An automated on-line method for simultaneous analysis of phenothiazines in human serum by high-performance liquid chromatography/sonic spray ionization mass spectrometry using backflush column switching

An automated on-line method for simultaneous analysis of five phenothiazine drugs by high-performance liquid chromatography (HPLC)/sonic spray ionization mass spectrometry (SSI-MS) has been established, using backflush column switching. A 400-μl portion of serum sample diluted 81-fold with distilled water was subjected to the on-line system. In the system, an Oasis HLB cartridge was used as the precolumn for extraction; large molecules such as proteins in serum were discarded by use of distilled water containing 0.1% formic acid as a mobile phase. After switching a valve, the analytes trapped in the precolumn were eluted in the backflush mode and separated by a Chromolith Performance RP-18e column, which is composed of C₁₈-bonded monolithic silica. The column effluents were then introduced into the SSI-MS. The present method provided successful separation and determination of six phenothiazines including an internal standard. Satisfactory linearities, reproducibility, and sensitivity were obtained at concentration levels that matched the toxic levels of phenothiazines. All drug peaks appeared within 18 min, and the system could be reequilibrated in only about 8 min for the next run. Because of the simplicity and rapidness of the method, it is likely to be useful in the fields of emergency medicine and forensic toxicology.