title: Development of a rapid normal-phase LC-positive ion APCI-MS method for simultaneous detection and quantitation of cholesterol, androst-4-ene-3,1 7-dione, and androsta-1,4-diene-3,17-dione.
abstract: 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-H2O+H]+ ions are considered for quantitation of cholesterol. The compounds are analyzed on a Si60 silica (5 microm, 125 x 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 (r2 > or = 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 microg/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.
authors.affiliation: Natural Products Chemistry Division, Regional Research Laboratory, Canal Road, Jammu-180001, India. khajuriark@yahoo.com
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Khajuria
authors.forename: Vikram
authors.initials: V
authors.lastname: Bhardwaj
authors.forename: Rajinder K
authors.initials: RK
authors.lastname: Gupta
authors.forename: Preeti
authors.initials: P
authors.lastname: Sharma
authors.forename: Priti
authors.initials: P
authors.lastname: Somal
authors.forename: Pradeep
authors.initials: P
authors.lastname: Mehta
authors.forename: Ghulam N
authors.initials: GN
authors.lastname: Qazi
mesh_terms: D000730:Androstadienes / Q000032:analysis*; D000735:Androstenedione / Q000032:analysis*; D001274:Atmospheric Pressure; D002784:Cholesterol / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000730:Androstadienes; C006573:1,4-androstadiene-3,17-dione; D000735:Androstenedione; D002784:Cholesterol