Abstract: A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and a high performance liquid chromatography (HPLC) method were established to simultaneously detect the contents of ergosterol and VD2 in white Hypsizygus marmoreus and methodological evaluation was performed to determine the linearity range, sensitivity and accuracy of the two detection methods under optimized detection parameters. The differences between the two detection methods in qualitative and quantitative analysis of ergosterol and VD2 extracted from samples by alcohol-alkali saponification reflux method were verified in order to provide technical support for the detection of effective bioactive components and the study of the conversion between ergosterol and VD2 in white H. marmoreus. In the HPLC-MS/MS method, the chromatographic peak retention times of ergosterol and VD2 were 4.303 min and 4.22 min, respectively, under optimized chromatographic conditions using Agilent SB-C8 Rapid Res column (2.1 mm × 50 mm, 3.5 μm) as the separation column. Ergosterol and VD2 were quantified by selecting m/z 379.3/125.3 and 397.3/125.3 as ion pairs under the multi-reaction monitoring (MRM) mode using an electrospray ionization (ESI) source operating in the positive ion mode. The results showed that the concentration and peak area presented a good linear relationship for ergosterol and VD2 in the range of 0.15–6 mg/L and 0.01–1 mg/L, respectively. The average recoveries of ergosterol and VD2 from spiked samples were 93.51% and 90.56%, respectively, and the relative standard deviations (RSDs) of intra-day and inter-day differences were both less than 7%. In the HPLC method, the peak retention times of ergosterol and VD2 were 12.891 min and 9.919 min, respectively, under optimized separation conditions on a COSMOSIL 5C18-MS-II column (4.6 mm × 250 mm, 5 μm) . The methodology evaluation results of HPLC showed a good linear relationship between concentration and peak area for ergosterol and VD2 in the range of 15–750 and 0.5–50 mg/L, respectively. The average recoveries of ergosterol and VD2 from spiked samples were 98.51% and 94.05%, respectively, and the RSDs of intraday and inter-day differences were lower than 1%. Some differences existed between the two methods. Compared with HPLC, the HPLC-MS/MS method established in this research exhibited the advantages of short detection time, low detection limit and high sensitivity.

Key words: liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), high performance liquid chromatography (HPLC), ergosterol, VD2, synchronous detection, white Hypsizygus marmoreus