MSACL 2019 EU Abstract

A Liquid Chromatography Tandem-Mass Spectrometry Method for Determination of 9 steroids in Human Serum for Diagnosis of CAH and Steroidogenic Biosynthesis Defects

Xiaolin Wu, Chris Sies, Ian Phillips
Canterbury Health Laboratories, New Zealand

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Xiaolin Wu (Presenter) Canterbury Health Laboratory >> POSTER (PDF)

Presenter Bio: I obtained my PhD degree in Chemistry at University of Otago, New Zealand before I came to work as the Scientific Officer in the Endocrinology and Steroids Department at Canterbury Health Laboratories in Christchurch.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION: Congenital adrenal hyperplasia (CAH), is a disorder which affects the function of adrenal glands in producing one or more of the steroid hormones. The commonest causes of CAH are the 21-hydroxylase deficiency (95%) and other rarer forms such as 11-beta hydroxylase deficiency. The analysis of 17-hydroxyprogesterone, androstenedione and cortisol is the best screening test for CAH, while the analysis of corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, progesterone and testosterone can either be the adjunct to evaluation of 21-hydroxylase deficiency or the evaluation of defects of adrenal steroid biosynthesis.

OBJECTIVES: The primary objective of this study was to develop a LC-MS/MS method for these 8 steroids.

METHODS: A sensitive and selective analytical method was developed and validated for the determination of 17-hydroxyprogesterone, androstenedione, cortisol, corticosterone, 11-deoxycortisol, 21-deoxycortisol,11-deoxycorticosterone, progesterone and testosterone in human serum. Protein precipitation and liquid-liquid extraction was used for sample preparation, followed by liquid chromatography-electrospray ionization-tandem mass spectrometry (Agilent 1290 Infinity LC System and Agilent 6490 Triple Quadrupole Mass Spectrometer). Steroids were separated on an Agilent ZORBAX SB-C18 LC column (2.1mm × 50mm × 1.8µm) with the mobile phase: water (with 2% methanol and 0.2% formic acid): methanol (53:47, v/v, initial ratio) at a flow rate of 0.475 mL/min.

RESULTS: The method was validated for accuracy (<15%), precision (<12.5%), carryover (<5%), linearity (R2>0.99), recovery (80.09-103.27%), matrix effect and stability.

CONCLUSION: This validated method for the 9 above mentioned steroids was deemed fit for purpose to be routinely used as part of the diagnostic investigation of CAH and other steroid disorders.