LC-DAD: A tool from the past to identify isomers of new psychoactives substances. Application to chloromethcathinones

Aim

To formally identify the isomers of chloromethcathinone (x-CMC).

Method

General toxicological unknown screening performed on peripheral blood of a man found dead at home in a context of chemsex revealed x-CMC on gas chromatography (GC) coupled with mass spectrometry (MS) using the Cayman commercial library. To formally identify the isomer, commercial standards of 3-CMC and 4-CMC were obtained and integrated in homemade spectrum libraries (mass and UV) by analyzing them using screening methods: GC-MS (after acetylation) and liquid chromatography (LC) coupled to diode array detection incremented to a mass spectrometer (DAD/MS), plus targeted methods dedicated to new psychoactive substances (NPSs) by tandem mass spectrometry: GC-MS/MS and LC-MS/MS. These methods are in daily use in laboratories in forensic contexts (determination of cause of death, drug abuse). Apolar columns were used for all methods. The conditions of the various methods have been previously published (Bottinelli, Toxac, 2017,29,123–129; Epain, Int Legal Med, 2024, 138, 1813–1820).

Results

Whatever the MS method and whether the compound was acetylated or not (GC), an identical spectrum was observed for 3-CMC and 4-CMC. The specific m/z ions selected for GC-MS were 111, 139 and 239. For GC-MS/MS, the transitions were 138.9 > 110.9, 138.9 > 75.0 and 112.9 > 75.0, and for LC-MS/MS 197.9 > 145.0 and 197.9 > 180.0. Under the chromatographic conditions tested, an identical retention time (RT) was observed for the two isomers using GC-MS and GC-MS/MS, making them completely indistinguishable, despite many temperature gradient tests. However, the LC methods obtained distinct RTs: 3.73 min and 3.52 min for 3-CMC and 4-CMC, respectively, on LC-MS/MS. As well as being discriminated by RT, 3-CMC (6.32 min) and 4-CMC (6.54 min) were clearly distinguishable on LC-DAD/MS by their absorption spectra. Absorbance maxima were observed at wavelengths of 210 and 250 nm for 3-CMC and 197 and 262 nm for 4-CMC. Concerning isomer identification in the postmortem case described here, 3-CMC was formally identified by LC-DAD/MS and then quantified (33.7 ng/mL) in the peripheral blood by LC-MS/MS.

Conclusion

In some cases, it is difficult to distinguish NPS isomers. It is generally expected that compounds with similar structures will react similarly under given analytic conditions. Therefore, it was initially suspected that the x-CMC isomers would behave identically to the x-MMC isomers: i.e., differentiable by RT on GC and undistinguishable on LC-MS/MS. The difference in these structures lies in the presence of a chlorine atom (CMC) replacing a methyl group (MMC). On LC, a chloride group allows additional interaction with the stationary phase, and thus facilitates isomer separation. On GC, the position of a chloride group, which has a higher molar mass than a methyl group, has less influence on isomer separation. In the absence of commercial standards, the LC-DAD/MS method alone made it possible to formally distinguish CMC isomers by their UV spectrum, as previously demonstrated for MMC isomers (Bottinelli, Toxac, 2017, 29, 123–129). In first-line analysis in toxicological unknown screening, LC-DAD/MS allowed immediate distinction in the cases analyzed. Quantification could be carried out secondarily using a specific LC-MS/MS method with greater sensitivity and accurate reproducible quantification. In 2023, six cases of 3-CMC, and no cases of 4-CMC, were identified in the laboratory, all in contexts of chemsex.