Analysis of 17 α-hydroxyprogesterone in bloodspots by liquid chromatography tandem mass spectrometry

Abstract

Background: Monitoring of treatment for patients diagnosed with congenital adrenal hyperplasia (CAH) can be performed by measuring the concentration of 17a-hydroxyprogesterone (17OHP) in bloodspots collected on filter papers. A method is described here for measuring 17OHP by liquid chromatography tandem mass spectrometry (LCMSMS). Methods: 17OHP was extracted by liquid-liquid extraction and analysed by LCMSMS. The method was validated for sensitivity, specificity, linearity, recovery, ion suppression, precision and bias. Results: The standard curve was linear from 0 to 400 nmol/L. Intra-assay %CVs were <10 and inter-assay %CVs were <15 over the range 10-200 nmol/L. Limit of quantitation was 6 nmol/L. No ion suppression was detected. The only interfering compound detected was deoxycorticosterone, an intermediate steroid with the same molecular weight as 17a-hydroxyprogesterone. The method was more accurate and precise than an existing radioimmunoassay. There was poor correlation between the two assays. Conclusions: We have developed a sensitive and specific assay suitable for quantitation of 17OHP in bloodspots. This method performs better than radioimmunoassay and allows smaller samples to be used.

Measurement of 17-Hydroxyprogesterone by LCMSMS Improves Newborn Screening for CAH Due to 21-Hydroxylase Deficiency in New Zealand

“…LCMSMS removes interference from other steroids but can be prone to interference from other isobaric steroids, a number of which can be present in newborn bloodspots. 11-deoxycorticosterone, an aldosterone precursor, has previously been shown to interfere with 17OHP measurements by LCMSMS [9] when present in large quantities. Although concentrations are normally much lower than 17OHP and even absent in 21-hydroxylase deficiency, we did not note any interfering peaks in our method in any of our specimens.…”

“…Salter et al [ 9 ] and Janzen et al [ 10 ] found poor correlation between LCMSMS and radioimmunoassay in a small number of CAH patient samples. When comparing the two techniques, Boelen et al reported that LCMSMS measurements in neonatal samples were up to 75% lower than immunoassay [ 15 ], while Dhillon et al showed that the difference between LCMSMS and immunoassay was much wider in very low birth weight babies [ 16 ].…”

“…Our goal was to develop a simple and reliable method to measure 17OHP in bloodspots as a second-tier test to reduce the false positive rate of newborn screening for CAH in New Zealand. Correlation between LCMSMS and the immunoassay for 17OHP is reported to be poor in neonates [9] but better in 17OHP bloodspot reference material [10]. Therefore, our approach was to develop a LCMSMS method and determine its performance using 17OHP bloodspot reference material, proficiency testing samples from a quality assurance scheme and bloodspots enriched with 17OHP prepared in our laboratory.…”

The diagnosis of nonclassic congenital adrenal hyperplasia due to 21‐hydroxylase deficiency, based on serum basal or post‐ACTH stimulation 17‐hydroxyprogesterone, can lead to false‐positive diagnosis

“…It was proven in newborn screening, however, that the use of LC/MS‐SIM has much higher accuracy in 17OHP determination . It can also be applicable in adults for the purposes of NCCAH diagnosis as shown by Salter et al . Lack of correlation between basal serum 17OHP level and urinary 17OHP metabolites in our study may speak for the hypothesis of the role of immunoassay reliability in the assessment of 17OHP.…”