Confirmation of cannabinoid use by forensic toxicology testing in urine has been traditionally focused on ∆9-tetrahydrocannabinol (∆9-THC) with analysis of its major metabolite, 11-nor-9-carboxy-∆9-THC (∆9-cTHC), in free and conjugated forms. Legalization of hemp, however, has led to the widespread production and sale of cannabidiol (CBD) derivatives with psycho-activity, including ∆8-THC and ∆10-THC isomers. The increasing availability and growing use of isomer derivatives necessitate an expanded scope of cannabinoid confirmation test protocols. We report a quantitative, isomer-selective method of cannabinoid confirmation by liquid chromatography–tandem mass spectrometry determination of parent drug isomers (∆8-THC, ∆9-THC, ∆10-THC and CBD) as well as isomeric metabolites (∆8-cTHC and ∆9-cTHC). An efficient C18 phase chromatography on 1.6-µm solid core particles was used with a step gradient for near isocratic separation of both early-eluting THC metabolite isomers and later-eluting CBD and THC isomers. A rapid method of hydrolysis, dilution and analysis was employed for the quantitative co-determination of free and conjugated analytes, using stable isotope internal standardization. Method validation is reported, along with interference assessment from a prior confirmation method. Casework experience with the isomer-selective method revealed a 14% prevalence of ∆8-cTHC positive cases with a pattern of concomitant ∆8-THC and ∆9-THC use. A comparison of ∆8-cTHC and ∆9-cTHC phase two metabolism is also reported.

Accurate identification and quantification of drugs and their metabolites (analytes) in biological matrices is an analytical foundation of clinical and forensic toxicology. For decades, liquid chromatography interfaced by electrospray ionization with tandem mass spectrometry (LC‐ESI‐MS/MS) has been a widely used technology for analysis in the field of toxicology, as well as in many other fields of bioscience. It is also known that ion response in LC‐ESI‐MS/MS analysis is influenced by coeluting biological compounds and that preanalytical sample clean‐up is often insufficient in removing these interferences. As a result, a normalization technique is commonly used for assessment and compensation of matrix effects encountered in routine analysis. Internal standardization with a stable isotope analog of the analyte is the predominant normalization technique used in LC‐ESI‐MS/MS analysis. The technique, however, requires commercial availability or costly custom synthesis of an isotopic analog specific for each analyte. Here we describe an alternative technique for matrix normalization for use in high‐volume, multianalyte testing without the need for isotope analogs. The technique involves analysis of the original sample (neat analysis) followed by analysis of a second sample aliquot (spike analysis) that has been fortified with a controlled amount of reference analyte. A calibration procedure similar to internal standardization is employed, using an ion response ratio of neat to fortified analyte. As a demonstration of the technique in multianalyte testing, we provide a detailed protocol for simultaneous detection and quantification of 102 drugs and drug metabolites in human urine. We also provide a support protocol for addition of new analytes to the multianalyte panel, allowing convenient collection of the validation data during routine testing. The matrix normalization technique and testing principles may be applicable to a wide range of analytes and biological matrices, not only those encountered in toxicology but also in other fields of bioscience. © 2023 Wiley Periodicals LLC. Basic Protocol: Detection and quantification of 102 toxicology analytes in urine by LC‐ESI‐MS/MS analysis using the threshold accurate calibration technique Support Protocol: Method for addition and validation of new analytes to expand the Basic Protocol