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This paper describes the development of a normal-phase liquid chromatograph-UV-diode array detection-positive ion atmospheric pressure chemical ionization-mass spectrometry method for the simultaneous identification and quantitation of cholesterol, androst-4-ene-3,17-dione (AD), and androsta-1,4- diene-3,17-dione (ADD) in fermentation broths. The compounds detected under positive ion atmospheric pressure chemical ionization on a mass spectrometer by selected ion monitoring are separated by normal-phase high-performance liquid chromatography. [M + H] +  ions are taken into consideration for quantitation of AD and ADD, and [M-H 2 O+H] +  ions are considered for quantitation of cholesterol. The compounds are analyzed on a Si 60  silica (5 µm, 125 × 4-mm i.d.) Merck column using a 2:3 isocratic mixture of isopropyl alcohol and hexane. The calibration curves resulting from the reference compounds in the concentration range of 100-5000 pg on column exhibit a good linear correlation ( r  2  ≥ 0.996). The method is validated by analyzing six replicates of broth samples fortified with three compounds, namely, cholesterol, AD, and ADD, at 0.050 and 0.5 µg/g levels. The mean recoveries for the fortifications range from 90% to 98% with relative standard deviations in the range of 3.36% to 9.78%. The method is developed to study the qualitative as well as quantitative conversion of cholesterol to AD and ADD by a microorganism identified as  Nocardia sp . These studies helped the investigation of the reaction kinetics, which showed that the molar biotransformation of cholesterol into AD and ADD was 21%, even when the reaction was prolonged for 96 h.

journal of chromatographic science

Development of a Rapid Normal-Phase LC-Positive Ion APCI-MS Method for Simultaneous Detection and Quantitation of Cholesterol, Androst-4-ene-3,17-dione, and Androsta-1,4-diene-3,17-dione