An automated sample pretreatment system for liquid–liquid extraction and its application in the analysis of four steroid hormones in human plasma

The aim of the current research was to develop a simple and rapid mass spectrometry-based assay for the determination of 15 steroid hormones in human plasma in a single run, which would be suitable for a routine practice setting. For this purpose, we designed a procedure based on the 2D-liquid chromatography-tandem mass spectrometry with a minimalistic sample pre-treatment. In our arrangement, the preparation of one sample takes only 10 min and can accommodate 40 samples per hour when tested in series. The following analytical run is 18 min long for all steroid hormones. In addition, we developed an independent analytical run for estradiol, significantly increasing the assay accuracy while taking an additional 10 min to perform an analytical run of a sample. The optimized method was applied to a set of human plasma samples, including chylous. Our results indicate the linearity of the method for all steroid hormones with squared regression coefficients R2 ≥ 0.995, within-run and between-run precision (RSD < 6.4%), and an accuracy of 92.9% to 106.2%. The absolute recovery for each analyzed steroid hormone ranged between 101.6% and 116.5%. The method detection limit for 15 steroid hormones ranged between 0.008 nmol/L (2.88 pg/mL) for aldosterone and 0.873 nmol/L (0.252 ng/mL) for DHEA. For all the analytes, the lowest calibration point relative standard deviation was less than 10.8%, indicating a good precision of the assay within the lowest concentration of interest. In conclusion, in this method article, we describe a simple, sensitive, and cost-effective 2D-LC/MS/MS method suitable for the routine analysis of a complex of steroid hormones allowing high analytical specificity and sensitivity despite minimal sample processing and short throughput times.

Magnetic molecularly imprinted polymers coupled with UPLC-MS/MS for simultaneous detection of 19 steroid hormones in human plasma.

Quantitative MALDI-MS assay of steroid hormones in plasma based on hydroxylamine derivatization

A gas chromatography-high resolution mass spectrometry (GC-HRMS) procedure for the simultaneous determination of 18 endogenous steroid hormones in blood plasma from teleost fish has been developed. Proteins were removed by precipitation in methanol and lipids were removed by a liquid–liquid extraction. The protein and lipid free extract was further purified by using two successive solid phase extraction (SPE) methods (C18 and NH2). The isolated steroid hormones were silylated with a mixture of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA): iodotrimethylsilane (TMSI):dithioerythritol (DTE) prior to determination with GC-HRMS working in selective ion recording mode. A volume of 200 µL blood plasma was sufficient for accurate determination of the steroid hormone levels, which enabled determination in individual fish. The detection limits ranged from 0.0003 to 0.2 ng g−1 blood plasma from perch (Perca fluviatilis), approximately 10 to 100 times lower then previously reported in this field. The recoveries for the entire procedure were in the range 58 to 150% with a variation, expressed as standard deviation (SD), below 10% with some exceptions. Despite the multi-step clean-up procedure, the intra-assay coefficient of variation, i.e. the within-day variation, for most steroid hormones was well below 14%. Finally, the procedure has been successfully applied to the determination of steroid hormones in blood plasma from female perch caught in two Swedish lakes.

Previous observations demonstrated that steroid hormones associate with plasma lipoproteins. The objective of this study was to estimate the relative importance of lipoproteins as steroid hormone binding agents in comparison to sex hormone binding globulin, corticosteroid binding globulin, and albumin in both normal and hyperlipidemic human plasma. The 16 steroid hormones and related metabolites included in the study were: androstanediol, androstenediol, androstenedione, androsterone, corticosterone, cortisol, dehydroepiandrosterone, deoxycorticosterone, dihydrotestosterone, estradiol, estriol, estrone, 17 alpha-hydroxyprogesterone, pregnenolone, progesterone, and testosterone. The binding activity of these 16 steroid hormones with purified high density lipoprotein (HDL), low density lipoprotein and very low density lipoprotein were separately evaluated by equilibrium dialysis incubations to yield 48 steroid hormone-lipoprotein combinations for further study. In incubations with HDL, six steroid hormones (androstenediol, dehydroepiandrosterone, dihydrotestosterone, estradiol, pregnenolone, and progesterone) were identified as non-equilibrium, apparently due to metabolic conversion of the steroid hormones. The metabolic activity for the three delta 5-3 beta hydroxy steroids and estradiol appears to be fatty acid esterification by lecithin:cholesterol acyltransferase. The computer program TRANSPORT, which was used to evaluate only the nonspecific steroid hormone-lipoprotein association levels in a 16 x 6 matrix at simultaneous equilibrium, indicated that lipoprotein-bound steroid hormones ranged from 1% for cortisol to 56% for pregnenolone in normal human blood. Simulated projections of the increase in nonspecific steroid hormone association with lipoproteins during hyperlipidemia are also presented. These results demonstrate how lipoproteins are likely to be important in the transport and metabolism of steroid hormones in human plasma.

Steroid hormones are associated in depth to cellular signaling, inflammatory immune responses, and reproductive functions, and their metabolism alterations incur various diseases. In particular, quantitative profiling of steroids in plasma of patients with gastric cancer can provide a vast information to understand development of gastric cancer, since both sex hormones and glucocorticoids might be correlated with the pathological mechanisms of gastric cancer. Here, we developed a gas chromatography-tandem mass spectrometry-dynamic multiple reaction monitoring (GC-MS/MS-dMRM) method combined with solid-phase extraction (SPE) and microwave-assisted derivatization (MAD) to determine 20 endogenous steroids in human plasma. In this study, MAD conditions were optimized with respect to irradiation power and time. The SPE enabled effective cleanup and extraction for profiling of steroid hormones in human plasma samples. The MAD could improve laborious and time-consuming derivatization procedure, since dielectric heating using microwave directly increase molecular energy of reactants by penetrating through medium. Furthermore, dMRM method provided more sensitive determination of 20 steroids, compared to traditional MRM detection. The limits of quantification of steroids were below 1.125 ng/mL and determination coefficients of calibration curves were higher than 0.9925. Overall precision and accuracy results were below 19.93% and within ±17.04%, respectively. The developed method provided sufficient detection sensitivities and reliable quantification results. The established method was successfully applied to profile steroid metabolism pathways in plasma of patients with chronic superficial gastritis (CSG), intestinal metaplasia (IM), and gastric cancer. Statistical significances of steroid plasma levels between gastric disorder groups were investigated. In conclusion, this method provided comprehensive profiling of 20 steroids in human plasma samples and will be helpful to discover potential biomarkers for the development of gastric cancer and to further understand metabolic syndrome.

A new, simple, rapid and sensitive preconcentration method was developed for separation of two neutral steroids (progesterone and testosterone) through an off-line extraction/preconcentration procedure using Au nanoparticles grafted on a 3-(trimethoxysilyl)-1-propanethiol modified magnetic nanoparticle adsorbent prior to their determination by HPLC-UV. The preconcentration step was optimized by a comprehensive study on the main factors affecting the extraction/preconcentration efficiency of the steroids such as pH value, amount of surfactant, amount of magnetic adsorbent, sample volume, desorption conditions and ionic strength. The validity of the method was investigated and good analytical performance was obtained including a wide dynamic range of 0.1–200 ng mL−1, low detection limits of 0.05 and 0.07 ng mL−1, and good precision (as RSD%) lower than 3.90 and 4.19% for progesterone and testosterone, respectively. The method was applied to determine the steroidal hormones in human plasma and urine samples. The levels were found to be 0.8 and 9.2 ng mL−1 for progesterone and 7.9 and 97.5 ng mL−1 for testosterone in plasma and urine samples, respectively, which are within the normal range reported in the literature.

Pre-analytical stability and physiological fluctuations affect plasma steroid hormone outcomes: A real-world study

Determination of Estrogens, Androgens, Progesterone, and Related Steroids in Human Plasma and Urine

Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian steroidogenesis. This paper presents two simple methods for the determination of the major steroid hormones in biological matrixes using liquid chromatography tandem mass spectrometry (LC-MS(2)). A novel method was developed for the determination of 14 steroids in the H295R in vitro assay without the need forsolid phase extraction (SPE) purification prior to LC-MS(2) analysis. The in vitro assay was validated by exposing H295R cells to prochloraz for inhibiting steroid hormone secretion and by exposing cells to forskolin for inducing steroid hormone secretion. The developed method fulfills the recommendations for the H295R assay suggested by the OECD. Furthermore, a simple off-line SPE methodology was developed for the necessary clean-up of in vivo assays. Samples, such as gonad tissue, plasma and serum, are complex biological matrixes, and the SPE methodology was optimized to remove salts and proteins prior to elution of target analytes. At the same time, lipophilic compounds were retained on the SPE cartridge during elution. This, combined with the multi-steroid LC-MS(2) method, made it possible to determine 10 steroids in male Sprague-Dawley rat gonad tissue. Furthermore, it was possible to quantify 6 steroids in the plasma. In general, the observed concentration of steroid hormones in plasma, testes, and H295R cell medium corresponded well with previous studies. The off-line SPE method was validated using spiked charcoal-stripped serum. Method recovery, accuracy, precision and robustness were all good. Instrument sensitivity was in the range of 55-530pg/mL (LLOQ).

Steroid hormones in plasma play an important role in reproductive cycles of animals especially during the final maturation stages. Steroid hormones synchronize gonad developments depending to fish species reproductive strategies. The wild Cyprinid fish, Kutum (Rutilus frisii kutum) is an ecologically and economically important fish species which inhabit in southern coastline of the Caspian Sea in Iran. Over the past few decades, natural reproduction of this species dramatically impaired due to the urbanization, civilization close to the land and shallow water in south western of the Caspian Sea. Therefore annual sex steroid hormones and gonads development were measured to assess the annual reproductive biology of female Kutum. In this study for the first time, our aims were to determine the annual variations in sex steroid hormones; 17 β Estradiol (E2), Progesterone (P) and testosterone (T) and gonad development of female Kutum. Our results showed that plasma steroid levels in females manifested in two phases in annual reproductive cycle; the resting phase (June - February) being characterized with the lowest level of steroid hormones and the peak reproduction activity phase (March–May) with simultaneously a significant increase in level of E2, P and T in plasma. Interestingly, comparing with other Teleost fish species the baseline level of E2 in plasma of Kutum during the resting phase to some extent was also huge. Increase in concentration of plasma E2 was in accordance with an increase of gonadosomatic index during spawning season. Our results contribute to our knowledge about the reproductive biology of Kutum and calls further long-term investigation.

Simultaneous quantification of endocannabinoids, oleoylethanolamide and steroid hormones in human plasma and saliva

Quantification of steroid hormones in low volume plasma and tissue homogenates of fish using LC-MS/MS

This paper presents the development, optimization and validation of a methodology to determine nine key steroid hormones (viz. pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol and 17β-estradiol) expressed in the steroidogenesis in biological fluids. The analytical method allows for the determination of steroid hormones in blood plasma and serum down to 0.08-0.16 ng/mL for estrogens, 0.20-0.36 ng/mL for androgens and 0.36-0.43 ng/mL for progestagens. These limits of detection were obtainable using a two-step solid-phase clean-up for fractionation and elimination of interfering lipids (fatty acids, phospholipids, glycerides and sterols) from the steroid hormones. The accuracy of the method was 50-112% in the range 0.10 to 2.00 ng/mL.

Determination of seven selected neuro- and immunomodulatory steroids in human cerebrospinal fluid and plasma using LC-MS/MS