The development of an ultra performance liquid chromatography-coupled atmospheric pressure chemical ionization mass spectrometry assay for seven adrenal steroids.
An ultra performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (UPLC-APCI-MS) method was developed for the separation and quantification of adrenal steroid metabolites from heterologous expression media. Steroids were extracted by liquid-liquid extraction, separated on a Waters UPLC BEH C18 column, ionized by APCI, and detected using a triple quadrupole mass spectrometer in APCI positive mode with single ion monitoring. The incorporation of UPLC enabled the detection of seven structurally closely related steroids at between 5 and 40 ng/ml using run times of 11 min. The adrenal steroidogenic enzyme cytochrome P450 17-hydroxylase/17,20-lyase (CYP17) was expressed in the yeast Pichia pastoris and in nonsteroidogenic COS-1 cells, and used as a model system to evaluate the detection and quantification of adrenal steroid metabolites by UPLC-APCI-MS.
Storbeck
Karl-Heinz
KH
Department of Biochemistry, University of Stellenbosch, Stellenbosch 7602, South Africa.
Kolar
Norbert W
NW
Stander
Marietjie
M
Swart
Amanda C
AC
Prevoo
Désirée
D
Swart
Pieter
P
D000311:Adrenal Glands / Q000737:chemistry*; D000818:Animals; D001274:Atmospheric Pressure; D019556:COS Cells; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D013256:Steroids / Q000032:analysis*
D013256:Steroids