Analysis of Quinocetone and its Four Metabolites in Swine Liver by Ultra–performance Liquid Chromatography Quadrupole Time–of–flight Mass Spectrometry

Abstract

A new method using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/ Q-TOF-MS) was developed for determination of quinocetone (QCT) and its major metabolites, 1–desoxyquinocetone (1– DQCT), 4–desoxyquinocetone (4–DQCT), 1,4–bisdesoxyquinocetone (DQCT) and methyl–3–quinoxaline–2–carboxylic acid (MQCA) in swine liver. Tissue samples were extracted by a two-step method. Firstly, all analytes except MQCA were extracted with ethyl acetate. Then a hydrochloric acid solution was added in the remained sediment for hydrolysis of combined MQCA. The MQCA in the acid extract was transferred to ethyl acetate by liquid-liquid extraction (LLE) approach. All the ethyl acetate extract were combined, evaporated, resolved in a phosphate buffer, and cleaned up using a Oasis MAX cartridges. The chromatographic separation of all the analytes was achieved in less than 5 min using UPLC. The identification and confirmation by accurate mass measurement and their different fragmentation pathways were performed on ESI-Q-TOF-MS (MS/MS). The quantitation was carried out in TOF mode using narrow window extracted ion chromatogram of each compound. In the range of 1-100 µg·kg–1, the calibration curve equations of MQCA, QCT, 4-DQCT, 1-DQCT and DQCT were y=0.7878x+10.49, y=4.656x+42.244, y=5.5825x+24.919, y=8.491x+21.195 and y=10.733x+160.72, respectively. And the relative coefficients of all calibration curves were higher than 0.98. The limit of detection and the limit of quantification were 1.4–4.8 µg·kg –1 and 4.6–15.9 µg·kg–1, respectively. The established method was validated for determination of incurred swine liver samples in an actual residue study.