What's in a whisker? High-throughput analysis of twenty-eight C(19) and C(21) steroids in mammalian whiskers by ultra-performance convergence chromatography-tandem mass spectrometry.

Obtaining longitudinal endocrinological data from free-ranging animals remains challenging. Steroid hormones can be extracted sequentially from non-invasively sampled biologically inert keratinous tissues, such as feathers, nails, hair and whiskers. However, uncertainty regarding the type and levels of steroids incorporated into such tissues complicates their utility in wildlife studies. Here, we developed a novel, comprehensive method to analyze fourteen C19 and fourteen C21 steroids deposited chronologically along the length of seal whiskers in a single, 6-minute chromatographic step, using ultra-performance convergence chromatography-tandem mass spectrometry. The limits of detection and quantification ranged from 0.01 to 2 ng/mL and from 0.1 to 10 ng/mL, respectively. The accuracy and precision were within acceptable limits for steroids at concentrations ≥2 ng/mL. The recovery (mean = 107.5% at 200 ng/mL), matrix effect and process efficiency of steroids evaluated, using blanked whisker matrix samples, were acceptable. The method was applied to the analysis of steroid hormone levels in adult female whisker segments obtained from southern elephant seals (Mirounga leonina), n = 10, and two fur seal species, Antarctic fur seals (Arctocephalus gazella; n = 5) and subantarctic fur seals (Arctocephalus tropicalis; n = 5), sampled between 2012 and 2017. In the whisker subsamples analyzed (n = 71), the median concentration of steroid hormones detected above the LOQ ranged from 2.0 to 273.7 pg/mg. This was the first extraction of multiple C19 and C21 steroids, including their C11-oxy metabolites, from the whiskers of mammals. Measuring hormones sequentially along the whisker lengths can contribute to our understanding of the impact of stress associated with environmental/climate changes that affect the health, survival of organisms, as well as to delineate the reproductive cycles of free-living mammals with cryptic life stages.

Lübcker N ，Bloem LM ，du Toit T ，Swart P ，de Bruyn PJN ，Swart AC ，Millar RP ... -  《-》

Analysis of 52 C(19) and C(21) steroids by UPC(2)-MS/MS: Characterising the C11-oxy steroid metabolome in serum.

The C11-oxy androgens have been implicated in the progression of many diseases and endocrine-linked disorders, such as polycystic ovarian syndrome (PCOS), congenital adrenal hyperplasia, specifically 21-hydroxylase deficiency (21OHD), castration resistant prostate cancer (CRPC), as well as premature adrenarche. While the C11-oxy C19 steroids have been firmly established in the steroid arena, the C11-oxy C21 steroids are now also of significance. The current study reports on a high-throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) method for the separation and quantification of 52 steroids in peripheral serum, which include the C11-oxy C19 and C11-oxy C21 steroids. Fifteen deuterium-labelled steroids were included for absolute quantification, which incorporates steroid extraction efficiency, together with one steroid and four non-steroidal compounds serving as quality controls (QC). The 15 min run-time per sample (16 min injection-to-injection time with an 8-step gradient) quantifies 68 analytes in a 2 µL injection volume. A single chromatographic step simultaneously identifies steroids in the mineralocorticoid, glucocorticoid and androgen pathways in adrenal steroidogenesis, together with steroid metabolites produced in the periphery, presenting an analytical method for the application of screening in vivo clinical samples. This study highlights cross-talk between the C11-oxy steroids, and describes the optimisation of multiple reaction monitoring required to measure steroids accurately. The limit of detection for the steroid metabolites ranged from 0.002 to 20 ng/mL and the limit of quantification from 0.02 to 100 ng/mL. The calibration range for the steroids ranged from 0.002 to 1000 ng/mL and for the QC compounds from 0.075 to 750 ng/mL. The method is fully validated in terms of accuracy (%RSD, <13%), precision (including inter-day variability across a three-day period) (%RSD, <16%), recovery (average 102.42%), matrix effect (ranging from -15.25 to 14.25%) and process efficiency (average 101.79%). The dilution protocol for the steroids, internal standards and QC compounds were validated, while the ion ratios of the steroid metabolites (%RSD, <16%) and QC compounds were monitored and the accuracy bias values (%RSD, <9%) were within acceptable limits. The method was subsequently used to quantify steroid levels in a cohort of healthy women. C11-oxy steroid metabolites produced as intermediates in steroidogenic pathways, together with end-products included in the method can potentially characterise the 11β-hydroxyandrostenedione-, C21- and C11-oxy backdoor pathways in vivo. The identification of these C11-oxy C19 and C11-oxy C21 intermediates would allow insight into active pathways, while steroid metabolism could be traced in patients and reference ranges established in both normal and abnormal conditions. Furthermore, conditions currently undefined in terms of the C11-oxy steroids would benefit from the analysis provided by this method, while the C11-oxy steroids could be further explored in PCOS, 21OHD, CRPC and adrenarche.

du Toit T ，van Rooyen D ，Stander MA ，Atkin SL ，Swart AC ... -  《-》

A high-throughput UPC(2)-MS/MS method for the separation and quantification of C(19) and C(21) steroids and their C11-oxy steroid metabolites in the classical, alternative, backdoor and 11OHA4 steroid pathways.

In the present study an ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) analytical method was developed and validated for the determination of 17 C19 and 14 C21 steroids, including C11-oxy C19 and C11-oxy C21 steroids. The limit of detection and limit of quantification ranged from 0.01 to 10 ng/mL and from 0.01 to 20 ng/mL, respectively, and the method shows the recovery, matrix effect and process efficiency of steroids isolated from a serum matrix to be within acceptable limits. Good accuracy, repeatability and reproducibility were also shown and the method provided excellent sensitivity and selectivity as stereoisomers and regioisomers were also resolved and quantified accurately. Clinical conditions such as congenital adrenal hyperplasia, polycystic ovary syndrome in females and disorders of sex development in neonates and in children, amongst others, are characterized by abnormal steroid levels. Steroid profiling is essential to accurately diagnose steroid levels in the above settings as well as in androgen excess or deficiency in adrenal-linked endocrine diseases. Our method, separating C19 and C21 steroids in a single chromatographic step, offers a reduced sample turnover rate in the clinical setting, while providing comprehensive steroid profiles of in vivo steroids in the nmol/L range. This is, to our knowledge, the first method reported to simultaneously separate C19 and C21 steroids, together with their C11-hydroxy and C11-keto metabolites -one which may hold promise in the identification of new steroid markers in steroid-linked endocrine diseases, in addition to profiling steroid metabolism and abnormal enzyme activity in patients.

du Toit T ，Stander MA ，Swart AC 《-》

Ultra-performance convergence chromatography tandem mass spectrometry analysis of adrenal and gonadal steroid hormones in southern white rhinoceros(Ceratotherium simum simum) faeces and serum.

Steroid hormone analysis is routinely undertaken in the assessment of stress response and reproductive function in the management of both captive and free-ranging wildlife species. Faecal samples have become the preferred sample type for analysis as collection is non-invasive and easily assessable. These investigations are generally aimed at aiding successful translocations, enhanced survival outcomes in captivity and improvement of reproductive rate. Immunoassays are the most common approach in the analysis of hormones, particularly in the case of the southern white rhinoceros (Ceratotherium simum simum). Non-specificity, attributed to structural similarity of steroid metabolites impedes accurate evaluations which can be eliminated by chromatographic techniques which are more specific, selective and provide comprehensive analyses. This study developed and validated three methods using ultra-performance convergence chromatography tandem mass spectrometry for the assessment of classical androgens, progestogens and adrenal steroids, as well as the C11-oxy androgens and C11-oxy progestogens in serum and faeces from white rhinoceros. The limit of detection and quantification were determined for each steroid, parameters such as accuracy (<19.8 % RSD) and precision (<20.2 % RSD) were established with recovery, matrix effect, and process efficiency within acceptable limits. Subsequent analysis of serum and faecal samples from five white rhinoceros identified novel steroids for the first time in this species. In addition to the classical adrenal steroids, the following C11-oxy steroids were detected in faecal samples: 11α-hydroxydihydroprogesterone (168 ng/g), 11α-hydroxyprogesterone (125.9 ng/g), 11β-hydroxyprogesterone (210.2 ng/g) and 11-ketoandrostenedione (3.3-19.6 ng/g) with 11-deoxycortisol being the major glucocorticoid (24.2-67.3 ng/g) together with 21-deoxycortisone (40.7 ng/g) and deoxycorticosterone (7.6-14.6 ng/g). In serum samples 11β-hydroxyandrostenedione (0.35-2.34 ng/mL) and 11β-hydroxytestosterone (0.18-1.62 ng/mL) were the predominant androgens with cortisol (5.8-20.5 ng/mL), the predominant glucocorticoid, while corticosterone, 18-hydroxycorticosterone and aldosterone were also detected. These methods can be applied independently to assess either androgens, progestogens, or adrenal steroid panels or in combination to assess the cohort of gonadal and adrenal steroids in faeces and/or serum, in southern white rhinoceros as well as other wildlife species. Analysis would enable the accurate assessment of reproductive health and stress responses while also distinguishing between stress and distress thus contributing to the conservation of wildlife species.

Gent R ，Barbier ID ，Atkin SL ，Newell-Fugate AE ，Swart AC ... -  《-》

High-throughput analysis of 19 endogenous androgenic steroids by ultra-performance convergence chromatography tandem mass spectrometry.

11-Oxygenated steroids such as 11-ketotestosterone and 11-ketodihydrotestosterone have recently been shown to play a putative role in the development and progression of castration resistant prostate cancer. In this study we report on the development of a high throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC(2)-MS/MS) method for the analysis of thirteen 11-oxygenated and six canonical C19 steroids isolated from a cell culture matrix. Using an Acquity UPC(2) BEH 2-EP column we found that UPC(2) resulted in superior selectivity, increased chromatographic efficiency and a scattered elution order when compared to conventional reverse phase ultra-performance liquid chromatography (UPLC). Furthermore, there was a significant improvement in sensitivity (5-50 times). The lower limits of quantification ranged between 0.01-10ngmL(-1), while the upper limit of quantification was 100ngmL(-1) for all steroids. Accuracy, precision, intra-day variation, recovery, matrix effects and process efficiency were all evaluated and found to be within acceptable limits. Taken together we show that the increased power of UPC(2)-MS/MS allows the analyst to complete in vitro assays at biologically relevant concentrations for the first time and in so doing determine the routes of steroid metabolism which is vital for studies of androgen responsive cancers, such as prostate cancer, and could highlight new mechanisms of disease progression and new targets for cancer therapy.

Quanson JL ，Stander MA ，Pretorius E ，Jenkinson C ，Taylor AE ，Storbeck KH ... -  《-》