Simultaneous quantification of drugs in whole blood by ultra-performance liquid chromatography-tandem mass spectrometry using solid-phase mini-cartridges (Smart-SPE) for sample preparation

ObjectiveDrug poisoning cases due to overdoses of over-the-counter (OTC) medications are increasing, and comprehensive measurement of blood drug concentrations, including OTC drugs, is important in emergency medicine and forensic science. In this study, we developed a simultaneous quantification method for blood drug levels using LC-MS/MS with solid-phase mini-cartridge SmartSPE for sample preparation. MethodsThe target analytes were acetaminophen, caffeine, flunitrazepam, 7-aminoflunitrazepam, risperidone, and phenobarbital. Internal standards (IS) used were acetaminophen-d4, caffeine-d9, diazepam-d5, and phenobarbital-d5. For solid-phase extraction, three types of Smart-SPE columns (AiSTI Science Co., Ltd.) were employed. 100 {micro}L of blank whole blood samples were subjected to protein precipitation using methanol and acetonitrile, and the supernatant obtained after centrifugation was processed using Smart-SPE for sample cleanup. Chromatographic separation was performed on a CAPCELL PAK INERT ADME-HR column (Osaka Soda), and detection was carried out in both positive and negative ESI modes. ResultsLinearity was observed for all drugs across the therapeutic to coma-death concentration ranges, with correlation coefficients exceeding 0.99 in all cases. Intra-day accuracy ranged from 98.06% to 110.87%, with precision between 0.19% and 17.37%. Inter-day accuracy ranged from 97.82% to 112.35%, with precision between 0.53% and 9.18%. Recovery rates varied from 72.9% to 95.1%, and matrix effects ranged from 91.2% to 113.0%. DiscussionThe validation results demonstrated satisfactory performance for all analytes, suggesting that this simultaneous quantification method may serve as a reliable analytical approach. Further investigations into sample stability are planned, and if analyses of actual specimens confirm its applicability, the method can be reported as a novel approach for simultaneous quantification of blood drug levels using a new sample pretreatment technique.