An improved micro-method for the measurement of steroid profiles by APPI-LC–MS/MS and its use in assessing diurnal effects on steroid concentrations and optimizing the diagnosis and treatment of adrenal insufficiency and CAH

Abstract

Our goals were to 1) develop an improved micro-method usable for neonates for steroid profile measurements and a method to measure androsterone, a key steroid in the recently described androgen backdoor pathway together, with dehydroepiandrosterone and 2) to assess if dehydroepiandrosterone diurnal concentration fluctuations exist potentially necessitating strict adherence to time of blood sample draw and requirement of separate time-dependent reference intervals. Liquid chromatography-tandem mass spectrometry was performed with an atmospheric pressure photoionization source [1]. For each sample 50 μL (100 μL for the backdoor pathway) of serum was deproteinized by adding 75 μL (150 μL for the backdoor pathway) of acetonitrile containing the internal standards. After centrifugation, 75 μL (150 μL for the backdoor pathway) of supernatant was diluted with 250 μL of water and injected onto a Poroshell 120 EC-C8 column (SB-C8 column for the backdoor pathway). Within-run coefficients of variation ranged from 2.4–10.4% and between-day coefficients of variation from 2.9–11.2%. Comparison studies yielded correlation coefficient between 0.97 and 1.00 with recoveries of 90% or greater. Our methods analyze a 9 steroid profile and an additional 2 steroid profile (backdoor pathway) with minimal sample volume (usable in neonates optimizing early diagnosis of endocrinopathies and genetic diseases). Low limits of quantitation make these methods ideal for steroid measurement in women and prepubertal children. As diurnal variations of dehydroepiandrosterone and other steroids [2] concentrations are clinically significant we recommend that separate reference intervals be developed for 8 am, 8 pm, and midnight sample draws. The use of this approach in improving the diagnosis of patients with adrenal insufficiency and congenital adrenal hyperplasia is discussed.

Diurnal variation of steroid hormones and their reference intervals using mass spectrometric analysis

“…Diurnal variations in the serum concentration of steroid hormones and their metabolism, as assessed by urinary excretion, have long been known for cortisol and testosterone ( 1 , 2 , 3 ). Less information exists for the other up or downstream hormones within the steroid synthesis pathway ( 4 , 5 , 6 ). A recent preliminary study comparing 8 AM and midnight samples on a small cohort of controls, suggests that significant diurnal variation in all but progesterone probably does exist ( 7 ).…”

“…The use of high-performance liquid chromatography (HPLC) in conjunction with tandem mass spectrometry (MS) is increasingly becoming the method of choice for clinical testing of hormone levels ( 5 , 6 , 15 , 16 , 17 ). Given its high sensitivity, specificity and precision for the identification and detection of analytes, its use is fundamental to disease diagnosis, management and treatment outcomes.…”

“…Here, we demonstrate significant diurnal fluctuations of most of these aforementioned steroids in a suitable cohort of healthy controls to emphasize the need to maintain strict adherence to time of blood sample draw and requirement of time-dependent reference intervals. Simultaneous measurement of nine steroid hormones (cortisone, cortisol, corticosterone, 11 deoxycortisol, androstenedione, testosterone, 17a-hydroxyprogesterone, DHEA and progesterone) was performed using our previously published micro HPLC–MS/MS methods ( 6 , 7 ). Diagnostic reliability is an important advantage gained by combining information for multiple steroids.…”

11‐Deoxycortisol may be Superior to Cortisol in Confirming a Successful Adrenal Vein Catheterization without Cosyntropin: A Pilot Study

“…The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to quantify nine steroids as described in our recent paper [16]. These steroids included cortisone, corticosterone, cortisol, 11-deoxycortisol, androstenedione, 17α-hydroxyprogesterone (17-OHPG), testosterone, progesterone and dehydroepiandrosterone (DHEA).…”

Acute Statin Administration Reduces Levels of Steroid Hormone Precursors

“…For the nine-steroid panel method, 1 ml aliquots of plasma samples were prepared with internal standards for LC-APPI-MS/MS analysis as previously described [16]. Compounds measured in plasma by 6490 Triple Quadrupole mass spectrometer with iFunnel technolo-gy (Agilent) operated in positive mode under multiple-reaction monitoring (MRM) conditions included: cortisol, cortisone, 11-deoxycortisol, corticosterone, androstenedione, total testosterone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, and cholesterol [16]. Plasma samples from 2 of the 14 subjects were not evaluated for cholesterol due to insufficient sample volume.…”