Published:Journal of Chromatographic Science,
ISSN 0021-9665 Volume
45, Number 8, September 2007, pp. 519-523
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
Ravi K. Khajuria[1], Vikram Bhardwaj[1], Rajinder
K. Gupta[1], Preeti Sharma[2], Priti Somal[2], Pradeep Mehta[3],
and Ghulam N. Qazi[2]
[1]Natural Products Chemistry Division, Regional Research Laboratory,
Canal Road, Jammu-180001-(INDIA),
[2]Biotechnology Division,
Regional Research Laboratory, Canal Road, Jammu-180001-(INDIA),
and
[3]Department of Botany, Saugar University, M. P. India
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 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 (r2 ≥ 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.
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