designer drugs. Frontiers in Behavioral Neuroscience, 5, 60. 6. Tuv, S.S., Krabseth, H., Karinen, R., Olsen, K.M., Øiestad, E.L., Vindenes, V. (2014) Prevalence of synthetic cannabinoids in blood samples from Norwegian drivers suspected of impaired driving during a seven weeks period. Accident Analysis and Prevention, 62, 26– 31. 7. Jang, M., Yang, W., Shin, I., Choi, H., Chang, H., Kim, E. (2014) Determination of AM-2201 metabolites in urine and comparison with JWH-018 abuse. International Journal of Legal Medicine, 128, 285– 294. 8. Moran, C.L., Le, V.H., Chimalakonda, K.C., Smedley, A.L., Lackey, F.D., Owen, S.N. et al. (2011) Quantitative measurement of JWH-018 and JWH-073 metabolites excreted in human urine. Analytical Chemistry, 83, 4228– 4236. 9. Chimalakonda, K.C., Moran, C.L., Kennedy, P.D., Endres, G.W., Uzieblo, A., Dobrowolski, P.J. et al. (2011) Solid-phase extraction and quantitative measurement of omega and omega-1 metabolites of JWH-018 and JWH-073 in human urine. Analytical Chemistry, 83, 6381– 6388. 10. ElSohly, M.A., Gul, W., Elsohly, K.M., Murphy, T.P., Madgula, V.L., Khan, S.I. (2011) Liquid chromatography-tandem mass spectrometry analysis of urine specimens for K2 (JWH-018) metabolites. Journal of Analytical Toxicology, 35, 487–495.
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Article
Detection Times of Carboxylic Acid Metabolites of the Synthetic Cannabinoids JWH-018 and JWH-073 in Human Urine Solfrid Hegstad1*, Andreas A. Westin1 and Olav Spigset1,2 1
Department of Clinical Pharmacology, St Olav University Hospital, Trondheim, Norway, and 2Department of Laboratory Medicine, Children’s and Women’s Health, Norwegian University of Science and Technology, Trondheim, Norway
*Author to whom correspondence should be addressed. Email:[email protected]
Over the past years, use of synthetic cannabinoids has become increasingly popular. To draw the right conclusions regarding new intake of these substances in situations of repeated urinary drug testing, knowledge of their elimination rate in urine is essential. We report data from consecutive urine specimens from five subjects after ingestion of synthetic cannabinoids. Urinary concentrations of the carboxylic acid metabolites JWH-018-COOH and JWH-073-COOH were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) with a limit of quantification of 0.1 ng/mL. In these subjects, specimens remained positive over a period of 20–43 (mean 27) days for JWH-018-COOH and over a period of 11–25 (mean 19) days for JWH-073-COOH. Detection times were shorter for subjects that appeared to have ingested only one, or a few, doses prior to urine collection in the study. Creatinine-normalized concentrations (CN-concentrations) slowly declined throughout the follow-up period in all subjects, suggesting that no new intake had taken place during this period. Mean elimination half-lives in urine were 14.0 (range 4.4 – 23.8) days for CN-JWH-018-COOH and 9.3 (range 3.6 –16.8) days for CN-JWH-073-COOH. These data show that urine specimens could be positive for JWH-018-COOH for more than 6 weeks and JWH-073-COOH for more than 3 weeks after ingestion. However, such long detection periods require a low limit of quantification.
Introduction Testing for drugs-of-abuse in urine is requested in various situations, including health care, workplace, military and criminal justice settings. Test results may be used to distinguish between a previous and a recent drug intake based upon changes in the (creatinine-normalized) drug concentration between two positive specimens from the same individual. However, urinary detection times depend both on the pharmacological properties of the drug, the sensitivity of the drug test and the subjects’ frequency of use. For some drugs, such as cannabis and some benzodiazepines, excretion can be prolonged, especially following chronic use (1, 2). Synthetic cannabinoids are a new and commonly abused class of designer drugs (3). They are often marketed as herbal blends or incense, with brand names such as ‘K2’ and ‘Spice’. Nicknames such as ‘legal highs’ and ‘herbal highs’ promote perceived safety, but in reality their pharmacological properties are largely unknown (3). The synthetic cannabinoid products are easily available on the Internet, are usually smoked like marijuana, and induce similar intoxicating effects (4). Despite international efforts to control these products, surveys and poison control data indicate that they are still readily available (3). The increasing number of available synthetic cannabinoid products and
metabolites pose a great challenge on analytical laboratories to have updated methods for the drug detection. JWH-018 and JWH-073 were among the first synthetic cannabinoids available on the market (5). In Norway, JWH-018 and AM-2201 were the most frequently synthetic cannabinoids detected in blood samples from drivers suspected of impaired driving during a 7 weeks’ period in 2011 –2012 (6). Several studies on the characterization of the metabolites of JWH-018 and JWH-073 in urine have been performed (7 – 17). These studies describe carboxylation and hydroxylation with subsequent glucuronidation as the metabolic pathways. In a recent study, metabolites of JWH-018 and JWH-073 were detected in urine for 2 –3 days with peak concentrations of 3 –16.5 h after smoking a single dose (18). Moreover, based on their experience with other samples at that laboratory (18), the authors suggest that these metabolites remain detectable in urine for about 2 – 3 weeks after ending a chronic use. Except for that report, we are not aware of any other published data on urinary detection times after intake of synthetic cannabinoids. The aim of this study is to present data on the concentration range and time span for detection of the metabolites JWH-018 N-pentanoic acid (JWH-018-COOH) and JWH-073 N-butanoic acid (JWH-073-COOH) in urine collected from the same individuals. The implications these findings may have for the interpretation related to new drug ingestion or not, are also discussed. Material and methods Chemicals and reagents The synthetic cannabinoid metabolites 5-(3-(1-naphthoyl)1H-indol-1-yl) pentanoic acid (JWH-018-COOH) and 4-(3(1-naphthoyl)-1H-indol-1-yl) butanoic acid (JWH-073-COOH), as well as the internal standard JWH-018-COOH-d4, were purchased from Chiron (Trondheim, Norway). The enzyme b-glucuronidase (Type 2 HP-2 from Helix pomatia, 100 000 units/mL) was obtained from Sigma-Aldrich (St Louis, MO). LC–MS grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany) and formic acid 98% from VWR (Leuven, Belgium). 96-well sample collection plates (1 mL) and polypropylene cap mat round wells for 96-well plates were purchased from Waters (Milford, MA). Sample preparation Calibrator, QC sample or urine sample (100 mL) was mixed with 20 mL internal standard (JWH-018-COOH-d4; 3 ng/mL), 60 mL 0.2 M ammonium acetate buffer ( pH 4.8) and 20 mL b-glucuronidase (25 000 U/mL), and incubated at 658C for 1 h, thereby hydrolyzing glucuronide conjugated analytes. An aliquot of 50 mL was diluted with 200 mL methanol/water (60/40, v/v), and centrifuged at 1800 g (Rotanta 460, Hettich Lab Technology,
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Tuttlingen, Germany) for 5 min. All dilution steps were done in 96-well plates using a Tecan pipetting robot. Instruments An Acquity UPLC I-Class FTN system (Waters) was used for separation, applying an Acquity HSS-T3 column (2.1 100 mm, 1.8 mm) maintained at 508C. The mobile phase consisted of 0.1% formic acid in water (A) and 100% acetonitrile (B). The system was run with a linear gradient from 40% B to 98% B for 2.5 min. The flow rate was 0.6 mL/min and the injection volume was 5 mL. Mass detection was performed by positive ion mode electrospray MS-MS with a Xevo TQ-S tandem-quadrupole mass spectrometer (Waters). The capillary voltage was set to 2.0 kV, the source block temperature was 1208C, and the desolvation gas nitrogen was heated to 6508C and delivered at a flow rate of 1000 L/h. The m/z 372.1 .155.0 (cone voltage: 60 V, collision energy: 25 eV) and m/z 372.1 .127.0 (cone voltage: 60 V, collision energy: 55 eV) transitions were monitored for JWH-018-COOH and the m/z 358.1 .155.0 (cone voltage: 30 V, collision energy: 23 eV) and m/z 358.1 .127.0 (cone voltage: 30 V, collision energy: 43 eV) transitions were monitored for JWH-073-COOH. For the internal standard JWH-018-COOH-d4 the m/z 376.1 .155.0 (cone voltage: 60 V, collision energy: 25 eV) transition was monitored. System operation and data acquisition were controlled using the Mass Lynx 4.1 software (Waters). A representative chromatogram is presented in Figure 1. Method validation The six-point calibration curves (three replicates of each standard) were based on peak-area ratios of the analyte relative to the internal standard using a weighted (1/x) linear line, which
excluded the origin. The correlation coefficient was above 0.999 with concentrations of 0.10, 0.30, 1.0, 3.0, 6.0 and 10.0 ng/mL. The limit of quantification (LOQ) was determined with a signal to noise ratio .10 at the lowest calibrator concentration (0.10 ng/mL). LOQ samples of 0.10 ng/mL were run in one replicate on 10 different days and the coefficients of variation (CV) and the bias were in the range 6.2 – 9.3 and 21.9 to 1.9%, respectively. Within-assay CVs were estimated by analysis of 10 separate replicates of quality control (QC) samples at three concentrations (0.50, 2.5 and 8.0 ng/mL) in a single assay, and were in the range 2.8 – 4.3%. Between-assay CVs were determined by analysis of aliquots of each QC concentration at 10 different days, one replicate in each assay and were in the range 2.2 – 6.7%. Bias was in the range 24.4 to þ1.0%. Matrix effects (ME) were evaluated by the method by Matuszewski et al. (19). ME in percent was calculated as ME% ¼ (Peak intensitymatrix/Peak intensitymethanol/water) 100. Relative ME (CV%) expresses the precision of peak intensity in matrix. ME% corrected with the internal standard (IS) was calculated as ¼ [(Peak intensitymatrix/Peak IS intensitymatrix)/(Peak intensitymethanol/water/Peak IS intensitymethanol/water)] 100. Relative ME corrected with the IS (CV %) expresses the precision of Peak intensity/Peak IS intensity in matrix. Six replicates of urine from six different individuals were analyzed at two concentrations level (0.50 and 8.0 ng/mL). ME % ranged from 70 to 90% and CVs from 7.7 to 17.5%. When corrected with internal standard the ME % were from 94 to 104% and CVs from 2.1 to 5.8%. Specificity of the methods was investigated by selecting substances with almost the same MHþ and MH– ions (+2 atomic mass units) as the analytes. The substances tested were amisulpride (MW 369.5), prochlorperazine (MW 373.9), thioridazine (MW 370.6) and JWH-203-COOH (MW 369.8). Potential endogenous
Figure 1. MRM-chromatograms of JWH-073-COOH (upper part) and JWH-018-COOH (lower part) with quantitative and qualitative transitions. Left panel represents the lowest quality control sample with concentrations of 0.5 ng/mL for both compounds. Right panel is an authentic sample with concentrations of 0.73 ng/mL for JWH-073-COOH and 1.03 ng/mL for JWH-018-COOH.
Detection Times of Carboxylic Acid Metabolites 281
interferences were assessed by analyzing 10 urine specimens from different individuals. No interferences were noted. The QC samples were found to be stable in urine for 7 days at 48C and for 6 weeks at 2208C, respectively. Diluted QC samples were found to be stable in the autosampler for 4 days at 48C. Subjects and specimens Every year our laboratory receives several hundred urinary samples for the analysis of synthetic cannabinoids, including JWH-018 and JWH-073. The results from these analyses are stored in a large database. After approval from the Regional Ethics Committee, we retrieved data from subjects with serial samples positive for JWH-018-COOH and JWH-073-COOH from our database. Five subjects in the database were of particular interest because there were numerous samples obtained during a limited period of time. All samples were sent from the same drug rehabilitation clinic. According to the staff at this clinic, they had over a short period of time experienced several episodes where suspected drug use among inpatients had been undetectable by regular urinary drug screening tests. The suspected inpatients resided in a closed ward, and all had their belongings searched prior to unit entry. However, they all had access to a common living room, and were only intermittently monitored during nighttime. Moreover, inpatients were allowed to leave the ward for short periods of time, such as for jogging, and could after special application get permission to leave for longer periods, from hours to days. Thus, drug use during hospitalization could not be ruled out, and the clinic staff decided to monitor the suspected inpatients with repeated urine samplings at least once or twice weekly, and had sent the specimens to our laboratory for additional testing. All urinary samples had been obtained under close surveillance. Calculations In order to control for differences in hydration and urine output, creatinine-normalized urinary concentrations (CN-concentrations) of JWH-018-COOH and JWH-073-COOH were determined by dividing the drug concentration (in ng/mL) by the creatinine concentration (in mg/dL). Then the result was multiplied by 100 in order to report the results in nanograms of drug per milligram of creatinine excreted (ng/mg). Creatinine was analyzed photometrically after complex formation with picric acid in an alkaline solution by a routine method (Creatinine Jaffe´ Gen.2 (CRJ2 U)) on a Cobas Intergra 400þ multianalyzer (Roche Diagnostics, Basel, Switzerland). Elimination half-lives (t1/2) of JWH-018-COOH and JWH-073COOH in urine were calculated from the CN-concentrations by the pharmacokinetic program package Kinetica, version 5.0 (Thermo Scientific, Waltham, MA, USA). By using a mixed loglinear model, the parameter estimate describing the decrease of the log-concentrations (lz) was calculated using the best-fit logregression line of the samples representing the elimination phase. The elimination half-life was calculated as ln 2/lz.
Results The subjects included were three males and two females, with a mean age of 28.6 (range 20– 40) years. Except for synthetic cannabinoids, no illicit drugs were detected (20). Concentrations of 282 Hegstad et al.
JWH-018-COOH and JWH-073-COOH in the collected urinary samples from each subject are presented in Table I. Specimens were positive over a period of 20 – 43 (mean 27.4) days for JWH-018-COOH and over a period of 11 – 25 (mean 19.4) days for JWH-073-COOH (Tables I and II). In addition to the positive samples, three of the five subjects had negative specimens within the sequence of sampling. Subject B had negative samples after 31 and 37 days, subject C had a negative sample after 19 days and subject D had a negative sample after 8 days (Table I). In all these samples, creatinine concentrations were low (in the range of 39 – 65 mg/dL), indicating intake of a large volume of fluid prior to sampling. Excluding samples with low creatinine levels, JWH-018-COOH was found alone in the last positive sample in four subjects (A, B, C, E), whereas in the fifth subject (D), both JWH-018-COOH and JWH-073-COOH were found in the last positive sample. CN-concentrations and corresponding regression lines representing lz are presented in Table I and Figure 2. Calculated urinary elimination half-lives were 4.4 – 23.8 (mean 14.0) days for CN-JWH-018-COOH and 3.6 – 16.8 (mean 9.3) days for CN-JWH-073-COOH (Table II). There were no previous samples available from subjects A and B before the first positive sample was obtained. In contrast, in subjects C, D and E, samples had been obtained earlier during their same stay at the clinic; 1, 4 and 3 days before the index sample, respectively. They were all negative (Table II). Thus, in these subjects it was verified that the drug use had taken place over no more than one to a few days. Discussion The principal finding in the present study is that urine specimens could be positive for JWH-018-COOH for more than 6 weeks and JWH-073-COOH for more than 3 weeks after ingestion of synthetic cannabinoids. Even when the duration of use prior to the index sampling was documented to be no more than 4 days, making tissue accumulation less likely, metabolites were detected for about 3 weeks. We consider it being a strength of this study that we could follow the excretion of JWH-018 and JWH-073 metabolites for a prolonged period of time with frequent urinary samplings. The index sample from all subjects dated from the same day, except for two subjects who were not tested that day, but were tested 2 days later. This indicates that the drug ingestion took place at approximately the same time, and possibly inside the institution. Thus, it cannot be ruled out that additional intakes had taken place after the baseline samples were obtained, despite the ward surveillance. However, the steadily falling slopes of the CN-concentrations (Figure 2) clearly indicate residual urinary excretion and no new ingestion during the follow-up period. This study has some weaknesses, mostly caused by its naturalistic design. There were only five subjects included, and we had no information regarding dose and type of herbal mixture consumed. Moreover, only one metabolite of each compound was analyzed. As various synthetic cannabinoids can produce overlapping metabolites, it might be necessary to analyze more than one metabolite of each compound to determine the intake of a specific substance with certainty (7). For example, in a recent study, two subjects consuming JWH-018 were found to excrete JWH-073 metabolites as well as JWH-018 metabolites (8). On
Table I. Concentrations of Creatinine, JWH-018-COOH and JWH-071-COOH in Individual Consecutive Urinary Samples From five Subjects (A –E) who had Ingested JWH-018 and JWH-073 Subject
Variable
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Sample 11
Sample 12
A
Day Creatinine JWH-018-COOH JWH-073-COOH CN-JWH-018-COOH CN-JWH-073-COOH Day Creatinine JWH-018-COOH JWH-073-COOH CN-JWH-018-COOH CN-JWH-073-COOH Day Creatinine JWH-018-COOH JWH-073-COOH CN-JWH-018-COOH CN-JWH-073-COOH Day Creatinine JWH-018-COOH JWH-073-COOH CN-JWH-018-COOH CN-JWH-073-COOH Day Creatinine JWH-018-COOH JWH-073-COOH CN-JWH-018-COOH CN-JWH-073-COOH
0 67 1.35 1.67 2.02 2.50 0 135 4.49 2.18 3.34 1.62 0 48 0.57 0.56 1.20 1.18 0 60 0.81 0.82 1.35 1.37 0 269 1.24 0.68 0.46 0.25
6 117 0.90 0.63 0.77 0.54 3 119 0.41 0.33 0.35 0.28 0.5 37 0.41 0.21 1.13 0.58 2 91 0.21 0.11 0.23 0.12 2 180 0.46 0.41 0.26 0.23
12 81 0.26 0.17 0.32 0.21 4 258 0.68 0.46 0.26 0.18 6 193 1.03 0.73 0.53 0.38 7 115 0.18 0.10 0.16 0.09 7 251 0.24 0.18 0.10 0.07
15 93 0.15 0.11 0.16 0.12 9 242 0.50 0.26 0.21 0.11 11 181 0.20 0.18 0.11 0.10 8 51 ,LOQ ,LOQ – – 16 300 0.22 0.21 0.07 0.07
19 71 0.14 0.12 0.20 0.17 11 157 0.25 0.13 0.16 0.08 19 39 ,LOQ ,LOQ – – 14 181 0.24 0.20 0.13 0.11 24 325 0.13 ,LOQ 0.04 –
22 213 0.27 0.23 0.13 0.11 17 355 0.42 0.27 0.12 0.08 20 154 0.10 ,LOQ 0.07 – 21 213 0.22 0.10 0.10 0.05 30 154 ,LOQ ,LOQ – –
25 290 0.27 0.23 0.09 0.08 23 214 0.25 0.16 0.12 0.07 25 42 ,LOQ ,LOQ – – 23 129 ,LOQ ,LOQ – – – – – – – –
29 143 0.10 ,LOQ 0.07 – 24 436 0.39 0.20 0.09 0.05 – – – – – – – – – – – – – – – – – –
36 83 ,LOQ ,LOQ – – 31 65 ,LOQ ,LOQ – – – – – – – – – – – – – – – – – – – –
– – – – – – 37 48 ,LOQ ,LOQ – – – – – – – – – – – – – – – – – – – –
– – – – – – 43 213 0.14 ,LOQ
– – – – – – 52 192 ,LOQ ,LOQ – – – – – – – – – – – – – – – – – – – –
B
C
D
E
– – – – – – – – – – – – – – – – – – –
Day zero is defined as the day when the first positive sample was obtained. Creatinine ¼ Urinary creatinine concentration (mg/dL). JWH-018-COOH ¼ Urinary JWH-018-COOH concentration (ng/mL). JWH-073-COOH ¼ Urinary JWH-073-COOH concentration (ng/mL). CN-JWH-018-COOH: Creatinine-normalized urinary JWH-018-COOH concentration (ng/mg). CN-JWH-073-COOH: Creatinine-normalized urinary JWH-073-COOH concentration (ng/mg). ,LOQ ¼ below limit of quantification.
Table II. Detection Times and Elimination Half-lives of JWH-018-COOH and JWH-073-COOH in Urine Based upon Data from five Subjects (A –E) Subject
Time since last negative sample
JWH-018-COOH
JWH-073-COOH
Time to last positive sample (days)
Elimination half-life (days)
Time to last positive sample (days)
Elimination half-life (days)
A B C D E Mean + SD
NA NA 1 day 4 days 3 days –
29 43 20 21 24 27 + 8
8.6 19.8 4.4 23.8 13.6 14.0 + 7.9
25 24 11 21 16 19 + 5
7.8 10.3 3.6 16.8 8.1 9.3 + 4.9
NA, not applicable (no previous sampling); SD, standard deviation.
the basis of this finding, the authors hypothesize that JWH-018 could be demethylated to JWH-073 in humans, although they cannot exclude that the subjects had had previous, non-reported intakes of JWH-073. Interestingly, the metabolite pattern in urine was the same in these two subjects as in the third subject who consumed a mixture of JWH-018 and JWH-073. Unfortunately, no ratios between these metabolites are presented in that study. In another study, a somewhat higher concentration of JWH-018-COOH than of JWH-073-COOH was found after a single smoke of a blend known to contain a mixture of JWH-018 and JWH-073 (18). In the present study, concentrations of JWH-018-COOH were, with the exception of two single samples, always higher than
those of JWH-073-COOH. The mean ratio was 1.47, with a range from 0.81 to 2.20. This range is very close to the urinary ratios (mean 1.35, range 0.81 – 2.16) than can be calculated based upon the concentrations found in five subjects who were accused for possession of herbal products shown to contain both JWH-018 and JWH-073 (15). In other subjects in that study, JWH-018-COOH/ JWH-073-COOH ratios can be calculated to 20 –40. We consider that these data indicate that those with the highest ratios had ingested JWH-018 only, whereas those with a ratio closer to 1 most likely had ingested a mix. It is well known also from other studies that JWH-018 and JWH-073 are often mixed in herbal products (8, 17, 21). Another indication of what has been ingested can be found from the urinary Detection Times of Carboxylic Acid Metabolites 283
Figure 2. Creatinine-normalized concentrations (CN-concentrations) of JWH-018-COOH () and JWH-073-COOH (W) in individual consecutive urinary samples from five subjects (A– E) who had ingested synthetic cannabinoids. The dashed and dotted lines show the regression lines for CN-018-COOH and CN-JWH-073-COOH, respectively.
elimination curves in our subjects, where the half-lives were shorter for CN-JWH-073-COOH than for CN-JWH-018-COOH (Figure 2, Table II). If JWH-073 were produced from JWH-018, 284 Hegstad et al.
only, the half-lives for CN-JWH-073-COOH could not have been shorter than for CN-JWH-018-COOH, as the production rate of JWH-073 from JWH-018 would then be the rate-limiting step.
Thus, JWH-073 most likely stems from another source. Although we cannot with certainty conclude whether the subjects in our study had ingested JWH-018 only or a mixture of JWH-018 and JWH-073, we therefore consider the latter being more likely. Biotransformation of the synthetic cannabinoid AM-2201 produces metabolites identical to those of JWH-018, including JWH-018-COOH (7). Therefore, hydroxylated metabolites specific to AM-2201 have been used to differentiate between intake of AM-2201 and JWH-018 (7). We included AM-2201 –5-hydroxy in our method (data not shown), but this metabolite was not detected in any of the samples. Consequently, there was no evidence that the blend consumed by the subjects in the present study contained AM-2201. An LOQ as low as 0.1 ng/mL is essential in order to be able to follow the excretion over weeks instead of days. For example, with a limit of 0.5 ng/mL, analytes would have been detected in about 1 week in subjects A and B, and in the first sample only for the other three subjects. One of the authors of a previous study (18) smoked a blend known to contain a mixture of JWH-018 and JWH-073. With an LOQ of 0.1 ng/mL, they were able to monitor the urinary excretion for 65 h, and suggest a detection window of 2 –3 days following a single intake. As this time frame was shorter than for subject C in our study (who also may have had a single intake) we suggest that the doses consumed by our subjects were higher than the one smoked by the drug-naive volunteer in the study by Jager et al. (18). Jager et al. also report that their experience with routine samples analyzed at their laboratory suggests that metabolites remain detectable for 2 – 3 weeks after cessation of use. That time frame is shorter than the detection times found in our study. Due to the prolonged urinary excretion of metabolites of JWH-018 and JWH-073, it could be a challenge to determine whether serial positive samples represent residual excretion from a previous intake, or a new intake. To clarify this issue, calculations of CN-concentrations are essential. During residual excretion, the elimination curves of CN-JWH-018-COOH and CN-JWH-073-COOH should follow a steadily declining slope without any spikes, as illustrated in Figure 2. As the patterns of urinary metabolite excretion are close to those seen after intake of conventional cannabis products, it seems logical to suggest that the disposition of JWH-018 and JWH-073 follow a twocompartment model, with an early distribution phase followed by a prolonged period of redistribution and metabolism, in the same way as D9-tetrahydrocannabinol (THC) (22, 23). Consequently, the rate-limiting step in the elimination process of JWH-018 and JWH-073 could be redistribution from tissue depots back into circulation. Related to this assumption, it is worth noting that subject A and subject B who had the longest metabolite elimination times of the subjects included in our study, lacked negative samples taken prior to the first positive specimen in the series (Table II). Thus, it is not unlikely that these two subjects were chronic users, and that their slow metabolite elimination was caused by prior tissue accumulation during chronic use. Interestingly, they were also both women. As a consequence of the prolonged elimination phase, a positive sample after a negative sample does not necessarily represent a new intake. Variations in hydration and urine output (indirectly measured by creatinine concentrations in urine) may cause the concentrations of JWH-018-COOH and/or JWH-073-COOH to fluctuate above and below the LOQ for the
analytical method. In our study, subject B had two negative specimens (on Day 31 and Day 37) during the course of sampling, whereas subject C and subject D had one negative specimen each (on Day 19 and Day 8, respectively) (Table I). In these samples the creatinine concentrations were low, varying from 39 to 65 mg/dL. Thus, when the urine is as dilute as in these cases, a negative sample may well be falsely negative. Without calculated CN-concentrations of the analytes, the subjects risk being wrongly accused of new drug intake. For cannabis, various algorithms have been suggested to aid the interpretation of serial positive findings in urine after single and chronic use (2, 24 –26). Taking into consideration the similarities in pharmacokinetics, it seems reasonable that similar algorithms could be applied when differentiating new synthetic cannabinoid use from residual excretion. However, far more background information, e.g., related to the amount and the exact type of synthetic cannabinoid ingested, is needed before reliable algorithms can be produced for these substances. In conclusion, the present study indicates that urinary specimens could be positive for JWH-018-COOH for more than 6 weeks and JWH-073-COOH for more than 3 weeks after ingestion of synthetic cannabinoids, based upon a method with an LOQ as low as 0.1 ng/mL. Even when the duration of use was no more than 4 days prior to the index sampling, making tissue accumulation less likely, metabolites were detected for about 3 weeks. References 1. Lennesta˚l, R., Lakso, H.A., Nilsson, M., Mjo¨rndal, T. (2008) Urine monitoring of diazepam abuse- new intake or not? Journal of Analytical Toxicology, 32, 402–407. 2. Schwilke, E.W., Gullberg, R.G., Darwin, W.D., Chiang, C.N., Cadet, J.L., Gorelick, D.A. et al. (2011) Differentiating new cannabis use from residual urinary cannabinoid excretion in chronic, daily cannabis users. Addiction, 106, 499– 506. 3. Gunderson, E.W. (2013) Synthetic cannabinoids: a new frontier of designer drugs. Annals of Internal Medicine, 159, 563– 564. 4. Brents, L.K., Prather, P.L. (2014) The K2/Spice phenomenon: emergence, identification, legislation and metabolic characterization of synthetic cannabinoids in herbal incense products. Drug Metabolism Reviews, 46, 72 –85. 5. Fattore, L., Fratta, W. (2011) Beyond THC: The new generation of cannabinoid designer drugs. Frontiers in Behavioral Neuroscience, 5, 60. 6. Tuv, S.S., Krabseth, H., Karinen, R., Olsen, K.M., Øiestad, E.L., Vindenes, V. (2014) Prevalence of synthetic cannabinoids in blood samples from Norwegian drivers suspected of impaired driving during a seven weeks period. Accident Analysis and Prevention, 62, 26– 31. 7. Jang, M., Yang, W., Shin, I., Choi, H., Chang, H., Kim, E. (2014) Determination of AM-2201 metabolites in urine and comparison with JWH-018 abuse. International Journal of Legal Medicine, 128, 285– 294. 8. Moran, C.L., Le, V.H., Chimalakonda, K.C., Smedley, A.L., Lackey, F.D., Owen, S.N. et al. (2011) Quantitative measurement of JWH-018 and JWH-073 metabolites excreted in human urine. Analytical Chemistry, 83, 4228– 4236. 9. Chimalakonda, K.C., Moran, C.L., Kennedy, P.D., Endres, G.W., Uzieblo, A., Dobrowolski, P.J. et al. (2011) Solid-phase extraction and quantitative measurement of omega and omega-1 metabolites of JWH-018 and JWH-073 in human urine. Analytical Chemistry, 83, 6381– 6388. 10. ElSohly, M.A., Gul, W., Elsohly, K.M., Murphy, T.P., Madgula, V.L., Khan, S.I. (2011) Liquid chromatography-tandem mass spectrometry analysis of urine specimens for K2 (JWH-018) metabolites. Journal of Analytical Toxicology, 35, 487–495.
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