Determination of 65 synthetic cannabinoids in whole blood by QuEChERS-ultra performance liquid chromatography- mass spectrometry

Synthetic cannabinoids （SCs） have become the most diverse and widely abused class of new psychoactive substances in the world. Blood and urine are classic sample matrices for in vivo toxin analysis， but some SCs have high lipophilicity， and the concentration of parent drugs in urine is extremely low， making them difficult to detect. Metabolites need to be investigated， and SCs with similar structures can produce the same metabolites. Therefore， establishing urine detection methods and interpreting results are relatively complex. In contrast， the detection of synthetic cannabinoid parent drugs in blood is more straightforward， and detecting parent drugs in blood can serve as direct evidence in legal cases. In order to detect the abuse of SCs， a QuEChERS-ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method was developed to simultaneously determine 65 SCs in blood. The sample preparation and detection conditions were optimized. Quantification was achieved using an internal standard and matrix-matched calibration curves， enabling rapid screening and quantitative analysis of the 65 SCs in blood. Following protein precipitation with acetonitrile， the blood extract was further extracted and purified using QuEChERS reagents. Chromatographic separation of all 65 SCs was performed on a Waters Acquity UPLC HSS T3 column （100 mm×2.1 mm， 1.8 μm） maintained at 40 ℃. Detection employed dynamic multiple reaction monitoring （dMRM） mode with a mobile phase consisting of 0.1% （v/v） formic acid aqueous solution and acetonitrile， delivered at a flow rate of 0.25 mL/min. The injection volume was 2 μL. The 65 SCs exhibited good linear relationships in the mass concentration range of 0.05-200 ng/mL with correlation coefficents （<italic>r</italic>） exceeding 0.992. The limits of detection （LODs） were between 0.01 and 0.2 ng/mL， and the limits of quantitation （LOQs） were between 0.05 and 0.5 ng/mL， respectively， which meet the requirements for analyzing SCs in blood sample. The intra-day and inter-day precisions （<italic>n</italic>=6） were determined to be 1.0%–9.9% by spiking blank blood samples with the 65 SCs at mass concentrations of 1， 5， and 50 ng/mL. The recoveries of the 65 SCs were between 62.2% and 116.9%， and the matrix effects were between 70.2% and 117.7%. All analytes demonstrated good stability and acceptable dilution integrity in blood samples. Using the established method， 10 blood samples from suspected drug use cases were successfully screened for SCs. The target compounds were detected in all 10 samples， specifically including ADB-BUTINACA， MDMB-4en-PINACA， MDMB-FUBICA， and 5F-MDMB-PICA with mass concentrations ranging from 1.9 to 23.1 ng/mL. The detection rates of ADB-BUTINACA and MDMB-4en-PINACA were 90% and 50%， respectively， suggesting their relatively high prevalence in China’s illegal drug market. In addition， two or more SCs were detected simultaneously in six blood samples. Compared with other existing literature methods， this study combines QuEChERS with precipitation protein method for blood sample pretreatment， greatly improving the detection efficiency and suitable for rapid screening of whole blood samples in batches. The results demonstrate that the established method offers accuracy， rapidity， sensitivity， and effective chromatographic separation. It can serve as a reliable tool for forensic laboratories to perform rapid screening and quantitative analysis of SCs in blood， providing robust technical support for combating drug-related crime and supporting social stability.