Determination of steroid hormones in blood by GC–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.     

Comparison of multiple API techniques for the simultaneous detection of microconstituents in water by on‐line SPE‐LC‐MS/MS

This study described a fully automated method using on‐line solid phase extraction of large volume injections coupled with high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) to simultaneously detect a group of recalcitrant microconstituents (pharmaceuticals and personal care products, steroid hormones and sterols) in aqueous matrices. Samples (1 mL to 20 mL) were loaded to the preconcentration column at 1 mL/min, and the column was washed with 1000 μL of 25% methanol in LC/MS water to remove polar and ionic interferences before LC‐MS/MS analysis. Three different atmospheric pressure ionization (API) techniques, including photoionization (APPI) with four different dopants (acetone, anisole, chlorobenzene and toluene), heated electrospray ionization (HESI) and atmospheric pressure chemical ionization (APCI), were evaluated on the basis of method detection limits (MDLs) and recoveries from different aqueous matrixes. Results indicated that APPI with toluene as dopant was the most sensitive ionization method for the majority of the analytes. When using 5 mL of sample, MDLs for pharmaceuticals and personal care products, including carbamazepine, DEET, caffeine, naproxen, acetaminophen and primidone, were between 0.3 ng/L and 15 ng/L. MDLs of hormones, including testosterone, equilenin, progesterone, equilin, 17β‐estradiol, 17α‐ethynylestradiol, estrone, androsterone, mestranol and estriol, were between 1.2 ng/L and 37 ng/L. The combination of APPI with dopant allowed the detection of two difficult to ionize fecal related sterols, such as coprostan‐3‐ol and coprostan‐3‐one with MDLs of 5.4 ng/L and 11 ng/L, respectively. Calculated MDLs are more than adequate for analysis of wastewater using 1 to 5 mL sample size and for surface waters using up to 20 mL sample size. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of 15 steroidal oral contraceptives in water using solid-phase disk extraction followed by high performance liquid chromatography–tandem mass spectrometry

A rapid, accurate and sensitive method for simultaneous determination of 15 steroidal hormones including four estrogens (estrone, 17β-estradiol, 17α-ethynylestradiol, estriol) and eleven progestogens (17β-estradiol-3-benzoate, 19-norethindrone, gestodene, levonorgestrel, medroxyprogesterone, cyproterone acetate, megestrol-17-acetate, progesterone, norethindrone acetate, chlormadinone-17-acetate, and hydroxy progesterone caproate) in environmental waters was developed by coupling solid-phase disk extraction (SPDE) to ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) with electrospray ionization. Among three types of extraction tested (C₈ SPDE, C₁₈ SPDE and C₁₈ SPE), the most satisfactory result was achieved using C₁₈ SPDE for its satisfactory recovery (75.6 to 101.4%) and short extraction time (15 min for 1 L deionised water). The validity of this method was investigated and good analytical performance for all the analytes was obtained, including low limits of method detection (0.5–3.4 ng/L) and excellent linear dynamic range (1.0–50.0 ng/L). The method was applied to determine the steroidal hormones in 10 environmental waters including tap water, river water, lake water and waste water in Beijing. No progestogen was detected in all samples and estrone, estriol, 17α-ethynylestradiol were found in most samples at levels between 1.8 and 127.9 ng/L.     

Confirmation of anabolic hormone residues in bovine blood by micro-HPLC–ion spray–tandem mass spectrometry

Sensitive, specific analytical methods for the determination of an-abolics in biological matrices are essential to control the illegal use of these substances in food-producing animals. Programs of residue control are performed annually in Italy for the determination of endogenous sex hormones (17β-estradiol, progesterone, testoster-one) for which maximum physiological levels have been established. At present the methods used in the Italian programs to determine natural hormones in bovine blood are based on the sensitive radio-immunoassay (RIA), due to relatively low levels of these substances in plasma/serum. In this study, we report a new method based on tandem mass spectrometry with on-line micro-high performance liquid chromatography (micro-HPLC-MS-MS) using an atmos-pheric pressure ionization (API) source and an ion spray (IS) interface for the specific direct detection of natural (progesterone and testosterone), and synthetic (17β19-nortestosterone and me-droxyprogesterone) hormone residues in bovine serum. 17-Methyl-testosterone was used as the internal standard. Analytes were extracted with acetate buffer, purified on C18 Solid Phase Extrac-tion (SPE) cartridge and separated on a reverse phase C18 micro-HPLC column (300 mm × 1 mm, 5 μm), using acetonitrile-water, 80:20 (v/v) containing 2mM ammonium acetate as the mobile phase, at a flow rate of 10 μl/min. When anabolic hormones were ionized in the IS interface operating in the positive ion mode, only the protonated molecules, [M+H]⁺, were generated, without evidence of any fragmentation. These served as precursor ions for collision induced dissociation (CID) and Diagnostic daughter ions for each analyte were identified in order to carry out analysis by micro-HPLC-MS-MS in the selected reaction monitoring (SRM) mode. For the analytes in question, the response of the mass detector was related linearly to the quantity of each analyte injected between 10 and 300 pg, in the SRM mode. The limit of detection, based on a 3:1 signal-to-noise ratio, is 6–7 pg for the hormones. Recoveries were higher than 83% for 17β-19-nortestosterone, testosterone, and 17-methyltestosterone, and 72% for the medroxyprogesterone, and progesterone. The micro-HPLC-MS-MS method for the determi-nation of anabolic hormones in bovine blood requires no sample derivatization, minimal sample preparation, and provides a sensi-tive, selective, rapid alternative to the existing purification, separa-tion, and detection techniques. At present this very sensitive method is being successfully applied to measure bovine serum concentra-tions of natural hormones, such as testosterone and progesterone, in order to then confirm any illegal administration of these sub-stances.     

GC-MS determination of steroids related to androgen biosynthesis in human hair with pentafluorophenyldimethylsilyl-trimethylsilyl derivatisation

An efficient method for the simultaneous determination of eight steroids, androstenedione, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), testosterone, androsterone, etiocholanolone, progesterone and pregnenolone, in human hair by gas chromatography-mass spectrometry (GC-MS) using d3-testosterone as internal standard is described. The method involves alkaline digestion, liquid-liquid extraction and subsequent conversion to mixed pentafluorophenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive analysis in the selected ion monitoring (SIM) mode. This method showed good overall repeatability and reproducibility of 4.88-11.24 and 3.19-9.58%, respectively. For the first time, the quantification of DHT, DHEA and pregnenolone in human hair has been achieved by GC-MS, testosterone was also quantified. The detection of four steroids in hair samples was possible in the concentration range 0.12-8.45 ng g-1. The other four steroids, androstenedione, androsterone, etiocholanolone and progesterone, were not detected. The detection limits for SIM of the steroids varied in the range 0.02-0.5 ng g-1, and the SIM responses were linear with correlation coefficients varying from 0.991 to 0.996 for most of the steroids studied. The concentrations of the four steroids detected were different in male and female hair samples.     

Stable isotope labeling – Liquid chromatography/mass spectrometry for quantitative analysis of androgenic and progestagenic steroids

Steroid hormones play important roles in mammal at very low concentrations and are associated with numerous endocrinology and oncology diseases. Therefore, quantitative analysis of steroid hormones can provide crucial information for uncovering underlying mechanisms of steroid hormones related diseases. In the current study, we developed a sensitive method for the detection of steroid hormones (progesterone, dehydroepiandrosterone, testosterone, pregnenolone, 17-hydroxyprogesterone, androstenedione and 17α-hydroxypregnenolone) in body fluids by stable isotope labeling coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. In this respect, a pair of isotopes labeling reagents, Girard reagent P (GP) and d₅-Girard reagent P (d₅-GP), were synthesized and utilized to label steroid hormones in follicular fluid samples and steroid hormone standards, respectively. The heavy labeled standards were used as internal standards for quantification to minimize quantitation deviation in MS analysis due to the matrix and ion suppression effects. The ionization efficiencies of steroid hormones were greatly improved by 4–504 folds through the introduction of a permanent charged moiety of quaternary ammonium from GP. Using the developed method, we successfully quantified steroid hormones in human follicular fluid. We found that the contents of testosterone and androstenedione exhibited significant increase while the content of pregnenolone had significant decrease in follicular fluid of polycystic ovarian syndrome (PCOS) patients compared with healthy controls, indicating that these steroid hormones with significant change may contribute to the pathogenesis of PCOS. Taken together, the developed stable isotope labeling coupled LC-ESI-MS/MS analysis demonstrated to be a promising method for the sensitive and accurate determination of steroid hormones, which may facilitate the in-depth investigation of steroid hormones related diseases.     

Application of multiwall carbon nanotubes-based matrix solid phase dispersion extraction for determination of hormones in butter by gas chromatography mass spectrometry

The multiwall carbon nanotubes (MWCNTs)-based matrix solid phase dispersion (MSPD) was applied for the extraction of hormones, including 17-α-ethinylestradiol, 17-α-estradiol, estriol, 17-β-estradiol, estrone, medroxyprogesterone, progesterone and norethisterone acetate in butter samples. The method includes MSPD extraction of the target analytes from butter samples, derivatization of hormones with heptafluorobutyric acid anhydride-acetonitrile mixture, and determination by gas chromatography-mass spectrometry. The mixture containing 0.30 g graphitized MWCNTs and 0.10 g MWCNTs was selected as absorbent. Ethyl acetate was used as elution solvent. The elution solvent volume and flow rate were 12 mL and 0.9 mL min(-1), respectively. The recoveries of hormones obtained by analyzing the five spiked butter samples were from 84.5 to 111.2% and relative standard deviations from 1.9 to 8.9%. Limits of detection and quantification for determining the analytes were in the range of 0.2-1.3 and 0.8-4.5 μg kg(-1), respectively. Compared with other traditional methods, the proposed method is simpler in the operation and shorter in the sample pretreatment time.     

Trace analysis of 28 steroids in surface water, wastewater and sludge samples by rapid resolution liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-MS/MS) method, combined with solid-phase extraction, ultrasonic extraction and silica gel cartridge cleanup, was developed for 28 steroids including 4 estrogens (estrone (E1), 17β-estradiol (E2), 17α-ethynyl estradiol (EE2), diethylstilbestrol (DES)), 14 androgens (androsta-1,4-diene-3,17-dione (ADD), 17α-trenbolone, 17β-trenbolone, 4-androstene-3,17-dione, 19-nortestoserone, 17β-boldenone, 17α-boldenone, testosterone (T), epi-androsterone (EADR), methyltestosterone (MT), 4-hydroxy-androst-4-ene-17-dione (4-OHA), 5α-dihydrotestosterone (5α-DHT), androsterone (ADR), stanozolol (S)), 5 progestagens (progesterone (P), ethynyl testosterone (ET), 19-norethindrone, norgestrel, medroxyprogesterone (MP)), and 5 glucocorticoids (cortisol, cortisone, prednisone, prednisolone, dexamethasone) in surface water, wastewater and sludge samples. The recoveries of surface water, influents, effluents and sludge samples were 90.6-119.0% (except 5α-DHT was 143%), 44.0-200%, 60.7-123% and 62.6-138%, respectively. The method detection limits for the 28 analytes in surface water, influents, effluents and freeze-dried sludge samples were 0.01-0.24 ng/L, 0.02-1.44 ng/L, 0.01-0.49 ng/L and 0.08-2.06 ng/g, respectively. This method was applied in the determination of the residual steroidal hormones in two surface water of Danshui River, 12 wastewater and 8 sludge samples from two wastewater treatment plants (Meihu and Huiyang WWTPs) in Guangdong (China). Ten analytes were detected in surface water samples with concentrations ranging between 0.4 ng/L (17β-boldenone) and 55.3 ng/L (5α-DHT); twenty analytes in the wastewater samples with concentrations ranging between 0.3 ng/L (P) and 621 ng/L (5α-DHT); and 12 analytes in the sludge samples with concentrations ranging between 1.6 ng/g (E1) and 372 ng/g (EADR).     

Inhibitors of testosterone biosynthetic and metabolic activation enzymes

The Leydig cells of the testis have the capacity to biosynthesize testosterone from cholesterol. Testosterone and its metabolically activated product dihydrotestosterone are critical for the development of male reproductive system and spermatogenesis. At least four steroidogenic enzymes are involved in testosterone biosynthesis: Cholesterol side chain cleavage enzyme (CYP11A1) for the conversion of cholesterol into pregnenolone within the mitochondria, 3β-hydroxysteroid dehydrogenase (HSD3B), for the conversion of pregnenolone into progesterone, 17α-hydroxylase/17,20-lyase (CYP17A1) for the conversion of progesterone into androstenedione and 17β-hydroxysteroid dehydrogenase (HSD17B3) for the formation of testosterone from androstenedione. Testosterone is also metabolically activated into more potent androgen dihydrotestosterone by two isoforms 5α-reductase 1 (SRD5A1) and 2 (SRD5A2) in Leydig cells and peripheral tissues. Many endocrine disruptors act as antiandrogens via directly inhibiting one or more enzymes for testosterone biosynthesis and metabolic activation. These chemicals include industrial materials (perfluoroalkyl compounds, phthalates, bisphenol A and benzophenone) and pesticides/biocides (methoxychlor, organotins, 1,2-dibromo-3-chloropropane and prochloraz) and plant constituents (genistein and gossypol). This paper reviews these endocrine disruptors targeting steroidogenic enzymes.     

Investigation of the feeding effect on the 13C/12C isotope ratio of the hormones in bovine urine using gas chromatography/combustion isotope ratio mass spectrometry

The effect of the feeding on the 13C/12C isotope ratio of four endogenous steroid hormones testosterone (T), epi-testosterone (epi-T), dehydroepiandrosterone (DHEA) and etiocholanolone (ETIO) in bovine urine was investigated. An analytical method to determine the accurate isotope ratio was developed including an extensive clean up followed by enrichment of the analytes in two steps of HPLC fractionation. Feeding experiments with four young animals were performed using C3 and C4 plants (grass, maize silage, hay, etc.) over a time period of about 280 days. One cattle was used as a control animal with no change of its diet over the full period. The detection of the 13C/12C isotope ratio of the acetylated extracts was performed by gas chromatography/combustion isotope ratio mass spectrometry. After the first change of the feeding from C4 to C3 plants significant changes of the delta 13C % values were observed from the -19 to -23% level to the -24 to -32% level for etiocholanolone and epi-testosterone in urine of three animals, whereas the DHEA values remained under the level of the two metabolites. Testosterone could not be detected with GC-C-IRMS due to its low concentration in young animals. After the second change of the diet from C3 to C4 plants (after 222 days), the measured delta 13C % values have been stabilised at the original level. The results show that in case of the feeding with only C3 plants the endogenous delta values of -32% can be reached. In this case the contribution of exogenous material with a delta value of -32% could not be detected independently of the concentration. If the diet contains C4 plants the difference or the ratio of the delta 13C % values becomes the determinant in the discriminatory power. For validation of the method a human and a cattle were treated with testosterone and the delta 13C % values were measured in incurred human and cattle urine.     

Solid-phase extraction using molecularly imprinted polymer for selective extraction of natural and synthetic estrogens from aqueous samples

A method is proposed for the clean-up and preconcentration of natural and synthetic estrogens from aqueous samples employing molecularly imprinted polymer (MIP) as selective sorbent for solid-phase extraction (SPE). The selectivity of the MIP was checked toward several selected natural and synthetic estrogens such as estrone (E1), 17β-estradiol (β-E2), 17α-estradiol (α-E2), estriol (E3), 17α-ethinylestradiol (EE2), dienestrol (DIES) and diethylstilbestrol (DES). Ultrahigh pressure liquid chromatography (UHPLC) coupled to a TSQ triple quadrupole mass spectrometry (QqQ) was used for analysis of target analytes. The chromatographic separation of the selected compounds was performed in less than 2 min under isocratic conditions. The method was applied to the analysis of estrogens in spiked river and tap water samples. High recoveries (>82%) for estrone, 17β-estradiol, 17α-estradiol, estriol and 17α-ethinylestradiol were obtained. Lower but still satisfactory recoveries (>48%) were achieved for dienestrol and diethylstilbestrol. The method was validated and found to be linear in the range 50-500 ng L(-1) with correlation coefficients (R(2)) greater than 0.995 and repeatability relative standard deviation (RSD) below 8% in all cases. For analysis of 100-mL sample, the method detection limits (LOD) ranged from 4.5 to 9.8 ng L(-1) and the limit of quantitation (LOQ) from 14.9 to 32.6 ng L(-1). To demonstrate the potential of the MIP obtained, a comparison with commercially available C(18) SPE was performed. Molecularly imprinted SPE showed higher recoveries than commercially available C(18) SPE for most of the compounds. These results showed the suitability of the MIP-SPE method for the selective extraction of a class of structurally related compounds such as natural and synthetic estrogens.     

Development and application of a UHPLC‐MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum

A new, fast and simple analytical method that is able to identify and quantify simultaneously 17 steroid hormones and metabolites (pregnenolone, 17‐OH‐pregnenolone, progesterone, 17‐OH‐progesterone, androsterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisol, corticosterone, aldosterone, 11‐deoxycortisol, 11‐deoxycorticosterone, dihydrotestosterone, estrone and estradiol) has been developed in equine serum using the ultra‐high‐performance liquid chromatography–tandem mass spectrometry technique. A total of 400 µl of sample was deproteinized with 1000 µl of acetonitrile, evaporated, restored with 50 µl of a solution of 25% methanol and injected in ultra‐high‐performance liquid chromatography–tandem mass spectrometry triple quadrupole. The recovery percentage obtained by spiking the matrix at two different concentrations with a standard mixture of steroid hormones was in all cases higher than 85.60% and with the percentage of coefficient of variation lower than 8.37%. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.9922–0.9986, and the limits of detection and limits of quantification were in the range of 0.002–2 and 0.0055–5.5 ng ml⁻¹, respectively. The detected limit of quantification for testosterone (i.e. 50 pg ml⁻¹) is twofold lower with respect to its threshold admitted in geldings plasma (100 pg ml⁻¹ free testosterone). The high sensitivity and the quantitative aspect of the method permitted to detect most of the steroids in equine serum. Once validated, the method was used to quantify 17 steroid hormones in mare, stallion and gelding serum samples. The main steroids detected were corticosterone (range 37.25–51.26 ng ml⁻¹) and cortisol (range 32.57–52.24 ng ml⁻¹), followed by 17‐OH‐pregnenolone, dihydrotestosterone and pregnenolone. Copyright © 2016 John Wiley & Sons, Ltd.     

Ion-pair single-drop microextraction versus phase-transfer catalytic extraction for the gas chromatographic determination of phenols as tosylated derivatives

A sensitive ultra-performance liquid chromatography-electrospray tandem mass spectrometry method, combined with solid-phase extraction and silica cartridge cleanup, was established for nine androgens (androstenedione, 19-nor-4-androstene-3,17-diol, androsterone, epiandrosterone, testosterone, methyltestosterone, trenbolone, nandrolone, stanozolol) and nine progestogens (progesterone, 17alpha-hydroxyprogesterone, 21alpha-hydroxyprogesterone, 6alpha-methyl-hydroxyprogesterone, 17alpha,20beta-dihydroxy-4-pregnene-3-one, megestrol acetate, norethindrone, norgestrel, medroxyprogesterone acetate) in environmental waters. For the various water matrices considered, the overall method recoveries were from 78 to 100%, and no apparent signal suppression was found. The method detection limits for the eighteen analytes in the influent, effluent and surface water samples were 0.20-50, 0.04-20 and 0.01-12 ng/L, respectively. This method was used to analyze the residual androgens and progestogens in the wastewater and surface water samples from Japan, and ten analytes (0.03 (medroxyprogesterone acetate)-1441 ng/L (androsterone)) were detected in the wastewater samples, and four analytes (0.06 (progesterone)-0.46 ng/L (androstenedione)) were detected in the surface water samples.     

Determination of the exogenous character of testosterone in bovine urine by gas chromatography-combustion-isotope ratio mass spectrometry.

A new approach was developed in order to control testosterone abuse in animal production. A gas chromatographic-combustion-isotope ratio mass spectrometric (GC-C-IRMS) method was used to distinguish the exogenous character from the endogenous character of the main metabolites of testosterone (epitestosterone and etiocholanolone) in cattle urine. This method is based on a comparison between the carbon isotope ratio (13C/12C) of testosterone metabolites and those of testosterone endogenous precursors. After urinary steroid purification, extracts were acetylated with acetic anhydride and injected into the GC-C-IRMS system. In order to validate the method, testosterone enanthate was administered to a 4 year old cow. The 13C/12C isotope ratios of testosterone exogenous metabolites appeared to be significantly different to the 13C/12C precursor ratios and were detected until 3 weeks after the anabolic administration. These preliminary results appear to be promising for the difficult control of natural hormones in livestock.     

气相色谱-质谱法检测动物肌肉组织中残留的甾类同化激素

建立了不同动物肌肉中甾类同化激素残留的气相色谱/质谱（GC/MS）检测方法。样品经酶解、组织捣碎和超声提取后,用叔丁基甲醚萃取和反相固相萃取柱净化,进一步衍生后进行GC/MS分析,分别测定雄激素（表睾酮（ETS）、丙酸睾酮（PTS）、19-去甲基睾酮（17β-NT）、甲基睾酮（MTS）)和雌激素（雌二醇（17β-ES）、雌三醇（EST）、炔雌醇（EES）、雌酮（ESN）、苯甲酸雌二醇（BES)）。实验结果表明,ETS、17β-NT、MTS、PTS、BES、ESN、17β-ES、EES和EST的定量检测限为1.0~2.0 μg/kg。在2.0 μg/kg的定量检测低限添加水平,上述9种激素的平均回收率为62.5%~80.5%,相对标准偏差为12.5%~26.8%。该法可实现对样品进行灵敏、准确的定性定量分析。     

Development and optimisation of a dopant assisted liquid chromatographic-atmospheric pressure photo ionisation-tandem mass spectrometric method for the determination of 15+1 EU priority PAHs in edible oils

European food legislation defines a set of 16 polycyclic aromatic hydrocarbons (PAHs) as of high concern for human health. The EU set contains structurally very similar PAHs with ring numbers between 4 and 6, and so raises some separation aspects and problems, which were not experienced with traditionally analysed PAHs. Many of the currently applied gas chromatographic mass spectrometric (GC-MS) methods suffer from separation problems, while high performance liquid chromatography with fluorescence detection (HPLC-FLD) is neither capable of detecting the whole set of EU priority PAHs nor does it (compared to GC-MS) allow structural identification. In addition HPLC-FLD shows limitations with difficult matrices due to interferences. The aim of this paper is to fill this gap by describing a liquid chromatographic dopant assisted atmospheric pressure photo ionisation tandem mass spectrometric (LC-DA-APPI-MS/MS) method for the determination of 15+1 EU priority PAHs in edible oil, which complies with the requirements set by European food legislation. Measurements were performed in positive ion mode. Anisole at a flow rate of 30 μl/min was used as dopant. Sample preparation was performed offline by donor-acceptor complex chromatography (DACC). Compared to HPLC-FLD methods the presented method enables the determination of all 15+1 EU priority PAHs at the low μg/kg concentration range including less fluorescence active compounds like benzo[j]fluoranthene and indeno[1,2,3-cd]pyrene. By analysing four reference materials it could be demonstrated that this method provides accurate results and is sufficiently sensitive for food control purposes. Statistically significant differences between the reference values and the measured analyte contents were not found. The method performs well also for very complex samples. Repeatability relative standard deviations (RSDr) of the determination of the target PAHs in olive oil were for most analytes below 5%. The limit of detection (LOD) of the method met the requirement set by EU legislation (0.3 μg/kg).     

Application of bar adsorptive microextraction to determine trace organic micro-pollutants in environmental water matrices

The present work proposes the application of bar adsorptive micro-extraction (BAµE) coated with an N-vinylpyrrolidone polymer (NVP) combined with micro-liquid desorption (200 µL) followed by high-performance liquid chromatography with diode array detection (BAµE(NVP)-µLD/HPLC-DAD) for the determination of trace levels of emerging organic micro-pollutants in environmental water matrices. The model compounds selected include an antibacterial/antifungal agent (triclosan), two pharmaceuticals (carbamazepine and diclofenac) and two steroid hormones (17α-ethinylestradiol and 17β-estradiol), in which the latter three were recently included in the European Union watch list of substances to be monitored in the field of water policy. Assays performed on 25 mL of ultrapure water samples spiked at the 8.0 µg L^?1 level yielded average recoveries ranging from 81.9 to 102.4% for the compounds studied using optimised experimental conditions. The proposed analytical methodology demonstrated suitable detection limits (0.02–0.10 µg L^?1) and good linear dynamic ranges (0.1–20.0 µg L^?1) with determination coefficients higher than 0.9909. Using the standard addition method (SAM), the present analytical approach was applied on environmental water matrices, including surface, sea, river and groundwaters. The proposed method proved to be a suitable and alternative sorption-based static micro-extraction technique for monitoring trace levels of organic micro-pollutants in environmental water matrices.     

Ionic liquid foam floatation coupled with solid phase extraction for separation and determination of hormones by high-performance liquid chromatography

The foaming property of ionic liquids (ILs) was found and the factors that can influence foamability of the ILs were investigated. Based on the property of the ILs, the foam floatation-solid phase extraction (FF-SPE) was developed. The IL-based FF-SPE was applied to the extraction and concentration of steroid hormones, including corticosterone, 17-β-estadiol, 17-α-estradiol, 19-nortestosterone, estrone, testosterone, 17-α-hydroxyprogesterone, medroxyprogesterone, chloromadinon 17-acetate, norethisterone acetate, medroxyprogesterone-17-acetate, progesterone, 17-β-estradiol 3-benzoate and testosteron 17-propionate in water samples and then the steroid hormones were determined by high-performance liquid chromatography. The extraction and concentration were performed synchronously in 10 min. Some experimental conditions were examined and optimized. The recoveries ranged from 50.6% to 95.2% for lake water sample and from 53.4% to 98.7% for rain water sample. The precision ranged from 2.43% to 7.43% for the lake water sample and 2.07-7.01% for rain water sample. Based on the foaming property of ILs, the application of foam floatation should be widened.     

A sensitive and selective LC-MS/MS analysis coupled with an online sample enrichment technique for H295R steroidogenesis assay and its application in the investigation of the effect of sildenafil on steroidogenesis

An in vitro steroidogenesis assay using H295R human adenocarcinoma cells is a useful tool for the fast identification of compounds that affect the production of testosterone and 17β-estradiol. Selective and sensitive hormone measurement by liquid chromatography–tandem mass spectrometry (LC–MS/MS) can make this assay more reliable. Therefore, in the present study, a sensitive and selective method for the quantification of testosterone and 17β-estradiol in the H295R steroidogenesis assay was developed and fully validated using LC–MS/MS coupled with an online sample enrichment technique. To prove its usefulness, the method developed was applied to investigate the effect of sildenafil on steroidogenesis. Cell medium samples were diluted and prepared using solid-phase extraction. The samples were prepared on ice and were not kept for more than 30 min to prevent degradation of hormones. The extracts were dried, reconstituted, filtered, and analyzed by LC–MS/MS with polarity switching electrospray ionization. The validation results for selectivity, matrix effect, recovery, linearity, precision, and accuracy were satisfactory. The limits of detection for testosterone and 17β-estradiol were 5 and 10 pg/mL, respectively, and the limit of quantification for both testosterone and 17β-estradiol was 10 pg/mL, which was in accordance with the OECD guideline. No degradation was observed under the storage conditions for 7 and 14 days at -80 °C as well as after three freeze–thaw cycles, whereas 17β-estradiol was degraded after 1 h on ice during sample processing. The method developed was successfully used for the investigation of the effect of sildenafil on steroidogenesis. This method can be very useful for the initial selection of drugs with androgenic and/or estrogenic effects for specific purposes, e.g., in the selection of drugs that are used to reverse the effects of chemical castration.     

Multiple monolithic fiber solid‐phase microextraction based on a polymeric ionic liquid with high‐performance liquid chromatography for the determination of steroid sex hormones in water and urine

The development of a simple and sensitive analytical approach that combines multiple monolithic fiber solid‐phase microextraction with liquid desorption followed by high‐performance liquid chromatography with diode array detection is proposed for the determination of trace levels of seven steroid sex hormones (estriol, 17β‐estradiol, testosterone, ethinylestradiol, estrone, progesterone and mestranol) in water and urine matrices. To extract the target analytes effectively, multiple monolithic fiber solid‐phase microextraction based on a polymeric ionic liquid was used to concentrate hormones. Several key extraction parameters including desorption solvent, extraction and desorption time, pH value and ionic strength in sample matrix were investigated in detail. Under the optimal experimental conditions, the limits of detection were found to be in the range of 0.027–0.12 μg/L. The linear range was 0.10–200 μg/L for 17β‐estradiol, 0.25–200 μg/L estriol, ethinylestradiol and estrone, and 0.50–200 μg/L for the other hormones. Satisfactory linearities were achieved for analytes with the correlation coefficients above 0.99. Acceptable method reproducibility was achieved by evaluating the repeatability and intermediate precision with relative standard deviations of both less than 8%. The enrichment factors ranged from 54‐ to 74‐fold. Finally, the proposed method was successfully applied to the analysis of steroid sex hormones in environmental water samples and human urines with spiking recoveries ranged from 75.6 to 116%.