A new method was developed for the identification and determination of L-ergothioneine in cosmetics based on ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The pretreatment method, chromatographic column, chromatographic conditions, and mass spectrometric conditions of cosmetic samples were optimized. Methanol was chosen as the extraction solvent, 85% acetonitrile with 0.1% FA was selected as the mobile phase, and the Waters CORTECS UPLC hydrophilic interaction liquid chromatography (HILIC) column was chosen for the separation. The sample was extracted with methanol and filtered, then separated by HILIC and detected by triple-quadrupole mass spectrometry. The quantitation was done under the matrix calibration curve using the external standard method. The results showed good linear relationships in the range of 5–200 ng/mL, and the correlation coefficient was greater than 0.999 in cosmetic samples. The limit of detection was in the range of 25–50 μg/kg and the limit of quantitation was in the range of 50–100 μg/kg. The recoveries of the method spiked ranged from 85.3% to 96.2% and the relative standard deviation (RSD) was in the range of 0.84%–2.08% (n = 6). The method is simple, quick, and accurate for the determination of L-ergothioneine in cosmetics, and has great practical value.
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