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.
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.
Khajuria
Ravi K
RK
Natural Products Chemistry Division, Regional Research Laboratory, Canal Road, Jammu-180001, India. khajuriark@yahoo.com
Bhardwaj
Vikram
V
Gupta
Rajinder K
RK
Sharma
Preeti
P
Somal
Priti
P
Mehta
Pradeep
P
Qazi
Ghulam N
GN
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
D000730:Androstadienes; C006573:1,4-androstadiene-3,17-dione; D000735:Androstenedione; D002784:Cholesterol