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 ... -  《-》

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 《-》

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 ... -  《-》

Profiling adrenal 11β-hydroxyandrostenedione metabolites in prostate cancer cells, tissue and plasma: UPC(2)-MS/MS quantification of 11β-hydroxytestosterone, 11keto-testosterone and 11keto-dihydrotestosterone.

Adrenal C19 steroids serve as precursors to active androgens in the prostate. Androstenedione (A4), 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT) are metabolised to potent androgen receptor (AR) agonists, dihydrotestosterone (DHT), 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). The identification of 11OHA4 metabolites, 11KT and 11KDHT, as active androgens has placed a new perspective on adrenal C11-oxy C19 steroids and their contribution to prostate cancer (PCa). We investigated adrenal androgen metabolism in normal epithelial prostate (PNT2) cells and in androgen-dependent prostate cancer (LNCaP) cells. We also analysed steroid profiles in PCa tissue and plasma, determining the presence of the C19 steroids and their derivatives using ultra-performance liquid chromatography (UHPLC)- and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). In PNT2 cells, sixty percent A4 (60%) was primarily metabolised to 5α-androstanedione (5αDIONE) (40%), testosterone (T) (10%), and androsterone (AST) (10%). T (30%) was primarily metabolised to DHT (10%) while low levels of A4, 5αDIONE and 3αADIOL (≈20%) were detected. Conjugated steroids were not detected and downstream products were present at <0.05μM. Only 20% of 11OHA4 and 11OHT were metabolised with the former yielding 11keto-androstenedione (11KA4), 11KDHT and 11β-hydroxy-5α-androstanedione (11OH-5αDIONE) and the latter yielding 11OHA4, 11KT and 11KDHT with downstream products <0.03μM. In LNCaP cells, A4 (90%) was metabolised to AST-glucuronide via the alternative pathway while T was detected as T-glucuronide with negligible conversion to downstream products. 11OHA4 (80%) and 11OHT (60%) were predominantly metabolised to 11KA4 and 11KT and in both assays more than 50% of 11KT was detected in the unconjugated form. In tissue, we detected C11-oxy C19 metabolites at significantly higher levels than the C19 steroids, with unconjugated 11KDHT, 11KT and 11OHA4 levels ranging between 13 and 37.5ng/g. Analyses of total steroid levels in plasma showed significant levels of 11OHA4 (≈230-440nM), 11KT (≈250-390nM) and 11KDHT (≈19nM). DHT levels (<0.14nM) were significantly lower. In summary, 11β-hydroxysteroid dehydrogenase type 2 activity in PNT2 cells was substantially lower than in LNCaP cells, reflected in the conversion of 11OHA4 and 11OHT. Enzyme substrate preferences suggest that the alternate pathway is dominant in normal prostate cells. Glucuronidation activity was not detected in PNT2 cells and while all T derivatives were efficiently conjugated in LNCaP cells, 11KT was not. Substantial 11KT levels were also detected in both PCa tissue and plasma. 11OHA4 therefore presents a significant androgen precursor and its downstream metabolism to 11KT and 11KDHT as well as its presence in PCa tissue and plasma substantiate the importance of this adrenal androgen.

du Toit T ，Bloem LM ，Quanson JL ，Ehlers R ，Serafin AM ，Swart AC ... -  《-》