TY - JOUR
AU1 - Williams,, Michelle
AU2 - Martin,, Jennifer
AU3 - Galettis,, Peter
AB - Abstract Workplace drug testing in Australia is governed by two standards AS/NZS 4308:2008 for testing in urine and AS 4760:2006 for oral fluid. These standards are prescriptive and describe the drugs tested, procedures for analysis and collection devices. However, the drugs listed are not exhaustive and workers may consume novel psychoactive substances without detection. Here we present a validated method for the detection and quantitation of 19 synthetic cannabinoids in oral fluid. These drugs are AM2233, JWH-200, AB-005, AB-FUBINACA, AB-PINACA, AB-CHMINACA, AM2201, RCS-4, JWH-250, STS-135, JWH-73, XLR-11, JWH-251, JWH-18, JWH-122, JWH-19, UR-144, JWH-20 and AKB-48. The sample volume is 100 μL and is subject to a rapid, simple, protein precipitation step prior to centrifugation and injection into the LC–MS/MS system. Chromatographic separation was achieved in 4 min on a Kinetex Biphenyl column (50 mm × 3 mm × 2.6 μm) using 0.1% formic acid in water and acetonitrile as the mobile phase. The method was validated with a limit of detection (1 ng/mL) limit of quantitation (2.5 ng/mL), selectivity, linearity (2.5–500 ng/mL), accuracy (90.5–112.5% of the target concentration) and precision (3–14.7%). This method provides for the rapid detection of synthetic cannabinoids in oral fluid which is readily applicable to a routine laboratory. Introduction Synthetic cannabinoid is a broad term for the class of novel psychoactive substance (NPS) designed to mimic the effects of traditional cannabis. These compounds were first characterized through the legitimate research of John W Huffman at Clemson University yet remained largely unknown until recently (1). The evolution of synthetic cannabinoids has been in response to market pressure and legislation (2). The first compounds identified, while different in structure to Δ-9 Tetrahydrocannabinol (THC), were somewhat similar in that they contained an alkyl tail; some of these were JWH-73, JWH-18 and HU-210. These compounds pose a significant health risk as they bind more effectively to the CB1 and CB2 receptors (3) requiring a lower dose to produce similar effects to THC. The metabolites also have pharmacological activity greater than THC (4, 5). More recent variants of this drug class are structurally diverse, with some compounds such as AB-CHMINACA and AB-PINACA shown to be significantly more potent than THC (6). The addition of a halogen may also increase the selectivity for the CB1 receptor over CB2, depending on the moiety it replaces and location (7). Adverse events from exposure to these drugs range in severity from agitation and confusion to psychosis, seizures and tachycardia (8). Synthetic cannabinoids have also been identified as the cause or a contributing factor in a number of deaths (9). These compounds are sold in a pure crystalline form as research chemicals (10) or in herbal blends designed for smoking, vaping or used to produce a tea. These blends are labeled as incense, frequently carry the warning “not for human consumption” (11) and are packaged in bright packets with creative names. The unregulated nature of these products means there are no labeling requirements relating to the specific drug or dose. The variability in these products means that K2 purchased 1 week may not contain the same drug as K2 purchased the next. Furthermore, analysis has indicated that some packages contain multiple drugs (12) and that differences in the distribution of the drug on the plant material give rise to “hot spots” within the package (13). These drugs have been analyzed in blood, or fractions thereof (14, 15), urine (16, 17), oral fluid (18, 19) and hair (20). Each of these matrices has advantages such as the ease of collection of oral fluid or the long detection window of hair, along with some drawbacks, notably where urine is used, the detection of parent drug is unlikely and the availability of metabolite analytical standards is limited. However, the ultimate decision as to the preferred matrix, in workplace drug testing, remains with the requesting authority. Currently, there are two standards that govern workplace drug testing performed in Australia AS/NZS 4308:2008 for the detection of drugs of abuse in urine and AS 4760:2006 for the detection of drugs of abuse in oral fluid. Both of these standards prescribe the drug classes tested (opiates, amphetamine type substances, cocaine and metabolites and cannabis, with benzodiazepines only in urine) the procedures that must be adhered to and laboratory testing protocols. Oral fluid drug testing is often selected by an employer as it is preferred by industrial unions favoring the shorter detection window indicative of recent use, the collection does not require a secure toilet facility and can be collected under direct observation with minimal invasion of the donors’ privacy. Oral fluid testing also typically detects the parent drug, which may be deposited during smoking or insufflation as well as excreted into the oral fluid, rather than the drug metabolites detected when analyzing urine (21). As the pharmacokinetics of synthetic cannabinoids are largely unknown, this was assumed based upon the known profile of THC metabolism with extremely low concentrations of THC–COOH being detected in oral fluid. Furthermore, consistency with AS 4760 was sought which also specifies the parent drug (THC) compared to metabolite (THC–COOH) listed in AS/NZS 4308:2008. Here we present a validated method for the detection of 19 synthetic cannabinoids in oral fluid with minimal sample preparation and a rapid LC–MS-MS analysis. The synthetic cannabinoids included in this method are AM2233, JWH-200, AB-005, AB-FUBINACA, AB-PINACA, AB-CHMINACA, AM2201, RCS-4, JWH-250, STS-135, JWH-73, XLR-11, JWH-251, JWH-18, JWH-122, JWH-19, UR-144, JWH-20 and AKB-48. Table I outlines the structures of the drugs tested, their chemical names and where relevant, synonyms, the name in bold will be used throughout this paper. Table I. Synthetic cannabinoid structures and names Structure Common name and synonyms AM-2233 (2-iodophenyl)[1-[(1-methyl-2-piperidinyl)methyl]-1H-indol-3-yl]-methanone JWH-200 [1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl]-1-naphthalenyl-methanone AB-005 [1-[(1-methyl-2-piperidinyl)methyl]-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)-methanone AB-FUBINACA N-[(1 S)-1-(aminocarbonyl)-2-methylpropyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide AB-PINACA (S)-N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide AB-CHMINACA N-[(1 S)-1-(aminocarbonyl)-2-methylpropyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide AM-2201 [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone RCS-4 (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone BTM-4 E-4 OBT-199 JWH-250 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone Structure Common name and synonyms AM-2233 (2-iodophenyl)[1-[(1-methyl-2-piperidinyl)methyl]-1H-indol-3-yl]-methanone JWH-200 [1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl]-1-naphthalenyl-methanone AB-005 [1-[(1-methyl-2-piperidinyl)methyl]-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)-methanone AB-FUBINACA N-[(1 S)-1-(aminocarbonyl)-2-methylpropyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide AB-PINACA (S)-N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide AB-CHMINACA N-[(1 S)-1-(aminocarbonyl)-2-methylpropyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide AM-2201 [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone RCS-4 (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone BTM-4 E-4 OBT-199 JWH-250 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone Table I. Synthetic cannabinoid structures and names Structure Common name and synonyms AM-2233 (2-iodophenyl)[1-[(1-methyl-2-piperidinyl)methyl]-1H-indol-3-yl]-methanone JWH-200 [1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl]-1-naphthalenyl-methanone AB-005 [1-[(1-methyl-2-piperidinyl)methyl]-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)-methanone AB-FUBINACA N-[(1 S)-1-(aminocarbonyl)-2-methylpropyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide AB-PINACA (S)-N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide AB-CHMINACA N-[(1 S)-1-(aminocarbonyl)-2-methylpropyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide AM-2201 [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone RCS-4 (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone BTM-4 E-4 OBT-199 JWH-250 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone Structure Common name and synonyms AM-2233 (2-iodophenyl)[1-[(1-methyl-2-piperidinyl)methyl]-1H-indol-3-yl]-methanone JWH-200 [1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl]-1-naphthalenyl-methanone AB-005 [1-[(1-methyl-2-piperidinyl)methyl]-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)-methanone AB-FUBINACA N-[(1 S)-1-(aminocarbonyl)-2-methylpropyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide AB-PINACA (S)-N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide AB-CHMINACA N-[(1 S)-1-(aminocarbonyl)-2-methylpropyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide AM-2201 [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone RCS-4 (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone BTM-4 E-4 OBT-199 JWH-250 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone The selection of drugs to include in this assay was difficult as there was no published data on the usage trends of specific drugs in Australia. The selection was based on the analytes listed in the Randox Toxicology Synthetic cannabinoids AB-PINACA, synthetic cannabinoids UR144/XLR11 and synthetic cannabinoids JWH- 250/RCS 8 ELISA kits. This list was refined further by eliminating the metabolites and the availability of reference materials in Australia. Methods Chemicals and reagents 1 mg/mL solutions of each AM2233, JWH-200, AB-005, AB-FUBINACA, AB-PINACA, AB-CHMINACA, AM2201, RCS-4, JWH-250, STS-135, JWH-73, XLR-11, JWH-251, JWH-18, JWH-122, JWH-19, UR-144, JWH-20 and AKB-48 were purchased from Lipomed (Arlesheim, Switzerland). JWH-250 d5, JWH-73 d7, JWH-18 d11 and JWH-122 d9 were purchased from Cayman Chemicals (Ann Arbour, MI, USA). Water was purified using a Merk Millipore, Milli-Q Advantage A10 system (Darmstadt, Germany). Reagent grade ≥95% formic acid and LC-MS grade Chromasolv® Acetonitrile were from Sigma-Aldrich (St Louis, MO, USA). Oral fluid samples Oral fluid samples were collected from laboratory staff by direct expectoration into a sterile tube, these samples were tipped into a larger vessel when pooled, leaving any sediment in the primary container for discard. The samples were not centrifuged prior to spiking. Preparation of internal standards, calibration solutions and quality controls A stock solution at 10 μg/mL of all analytes in ACN was prepared from the individual primary standards of 1 mg/mL. The calibration curve was generated by adding 50, 20, 10 or 5 μL stock solutions to 1 mL blank oral fluid producing the highest points on the calibrations curve (500, 200, 100 and 50 ng/mL, respectively). These were diluted further in blank oral fluid to produce the lower end of the calibration curve at 20, 10, 7.5, 5 and 2.5 ng/mL. High (300 ng/mL) and medium (30 ng/mL) QC solutions were prepared by adding 90 or 9 μL stock solution to 3 mL blank oral fluid. Low QC (3 ng/mL) was prepared by diluting 30 μL of the high QC in 3 mL of blank oral fluid. Internal standard solution was prepared by adding 20 μL of each JWH-18 d11, JWH-73 d7, JWH-122 d9 and JWH-250 d5 (100 μg/mL) to 100 mL of ACN giving a final concentration of 20 ng/mL. Sample preparation Samples were prepared by adding 100 μL spiked oral fluid to 300 μL water and 200 μL ACN containing the internal standards. The tubes were centrifuged at 5,000 r.p.m. (2,300 g) for 5 min. A 100 μL aliquot of the supernatant was transferred to autosampler vials and 1 μL was injected into an LC–MS-MS system. LC–MS-MS system The UHPLC system was a Shimadzu, Nexera X2 LC-30AD pumps, SIL-30 AC autosampler with a DGU-20A5 degassing unit and CTO-20A column oven (Kyoto, Japan). Chromatographic separation was performed on a Kinetix Biphenyl column (50 mm × 3 mm × 2.6 μm) purchased from Phenomenex (Torrence, CA, USA) held at 40°C. Formic acid 0.1% in water (A) and acetonitrile (B) were used as the mobile phases at a flow rate of 0.5 mL/min. The first stage of the chromatographic gradient is isocratic at 55%B for 1 min with a step up to 75%, then with a linear gradient increasing to 95%B at 4 min and holding for 1 min before returning to 55% at 5 min for 1 min column re-equilibration. The mass spectrometer was a 6,500 QTRAP (SCIEX, Framingham, MA, USA). The MS operated in electrospray positive mode with the following settings: Curtain gas—20, collision gas—medium. Ion spray voltage—5,500, source temperature—450°C, ion source gas—1–15 and ion source gas—2–20. Scheduled MRM mode was used for compound detection with a detection window set to 20 s around the expected retention time. Data acquisition was controlled by Analyst 1.6.3 and processed with MultiQuant 3.0 (SCIEX, Framingham, MA, USA). Validation of method Each analyte was optimized individually by direct injection into the MS via the inbuilt syringe drive. The parameters in Table II are those obtained from the compound optimization function in Analyst. Method validation was performed in accordance with National Association of Testing Authorities (NATA) guidelines (22), where limit of detection (LOD), limit of quantitation (LOQ), selectivity, linearity of calibration, precision, repeatability, ion suppression and measurement of uncertainty (MOU) were evaluated. Table II. Analyte MS parameters Analyte Precursion ion Product ion Retention time DP (volts) CE (volts) CXP (volts) Internal Std Ion ratio Ion supression % AM2233 458.9 112.1 0.43 91 27 12 JWH-18 d11 94.7 39.9 458.9 98.1 0.43 91 61 10 JWH-200 385.1 155.1 0.49 197 29 10 JWH-122 d9 43.1 29.46 385.1 114.1 0.49 197 31 12 AB-005 353.1 112.1 0.49 96 31 14 JWH-18 d11 63.1 24 353.1 125.1 0.49 96 29 14 AB-FUBINACA 368.8 253.1 0.8 51 33 16 JWH-73 d7 93.9 27.4 368.8 324.2 0.8 51 23 20 AB-PINACA 331.0 215.1 1.06 51 33 14 JWH-250 d5 96.9 26.4 331.0 286.2 1.06 51 21 20 AB-CHMINACA 357.1 241.1 1.38 46 37 14 JWH-122 d9 97.2 27.1 357.1 312.2 1.38 46 23 16 AM2201 360.0 232.1 1.96 176 33 16 JWH-250 d5 56.1 22.2 360.0 127.1 1.96 176 63 14 RCS-4 322.0 135.0 2.05 161 31 14 JWH-73 d7 20.1 21.4 322.0 77.1 2.05 161 69 8 JWH-250 336.0 121.1 2.13 156 25 14 JWH-250 d5 6.9 20.8 336.0 144.1 2.13 156 43 8 STS-135 383.1 135.1 2.17 31 39 8 JWH-250 d5 13.4 28 383.1 77.1 2.17 31 119 8 JWH-73 328.0 127.1 2.18 166 55 16 JWH-73 d7 45.8 20.5 328.0 155.1 2.18 166 31 12 XLR-11 330.1 125.2 2.23 171 29 8 JWH-73 d7 49.7 20.5 330.1 232.1 2.23 171 33 22 JWH-251 320.0 214.1 2.27 156 33 12 JWH-250 d5 89.8 14.2 320.0 105.1 2.27 156 31 12 JWH-18 342.0 155.1 2.41 171 33 10 JWH-18 d11 59.5 19.1 342.0 127.1 2.41 171 63 14 JWH-122 356.0 214.1 2.62 191 33 18 JWH-122 d9 72.1 16.3 356.0 141.1 2.62 191 51 8 JWH-19 356.0 155.1 2.66 191 33 8 JWH-18 d11 40.6 15.5 356.0 127.1 2.66 191 65 16 UR-144 311.9 125.1 2.76 156 25 12 JWH-73 d7 53.9 17.4 311.9 214.1 2.76 156 31 16 JWH-20 370.1 155.1 2.95 196 35 16 JWH-122 d9 45.8 31.9 370.1 127.1 2.95 196 67 12 AKB-48 366.0 135.1 3.31 136 51 14 JWH-250 d5 24.1 13.3 366.0 93.0 3.31 136 63 10 JWH-250 d5 341.0 121.0 2.11 126 27 14 JWH-73 d7 335.0 155.0 2.16 156 49 6 JWH-18 d11 353.0 155.0 2.37 100 33 16 JWH-122 d9 365.0 169.0 2.59 100 33 10 Analyte Precursion ion Product ion Retention time DP (volts) CE (volts) CXP (volts) Internal Std Ion ratio Ion supression % AM2233 458.9 112.1 0.43 91 27 12 JWH-18 d11 94.7 39.9 458.9 98.1 0.43 91 61 10 JWH-200 385.1 155.1 0.49 197 29 10 JWH-122 d9 43.1 29.46 385.1 114.1 0.49 197 31 12 AB-005 353.1 112.1 0.49 96 31 14 JWH-18 d11 63.1 24 353.1 125.1 0.49 96 29 14 AB-FUBINACA 368.8 253.1 0.8 51 33 16 JWH-73 d7 93.9 27.4 368.8 324.2 0.8 51 23 20 AB-PINACA 331.0 215.1 1.06 51 33 14 JWH-250 d5 96.9 26.4 331.0 286.2 1.06 51 21 20 AB-CHMINACA 357.1 241.1 1.38 46 37 14 JWH-122 d9 97.2 27.1 357.1 312.2 1.38 46 23 16 AM2201 360.0 232.1 1.96 176 33 16 JWH-250 d5 56.1 22.2 360.0 127.1 1.96 176 63 14 RCS-4 322.0 135.0 2.05 161 31 14 JWH-73 d7 20.1 21.4 322.0 77.1 2.05 161 69 8 JWH-250 336.0 121.1 2.13 156 25 14 JWH-250 d5 6.9 20.8 336.0 144.1 2.13 156 43 8 STS-135 383.1 135.1 2.17 31 39 8 JWH-250 d5 13.4 28 383.1 77.1 2.17 31 119 8 JWH-73 328.0 127.1 2.18 166 55 16 JWH-73 d7 45.8 20.5 328.0 155.1 2.18 166 31 12 XLR-11 330.1 125.2 2.23 171 29 8 JWH-73 d7 49.7 20.5 330.1 232.1 2.23 171 33 22 JWH-251 320.0 214.1 2.27 156 33 12 JWH-250 d5 89.8 14.2 320.0 105.1 2.27 156 31 12 JWH-18 342.0 155.1 2.41 171 33 10 JWH-18 d11 59.5 19.1 342.0 127.1 2.41 171 63 14 JWH-122 356.0 214.1 2.62 191 33 18 JWH-122 d9 72.1 16.3 356.0 141.1 2.62 191 51 8 JWH-19 356.0 155.1 2.66 191 33 8 JWH-18 d11 40.6 15.5 356.0 127.1 2.66 191 65 16 UR-144 311.9 125.1 2.76 156 25 12 JWH-73 d7 53.9 17.4 311.9 214.1 2.76 156 31 16 JWH-20 370.1 155.1 2.95 196 35 16 JWH-122 d9 45.8 31.9 370.1 127.1 2.95 196 67 12 AKB-48 366.0 135.1 3.31 136 51 14 JWH-250 d5 24.1 13.3 366.0 93.0 3.31 136 63 10 JWH-250 d5 341.0 121.0 2.11 126 27 14 JWH-73 d7 335.0 155.0 2.16 156 49 6 JWH-18 d11 353.0 155.0 2.37 100 33 16 JWH-122 d9 365.0 169.0 2.59 100 33 10 Table II. Analyte MS parameters Analyte Precursion ion Product ion Retention time DP (volts) CE (volts) CXP (volts) Internal Std Ion ratio Ion supression % AM2233 458.9 112.1 0.43 91 27 12 JWH-18 d11 94.7 39.9 458.9 98.1 0.43 91 61 10 JWH-200 385.1 155.1 0.49 197 29 10 JWH-122 d9 43.1 29.46 385.1 114.1 0.49 197 31 12 AB-005 353.1 112.1 0.49 96 31 14 JWH-18 d11 63.1 24 353.1 125.1 0.49 96 29 14 AB-FUBINACA 368.8 253.1 0.8 51 33 16 JWH-73 d7 93.9 27.4 368.8 324.2 0.8 51 23 20 AB-PINACA 331.0 215.1 1.06 51 33 14 JWH-250 d5 96.9 26.4 331.0 286.2 1.06 51 21 20 AB-CHMINACA 357.1 241.1 1.38 46 37 14 JWH-122 d9 97.2 27.1 357.1 312.2 1.38 46 23 16 AM2201 360.0 232.1 1.96 176 33 16 JWH-250 d5 56.1 22.2 360.0 127.1 1.96 176 63 14 RCS-4 322.0 135.0 2.05 161 31 14 JWH-73 d7 20.1 21.4 322.0 77.1 2.05 161 69 8 JWH-250 336.0 121.1 2.13 156 25 14 JWH-250 d5 6.9 20.8 336.0 144.1 2.13 156 43 8 STS-135 383.1 135.1 2.17 31 39 8 JWH-250 d5 13.4 28 383.1 77.1 2.17 31 119 8 JWH-73 328.0 127.1 2.18 166 55 16 JWH-73 d7 45.8 20.5 328.0 155.1 2.18 166 31 12 XLR-11 330.1 125.2 2.23 171 29 8 JWH-73 d7 49.7 20.5 330.1 232.1 2.23 171 33 22 JWH-251 320.0 214.1 2.27 156 33 12 JWH-250 d5 89.8 14.2 320.0 105.1 2.27 156 31 12 JWH-18 342.0 155.1 2.41 171 33 10 JWH-18 d11 59.5 19.1 342.0 127.1 2.41 171 63 14 JWH-122 356.0 214.1 2.62 191 33 18 JWH-122 d9 72.1 16.3 356.0 141.1 2.62 191 51 8 JWH-19 356.0 155.1 2.66 191 33 8 JWH-18 d11 40.6 15.5 356.0 127.1 2.66 191 65 16 UR-144 311.9 125.1 2.76 156 25 12 JWH-73 d7 53.9 17.4 311.9 214.1 2.76 156 31 16 JWH-20 370.1 155.1 2.95 196 35 16 JWH-122 d9 45.8 31.9 370.1 127.1 2.95 196 67 12 AKB-48 366.0 135.1 3.31 136 51 14 JWH-250 d5 24.1 13.3 366.0 93.0 3.31 136 63 10 JWH-250 d5 341.0 121.0 2.11 126 27 14 JWH-73 d7 335.0 155.0 2.16 156 49 6 JWH-18 d11 353.0 155.0 2.37 100 33 16 JWH-122 d9 365.0 169.0 2.59 100 33 10 Analyte Precursion ion Product ion Retention time DP (volts) CE (volts) CXP (volts) Internal Std Ion ratio Ion supression % AM2233 458.9 112.1 0.43 91 27 12 JWH-18 d11 94.7 39.9 458.9 98.1 0.43 91 61 10 JWH-200 385.1 155.1 0.49 197 29 10 JWH-122 d9 43.1 29.46 385.1 114.1 0.49 197 31 12 AB-005 353.1 112.1 0.49 96 31 14 JWH-18 d11 63.1 24 353.1 125.1 0.49 96 29 14 AB-FUBINACA 368.8 253.1 0.8 51 33 16 JWH-73 d7 93.9 27.4 368.8 324.2 0.8 51 23 20 AB-PINACA 331.0 215.1 1.06 51 33 14 JWH-250 d5 96.9 26.4 331.0 286.2 1.06 51 21 20 AB-CHMINACA 357.1 241.1 1.38 46 37 14 JWH-122 d9 97.2 27.1 357.1 312.2 1.38 46 23 16 AM2201 360.0 232.1 1.96 176 33 16 JWH-250 d5 56.1 22.2 360.0 127.1 1.96 176 63 14 RCS-4 322.0 135.0 2.05 161 31 14 JWH-73 d7 20.1 21.4 322.0 77.1 2.05 161 69 8 JWH-250 336.0 121.1 2.13 156 25 14 JWH-250 d5 6.9 20.8 336.0 144.1 2.13 156 43 8 STS-135 383.1 135.1 2.17 31 39 8 JWH-250 d5 13.4 28 383.1 77.1 2.17 31 119 8 JWH-73 328.0 127.1 2.18 166 55 16 JWH-73 d7 45.8 20.5 328.0 155.1 2.18 166 31 12 XLR-11 330.1 125.2 2.23 171 29 8 JWH-73 d7 49.7 20.5 330.1 232.1 2.23 171 33 22 JWH-251 320.0 214.1 2.27 156 33 12 JWH-250 d5 89.8 14.2 320.0 105.1 2.27 156 31 12 JWH-18 342.0 155.1 2.41 171 33 10 JWH-18 d11 59.5 19.1 342.0 127.1 2.41 171 63 14 JWH-122 356.0 214.1 2.62 191 33 18 JWH-122 d9 72.1 16.3 356.0 141.1 2.62 191 51 8 JWH-19 356.0 155.1 2.66 191 33 8 JWH-18 d11 40.6 15.5 356.0 127.1 2.66 191 65 16 UR-144 311.9 125.1 2.76 156 25 12 JWH-73 d7 53.9 17.4 311.9 214.1 2.76 156 31 16 JWH-20 370.1 155.1 2.95 196 35 16 JWH-122 d9 45.8 31.9 370.1 127.1 2.95 196 67 12 AKB-48 366.0 135.1 3.31 136 51 14 JWH-250 d5 24.1 13.3 366.0 93.0 3.31 136 63 10 JWH-250 d5 341.0 121.0 2.11 126 27 14 JWH-73 d7 335.0 155.0 2.16 156 49 6 JWH-18 d11 353.0 155.0 2.37 100 33 16 JWH-122 d9 365.0 169.0 2.59 100 33 10 Limit of detection was defined as the value where the analyte of interest could be identified with a signal to noise ratio greater than three, while having the appropriate retention time and ion ratio. Limit of quantitation was defined as the lowest value, where the analyte could be identified with a signal to noise ratio greater than 10 with adequate precision and accuracy (<20%CV (coefficient of variation) and ±20% target concentration). The LOQ was determined by analysis of seven replicates of the lowest concentration of the calibration curve. LOQ, LOD and linearity were determined around an administratively defined cutoff value of 5 ng/mL. Linearity was determined by the generation of four calibration curves from 2.5 to 500 ng/mL on four different days. The slope was plotted using the signal finder algorithm in SCIEX Multiquant Software. Imprecision, accuracy and measurement of uncertainty were determined by evaluating low (3 ng/mL), medium (30 ng/mL) and high (300 ng/mL) QC concentrations. Each was repeated three times on four different days with an additional seven on a fifth day for intraday precision. Imprecision was calculated by the %CV, and accuracy was calculated as the percent of the target concentration. CV was required to be within 15% (or 20% at LODQ) and accuracy was required to be within 85–115%. Instrumental repeatability was evaluated by injecting each QC three times and calculating the mean and standard deviation of the calculated value. MOU was calculated as two times this standard deviation. Carryover was assessed using a blank sample following each of the highest calibrators and QC samples; no carryover was detected. Ruggedness was not specifically evaluated though a number of parameters were changed during the routine preparation of samples. These included the order in which water, oral fluid and ACN were added, the temperature of the ACN internal standard and the time taken to centrifuge, up to 30 min. None of these parameters had any effect on the outcome of the test. Selectivity was evaluated by the assessment of interferences caused by endogenous and exogenous factors. Endogenous interferences were evaluated by investigating blank oral fluid collected from laboratory staff (n = 7) by direct expectoration into a tube. Exogenous inferences were investigated by the addition of 500 ng/mL drugs to blank oral fluid. The drugs tested were cathinone, ephedrone, methylone, flephedrone, 3,4–Methylenedioxyamphetamine, para-Methoxyamphetamine, methedrone, 3,4,5–Trimethoxyamphetamine, Methylenedioxyamphetamine, butylone, mephedrone, 3,4–Methylenedioxyethylamphetamine, 4-Methylethcathinone, pentedrone, N-Methyl-1,3–Benzodioxolylbutanamine, 4-Methylthioamphetamine, α-Pyrrolidinovalerophenone, 1-(4-methylphenyl)-2-(1-pyrrolidinyl)-1-butanone, 4-Bromo-2,5-dimethoxyphenethylamine (2C-B),3,4-Methylenedioxy-pyrovalerone, dimethoxybromoamphetamine, 4-(ethylthio)-2,5- dimethoxy-benzeneethanamine (2C-T-2), 1-[3-(trifluoromethyl)phenyl]-piperazine, dihydrochloride, 4-ethyl-2,5-dimethoxy-α-methyl-benzeneethanamine, 2,5-dimethoxy-4-(propylthio)-benzeneethanamine (2C-T-7), naphyrone, 5,6-Methylenedioxy-2-aminoindane, 2-Fluoromethamphetamine, 2,5-Dimethoxyamphetamine, 2-(4-chloro-2,5-dimethoxyphenyl)-N-(2- methoxybenzyl)ethanamine (25C-NBOMe), 4-bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]- benzeneethanamine (25B-NBOMe), 2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-4-[(1-methylethyl)thio]- benzeneethanamine (25T4-NBOMe), Δ-9-THC, cannabidiol and 3,4-dichloro- N-[[1-(dimethylamino)cyclohexyl]methyl]-benzamide (AH-7,921). Additionally, bias was evaluated by assessing the calculated concentration to the actual concentration over five runs with a minimum of three samples in each. The acceptable bias is ±20%. Ion suppression and enhancement were evaluated by comparing 10 post extraction spiked samples with six pure standards in ACN. Ion suppression was found to range from 13.3 to 39.9%. Results Chromatographic separation was achieved under 4 min with a total run time of 6 min including column re-equilibration time (Figure 1). The step from 55 to 75% B at 1 min improved chromatography and decreased run time compared to a continuous gradient from 55 to 95%B over 5 or 6 min. The %CV for retention time across four validation runs was less than 2%. Quantification of each analyte was based on the most prominent peak with a second qualifier transition monitored to ensure accurate identification. Table II outlines the Analyte, Q1 and Q3 mass, retention time and optimized parameters for each analyte, where the first transition is used for quantification. Figure 1. View largeDownload slide (A) Chromatogram of all analytes 1-AM 2233, 2-JWH-200, 3-AB-005, 4-AB-FUBINACA, 5- AB-PIANCA, 6-AB-CHMINACA, 7-AM 2201, 8-RCS-4, 9-JWH-250, 10-STS-135, 11-JWH-73, 12-XLR-11, 13-JWH-250, 14-JWH-18, 15-JWH-122, 16-JWH-19, 17-UR-144, 18-JWH-20, 19-AKB-48. Internal standards (not displayed) JWH-250 d5, JWH-73 d7, JWH-18 d11, JWH-122 d9 elute at 2.11 min, 2.16 min, 2.37 min, 2.59 min, respectively (B) chromatogram of all analytes at LOQ and (C) negative genuine oral fluid sample. Figure 1. View largeDownload slide (A) Chromatogram of all analytes 1-AM 2233, 2-JWH-200, 3-AB-005, 4-AB-FUBINACA, 5- AB-PIANCA, 6-AB-CHMINACA, 7-AM 2201, 8-RCS-4, 9-JWH-250, 10-STS-135, 11-JWH-73, 12-XLR-11, 13-JWH-250, 14-JWH-18, 15-JWH-122, 16-JWH-19, 17-UR-144, 18-JWH-20, 19-AKB-48. Internal standards (not displayed) JWH-250 d5, JWH-73 d7, JWH-18 d11, JWH-122 d9 elute at 2.11 min, 2.16 min, 2.37 min, 2.59 min, respectively (B) chromatogram of all analytes at LOQ and (C) negative genuine oral fluid sample. All analytes were found to have a LOD of 1 ng/mL and LOQ of 2.5 ng/mL. The calibration curve was linear from 2.5 to 500 ng/mL when using a 1/x regression not forced through zero and all calibration curves had an R value greater than 0.97, no endogenous or exogenous interferences were detected. Interday, intraday precision and accuracy and SD values for the low (3), mid (30) and high (300) QCs are presented in Table III. Table III. Accuracy and precision parameters Intraday %CV n = 7 Interday %CV n = 12 Accuracy %CV n = 12 Mean ± SD LQC MQC HQC LQC MQC HQC LQC MQC HQC LQC MQC HQC STS-135 T1 383/135 3.7 5.0 10.8 14.7 5.2 5.7 112.5 102.6 99.2 3.38 ± 0.5 30.8 ± 1.6 297.6 ± 17.0 JWH-250 T1 336/121 4.2 6.2 10.6 3.5 5.5 9.8 102.4 102.2 97.5 3.07 ± 0.11 30.7 ± 1.6 292.4 ± 28.6 JWH-251 T1 320/214 2.7 6.8 9.1 5.6 2.3 3.9 103.7 101.3 97.2 3.11 ± 0.18 30.4 ± 0.7 291.5 ± 11.3 UR-144 T1 312/125 2.3 5.9 3.6 6.7 9.3 12.8 97.3 98.2 97.5 2.92 ± 0.20 29.4 ± 2.7 292.3 ± 37.4 RCS-4 T1 332/135 5.6 5.7 6.9 8.2 3.8 7.2 98.4 98.7 97.4 2.95 ± 0.24 29.6 ± 1.1 292.3 ± 21.0 JWH-73 T1 328/127 2.9 7.9 6.8 5.7 5.7 3.7 101.0 100.4 101.2 3.03 ± 0.17 30.1 ± 1.7 303.7 ± 11.3 XLR-11 T1 330/125 4.4 5.3 5.7 10.5 4.0 4.2 96.7 103.1 99.6 2.90 ± 0.30 30.9 ± 1.2 298.7 ± 12.7 JWH-18 T1 342/155 4.1 4.2 8.9 7.0 4.9 4.9 97.1 103.6 102.1 2.91 ± 0.20 31.1 ± 1.5 306.2 ± 15.1 AM2201 T1 360/232 3.4 6.9 6.0 4.3 5.8 3.7 100.2 101.1 101.1 3.10 ± 0.13 30.3 ± 1.8 303.2 ± 11.1 AKB-48 T1 366/135 1.8 4.7 6.9 4.3 2.3 5.7 98.4 99.6 102.9 2.95 ± 0.13 29.9 ± 0.7 308.6 ± 17.7 JWH-19 T1 356/155 4.3 5.3 5.8 11.0 6.0 9.5 92.9 98.4 100.8 2.79 ± 0.31 29.5 ± 1.8 302.4 ± 28.7 JWH-122 T2 356/214 3.8 3.1 6.7 7.3 2.9 9.0 98.2 99.1 103.2 2.95 ± 0.22 29.7 ± 0.9 309.5 ± 27.8 JWH-20 T2 370/155 3.6 5.5 7.0 11.6 4.9 12.0 90.5 101.6 95.2 2.72 ± 0.32 30.5 ± 1.5 286.7 ± 34.4 JWH-200 T1 285/155 8.1 6.6 9.0 10.9 6.8 4.9 103.9 104.3 101.0 3.12 ± 0.34 31.1 ± 2.1 303.1 ± 14.8 AB-CHMINACA T1 365/241 4.7 3.7 6.9 11.3 3.2 3.4 98.6 98.6 99.6 2.96 ± 0.34 29.6 ± 0.9 298.9 ± 10.2 AB-FUBINACA T1 369/235 9.8 5.2 7.4 7.4 5.4 3.2 97.7 102.1 100.3 2.93 ± 0.22 30.6 ± 1.7 301.0 ± 9.5 AB-PINACA T1 331/215 7.3 6.8 7.8 6.0 3.4 3.0 103.1 99.9 99.8 3.09 ± 0.18 30.0 ± 1.0 299.4 ± 8.9 AM2233 T2 458/112 8.8 5.6 10.8 11.7 11.1 6.6 98.1 94.4 103.9 2.94 ± 0.35 28.3 ± 3.1 311.7 ± 20.6 AB-005 T1 353/112 3.8 6.1 9.6 14.1 7.5 3.8 108.6 97.5 100.4 3.26 ± 0.46 29.3 ± 2.2 301.1 ± 11.4 Intraday %CV n = 7 Interday %CV n = 12 Accuracy %CV n = 12 Mean ± SD LQC MQC HQC LQC MQC HQC LQC MQC HQC LQC MQC HQC STS-135 T1 383/135 3.7 5.0 10.8 14.7 5.2 5.7 112.5 102.6 99.2 3.38 ± 0.5 30.8 ± 1.6 297.6 ± 17.0 JWH-250 T1 336/121 4.2 6.2 10.6 3.5 5.5 9.8 102.4 102.2 97.5 3.07 ± 0.11 30.7 ± 1.6 292.4 ± 28.6 JWH-251 T1 320/214 2.7 6.8 9.1 5.6 2.3 3.9 103.7 101.3 97.2 3.11 ± 0.18 30.4 ± 0.7 291.5 ± 11.3 UR-144 T1 312/125 2.3 5.9 3.6 6.7 9.3 12.8 97.3 98.2 97.5 2.92 ± 0.20 29.4 ± 2.7 292.3 ± 37.4 RCS-4 T1 332/135 5.6 5.7 6.9 8.2 3.8 7.2 98.4 98.7 97.4 2.95 ± 0.24 29.6 ± 1.1 292.3 ± 21.0 JWH-73 T1 328/127 2.9 7.9 6.8 5.7 5.7 3.7 101.0 100.4 101.2 3.03 ± 0.17 30.1 ± 1.7 303.7 ± 11.3 XLR-11 T1 330/125 4.4 5.3 5.7 10.5 4.0 4.2 96.7 103.1 99.6 2.90 ± 0.30 30.9 ± 1.2 298.7 ± 12.7 JWH-18 T1 342/155 4.1 4.2 8.9 7.0 4.9 4.9 97.1 103.6 102.1 2.91 ± 0.20 31.1 ± 1.5 306.2 ± 15.1 AM2201 T1 360/232 3.4 6.9 6.0 4.3 5.8 3.7 100.2 101.1 101.1 3.10 ± 0.13 30.3 ± 1.8 303.2 ± 11.1 AKB-48 T1 366/135 1.8 4.7 6.9 4.3 2.3 5.7 98.4 99.6 102.9 2.95 ± 0.13 29.9 ± 0.7 308.6 ± 17.7 JWH-19 T1 356/155 4.3 5.3 5.8 11.0 6.0 9.5 92.9 98.4 100.8 2.79 ± 0.31 29.5 ± 1.8 302.4 ± 28.7 JWH-122 T2 356/214 3.8 3.1 6.7 7.3 2.9 9.0 98.2 99.1 103.2 2.95 ± 0.22 29.7 ± 0.9 309.5 ± 27.8 JWH-20 T2 370/155 3.6 5.5 7.0 11.6 4.9 12.0 90.5 101.6 95.2 2.72 ± 0.32 30.5 ± 1.5 286.7 ± 34.4 JWH-200 T1 285/155 8.1 6.6 9.0 10.9 6.8 4.9 103.9 104.3 101.0 3.12 ± 0.34 31.1 ± 2.1 303.1 ± 14.8 AB-CHMINACA T1 365/241 4.7 3.7 6.9 11.3 3.2 3.4 98.6 98.6 99.6 2.96 ± 0.34 29.6 ± 0.9 298.9 ± 10.2 AB-FUBINACA T1 369/235 9.8 5.2 7.4 7.4 5.4 3.2 97.7 102.1 100.3 2.93 ± 0.22 30.6 ± 1.7 301.0 ± 9.5 AB-PINACA T1 331/215 7.3 6.8 7.8 6.0 3.4 3.0 103.1 99.9 99.8 3.09 ± 0.18 30.0 ± 1.0 299.4 ± 8.9 AM2233 T2 458/112 8.8 5.6 10.8 11.7 11.1 6.6 98.1 94.4 103.9 2.94 ± 0.35 28.3 ± 3.1 311.7 ± 20.6 AB-005 T1 353/112 3.8 6.1 9.6 14.1 7.5 3.8 108.6 97.5 100.4 3.26 ± 0.46 29.3 ± 2.2 301.1 ± 11.4 Table III. Accuracy and precision parameters Intraday %CV n = 7 Interday %CV n = 12 Accuracy %CV n = 12 Mean ± SD LQC MQC HQC LQC MQC HQC LQC MQC HQC LQC MQC HQC STS-135 T1 383/135 3.7 5.0 10.8 14.7 5.2 5.7 112.5 102.6 99.2 3.38 ± 0.5 30.8 ± 1.6 297.6 ± 17.0 JWH-250 T1 336/121 4.2 6.2 10.6 3.5 5.5 9.8 102.4 102.2 97.5 3.07 ± 0.11 30.7 ± 1.6 292.4 ± 28.6 JWH-251 T1 320/214 2.7 6.8 9.1 5.6 2.3 3.9 103.7 101.3 97.2 3.11 ± 0.18 30.4 ± 0.7 291.5 ± 11.3 UR-144 T1 312/125 2.3 5.9 3.6 6.7 9.3 12.8 97.3 98.2 97.5 2.92 ± 0.20 29.4 ± 2.7 292.3 ± 37.4 RCS-4 T1 332/135 5.6 5.7 6.9 8.2 3.8 7.2 98.4 98.7 97.4 2.95 ± 0.24 29.6 ± 1.1 292.3 ± 21.0 JWH-73 T1 328/127 2.9 7.9 6.8 5.7 5.7 3.7 101.0 100.4 101.2 3.03 ± 0.17 30.1 ± 1.7 303.7 ± 11.3 XLR-11 T1 330/125 4.4 5.3 5.7 10.5 4.0 4.2 96.7 103.1 99.6 2.90 ± 0.30 30.9 ± 1.2 298.7 ± 12.7 JWH-18 T1 342/155 4.1 4.2 8.9 7.0 4.9 4.9 97.1 103.6 102.1 2.91 ± 0.20 31.1 ± 1.5 306.2 ± 15.1 AM2201 T1 360/232 3.4 6.9 6.0 4.3 5.8 3.7 100.2 101.1 101.1 3.10 ± 0.13 30.3 ± 1.8 303.2 ± 11.1 AKB-48 T1 366/135 1.8 4.7 6.9 4.3 2.3 5.7 98.4 99.6 102.9 2.95 ± 0.13 29.9 ± 0.7 308.6 ± 17.7 JWH-19 T1 356/155 4.3 5.3 5.8 11.0 6.0 9.5 92.9 98.4 100.8 2.79 ± 0.31 29.5 ± 1.8 302.4 ± 28.7 JWH-122 T2 356/214 3.8 3.1 6.7 7.3 2.9 9.0 98.2 99.1 103.2 2.95 ± 0.22 29.7 ± 0.9 309.5 ± 27.8 JWH-20 T2 370/155 3.6 5.5 7.0 11.6 4.9 12.0 90.5 101.6 95.2 2.72 ± 0.32 30.5 ± 1.5 286.7 ± 34.4 JWH-200 T1 285/155 8.1 6.6 9.0 10.9 6.8 4.9 103.9 104.3 101.0 3.12 ± 0.34 31.1 ± 2.1 303.1 ± 14.8 AB-CHMINACA T1 365/241 4.7 3.7 6.9 11.3 3.2 3.4 98.6 98.6 99.6 2.96 ± 0.34 29.6 ± 0.9 298.9 ± 10.2 AB-FUBINACA T1 369/235 9.8 5.2 7.4 7.4 5.4 3.2 97.7 102.1 100.3 2.93 ± 0.22 30.6 ± 1.7 301.0 ± 9.5 AB-PINACA T1 331/215 7.3 6.8 7.8 6.0 3.4 3.0 103.1 99.9 99.8 3.09 ± 0.18 30.0 ± 1.0 299.4 ± 8.9 AM2233 T2 458/112 8.8 5.6 10.8 11.7 11.1 6.6 98.1 94.4 103.9 2.94 ± 0.35 28.3 ± 3.1 311.7 ± 20.6 AB-005 T1 353/112 3.8 6.1 9.6 14.1 7.5 3.8 108.6 97.5 100.4 3.26 ± 0.46 29.3 ± 2.2 301.1 ± 11.4 Intraday %CV n = 7 Interday %CV n = 12 Accuracy %CV n = 12 Mean ± SD LQC MQC HQC LQC MQC HQC LQC MQC HQC LQC MQC HQC STS-135 T1 383/135 3.7 5.0 10.8 14.7 5.2 5.7 112.5 102.6 99.2 3.38 ± 0.5 30.8 ± 1.6 297.6 ± 17.0 JWH-250 T1 336/121 4.2 6.2 10.6 3.5 5.5 9.8 102.4 102.2 97.5 3.07 ± 0.11 30.7 ± 1.6 292.4 ± 28.6 JWH-251 T1 320/214 2.7 6.8 9.1 5.6 2.3 3.9 103.7 101.3 97.2 3.11 ± 0.18 30.4 ± 0.7 291.5 ± 11.3 UR-144 T1 312/125 2.3 5.9 3.6 6.7 9.3 12.8 97.3 98.2 97.5 2.92 ± 0.20 29.4 ± 2.7 292.3 ± 37.4 RCS-4 T1 332/135 5.6 5.7 6.9 8.2 3.8 7.2 98.4 98.7 97.4 2.95 ± 0.24 29.6 ± 1.1 292.3 ± 21.0 JWH-73 T1 328/127 2.9 7.9 6.8 5.7 5.7 3.7 101.0 100.4 101.2 3.03 ± 0.17 30.1 ± 1.7 303.7 ± 11.3 XLR-11 T1 330/125 4.4 5.3 5.7 10.5 4.0 4.2 96.7 103.1 99.6 2.90 ± 0.30 30.9 ± 1.2 298.7 ± 12.7 JWH-18 T1 342/155 4.1 4.2 8.9 7.0 4.9 4.9 97.1 103.6 102.1 2.91 ± 0.20 31.1 ± 1.5 306.2 ± 15.1 AM2201 T1 360/232 3.4 6.9 6.0 4.3 5.8 3.7 100.2 101.1 101.1 3.10 ± 0.13 30.3 ± 1.8 303.2 ± 11.1 AKB-48 T1 366/135 1.8 4.7 6.9 4.3 2.3 5.7 98.4 99.6 102.9 2.95 ± 0.13 29.9 ± 0.7 308.6 ± 17.7 JWH-19 T1 356/155 4.3 5.3 5.8 11.0 6.0 9.5 92.9 98.4 100.8 2.79 ± 0.31 29.5 ± 1.8 302.4 ± 28.7 JWH-122 T2 356/214 3.8 3.1 6.7 7.3 2.9 9.0 98.2 99.1 103.2 2.95 ± 0.22 29.7 ± 0.9 309.5 ± 27.8 JWH-20 T2 370/155 3.6 5.5 7.0 11.6 4.9 12.0 90.5 101.6 95.2 2.72 ± 0.32 30.5 ± 1.5 286.7 ± 34.4 JWH-200 T1 285/155 8.1 6.6 9.0 10.9 6.8 4.9 103.9 104.3 101.0 3.12 ± 0.34 31.1 ± 2.1 303.1 ± 14.8 AB-CHMINACA T1 365/241 4.7 3.7 6.9 11.3 3.2 3.4 98.6 98.6 99.6 2.96 ± 0.34 29.6 ± 0.9 298.9 ± 10.2 AB-FUBINACA T1 369/235 9.8 5.2 7.4 7.4 5.4 3.2 97.7 102.1 100.3 2.93 ± 0.22 30.6 ± 1.7 301.0 ± 9.5 AB-PINACA T1 331/215 7.3 6.8 7.8 6.0 3.4 3.0 103.1 99.9 99.8 3.09 ± 0.18 30.0 ± 1.0 299.4 ± 8.9 AM2233 T2 458/112 8.8 5.6 10.8 11.7 11.1 6.6 98.1 94.4 103.9 2.94 ± 0.35 28.3 ± 3.1 311.7 ± 20.6 AB-005 T1 353/112 3.8 6.1 9.6 14.1 7.5 3.8 108.6 97.5 100.4 3.26 ± 0.46 29.3 ± 2.2 301.1 ± 11.4 Carryover was assessed using a blank sample following each of the highest calibrators and QC samples; no carryover was detected. Ruggedness was not specifically evaluated though a number of parameters were changed during the routine preparation of samples. These included the order in which water, oral fluid and ACN were added, the temperature of the ACN internal standard and the time taken to centrifuge, up to 30 min. None of these parameters had any effect on the outcome of the test. Discussion The method described in this study detects the presence of 19 synthetic cannabinoids in oral fluid, these drugs are AM2233, JWH-200, AB-005, AB-FUBINACA, AB-PINACA, AB-CHMINACA, AM2201, RCS-4, JWH-250, STS-135, JWH-73, XLR-11, JWH-251, JWH-18, JWH-122, JWH-19, UR-144, JWH-20 and AKB-48. Workplace drug testing in Australia is usually limited to the drugs listed in AS/NZS 4308:2008 or AS 4760:2006. As NPS are not prescribed under either standard, and testing options are limited, they are rarely tested for and thus detection rates are unknown though the resource sector first noted these drugs as a problem (23). This method has been specifically designed to meet the needs of workplaces wishing to test for this class of drug. The decision to use oral fluid as a matrix was based on two factors; policy and ease of development. A workplace is constrained in the development of a drug testing policy largely by industrial unions which view the sampling of urine as invasive to the donors' privacy both during collection and due to the longer detection window, particularly of cannabis. A workplace whose policy specifies oral fluid as the matrix will be unable to collect a urine sample however a workplace that uses urine routinely will have few issues implementing oral fluid testing as an additional test for this class. Second, analytically the detection of parent drug in urine is limited and rather than wait for analytical standards to become available we elected to detect the parent drug, more commonly found in oral fluid. This method offers a wide range of drugs, is rapid enough to limit the time a worker is stood down, can be adapted to include new drugs as they become available and will function as a deterrent to the continued use whilst at work. A brief literature search yielded eight published methods where synthetic cannabinoids have been detected in oral fluid (18, 19, 24–30). These methods are outlined in Table IV and have been comprehensively reviewed elsewhere (31). Two of the methods include more cannabinoids (19, 26) than the one presented here, however ours includes the more recent drugs such as AB-CHMINACA and STS-135. Six of the methods require significant sample preparation including SPE (24, 25, 29, 30) or a preparatory step as in followed by evaporation then reconstitution (19, 26). The remaining two methods use dilution only. All of the evaluated methods with one exception use a large sample volume. In these cases, where the test uses the entire sample produced, this would prevent reanalysis in the event of a challenge to the results or additional tests to be performed such as adulteration. The remaining method uses a small volume of sample and has a relatively short chromatographic run time, however only includes seven of the original synthetic cannabinoids (28). Table IV. Methods for the detection of synthetic cannabinoids in oral fluid Year published Cannabinoids Sample preparation Chromatographic run time (min) Sample volume LOD/LOQ Ref. 2011 JWH-18, JWH-73, JWH-250, CP47-497, CP47-497-C8, HU-210 SPE 12.2 inc re-equil 1 mL Quantisal LOQ 0.5 ng/mL (24) 2012 JWH-18, JWH-19, JWH-73, JWH-122, JWH-200, JWH-250, HU-210, CP47-497 AM 694, Nabilone 1:1 dilution with mobile phase A 9 250 μL LOD 1–20 ng/mL (18) 2013 JWH-18, JWH-73, JWH-200, JWH-250, HU-211, CP47-497, CP47-497-C8 SPE Pos mode 8Neg mode 6 500 μL LOD 0.025–1 ng/mL LOQ 0.1–2.5 ng/mL (25) 2013 AM-1220, AM-2201, AM-2233, AM-694, JWH-007, JWH-15, JWH-18, JWH-19, JWH-20, JWH-73, JWH-81, JWH-122, JWH-200, JWH-203, JWH-210, JWH-250, JWH-251, JWH-307, JWH-387, JWH-398, JWH-412, MAM-2201, Methanandamide, RCS-4, RCS-4 ortho isomer, RCS-8, WIN 48.098, WIN 55,212-2 Protein precipitation evaporation and reconstitution in 100 μL 12 200 μL LOD 0.02–0.4 ng/mL LOQ 0.2–4 ng/mL (26) Year published Cannabinoids Sample preparation Chromatographic run time (min) Sample volume LOD/LOQ Ref. 2011 JWH-18, JWH-73, JWH-250, CP47-497, CP47-497-C8, HU-210 SPE 12.2 inc re-equil 1 mL Quantisal LOQ 0.5 ng/mL (24) 2012 JWH-18, JWH-19, JWH-73, JWH-122, JWH-200, JWH-250, HU-210, CP47-497 AM 694, Nabilone 1:1 dilution with mobile phase A 9 250 μL LOD 1–20 ng/mL (18) 2013 JWH-18, JWH-73, JWH-200, JWH-250, HU-211, CP47-497, CP47-497-C8 SPE Pos mode 8Neg mode 6 500 μL LOD 0.025–1 ng/mL LOQ 0.1–2.5 ng/mL (25) 2013 AM-1220, AM-2201, AM-2233, AM-694, JWH-007, JWH-15, JWH-18, JWH-19, JWH-20, JWH-73, JWH-81, JWH-122, JWH-200, JWH-203, JWH-210, JWH-250, JWH-251, JWH-307, JWH-387, JWH-398, JWH-412, MAM-2201, Methanandamide, RCS-4, RCS-4 ortho isomer, RCS-8, WIN 48.098, WIN 55,212-2 Protein precipitation evaporation and reconstitution in 100 μL 12 200 μL LOD 0.02–0.4 ng/mL LOQ 0.2–4 ng/mL (26) Table IV. Methods for the detection of synthetic cannabinoids in oral fluid Year published Cannabinoids Sample preparation Chromatographic run time (min) Sample volume LOD/LOQ Ref. 2011 JWH-18, JWH-73, JWH-250, CP47-497, CP47-497-C8, HU-210 SPE 12.2 inc re-equil 1 mL Quantisal LOQ 0.5 ng/mL (24) 2012 JWH-18, JWH-19, JWH-73, JWH-122, JWH-200, JWH-250, HU-210, CP47-497 AM 694, Nabilone 1:1 dilution with mobile phase A 9 250 μL LOD 1–20 ng/mL (18) 2013 JWH-18, JWH-73, JWH-200, JWH-250, HU-211, CP47-497, CP47-497-C8 SPE Pos mode 8Neg mode 6 500 μL LOD 0.025–1 ng/mL LOQ 0.1–2.5 ng/mL (25) 2013 AM-1220, AM-2201, AM-2233, AM-694, JWH-007, JWH-15, JWH-18, JWH-19, JWH-20, JWH-73, JWH-81, JWH-122, JWH-200, JWH-203, JWH-210, JWH-250, JWH-251, JWH-307, JWH-387, JWH-398, JWH-412, MAM-2201, Methanandamide, RCS-4, RCS-4 ortho isomer, RCS-8, WIN 48.098, WIN 55,212-2 Protein precipitation evaporation and reconstitution in 100 μL 12 200 μL LOD 0.02–0.4 ng/mL LOQ 0.2–4 ng/mL (26) Year published Cannabinoids Sample preparation Chromatographic run time (min) Sample volume LOD/LOQ Ref. 2011 JWH-18, JWH-73, JWH-250, CP47-497, CP47-497-C8, HU-210 SPE 12.2 inc re-equil 1 mL Quantisal LOQ 0.5 ng/mL (24) 2012 JWH-18, JWH-19, JWH-73, JWH-122, JWH-200, JWH-250, HU-210, CP47-497 AM 694, Nabilone 1:1 dilution with mobile phase A 9 250 μL LOD 1–20 ng/mL (18) 2013 JWH-18, JWH-73, JWH-200, JWH-250, HU-211, CP47-497, CP47-497-C8 SPE Pos mode 8Neg mode 6 500 μL LOD 0.025–1 ng/mL LOQ 0.1–2.5 ng/mL (25) 2013 AM-1220, AM-2201, AM-2233, AM-694, JWH-007, JWH-15, JWH-18, JWH-19, JWH-20, JWH-73, JWH-81, JWH-122, JWH-200, JWH-203, JWH-210, JWH-250, JWH-251, JWH-307, JWH-387, JWH-398, JWH-412, MAM-2201, Methanandamide, RCS-4, RCS-4 ortho isomer, RCS-8, WIN 48.098, WIN 55,212-2 Protein precipitation evaporation and reconstitution in 100 μL 12 200 μL LOD 0.02–0.4 ng/mL LOQ 0.2–4 ng/mL (26) We believe this method has a number of advantages over tests that are currently available due to the number of compounds detected, the speed of the run, minimal sample volume required and minimal sample preparation. Range of compounds detected One limitation of this method is that it is MRM data acquisition as opposed to high-resolution MS. Therefore, it will require regular updates to maintain currency with new drugs discovered in the market. However, it does contain a number of the well-known “original” synthetic cannabinoids such as JWH-18 and JWH-73 as well as more recent compounds such as AB-CHMINACA. The prevalence of these drugs in the workplace is not known however a number of ELISA assays would be required to cover this range of drugs. Multiple tests would bring with it the issue of sample volume as the typical volume collected is less than 1 mL. Minimal sample volume and preparation Oral fluid is the preferred matrix for industrial unions as it can be collected under direct observation, while still maintaining the privacy of the donor. It is also more indicative of recent drug use and by extension impairment, which is the primary concern in the workplace. As discussed above, the volume collected is minimal, particularly among donors who have dry mouth or use stimulant drugs including caffeine, nicotine and pseudoephedrine. Further requirements for separate testing and referee samples as well as enough sample specimen to perform screening and confirmatory tests and the proposed addition of adulteration tests mean that all analyses must be completed using small aliquots of original sample, which is usually less than 1 mL. This method uses 100 μL of neat oral fluid which is diluted and subject to a protein precipitation step. There is no SPE, or concentration making the total preparation time for a single sample around 7 min including a 5-min centrifugation step. From this, 1 μL is injected into the column, thus the actual volume of neat oral fluid that could contaminate the column and mass spectrometer is very small. This, combined with the use of a guard column has produced consistent results in over 500 injections to date. Speed and adaptability and speed of run The run time of 6 min makes it useful in a high throughput laboratory. Because it is a relatively simple gradient, additional drugs can be easily added. Prior to publication, this assay has been setup in a commercial laboratory virtually unchanged and includes over 30 drugs though NATA accreditation is attained will not be applied to routine samples. Assay design note This assay was designed to function as a confirmatory test in the same manner as tests undertaken as part of AS 4760:2006 or AS/NZS 4308:2008 using a proposed cutoff value of 5 ng/mL. To meet the requirements of this standard, this would require QC concentrations to be within ±50% of this value and a typical ULOQ of 100 ng/mL. There is little known about the expected concentrations in genuine samples, the decision was made to make the working range as wide as possible and to space the QCs along the range. It is expected that this method will act as a starting point for entities wishing to undertake this testing to begin from and adapt to their particular regulatory framework. It is also expected that laboratories which use different techniques for data acquisition such as SWATH would need to validate the method according to their requirements. Limits of study Stability of novel psychoactive substances is poorly characterized and did not form part of this study. However, the stability of cathinones and synthetic cannabinoids was investigated following validation of these methods. This work investigated the stability and recovery of NPS in glass tubes with neat oral fluid as well as using a buffered system both with and without the collection pad. These samples were evaluated at a number of time points throughout the month and in three temperature conditions (room temperature, refrigerated and frozen) at three concentrations (H = 300 ng/mL, M = 30 ng/mL L = 3 ng/mL). Overall synthetic cannabinoids are most stable when stored in glass tubes (without buffer) either refrigerated or frozen. This method was applied to 12 authentic samples previously submitted for routine testing, however no cannabinoids were detected. By conducting the validation in authentic oral fluid, we believe the method produced is more robust than had it relied on synthetic oral fluid, however the lack of confirmed positive samples for comparison or an external quality assurance program is a challenge The step from 55 to 75%B at 1 min allows for a faster run time, however, should any compounds be added that elute during this rapid change in the gradient caution should be exercised to ensure the repeatability of the chromatography. Conclusion This method details the validation for the detection of 19 synthetic cannabinoids in oral fluid. 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TI - A Validated Method for the Detection of Synthetic Cannabinoids in Oral Fluid
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bky043
DA - 2019-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-validated-method-for-the-detection-of-synthetic-cannabinoids-in-oral-XKETyncuxG
SP - 10
VL - 43
IS - 1
DP - DeepDyve
ER -