Ultrafast determination of drugs in biological samples by column-switching LC/MS

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Theophylline and caffeine are natural alkaloids widely being ingested, and show various pharmacological effects in humans. Caffeine is a constituent of coffee and other beverages and included in many medicines. Theophylline is used as a bronchodilator and also formed as a metabolite of caffeine in humans ; theophylline poisoning is occasionally encountered in the field of forensic toxicology, because of its relatively narrow safe range of therapeutic doses. They have been analyzed by various methods, such as gas chromatography (GC), GC-mass spectrometry (MS), high-performance (HP) liquid chromatography (LC)-MS. In the existing analytical methods for the drugs, the sample pretreatments such as liquid-liquid extraction, solid-phase extraction, filtration or centrifugation were usually required to remove proteins and other impurities contained in biological matrices. The analytical methods including time-consuming multi-step sample pretreatments are unsuitable for rapid detection of poisonous materials in human specimens. To avoid such sample pretreatments, special HPLC columns, which enabled direct injection of crude biological samples, were developed ; our group applied them to compounds of forensic toxicological interest. Furthermore, backflush column-switching system using one of special HPLC columns have become a powerful analytical tool for simultaneous determinations of various drugs in biological samples. In this study, we have established an automated on-line method for ultra-fast determination of theophylline and caffeine in human serum by HPLC-MS with backflush column-switching and a monolithic separation column. To our knowledge, our method presented in this study is the fastest MS determination technique for theophylline and caffeine in human serum using the conventional HPLC instrument.