MSACL 

31. Quantification of 17-Hydroxyprogesterone from Dried Blood Spots using Liquid Chromatography Tandem Mass Spectrometry
Poster: Tue 2:00-3:00PM
Donald Mason
Waters
C Rossi 1, HA Brown 2, LJ Calton 2, SD Gillingwater2, D Mason4 and S Wilson3

1 University of Chieti-Pescara, Italy
2 Waters Corporation, Manchester, UK
3 Waters Australia
4 Waters Corporation, Beverly, MA
Introduction
Congenital Adrenal Hyperplasia (CAH) is caused by inherited defects in steroid biosynthesis, in particular 21-hydroxylase deficiency, the classical form of CAH. Hormonal imbalances are reflected in decreased levels of aldosterone and cortisol and excessive secretion of 17-hydroxyprogesterone (17-OHP) and androstenedione. Diagnosis of CAH is based on the quantification of 17-OHP, usually by immunoassay. Compared with other neonatal screening tests, the specificity of screening for 21-CAH by immunoassays is low, characterized by high false-positive results. This is due to cross-reactivity with steroids other than 17-OHP, especially in pre-term neonates and in critically ill newborns. We have developed a simple method to determine the levels of 17-OHP in a dried blood spot (DBS) using ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS).

Methods
Patient and control DBS samples equalling 6µL whole blood were extracted in acetone/acetonitrile. Extract was analysed by UPLC tandem quadrupole mass spectrometry (ACQUITY UPLC®, Xevo TQ) using multiple reaction monitoring acquisition. 17-OHP was quantified by calibration against DBS standards prepared at Glasgow Royal Infirmary from washed red blood cells (3-420 ng/mL). Method precision and accuracy was ensured using CDC proficiency testing DBS material measuring 25, 50 and 100 ng/mL.

Results
The assay was shown to be linear over each analyte concentration range with all correlation co-efficient (R2) >0.997. Inter and intra-day imprecision of the assay was <6.5%CV and measured values were ±10 % of target across the analytical range.

Conclusions
A method for quantification of 17-OHP in DBS over the range of the clinical assay with good linearity, sensitivity and precision has been developed.
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