Journal of Analytical Toxicology 2014;38:466 –478 doi:10.1093/jat/bku092

Special Issue

A Rapid Quantitative Method for the Analysis of Synthetic Cannabinoids by Liquid Chromatography– Tandem Mass Spectrometry Tom D. Freijo Jr, Steve E. Harris and Subbarao V. Kala* One Source Toxicology Laboratory, 1213 Genoa Red Bluff, Pasadena, TX 77504, USA *Author to whom correspondence should be addressed. Email: [email protected]

Synthetic cannabinoids represent an emerging drug problem in the USA, as these compounds are constantly being modified and rapidly sold as soon as they become available. Laboratories around the world are constantly improving the analytical methods to detect and identify these newly available designer drugs. This study used a simple approach to detect and quantify a variety of synthetic cannabinoids (14 parent compounds and 15 metabolites including series XLR, AM, JWH, UR, RCS, PB, HU and AB-FUBINACA) using LC– MS-MS. Drug-free urine samples spiked with various synthetic cannabinoids and their metabolites were separated on a C18-Hypersil Gold column using an Agilent 1290 ultra-high performance liquid chromatography and detected by an AB Sciex API 4000 tandem mass spectrometer. Studies were carried out to determine limit of detection, limit of quantitation, upper limit of linearity, ion suppression, interference, precision and accuracy to validate the method. Urine samples from patients and known users were hydrolyzed with b-glucuronidase prior to the analysis by LC – MS-MS, and the data are presented. The method described here is rapid, highly sensitive and specific for the identification of a variety of synthetic cannabinoids.

Introduction Synthetic cannabinoids are compounds that were originally synthesized by scientists around the world to study the interaction of these compounds with endocannabinoid CB1 and CB2 receptors. As these compounds became more readily available, a market for recreational drug ensued. These compounds are constantly being modified and mixed with herbal products and are sold as incense under many brand names such as Spice, Spice Gold, Aroma, K2, Kush, Spike 99, etc. These compounds have a high potency for cannabinoid receptors and cause a variety of toxicological symptoms (1, 2). In 2012, the United States Drug Enforcement Agency classified the naphthoylindole series (JWH-015, JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210 and JWH-398), phenylacetylindoles (JWH203, JWH-250, JWH-251 and RCS-8), benzoylindoles (RCS-4 and AM694), cyclohexylphenols (CP 47,497, C7 and C8 analogs) and dibenzopyrans (HU-210) as Schedule I controlled substances (3) and most recently added UR-144, XLR11 and AKB48 to this list (4, 5). LC – MS-MS has been the analytical tool of choice for many scientists to detect and identify these compounds (6 – 11). The classification of various synthetic cannabinoids, their structural similarities and evolution are described in detail by Carroll et al. (2). The study on the analysis of JWH series of synthetic cannabinoids using LC–MS-MS was published in 2011 (6) followed by several other studies using the same technique for the identification of compounds in urine and serum (7 – 9). All these studies used liquid –liquid extraction prior to the analysis by LC–MS-MS. Recently, Huestis’s group (10, 11) published

two articles describing qualitative and quantitative analysis of .20 synthetic cannabinoids and their metabolites using liquid – liquid or solid-phase extraction prior to the analysis by LC–MS-MS. The current study describes the detection and quantification of 29 synthetic cannabinoids (both parent compounds and metabolites) using LC–MS-MS with a simple dilute and shoot procedure without any extraction. In order to improve the detection of various synthetic cannabinoids from urine samples collected at different times from actual drug abusers, both parent and metabolite compounds are included in the analysis. The procedure described herein does not involve extraction; thus eliminating the variation in the recovery of these different synthetic cannabinoids. Additionally, this method is highly sensitive in detecting synthetic cannabinoids at low nanogram per milliliter range (1– 5 ng/mL); it is highly specific and can easily be modified to incorporate upcoming newly available designer drugs.

Experimental materials and methods Chemicals and reagents All the synthetic cannabinoids and metabolites including the deuterated internal standards were purchased from Cerilliant (Round Rock, TX, USA) and Cayman Chemical Company (Ann Arbor, MI, USA). HPLC grade formic acid, methanol and other reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA). b-Glucuronidase (100,000 units/mL) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The HPLC columns (Hypersil Gold C18 column 100  4.6 mm, 5 mm) were purchased from Thermo Fisher Scientific. The reagents for screening were purchased from Immunalysis (Pomona, CA, USA).

Calibrators and controls All calibrators and controls were prepared in drug-free urine and spiked with appropriate standards. For all drugs, a calibrator, a low control (40% of cutoff ), a high control (25% above cutoff ) and a drug-free urine control were used for the analyses of urine samples from donors. A single point calibration including the origin was used to quantify the synthetic cannabinoid concentrations. For the determination of limit of detection (LOD)/ limit of quantitation (LOQ), drug-free urine samples were fortified with different concentrations of synthetic cannabinoids and their metabolites and the samples were analyzed in triplicate by LC–MS-MS. Deuterated internal standards were used to quantitate the analytes. Several criteria including the shape of the chromatographic peak (90% resolution, .3 : 1 signal-to-noise ratio; tailing factor 3 : 1), retention time (+0.05) and qualifying ion ratios (within+ 20%) were used for acceptability criteria of the analysis.

# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Sample preparation One-milliliter samples were spiked with 100 mL of 200 ng/mL deuterated internal standard and treated with 100 mL of b-glucuronidase (abalone) for 1 h at 758C. At the end of incubation, samples were diluted with 1.0 mL of methanol (1 : 1) and centrifuged at 4,000 rpm. The supernatants were transferred to polypropylene vials for the analysis of synthetic cannabinoids and their metabolites by LC –MS-MS.

Table I Flow Rate and Gradient Conditions Total time (min)

Flow rate (mL/min)

A (%)

B (%)

0.0 4.5 5.0 7.9 8.0 10

1.2 1.2 1.2 1.2 1.2 1.2

80 20 5 5 80 80

20 80 95 95 20 20

Mobile phase A: 0.1% formic acid in deionized water. Mobile phase B: 0.1% formic acid in methanol.

Screen Commercially available individual synthetic cannabinoids and their metabolites were screened by immunoassay (Immunalysis) to examine the specificity of the assay for these compounds. Commercially available controls and calibrators used for the immunoassay screen were at a 10 ng/mL cutoff. LC–MS-MS screen was used simultaneously to compare to the immunoassay screen results.

Ultra-high performance liquid chromatography All the ultra-high performance liquid chromatography (UHPLC) work was performed using an Agilent 1290 UHPLC system equipped with dual pumps, an autosampler and a column heater. The separation of synthetic cannabinoids was performed using a Hypersil Gold C18 column (100  4.6 mm, 5 mm from Thermo Scientific). The gradient for the separation of synthetic cannabinoids consisted of mobile phase A: 0.1% formic acid in deionized water and mobile phase B: 0.1% formic acid in methanol. The flow rate was set at 1.2 mL/min. See details of gradient in Table I and the list of synthetic cannabinoids and their metabolites analyzed in this study in Table II.

Table II MRM Transitions of Synthetic Cannabinoids Analyte name

Q1 masses (AMU)

Q3 masses (AMU)

DP (V)

EP (V)

CE (V)

CXP (V)

AB-FUBINACA (4) AM 694 Pentanoic acid (5) AM-1248 (3) AM-2201 (6) AM2201 N-(4 OH pentyl) (6) HU-210 (5) JWH 018 4 OH pentyl (2) JWH 019 (2) JWH 019 5 OH hexyl (2) JWH 200-6 OH indole (3) JWH 210 4 OH pentyl (5) JWH 250 4 OH pentyl (3) JWH-018 N-pentanoic acid (2) JWH-073-3 OH butyl (1) JWH-073-4-butanoic acid (1) JWH-081 (4) JWH-081-N-5 OH pentyl (1) JWH-122 (4) JWH-122 5 OH pentyl (4) JWH-210 (5) PB-22 (3) RCS 8 (1) RCS-4 (1) RCS-4-N-(5-OH pentyl) (1) UR 144 (4) UR-144 Pentanoic (1) XLR11 (1) XLR-11 6 OH indole (1) XLR-12 (1) Internal standards 1. JWH-073 3 OH butyl D5 IS 2. JWH-018 4 OH pentyl-D5 IS 3. JWH-250-4 OH pentyl D5 IS 4. JWH 122-4-OH pentyl D5 IS 5. JWH-210-4 OH pentyl D5 IS 6. AM2201-4-OH pentyl D5 IS

369.0 447.9 391.2 360.0 376.1 387.1 358.0 355.9 372.1 401.1 386.2 352.2 372.2 344.1 358.1 372.1 388.2 356.1 372.1 370.1 359.0 376.0 322.1 338.1 312.0 342.1 330.1 346.0 352.0

324.1, 253.1 230.9, 202.8 135.2, 112.1 155.1, 127.0 155.0, 126.9 71.0, 43.0 155.1, 127.0 154.9, 127.0 155.1, 127.0 155.1, 127.0 183.1, 155.2 121.1, 91.2 155.0, 126.9 155.0, 127.0 155.0, 127.0 185.1, 157.2 185.1, 157.1 169.0, 115.0 169.2, 115.1 183.1, 214.1 214.1, 144.1 120.9, 90.9 135.1, 77.2 135.0, 77.0 124.9, 214.1 125.0, 55.08 125.1, 232.0 125.0, 55.0 125.0, 254.0

51 86 116 101 44 91 96 50 106 91 51 51 40 40 50 40 116 40 53 60 61 35 40 86 60 101 56 106 101

10 7.5 10 10 10 10 10 10 6 10 8 3.5 10 10 10 10 10 10 3 10 10 10 10 10 10 10 10 2.5 10

21, 35, 43, 37, 38, 41, 35, 33, 27, 31, 29, 31, 32, 34, 33, 33, 33, 33, 29, 33, 17, 31, 31, 27, 33, 31, 30, 33, 35,

20, 40, 22, 28, 10, 12, 26, 10, 26, 26, 13, 13, 16, 12, 12, 16, 32, 16, 13, 18, 32, 12, 12, 24, 18, 22, 15, 22, 22,

349.1 363.0 357.0 377.1 391.1 381.1

155.2 155.1 121.0 169.1 183.1 155.1

96 71 91 111 101 106

10 10 10 10 10 10

33 31 31 31 37 33

33 67 45 71 70 75 67 57 67 69 45 67 68 60 65 51 59 91 91 33 53 63 73 81 33 71 40 67 35

44 34 10 22 10 8 22 10 22 22 13 13 10 14 14 10 28 12 13 18 24 14 10 14 18 10 15 10 14

28 26 20 28 34 13

Internal standards used in this study are shown in italics. The MRM transition Q1/Q3 –1 mass is used for quantitation. The ratio of Q3-1 mass/Q3-2 mass is used as the qualifier ratio to identify the compounds. JWH-073-3-OH butyl D5 IS (1), JWH-018 4-OH pentyl-D5 IS (2), JWH250-4-OH pentyl D5 IS (3), JWH122-4-OH pentyl D5 IS (4), JWH-210-4 OH pentyl D5 IS (5) and AM2201-4-OH pentyl D5 IS (6) were used as internal standards in this study. The number in the parenthesis next to the analyte in the first column of the table shows the internal standard used for the quantitation of that particular metabolite. AMU, atomic mass unit; V, voltage; IS, internal standard. MRM, multiple reaction monitoring; DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential.

Analysis of Synthetic Cannabinoids by LC– MS-MS 467

Table III LOD/LOQ, ULOL, Precision and Accuracy Study of Synthetic Cannabinoids in Urine Samples by LC –MS-MS Using SAMHSA Guidelines

Table IV Determination of LOD/LOQ and Inter- and Intraday Variation in the Analysis of Synthetic Cannabinoids in Urine Samples by LC –MS-MS Using SWGTOX Guidelines

Analyte name

LOD (ng/mL)

LOQ (ng/mL)

ULOL (ng/mL)

% CV 40% of cutoff

% CV cutoff

Analyte name

LOD/LOQ (ng/mL)

AB-FUBINACA AM 694 pentanoic acid AM-1248 AM-2201 AM-2201 N-(4-OH pentyl) HU-210 JWH 018 4 OH pentyl JWH 019 JWH 019 5 OH hexyl JWH 122 5 OH pentyl JWH 200-6 OH indole JWH 210-4 OH pentyl JWH 250 4 OH pentyl JWH-018 N-pentanoic acid JWH-073-4-OH butyl JWH-073-N-butanoic acid JWH-081 JWH-081-N-(5-OH-pentyl) JWH-122 JWH-210 PB-22 RCS 8 RCS-4 RCS-4-N-(5-OH-pentyl) UR 144 UR 144 pentanoic acid XLR 11 XLR 11-6 OH indole XLR 12

5.0 4.0 3.0 3.0 5.0 5.0 3.0 2.0 3.0 4.0 4.0 3.0 3.0 3.0 1.0 5.0 3.0 3.0 3.0 3.0 3.0 3.0 2.0 3.0 1.0 3.0 3.0 3.0 5.0

5.0 4.0 4.0 5.0 5.0 5.0 4.0 3.0 5.0 5.0 4.0 5.0 4.0 4.0 4.0 5.0 4.0 4.0 3.0 4.0 4.0 3.0 5.0 3.0 4.0 5.0 5.0 5.0 5.0

500 500 200 500 500 100 500 200 300 500 500 500 500 500 300 500 300 300 300 500 400 500 500 500 500 500 300 500 200

13.0 8.9 8.9 8.2 11.1 13.2 13.7 7.5 4.4 6.8 10.2 6.9 6.3 9.7 8.1 7.1 6.7 9.6 7.6 8.9 10.3 6.8 7.5 10.4 13.4 7.7 7.3 12.6 12.1

13.8 9.5 5.6 6.7 9.3 14.2 5.7 6.2 8.8 6.0 4.9 5.5 3.1 8.2 9.7 6.9 4.5 8.4 3.9 6.8 5.0 5.4 4.1 8.0 5.3 4.7 6.9 10.3 6.7

Mean AB-FUBINACA AM 694 Pentanoic acid AM-1248 AM-2201 AM2201 N-(4 OH pentyl) HU-210 JWH 018 4 OH pentyl JWH 019 JWH 019 5 OH hexyl JWH 200-6 OH indole JWH 210 4 OH pentyl JWH 250 4 OH pentyl JWH-018 N-Pentanoic acid JWH-073-3 OH butyl JWH-073-4-butanoic acid JWH-081 JWH-081-N-5 OH pentyl JWH-122 JWH-122 5 OH pentyl JWH-210 PB-22 RCS 8 RCS-4 RCS-4-N-(5-OH-pentyl) UR 144 UR-144 Pentanoic XLR11 XLR-11 6 OH indole XLR-12

4.3 4.1 4.1 4.6 4.0 4.0 4.2 4.0 4.1 4.6 4.2 4.1 4.1 4.2 4.2 4.2 4.1 4.4 4.2 3.8 4.2 4.3 4.1 4.2 3.5 3.8 4.0 4.4 3.8

LOD/LOQ values are expressed as nanogram per milliliter of urine. Precision and accuracy studies were carried out by analyzing 10 samples (N ¼ 10) for each drug at 4 ng/mL (40% cutoff) and at 10 ng/mL (cutoff). LOD, lower limit of detection; LOQ, lower limit of quantitation; ULOL, upper limit of linearity; CV, coefficient of variation; ng/mL, nanogram/milliliter; SAMHSA, Substance Abuse Mental Health Services Administration.

MS-MS analysis Individual synthetic cannabinoids were infused through an AB Sciex API 4000 tandem mass spectrometer to identify all the required parameters. The mass spectrometer and the electrospray unit were optimized to detect all these compounds. The turbo spray conditions were identical for analysis of all the cannabinoids (curtain gas: 30 psi; collision gas: 7 psi; ion spray voltage: 22,500 V, temperature: 6508C; ion source gas: 60 psi; ion source gas 2: 70 psi and dwell time-10 ms), and two multiple reaction monitoring (MRM) transitions were monitored for each drug. Only one MRM transition was monitored for the deuterated internal standards. The details of MRM transitions used for each drug, and internal standards are given in Table II. The cutoff concentrations for synthetic cannabinoids were set at 10 ng/mL. Data analysis was carried out using Applied Biosystems Analyst Software (version 1.6.1).

LOD/LOQ, upper limit of linearity, carryover, precision and accuracy studies Studies were performed to determine LOD, LOQ, upper limit of linearity (ULOL), carryover, precision and accuracy. These studies were performed according to the Substance Abuse Mental Health Services Administration (SAMHSA) guidelines (12). LOD, LOQ and ULOL were evaluated using drug-free urine samples 468 Freijo et al.

Interday variation

Intraday variation

CV (%)

Mean

CV (%)

Mean

CV (%)

18.3 19.9 11.7 15.6 12.4 14.5 8.8 14.1 10.1 15.2 8.0 7.6 9.8 11.3 9.0 13.8 8.9 15.9 9.9 9.0 7.4 11.3 6.2 15.0 11.7 15.0 13.9 16.1 18.0

9.4 9.2 9.4 10.6 9.9 9.9 10.2 10.7 10.5 9.7 10.0 10.1 10.9 10.9 10.3 10.4 10.6 10.7 10.8 9.3 10.5 10.7 11.1 9.7 10.4 10.2 10.7 10.0 10.8

17.0 13.2 14.7 17.2 12.2 17.6 8.9 15.1 11.0 16.5 7.1 8.4 12.4 9.4 14.0 14.7 12.4 15.1 8.8 8.2 13.0 13.7 14.3 12.0 15.2 16.5 13.7 13.9 7.5

9.8 9.3 9.0 11.0 10.2 8.7 9.9 9.5 10.1 9.9 10.3 9.6 10.5 10.2 10.5 9.6 9.8 10.8 10.5 9.9 9.3 10.1 10.3 10.0 9.7 9.9 10.4 9.9 9.4

3.3 7.1 7.3 10.1 4.5 2.9 4.6 11.9 6.1 4.9 2.7 3.9 9.3 4.5 4.0 9.5 7.6 7.9 7.6 6.4 7.3 3.4 7.1 1.1 10.7 6.2 11.6 6.2 14.4

LOD/LOQ studies were conducted using urine samples from three different sources over a period of 3 days. Inter- and intraday variation in synthetic cannabinoid determinations were performed using at least triplicate runs over a period of 5 days (n ¼ 15). LOD, limit of detection; LOQ, limit of quantitation; SWGTOX, Scientific Working Group for Forensic Toxicology.

(1.0 mL) spiked to various concentrations of synthetic cannabinoids, ranging from 1 to 500 ng/mL. These samples were run in triplicate. Samples were treated with b-glucuronidase prior to centrifugation, and the supernatants were diluted (1 : 2) before analysis by LC–MS-MS (Table III). For precision and accuracy studies, two batches of 10 individual drug-free urine (negative) samples were spiked with synthetic cannabinoids at concentrations of 4 ng/mL (40% cutoff) and 10 ng/mL (cutoff ) and processed as described above for analysis by LC–MS-MS and the data are presented in Table III. Carryover studies were performed by analyzing urine samples containing synthetic cannabinoids at concentrations above ULOL (500 ng/mL) followed by drug-free urine samples to detect any carryover of the individual synthetic cannabinoids. These studies were carried out in triplicate (data not presented). LOD is defined as the concentration producing a peak eluting within +0.05 min of the analyte’s retention time for the lowest concentration of the drug with a signal-to-noise ratio of at least 3 : 1 and the qualifier ion ratios within +20% of the calibration standard. For LOQ, the quantitative results must also be within 20% of target value in addition to satisfying all the above criteria of LOD. ULOL is defined as the concentration producing a peak eluting within +0.05 min of the analyte’s retention time for the highest concentration of the drug, the qualifier ion ratios within +20% of the calibration standard and the quantitative results must meet the criteria of being within +20% of target value.

Table V Dilution Integrity and Interference Studies on Synthetic Cannabinoid Analyses by LC –MS-MS Analyte

AB-FUBINACA AM 694 pentanoic acid AM-1248 AM-2201 AM2201 N-(4 OH pentyl) HU-210 JWH 018 4 OH pentyl JWH 019 JWH 019 5 OH hexyl JWH 200-6 OH indole JWH 210 4 OH pentyl JWH 250 4 OH pentyl JWH-018 N-pentanoic acid JWH-073-3 OH butyl JWH-073-4-butanoic acid JWH-081 JWH-081-N-5 OH pentyl JWH-122 JWH-122 5 OH pentyl JWH-210 PB-22 RCS 8 RCS-4 RCS-4-N-(5-OH-pentyl) UR 144 UR-144 pentanoic XLR11 XLR-11 6 OH indole XLR-12

Dilution integritya

Table VI Effect of Matrix on Synthetic Cannabinoid Analyses by LC –MS-MS Analyte

Mean (ng/mL)

SD

CV (%)

555.4 536.8 461.8 532.5 499.6 553.0 576.4 555.5 512.6 490.9 510.8 537.4 505.8 483.7 507.3 546.1 426.7 544.7 506.3 627.8 512.5 592.1 403.6 518.4 570.6 510.0 545.0 538.3 557.0

25.2 31.0 112.4 57.4 48.8 23.5 18.0 30.6 43.4 57.2 21.5 47.0 25.5 24.2 52.4 37.1 22.0 27.6 30.5 28.2 64.9 65.1 36.1 75.5 30.5 26.2 22.5 26.0 30.4

4.5 5.7 24.3 10.7 9.7 4.2 3.1 5.5 8.4 11.6 4.2 8.7 5.0 5.0 10.3 6.8 5.1 5.0 6.0 4.5 12.6 11.0 8.9 14.5 5.3 5.1 4.1 4.8 5.4

a Dilution integrity studies were carried out using urine samples much greater than ULOL (500 ng/mL). Samples were diluted 10 times prior to the analysis by LC – MS-MS. Endogenous interference studies were carried out using at least 20 drug-free sources of urine to be sure no endogenous interference produced false-positive results for any of the above analytes (n ¼ 20). Exogenous interference studies were carried out using 20 drug-free urine samples containing at least 5,000 ng/mL of codeine, morphine, hydromorphone, hydrocodone, oxycodone, oxymorphone, tetrahydrocannabinol, fentanyl, oxazepam, temazepam, meperidine, amphetamine, methamphetamine, benzoylecgonine, phencyclidine, a-hydroxyalprazolam, tapentadol, tramadol, 7-aminoclonazepam, fluoxetine and imipramine. No endogenous or exogenous interferences were found with any of the above synthetic cannabinoids (n ¼ 20). SD, standard deviation; CV, coefficient of variation.

Additional studies were performed according to Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines (13) to further evaluate LOD and LOQ. These studies were conducted in triplicate from three different sources analyzed on three different days. For this, drug-free urine samples from three different sources were spiked with synthetic cannabinoid mixture (4 ng/mL—a concentration chosen from studies presented in Table III) and LC – MS-MS analyses were performed (Table IV). Inter- and intra-day variation studies were also performed using drug-free urine samples from three different sources spiked with synthetic cannabinoids (10 ng/mL), and LC – MS-MS analyses were performed over a 5-day period (n ¼ 15). Data are presented in Table IV.

Dilution integrity and interference studies Drug-free urine samples containing concentrations of synthetic cannabinoids greater than ULOL (500 ng/mL) were used for the dilution integrity studies. Samples from 10 different sources were spiked to a final concentration of 500 ng/mL with a synthetic cannabinoid mixture and were diluted 10 times prior to

AB-FUBINACA AM 694 pentanoic acid AM-1248 AM-2201 AM2201 N-(4-OH pentyl) HU-210 JWH 018 4 OH pentyl JWH 019 JWH 019 5 OH hexyl JWH 200-6 OH indole JWH 210 4 OH pentyl JWH 250 4 OH pentyl JWH-018 N-pentanoic acid JWH-073-3 OH butyl JWH-073-4-butanoic acid JWH-081 JWH-081-N-5 OH pentyl JWH-122 JWH-122 5-OH pentyl JWH-210 PB-22 RCS-8 RCS-4 RCS-4-N-(5-OH-pentyl) UR 144 UR-144 pentanoic XLR-11 XLR-11 6 OH indole XLR-12

Analyte suppression or enhancement

ISTD suppression or enhancement

Low (%)

High (%)

Low (%)

High (%)

17.7 17.6 4.0 41.9 21.7 46.7 20.3 52.3 21.2 214.1 18.3 16.6 18.5 18.4 18.6 48.3 24.6 43.9 18.0 42.0 15.2 48.4 22.1 8.3 23.9 18.0 34.5 24.6 15.6

20.9 23.0 3.0 14.14 5.8 11.5 7.2 4.3 16.8 234.2 4.3 4.3 15.8 11.1 16.2 20.8 15.6 0.4 12.2 20.8 0.1 11.9 7.5 6.4 12.9 3.7 28.3 15.9 15.6

22.2 20.2 16.1 27.7 27.7 20.2 21.5 21.5 21.5 16.1 20.2 16.1 21.5 19.1 19.1 22.2 19.1 22.2 22.2 20.2 16.1 19.1 19.1 19.1 22.2 19.1 19.1 19.1 19.1

7.3 3.6 5.4 12.5 12.5 3.6 8.6 8.6 8.6 5.4 3.6 5.4 8.6 11.0 11.0 7.3 11.0 7.3 7.3 3.6 5.4 11.0 11.0 11.0 7.3 11.0 11.0 11.0 11.0

Ion suppression studies were carried out using drug-free urine samples from 10 different sources (N ¼ 10) analyzed in duplicate. Urine samples were spiked with low (10 ng/mL) and high (500 ng/ mL) concentrations of synthetic cannabinoid mixture and were analyzed by LC – MS-MS. Spiked water samples served as controls. The peak areas of analytes and internal standards obtained in urine matrix were compared to the corresponding peak areas obtained in water, and the data are presented as a percent suppression or enhancement. Negative numbers (2) indicate suppression; positive numbers indicate enhancement.

the analysis by LC – MS-MS, and the data are presented in Table V. Both endogenous and exogenous interference studies were performed to determine possible interference (falsepositive contributions) from endogenous urine as well as routinely used drugs of abuse and prescription pain management drugs. Drug-free urine samples from 10 different sources were analyzed to determine possible endogenous interference of urine matrix. For exogenous interferences, drug-free urine samples from 10 different sources were spiked to a final concentration of 5,000 ng/mL with various drugs (codeine, morphine, hydromorphone, hydrocodone, oxycodone, oxymorphone, tetrahydrocannabinol, fentanyl, oxazepam, temazepam, meperidine, amphetamine, methamphetamine, benzoylecgonine, phencyclidine, a-hydroxyalprazolam, tapentadol, tramadol, 7-aminoclonazepam, fluoxetine and imipramine) and the analyses were performed to identify any possible interference with synthetic cannabinoid analyses. Data are presented in Table V.

Stability The synthetic cannabinoids used in this study were found to be stable at room temperature for a period of 24 h at 4 –68C. For longer delays, the samples were stored at 2208C. Analysis of Synthetic Cannabinoids by LC– MS-MS 469

Figure 1. Representative ion chromatograms obtained from the analysis of a urine sample containing 10 ng/mL synthetic cannabinoid mixtures (JWH series). Extracted ion chromatogram of 10 ng/mL synthetic cannabinoid mixtures for JWH series are shown. The MRM transition 1 for each drug is shown on the left panel, and MRM transition 2 for each drug is shown on the right panel. The individual compounds analyzed during this run are JWH 019, JWH-081, JWH-122, JWH-210, JWH-073-4-butanoic acid, JWH-018 N-pentanoic acid, JWH 018 4 OH pentyl, JWH 019 5 OH hexyl, JWH 122 5 OH pentyl, JWH 200-6 OH indole, JWH 210-4 OH pentyl, JWH 250 4 OH pentyl, JWH-073-4-OH butyl and JWH-081-N-5-OH pentyl.

Matrix effects The matrix effects (ion suppression or ion enhancement) were performed by using drug-free urine obtained from 10 470 Freijo et al.

different sources, and the analyses were conducted both at low (10 ng/mL) and high (500 ng/mL) concentrations of synthetic cannabinoids. Corresponding deionized water spiked

Figure 1. Continued

samples were used as the controls. The average peak areas of individual synthetic cannabinoids obtained in these urine samples during LC – MS-MS analyses were compared with those of water samples (controls) to determine the matrix effects.

Data presented are shown as a percentage change from water controls (Table VI). Ion suppression values are indicated with a negative sign, whereas ion enhancement data are presented as positive values. Analysis of Synthetic Cannabinoids by LC– MS-MS 471

Figure 1. Continued

Results and discussion Drug-free urine samples were fortified with various synthetic cannabinoids and metabolites (see Table II) to check if all of these individual compounds could be detected by immunoassay. Drug-free urine samples spiked with 10 ng/mL individual synthetic cannabinoids were screened using homogenous immunoassay reagents purchased from Immunalysis. Although the reagent had cross-reactivity with most of the compounds, 13 compounds (XLR-12, UR-144-pentanoic acid, XLR-11-6-hydroxyindole, JWH-210-4-hydroxypentyl, JWH-250-4-hydroxypentyl, JWH-081-5-hydroxypentyl, RCS-4-hydroxypentyl, AM-1248, XLR-11, PB-22, HU-210, UR-144 and AB-FUBINACA) out of 29 were not detected by the immunoassay. Simultaneously, LC – MS-MS was used to detect and quantify the above 13 compounds along with the other 16 synthetic cannabinoids 472 Freijo et al.

that were detected by immunoassay (AM 694 pentanoic acid, AM 2201 N-(4-hydroxypentyl), JWH-073-4-butanoic acid, JWH 018 4-hydroxypentyl, AM-2201, JWH-018 N-pentanoic acid, JWH 019 5-hydroxyhexyl, JWH-122 5-hydroxypentyl, JWH 200-6-hydroxyindole, JWH-073 3-hydroxybutyl, JWH-019, JWH-081, JWH-122, JWH-210, RCS-8 and RCS-4). Individual synthetic cannabinoid solutions (10 – 200 ng/mL) were infused into the mass spectrometer at 10 mL/min to optimize the LC – MS-MS parameters listed in Table II. Electrospray positive ionization was used for the detection of all synthetic cannabinoids in urine samples. Two product ions were selected for each drug, and an MRM method was established to monitor transitions of each drug during the LC – MS-MS analysis. One MRM transition was used for the internal standards (see Table II).

Figure 2. Representative ion chromatograms obtained from the analysis of a urine sample containing 10 ng/mL synthetic cannabinoid mixtures (XLR, AM, PB and HU-series). Extracted ion chromatogram of 10 ng/mL synthetic cannabinoid mixtures for XLR, AM, PB and HU-series are shown. The MRM transition 1 for each drug is shown on the left panel, and MRM transition 2 for each drug is shown on the right panel. The individual compounds analyzed during this run are XLR-12, XLR-11 6 OH indole, XLR11, AM 694 Pentanoic acid, AM2201 N-4 OH pentyl, AM-2201, AM-1248, PB-22 and HU-210.

The information on the flow rate and gradient system used for the separation of synthetic cannabinoids is presented in Table I. The samples were analyzed using the above conditions with

a C18-Hypersil Gold column, and the individual synthetic cannabinoids were identified by LC– MS-MS (see Figures 1 –3). All compounds were well separated, and their identification was highly Analysis of Synthetic Cannabinoids by LC– MS-MS 473

Figure 2. Continued

specific. The few analytes that have similar transitions can be differentiated by their retention times, as can be seen in Figure 1 for JWH 073 4-butanoic acid (358.1/155.0) with a retention time of 5.42 min versus JWH 018 4-hydroxypentyl (358.0/155.2) at 5.58 min and JWH 018 N-pentanoic acid (372.2/155.0) with a retention time of 5.52 min versus JWH 019 5-hydroxyhexyl (372.1/ 155.1) at 5.75 min. The validation studies were conducted as described earlier in the ‘Methods’ section, and the data are presented in Tables III–VI. The LOD and LOQs ranged from 1 to 5 ng/mL for these synthetic cannabinoids, and the coefficient of variation both at cutoff (10 ng/mL) and 40% of cutoff (4 ng/mL) was ,10% for most of the compounds (Table III). These results are consistent with earlier reports (10, 11). Additional studies were conducted over a period of 3 days to determine the variation in the LOD values obtained, and the data are presented in Table IV. 474 Freijo et al.

Inter and intra-day variation data generated over a period of 5 days are also presented in Table IV. The lower detection limits and the inter- and intraday variations found in this study were similar when using either SAMHSA or SWGTOX guidelines (Tables III and IV). Data on dilution integrity and interference (endogenous and exogenous) are presented in Table V. No significant effects of dilution or interference were observed on the quantitation of synthetic cannabinoids. The results of the matrix effect studies on synthetic cannabinoids are presented in Table VI. Varying matrix effects (ranging from 0.1 to 52%) were seen on the analyte and internal standard of different synthetic cannabinoids. However, both the analyte and the internal standards varied to a similar degree in most cases resulting in 99% accuracy in quantitation (data not shown).

Figure 3. Representative ion chromatograms obtained from the analysis of a urine sample containing 10 ng/mL synthetic cannabinoid mixtures (RCS, UR and FUBINACA-series). Extracted ion chromatogram of 10 ng/mL synthetic cannabinoid mixtures for RCS, UR and FUBINACA-series are shown. The MRM transition 1 for each drug is shown on the left panel, and MRM transition 2 for each drug is shown on the right panel. The individual compounds analyzed during this run are RCS-4-N-5-OH-pentyl, RCS-8, RCS-4, UR-144 pentanoic and AB-FUBINACA.

A representative ion chromatogram from a urine sample obtained from a patient is presented in Figure 4. UR-144-pentanoic acid (1,370 ng/mL) and XLR-11-6-hydroxyindole (311 ng/mL) were detected and quantified. In addition, studies performed on the urine samples of known cannabinoid users (users of Kush) showed two AB-PINACA metabolites. Original analysis of the contents of Kush (a commercially available synthetic cannabinoid) revealed the presence of AB-PINACA parent compound. However, the analyses did not show parent compound AB-PINACA in the donor samples. In order to identify the unknown peaks that were found in the urine samples of these donors, the metabolites of AB-PINACA were purchased and included in the analyses to

determine the presence of AB-PINACA metabolites, namely, AB PINACA pentanoic acid and AB PINACA N (4-hydroxylpentyl). Data are presented in Figure 5.

Comments and conclusions Detection and quantitation of newly designed synthetic cannabinoids are a continuous challenge for scientists around the world. As more and more synthetic cannabinoids are mixed with herbal products and sold as incense under many brand names, modifications of existing methods to include new metabolites are Analysis of Synthetic Cannabinoids by LC– MS-MS 475

Figure 3. Continued

Figure 4. Extracted ion chromatogram of a urine sample from a patient positive for synthetic cannabinoids. Two synthetic cannabinoids were identified and quantified: UR-144 pentanoic acid (1,370 ng/mL) and XLR-11-6-hydroxyindole (311 ng/mL). The MRM transition 1 is shown on the left panel, and MRM transition 2 is shown on the right panel.

required to enable the identification of these compounds. In order to overcome the analytical difficulties of identifying continuously changing synthetic cannabinoids, a rapid and sensitive method was developed to detect and identify a comprehensive list of synthetic cannabinoids (14 parent compounds and 15 metabolites) in urine samples. All 29 synthetic cannabinoids can easily be detected and quantitated by LC–MS-MS. Additionally, new compounds can be added and analyzed with ease using this method as shown by the inclusion of AB PINACA analysis to 476 Freijo et al.

the panel (Figure 5). The described method is a simple dilute and shoot procedure that is both sensitive and specific and can identify a variety of synthetic cannabinoids at low nanogram levels in urine samples. It is beyond the scope of the present investigation to evaluate the interference of all the synthetic cannabinoid compounds and their metabolites. The possible interference due to the similarities in the structures of various synthetic cannabinoids and their metabolites warrants further investigation.

Figure 5. Identification of metabolites of AB-PINACA in a urine sample obtained from a known synthetic cannabinoid user. Urine samples obtained from donors using Kush (commercially available synthetic cannabinoid compound) were analyzed by LC – MS-MS. Two metabolites, AB PINACA pentanoic acid (2,460 ng/mL) and AB PINACA N (4-hydroxylpentanyl) (38 ng/mL), were identified in these samples. The MRM transition 1 of AB PINACA metabolites are shown on the left panel, and MRM transition 1 for the internal standards (ABPINACA, D9) are shown on the right panel.

Acknowledgments We thank Mr Romeo Laurel, CEO of One Source Toxicology, for his continuous encouragement and financial support of this project. We also thank others including, Margaret Gilbert, Autumn Courtney, Tim Madeksho and Brandy Marquez for their support and assistance.

3. 4. 5. 6.

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11. Scheidweiler, K.B., Huestis, M.A. (2014) Simultaneous quantification of 20 synthetic cannabinoids and 21 metabolites and semiquantification of 12 alkyl hydroxyl metabolites in human urine by liquid chromatography – tandem mass spectrometry. Journal of Chromatography A, 1327, 105–117. 12. National Laboratory Certification Program (NLCP). (2010) Manual for Urine Laboratories. RTI International, Research Triangle Park, NC, October 1. 13. Scientific Working Group for Forensic Toxicology (SWGTOX). (2013) Standard practices for method validation in forensic toxicology, SWGTOX Doc 003, Rev 1, May 20.

A rapid quantitative method for the analysis of synthetic cannabinoids by liquid chromatography-tandem mass spectrometry.

Synthetic cannabinoids represent an emerging drug problem in the USA, as these compounds are constantly being modified and rapidly sold as soon as the...
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