Determination of six progesterones in milk by on-line cleanup liquid chromatography-tandem mass spectrometry

Abstract

A new method using TurboFlow on-line cleanup liquid chromatography combined with tandem mass spectrometry (MS/MS) has been developed for simultaneous determination of six progesterones including 19-norethindrone, 17alpha-hydroxyprogesterone, medroxyprogesterone, megestrol acetate, progesterone and melengestrol acetate in milk. Samples were extracted by acetonitrile. The analytes in extract were on-line purified directly on Cyclone-P purification column where the sample matrices were washed away. Subsequently, the analytes which were eluted from the extraction column onto Phenyl-Hexyl column were separated with a gradient elution, and detected with electrospray ionization mass spectrometry in the positive scan mode using multiple reaction monitoring (MRM). The isotope internal standards were employed for quantification. As a result, the linearities were satisfactory with the correlation coefficients of > 0.999 at concentrations ranging from 0.1 microg/L to 50 microg/L. Based on the repeated analysis of a known blank sample, the limit of quantification (LOQ) is 0.5 microg/kg. Average recoveries of the analytes fortified at three levels (1, 5 and 25 microg/kg) ranged from 90.8% to 107.5%, and the relative standard deviations (RSDs) ranged from 6.3% to 11.8%. This proposed method is simple, rapid, sensitive and highly selective, and can be applied to simultaneous identification an quantification of the six progesterones in milk.