Simultaneous Determination of Diphenhydramine and Lidocaine in Antifungal Disinfection Products by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry

An aliquot (0.200 g) of antifungal disinfection product sample was taken, and mixed with 100 μL of 1.00 mg·L^-1 mixed internal standard solution of diphenhydramine-d₃ and lidocaine-d₁₀ by vortex. The mixture was settled for 30 min, and diluted to 5.00 mL with 70% (volume fraction) acetonitrile solution. The solution obtained was mixed by vortex for 2 min, and 1.0 mL of n-hexane solution saturated with acetonitrile and 1.0 mL of sodium chloride saturated solution were added. After ultrasonicating for 20 min, and vertexing for 15 min, the mixed solution was stored at -20℃ for 1 h. The upper n-hexane phase was discarded, and the middle layer phase was collected, and diluted by equal volume of water. After centrifuging for 5 min, the supernatant was taken and passed through a 0.22 μm filter membrane. The filtrate was determined by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), and quantified by internal standard method. In chromatographic analysis, Waters Equity UPLC^ BEH C₁₈ column was used as the stationary phase, and gradient elution separation was performed with the mixtures of 0.1% (volume fraction) formic acid solution and methanol solution at different volume ratios as the mobile phase. In mass spectrometry analysis, the positive ion mode of the electrospray ion source was used for ionization, and the multiple reaction monitoring mode was used for detection. It was shown that linear relationships between values of the mass concentration and the peak area ratio of the target component to the internal standard of diphenhydramine and lidocaine were kept in the range of 0.50-40.0 μg·L^-1, with detection limits (3S/N) of 8.12, 3.27 μg·kg^-1, respectively. Test for recovery was made by the standard addition method, giving results in the range of 88.5%-105%. Tests for intra-day and inter-day precision were performed on spiked negative samples, with RSDs (n=6) of the peak area ratios less than 6.0%. The proposed method was applied for the analysis of 84 commercially available antifungal disinfection products. The total detection rate of diphenhydramine and lidocaine was 4.76%, of which the detection amount of diphenhydramine in one sample was 2.86×10⁶μg·kg^-1.