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

Abstract Objective Drug 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. Methods The target analytes were acetaminophen, caffeine, flunitrazepam, 7-aminoflunitrazepam, risperidone, and phenobarbital. Internal standards (IS) used were acetaminophen- d 4 , caffeine- d 9 , diazepam- d 5 , and phenobarbital- d 5 . For solid-phase extraction, three types of Smart-SPE columns (AiSTI Science Co., Ltd.) were employed. 100 µL 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. Results Linearity 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%. Discussion The 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.