Multisteroid LC–MS/MS assay for glucocorticoids and androgens and its application in Addison's disease

Abstract

ObjectiveLiquid chromatography–tandem mass spectrometry (LC–MS/MS) offers superior analytical specificity compared with immunoassays, but it is not available in many regions and hospitals due to expensive instrumentation and tedious sample preparation. Thus, we developed an automated, high-throughput LC–MS/MS assay for simultaneous quantification of ten endogenous and synthetic steroids targeting diseases of the hypothalamic–pituitary–adrenal axis and gonads.MethodsDeuterated internal standards were added to 85 μl serum and processed by liquid–liquid extraction. Cortisol, cortisone, prednisolone, prednisone, 11-deoxycortisol, dexamethasone, testosterone, androstenedione and progesterone were resolved by ultra-high-pressure chromatography on a reversed-phase column in 6.1 min and detected by triple-quadrupole mass spectrometry. The method was used to assess steroid profiles in women with Addison's disease (AD, n=156) and blood donors (BDs, n=102).ResultsPrecisions ranged from 4.5 to 10.1% relative standard deviations (RSD), accuracies from 95 to 108% and extraction recoveries from 60 to 84%. The method was practically free of matrix effects and robust to individual differences in serum composition. Most postmenopausal AD women had extremely low androstenedione concentrations, below 0.14 nmol/l, and median testosterone concentrations of 0.15 nmol/l (interquartile range 0.00–0.41), considerably lower than those of postmenopausal BDs (1.28 nmol/l (0.96–1.64) and 0.65 nmol/l (0.56–1.10) respectively). AD women in fertile years had androstenedione concentrations of 1.18 nmol/l (0.71–1.76) and testosterone concentrations of 0.44 nmol/l (0.22–0.63), approximately half of those found in BDs of corresponding age.ConclusionThis LC–MS/MS assay provides highly sensitive and specific assessments of glucocorticoids and androgens with low sample volumes and is suitable for endocrine laboratories and research. Its utility has been demonstrated in a large cohort of women with AD, and the data suggest that women with AD are particularly androgen deficient after menopause.

Multiplexed steroid profiling of gluco- and mineralocorticoids pathways using a liquid chromatography tandem mass spectrometry method

“…The lower limit of quantification was in agreement with the sensitivities previously described [14][15][16][17]19] and was sufficient enough to establish the diagnosis or to monitor the treatment of CAH in neonates using even a limited volume of serum [21]. The analytical measurement range of each steroid was adapted to each's particular clinical needs but sometimes samples containing high concentrations of abundant steroids such as cortisol, progesterone or 17OHP required dilution.…”

“…The development of steroidomic profiles is particularly helpful for differential diagnosis of non-classical forms of congenital adrenal hyperplasia (CAH) [12]. Previous reports have described LC-MS/MS methods for simultaneous determination of the main four steroids on newborn blood spots [13], eight steroids in children between birth and 18 years [14] and nine glucocorticosteroids and androgens in Addison's disease [15]. Steroid hormone profile-analyses, including aldosterone, were reported after a simple protein precipitation step [16], using the Biocrates extraction kit [17] or after a solid phase extraction from culture supernatants of human adrenocortical H295R cells [18].…”

A sensitive, simple and robust LC–MS/MS method for the simultaneous quantification of seven androgen- and estrogen-related steroids in postmenopausal serum

“…The UPLC column with more separation power potentially improves the specificity of the LC-MS/MS method. For some compounds having high ionization efficiency due to their large proton affinity such as testo, progesterone and 4-dione, the low limit of quantification (LLOQ) can be achieved at the 5 pg/mL level for Testo and 40 pg/mL for 4-dione without derivatization using LC-MS/MS [12]. For compounds such as estrone (E 1 ) and estradiol (E 2 ) having low ionization efficiency, the pg/mL level of the LLOQ can be best achieved with derivatization [13].…”

In the overnight dexamethasone suppression test, 1.0 mg loading is superior to 0.5 mg loading for diagnosing subclinical adrenal Cushing’s syndrome based on plasma dexamethasone levels determined using liquid chromatography-tandem mass spectrometry

“…The specified accuracy of measurement at 50 pg, the lower limit quantification (LLQ), was 80-120% [18], and the specified precision (the precisions below were shown coefficient validation) for human plasma samples was ±15%. The QC samples (three of each level, for a total of six samples) were analyzed before and after the human plasma.…”