SOFT-TIAFT 2011

Abuse of substances marketed as “research chemicals” often sold for research purposes only or added to consumer products labeled “not for human consumption” has become increasingly popular. Cathinones, including mephedrone, methylone, and MDPV, are one class of compounds that have appeared on the market as part of the “research chemical” movement. These compounds are commonly sold as bath salts; however, drug users often snort or ingest these compounds to induce an amphetamine-like high.

Although these compounds have not yet been scheduled, some laboratories have added testing for cathinones to their analyses. If these substances become scheduled drugs, demand will increase for routine testing for cathinones. Since these compounds are structurally similar to amphetamine and may be taken in lieu of amphetamine, adding them to an existing amphetamine screen can save laboratories time and expense. The objective of this project was to develop a fast, quantitative LC/MS/MS method for the simultaneous determination of mephedrone, methylone, MDPV, amphetamine, methamphetamine, MDA, MDMA, and MDEA in urine.

An LC/MS/MS method was developed for the determination of cathinones and amphetamines in urine. The LC/MS/MS method employed a 50 mm x 2.1 mm x 5 µm Ultra Biphenyl column. Liquid chromatography utilized a Shimadzu UFLC_XR, and tandem mass spectrometry was achieved on an AB SCIEX API 4000™ system operating in ESI positive mode. Three transitions were used for each analyte. Mobile phase A consisted of water with 0.2% formic acid, and mobile phase B contained methanol with 0.2% formic acid. The flow rate used for analysis was 0.6 mL/min, and total analysis time including re-equilibration was 4.5 minutes. Samples were prepared with a 10:1 dilution in starting mobile phase containing 5 ng/mL of amphetamine-D₆. The calibration curve used for analysis ranged from 1 ng/mL to 500 ng/mL.

Linearity (r) for all compounds across the calibration range was 0.9980 – 0.9995. Signal-to-noise for all compounds at 1 ng/mL ranged from 4:1 to 67:1. Accuracy for QC samples (n=3, 40 ng/mL) was 92% – 112%. RSD for QC samples ranged from 4% – 7%. RSD for internal standard response across all injections was 4%. Urine from a user who admitted to ingesting “Ivory Wave” approximately 8 hours prior to sample collection showed an MDPV level of 116 ng/mL.

The method presented here allows for fast, routine analysis of mephedrone, methylone, and MDPV simultaneously with amphetamines by LC/MS/MS.

Since synthetic cannabinoids are relatively new, limited research has been performed to determine their urine metabolite profiles. Adding to analysis complexity, many metabolites are mono-hydroxylated isomers of their parent compounds. These metabolites are isobaric compounds that share a similar fragmentation pattern. These isomers are sometimes analyzed as one group with little to no chromatographic resolution, which makes specific metabolite identification impossible. The objective was to develop an analysis method for the primary metabolites of JWH-018 and JWH-073 and to apply the methods for the analysis of these compounds in authentic samples.

The extraction used for all samples employed a high-load, end-capped 500 mg, 6 mL C18 SPE column. SPE resulted in an overall 2x sample concentration. A Shimadzu UFLC_XR equipped with a Restek 50 mm x 2.1 mm x 5 µm Ultra Biphenyl column was employed for chromatographic separation. Mobile phases were (A) water + 0.05% acetic acid (pH ≈ 4.0) and (B) acetonitrile + 0.05% acetic acid. A gradient analysis with a flow rate of 0.5 mL/min was utilized. The detector was an AB SCIEX API 4000™ MS/MS system operated in ESI positive mode. Three multiple reaction monitoring (MRM) transitions were monitored for each metabolite based on spectra from reference standards (Cayman Chemical). Calibration curves ranged from 1–500 ng/mL for all metabolites, and quantification was accomplished using JWH-018 n-pentanoic acid-d4 as an internal standard.

The N-pentanoic acid metabolite of JWH-018 and the 5-hydroxypentyl metabolite of JWH-018 were identified in authentic urine specimens. In addition to these metabolites, two previously undocumented metabolites were observed at substantial levels in the samples. These metabolites are tentatively identified as 4-hydroxypentyl metabolite of JWH-018 and the 3-hydroxybutyl metabolite of JWH-073. Results obtained for authentic samples are listed below (Table I).

Table I  Authentic urine specimen results (n=6) and MRM transitions for synthetic cannabinoid metabolites.

The method presented here resolves 12 metabolites for JWH-018 and JWH-073. This method was used along with a straightforward SPE extraction to identify major metabolites in authentic samples. By using an SPE method rather than liquid/liquid extraction at high pH, the carboxylic acid metabolites of both JWH-018 and JWH-073 were recovered from fortified samples. This preliminary method may be suitable for the determination of JWH-018 and JWH-073 primary metabolites.