3.1. Chromatography and mass spectrometry
We optimized the chromatographic conditions by using various proportions of methanol in water to achieve optimal peak separation and peak shape with little peak tailing in a short analysis time, but very poor chromatography was achieved. Acetonitrile and 0.1% formic acid in water (25:75, v/v) were employed. The final chromatographic conditions showed that PPD behaved similarly to RA in terms of retention and ionization, and was adopted as the IS.
Methanol was first tested as a pretreatment method for protein precipitation. However, methanol caused poor efficiency in extracting A1 in this study. Hence, we introduced isopropanol:ethyl acetate (50:50, v/v) to increase the efficiency of sample extraction, which provided cleaner extracts and higher IS recoveries. By adjusting the ratio of isopropanol and ethyl acetate (20:80, v/v) to extracts, the recovery rates were further increased without compromising extract cleanliness.
A more accurate response for the two analytes was achieved using the ESI source when compared with the response using the APCI source. In full scan precursor ion spectra, the most abundant ions of A1, RA, and IS should be deprotonated molecules with m/z 358, 359, and 164, respectively (Fig. 2). Parameters such as the ESI source temperature and flow rate of the desolvation gas have been enhanced to obtain the maximum response of deprotonated molecules. To obtain the maximum response of the compound fragments, collision-induced decomposition (CID) and the SRM collision gas pressure and energy were selected through the precursor → product ion transition. The product ion spectra revealed abundant ions in fragments at m/z 161 for RA and A1 and 146 for the IS. The characterization of the analyte fragments is shown in Fig. 3.