Development and Performance Evaluation of a Tandem Mass Spectrometry Assay for 4 Adrenal Steroids

Abstract

Background:Congenital adrenal hyperplasia is a group of autosomal recessive disorders caused by a deficiency of 1 of 4 enzymes required for the synthesis of glucocorticoids, mineralocorticoids, and sex hormones. Analysis of 11-deoxycortisol (11DC), 17-hydroxyprogesterone (17OHP), 17-hydroxypregnenolone (17OHPr), and pregnenolone (Pr) in blood allows detection of these enzyme defects. Methods: The steroids were extracted from 200 L of serum or plasma by solid-phase extraction, derivatized to form oximes, and extracted again with methyl t-butyl ether. Instrumental analysis was performed on an API 4000 tandem mass spectrometer with electrospray ionization in positive mode and multiple reaction-monitoring acquisition. Results: The limits of detection were 0.025 g/L for 11DC, 17OHP, and Pr and 0.10 g/L for 17OHPr. The method was linear to 100 g/L for 11DC, 17OHP, and Pr, respectively, and to 40 g/L for 17OHPr. Within-and between-run (total) imprecision (CVs) were <7.1% and 11%, respectively. Reference intervals for children in Tanner stages 1 through 5 and adult males and females for 17OHP, 11DC, Pr, and 17OHPr were established. Prepared samples were stable for >72 h. Conclusions: The detection limit and selectivity of this method and its small sample volume requirement allow analysis of endogenous concentrations of adrenal ste-

Liquid chromatography-tandem mass spectrometry applications in endocrinology

“…As for other keto-steroids (Kushnir et al, 2006b), sensitivity of detection of Te can be enhanced with the use of oxime derivative (Kushnir et al, 2006a). A chromatogram of Te extracted from the serum of a healthy woman is shown in Figure 6, and the method's performance characteristics are listed in Table 2.…”

Steroid measurement with LC–MS/MS in pediatric endocrinology

“…Despite structural similarities among steroids, the efficiency of soft ionization methods for their detection is very different. The ionization efficiency of each steroid must be studied and can be different using different ion sources and instruments (Janzen et al, 2008;Kushnir et al, 2004;Kushnir et al, 2006b;Vogeser et al, 2001a). Kushnir, for example, observed the highest absolute intensity for 17-hydroxyprogesterone with APPI followed by APCI and ESI.…”

“…Derivatization can enhance the ionization efficiencies, leading to higher sensitivity and specific detection (Higashi and Shimada, 2004). Improvements in sensitivity of detection could be achieved by the formation of oxime or picolinoyl derivatives, and by optimization of the sample preparation and ionization (Kushnir et al, 2006b;Kushnir et al, 2008;Yamashita et al, 2007).…”

“…The lack of specificity is particularly unfortunate in the newborn period and early infancy. LC-MS/MS methods have been developed for quantification (Etter et al, 2006;Kushnir et al, 2006b;Rauh et al, 2006;Turpeinen et al, 2005;Wudy et al, 2000). The discrepancy between the LC-MS/MS method and the immunoassay is highly variable.…”

“…Steroid profiles Guo et al, 2006;Guo et al, 2008;Janzen et al, 2008;Kao et al, 2001;Kushnir et al, 2006b) allow for the acquisition of more clinically useful data than can be obtained through the measurement of a single steroid. The spectrum of diagnostically important metabolites in a plasma steroid profile is particularly helpful for a correct diagnosis and is also useful for the differential diagnosis of nonclassical forms of CAH, which may be indistinguishable using clinical presentation and sonography alone.…”