Feasibility of a liquid-phase microextraction sample clean-up and liquid chromatographic/mass spectrometric screening method for selected anabolic steroid glucuronides in biological samples

Anabolic androgenic steroids (AAS) are metabolized extensively in the human body, resulting mainly in the formation of glucuronide conjugates. Current detection methods for AAS are based on gas chromatographic/mass spectrometric (GC/MS) analysis of the hydrolyzed steroid aglycones. These analyses require laborious sample preparation steps and are therefore time consuming. Our interest was to develop a rapid and straightforward method for intact steroid glucuronides in biological samples, using liquid-phase microextraction (LPME) sample clean-up and concentration method combined with liquid chromatographic/tandem mass spectrometric (LC/MS/MS) analysis. The applicability of LPME was optimized for 13 steroid glucuronides, and compared with conventional liquid-liquid extraction (LLE) and solid-phase extraction (SPE) procedures. An LC/MS/MS method was developed for the quantitative detection of AAS glucuronides, using a deuterium-labeled steroid glucuronide as the internal standard. LPME, owing to its high specificity, was shown to be better suited than conventional LLE and SPE for the clean-up of urinary AAS glucuronides. The LPME/LC/MS/MS method was fast and reliable, offering acceptable reproducibility and linearity with detection limits in the range 2-20 ng ml(-1) for most of the selected AAS glucuronides. The method was successfully applied to in vitro metabolic studies, and also tested with an authentic forensic urine sample. For a urine matrix the method still has some unsolved problems with specificity, which should be overcome before the method can be reliably used for doping analysis, but still offering additional and complementary data for current GC/MS analyses.     

Feasibility of desorption atmospheric pressure photoionization and desorption electrospray ionization mass spectrometry to monitor urinary steroid metabolites during pregnancy

Steroids have important roles in the progress of pregnancy, and their study in maternal urine is a non-invasive method to monitor the steroid metabolome and its possible abnormalities. However, the current screening techniques of choice, namely immunoassays and gas and liquid chromatography–mass spectrometry, do not offer means for the rapid and non-targeted multi-analyte studies of large sample sets. In this study, we explore the feasibility of two ambient mass spectrometry methods in steroid fingerprinting. Urine samples from pregnant women were screened by desorption electrospray ionization (DESI) and desorption atmospheric pressure photoionization (DAPPI) Orbitrap high resolution mass spectrometry (HRMS). The urine samples were processed by solid phase extraction for the DESI measurements and by enzymatic hydrolysis and liquid–liquid-extraction for DAPPI. Consequently, steroid glucuronides and sulfates were detected by negative ion mode DESI–HRMS, and free steroids by positive ion mode DAPPI–HRMS. In DESI, signals of eleven steroid metabolite ions were found to increase as the pregnancy proceeded, and in DAPPI ten steroid ions showed at least an order of magnitude increase during pregnancy. In DESI, the increase was seen for ions corresponding to C18 and C21 steroid glucuronides, while DAPPI detected increased excretion of C19 and C21 steroids. Thus both techniques show promise for the steroid marker screening in pregnancy.     

Steroid and steroid glucuronide profiles in urine during pregnancy determined by liquid chromatography–electrospray ionization-tandem mass spectrometry

An ultra performance liquid chromatography–electrospray ionization-tandem mass spectrometry (UPLC–MS/MS) method was developed for the analysis of steroids and their glucuronides in urine samples. The method provides high sensitivity and fast analysis, as both steroids and their glucuronides can be analyzed directly without hydrolysis or complex sample preparation. The method was applied in profiling of targeted and nontargeted steroids and steroid glucuronides during pregnancy. The concentrations of 11 of 27 targeted steroids and steroid glucuronides and the concentrations of 25 nontargeted steroid glucuronides increased about 10–400 fold during the pregnancy. The concentrations of most of these 36 compounds began to increase in the first days of the pregnancy, increased gradually during the pregnancy, achieved a maximum in late pregnancy, and decreased sharply after delivery. Exceptionally, the concentrations of allopregnanolone and 17-hydroxypregnenolone started to increase later than those of the other steroids. Moreover, the concentrations of E2 glucuronides began to decrease one week before the delivery, in contrast to most of the steroids and steroid glucuronides, whose concentrations dropped sharply during the delivery. Concentrations of 34 compounds decreased noticeably when the subject was on sick leave owing a series of painful contractions. The results suggest that steroids and especially steroid glucuronides may provide a valuable diagnostic tool to follow the course of pregnancy.     

Electrospray and atmospheric pressure chemical ionization tandem mass spectrometric behavior of eight anabolic steroid glucuronides

Mass spectrometric and tandem mass spectrometric behavior of eight anabolic steroid glucuronides were examined using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in negative and positive ion mode. The objective was to elucidate the most suitable ionization method to produce intense structure specific product ions and to examine the possibilities of distinguishing between isomeric steroid glucuronides. The analytes were glucuronide conjugates of testosterone (TG), epitestosterone (ETG), nandrolone (NG), androsterone (AG), 5alpha-estran-3alpha-ol-17-one (5alpha-NG), 5beta-estran-3alpha-ol-17-one (5beta-NG), 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol (5alpha-MTG), and 17alpha-methyl-5beta-androstane-3alpha,17beta-diol (5beta-MTG), the last four being new compounds synthesized with enzyme-assisted method in our laboratory. High proton affinity of the 4-ene-3-one system in the steroid structure favored the formation of protonated molecule [M + H]+ in positive ion mode mass spectrometry (MS), whereas the steroid glucuronides with lower proton affinities were detected mainly as ammonium adducts [M + NH4]+. The only ion produced in negative ion mode mass spectrometry was a very intense and stable deprotonated molecule [M - H]- . Positive ion ESI and APCI MS/MS spectra showed abundant and structure specific product ions [M + H - Glu]+, [M + H - Glu - H2O]+, and [M + H - Glu - 2H2O]+ of protonated molecules and corresponding ions of the ammonium adduct ions. The ratio of the relative abundances of these ions and the stability of the precursor ion provided distinction of 5alpha-NG and 5beta-NG isomers and TG and ETG isomers. Corresponding diagnostic ions were only minor peaks in negative ion MS/MS spectra. It was shown that positive ion ESI MS/MS is the most promising method for further development of LC-MS methods for anabolic steroid glucuronides.     

Determination of estrogens and their conjugates in water using solid-phase extraction followed by liquid chromatography-tandem mass spectrometry

An analytical procedure for the determination of steroid estrogens and their conjugates was developed and applied to aqueous environmental samples. The analytes of 15 compounds were solid-phase extracted and fractionated into two fractions: one containing unconjugated (free) steroids and the other containing conjugates (sulfates and glucuronides). Identification and quantification were carried out using liquid chromatography coupled with tandem mass spectrometry. The recoveries for each compound ranged from 57 to 116% and reproducibilities represented as RSD ranged from 2.9 to 17%. Some of the sulfates and free compounds were detected in environmental samples, whereas most of the conjugates were below the detection limits.     

Extraction of steroid sulphates from plasma by use of quaternary ammonium salts

Conclusions Steroid sulphates are extracted from plasma in good yield by benzene/benzyltributylammonium chloride. Steroid glucuronides are not extracted. This method offers some advantages as compared with the direct solvolysis which is only to be recommended for the determination of steroid sulphates such as dehydroepiandrosterone sulphate which are present in plasma in higher concentrations. The coagulation of plasma proteins with ethanol has the disadvantage that steroid sulphates are adsorbed to the protein precipitated. Furthermore, there is no separation of steroid glucuronides. 17-Hydroxyprogesterone sulphate has not been determined until now in plasma.

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Extraktion von Steroidsulfaten aus dem Plasma mit Hilfe quaternärer Ammoniumsalze

     

Retention behavior of bile acid derivatives using cyclodextrin in the mobile phase in high-performance liquid chromatography

The retention behavior of 3-(1-anthroyl)bile acids together with bile acid glucuronides, sulfates, and 12-dehydro derivatives is examined by the addition of cyclodextrin to the mobile phase in reversed-phase high-performance liquid chromatography. The data suggest that the functional group at the 12 position of the steroid moiety may be the important factor for the formation of the inclusion complex from the solute and cyclodextrin. The separation of these bile acid derivatives is much improved by this inclusion chromatography.     

Synthesis of 1-beta-O-acyl glucuronides of diclofenac, mefenamic acid and (S)-naproxen by the chemo-selective enzymatic removal of protecting groups from the corresponding methyl acetyl derivatives

Using a straightforward chemo-enzymatic procedure, 1-beta-O-acyl glucuronides of three non-steroidal anti-inflammatory drugs, diclofenac (DF) 5, mefenamic acid (MF) 6 and (S)-naproxen (NP) 7, were prepared. Caesium salts of these carboxylic acid drugs reacted with commercially available methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate 4 to give exclusively the corresponding 1-beta-O-acyl glucuronides 8-10 in moderate yields. The protecting acetyl (for -OH group) and methyl ester (for -CO2H group) groups of each sugar moiety were easily removed to provide the corresponding free 1-beta-O-acyl glucuronides 1-3 in high yields. Deprotection was achieved through effective enzyme-catalysed chemo-selective hydrolyses of the acetyl groups using lipase AS Amano (LAS), and of the methyl ester group using esterase from porcine liver (PLE).     

Determination of sulfates and glucuronides of endogenic steroids in biofluids by high-performance liquid chromatography/orbitrap mass spectrometry

the possibility of selective determination of testosterone and epitestosterone glucuronides in urine by high-performance liquid chromatography/high-resolution mass spectrometry using solid phase microextraction on a meps cartridge was studied. the effect of the biological matrix on the spectra of conjugated steroids can be taken into account by using the spectra of conjugates recorded for urine samples after hydrolysis as reference spectra. the conditions of fragmentation in the ion source were optimized for separate analytes. this method was used for analyzing real samples with different testosterone/epitestosterone ratios. variations in conjugate contents and qualitative changes in the steroid profile of endogenic compounds were observed.     

Liquid chromatographic-mass spectrometric analysis of glucuronide-conjugated anabolic steroid metabolites: method validation and interlaboratory comparison

Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine is described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the AAS glucuronides was 89-100% and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts.     

Ultra high performance liquid chromatography tandem mass spectrometric detection of glucuronides resistant to enzymatic hydrolysis: Implications to doping control analysis

Controversial results have been reported in the literature regarding the behavior of two testosterone (T) metabolites (3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone) excreted after T administration. Due to their potential as biomarkers of T misuse, a UHPLC–MS/MS method for the direct quantification of these glucuronides was developed and validated. In addition, the main phase II metabolites of T that compose the steroid profile used for doping control purposes (glucuronides of T, epitestosterone, androsterone and etiocholanolone) were included. The method was found to be linear and with suitable LODs and LOQs for all metabolites. The average accuracies were between 86% and 120%, the RSDs for the intra- and inter-day precision were below 15% and 25% respectively. The method showed low matrix effect. Samples obtained before and after the administration of T were analyzed by both the developed UHPLC–MS/MS method and the GC–MS/MS method currently used by anti-doping laboratories. Relevant disagreements between the results obtained for 3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone quantitation were observed. These markers seemed to be more suitable for the screening of T misuse when detected by UHPLC–MS/MS. These discrepancies were further investigated in 50 urine samples from healthy volunteers. The two methods gave highly correlated results for all metabolites that are currently included in the athlete's steroid profile confirming the reliability of the UHPLC–MS/MS method. However, the quantification of 3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone, was only possible by using the UHPLC–MS/MS method since three interfering compounds were observed when performing the GC–MS/MS analysis with the most intense ion transitions. These results confirm the potential of the resistant glucuronides as biomarkers of T misuse. Additionally, they suggest that previously reported reference ranges for these metabolites should be reevaluated.     

Synthesis and chromatographic evaluation of molecularly imprinted polymers prepared by the substructure approach for the class-selective recognition of glucuronides

Two series of molecularly imprinted polymers (MIPs) for the class-selective recognition of glucuronides have been prepared by using lipophilic substructures of the target analyte as template molecule and potent host monomers against oxyanions, that are expected to establish a strong stoichiometric interaction with the single carboxylic group of the template. The polymers were tested as stationary phases in liquid chromatography for specific recognition. A preliminary investigation of the imprinting properties of eleven MIPs was carried out, by comparing the retention time of the template and of structurally related compounds on the MIP column with that on the corresponding non-imprinted polymer (NIP). The two polymers showing the best performance were selected to further test cotinine, mycophenolic acid, testosterone and their respective glucuronides as model compounds. The high specificity obtained against glucuronides and the different chemical structure of the parent drug make the two MIPs class-selective imprinted receptors, also suitable for SPE application.     

Cone voltage induced in-source dissociation of glucuronides in electrospray and implications in biological analyses

Glucuronidation is a common mechanism in drug metabolism. In-source dissociation of glucuronides in electrospray generates fragment ions identical to those of the precursor ions of the original drugs. The effect of experimental parameters on the process was investigated in the present study using both commercially available compounds and glucuronides generated from microsomal glucuronidation incubations. It was found that cone voltage was the most critical parameter contributing to in-source fragmentation of both O- and N-glucuronides, whereas both the desolvation temperature and the source temperature had little effect. Additionally, the extent of in-source dissociation varied for different glucuronides and could be minimized by lowering cone voltage. As demonstrated in real examples, minimizing in-source dissociation can lead to higher sensitivity in detecting glucuronides in biological samples. In addition, product ions resulting from in-source dissociation of glucuronides potentially interfere with accurate determinations of corresponding drug levels if chromatographic separation is not adequate. For cases in which chromatographic separation of glucuronides from the original drugs is not readily achieved or high-throughput analyses are desired, interference caused by in-source dissociation can usually be eliminated simply by using lower cone voltage. This approach has been proven to be effective in the analysis of more than 100 glucuronides generated from in vitro microsomal incubations.     

Quantification of appetite suppressing steroid glycosides from Hoodia gordonii in dried plant material, purified extracts and food products using HPLC-UV and HPLC-MS methods

High-performance liquid chromatography (HPLC)-UV and HPLC-Mass Spectrometry (MS) methods were developed for the quantitative analysis of the family of Hoodia gordonii steroid glycosides with appetite suppressing properties in dried plant material, in purified and enriched extracts and in various prototype food-products fortified with H. gordonii extracts. For solid materials, e.g. dried plants or for non-fatty foods, extraction of the steroid glycosides is performed using methanol. For products where the steroid glycosides are present in an oil matrix, direct injection of the oil after dilution in tetrahydrofuran is applied. The HPLC separation is performed on an octyl-modified reversed-phase column in the gradient mode with UV detection at lambda = 220 nm. Quantification is performed against an external calibration line prepared using either one of the pure steroid glycosides or geranyl-tiglate. Short- and long-term repeatabilities of the methods are better than 3 and 6%, respectively. Recoveries are better than 85%, even in the analysis of the least abundant steroid glycosides in a complex yoghurt drink. Linearity is better than 3-4 orders of magnitude and the detection limits are below approximately 2 microg g(-1) for the individual steroid glycosides in dried plant material and food products. HPLC-MS is used to confirm that the steroid glycosides contain the characteristic steroid core, the carbohydrate chain and the tigloyl group.     

Discovery of neurosteroid glucuronides in mouse brain

Neurosteroid glucuronides were found for the first time in brain samples. The intact glucuronides were extracted from the cortex, hippocampus, hypothalamus, and mid-brain tissues of nicotine- and water-treated mice, and detected with capillary liquid chromatography-electrospray-tandem mass spectrometry (CapLC-ESI-MS/MS). The glucuronides of estradiol, cortisol, corticosterone, tetrahydrodeoxycorticosterone, pregnenolone, and isopregnanolone were identified by comparing retention times in selected reaction monitoring (SRM) chromatograms and the relative abundances of two SRM transitions of each neurosteroid glucuronide between the reference and authentic samples, thus providing reliable identification. In vitro experiments, carried out by using S9 fractions from mouse and rat brains, showed a formation of glucuronides with selected test compounds (corticosterone, pregnenolone, and dehydroepiandrosterone), suggesting that biosynthesis of neurosteroid glucuronides is possible in rodent brain.     

Key fragments for identification of positional isomer pair in glucuronides from the hydroxylated metabolites of RT-3003 (Vintoperol) by liquid chromatography/electrospray ionization mass spectrometry.

The mass spectral properties of glucuronides of the 9- and 10-hydroxylated metabolites of RT-3003 (Vintoperol; (-)-1beta-ethyl-1alpha-hydroxymethyl-1,2,3,4,6,7, 12balpha-octahydroindolo[2,3-a]quinolizine), which were fractionated by high-performance liquid chromatography with fluorescence detection, were investigated using the positive ion electrospray ionization mode. These glucuronides showed predominantly the protonated molecular ion ([M + H](+) ion), and the [M + H](+) ion provided a characteristic product ion spectrum in which abundant ions were obtained at m/z 301, 160 and 142. The first ion, corresponding to the [aglycone + H](+) ion, was produced by neutral loss of the glucuronic acid moiety from the [M + H](+) ion. The product ion spectrum of the [M + H](+) ion of hydroxy-RT-3003 revealed a number of ions common to the glucuronide spectra, suggesting that other two ions observed most likely represent fragmentation of hydroxy-RT-3003. In turn, these glucuronides were positional isomers with respect to the binding site of glucuronic acid. The structures of the isomer pairs were discriminated by the presence of the ion of m/z 318 or 336 in the product ion spectrum. These ions were produced by fission of the C-ring, the same as for the formation of the ions of m/z 160 and 142, as were observed in the product ion spectrum from the [M + H](+) ion of hydroxy-RT-3003. For the formation of these ions, an unusual fragmentation process was proposed, and these ion structures were supported by evidence from the accurate mass measurement data. Additionally, in the sulfates of hydroxylated metabolites, a similar product ion corresponding to the ion of m/z 336 found in the phenolic glucuronides was observed, and was applied for identification of the sulfate metabolites.     

含羟基化合物及其葡萄糖醛酸苷类反相高效液相色谱法保留指数预测

采用体外孵有生物合成方法,获得葡萄糖醛酸耷类代谢物,并建立了其保留指数预测方法,在实验条件下,误差小于12％。     

Determination of urinary androgen glucuronides by capillary electrophoresis with electrospray tandem mass spectrometry

Capillary electrophoresis–electrospray tandem mass spectrometry (CE‐ESI/MS/MS) is a simple and highly sensitive method for quantifying seven urinary androgen glucuronides. The urine samples were diluted and filtered through a membrane filter, and the filtrate was injected into a CE‐MS/MS system without further sample preparation steps such as extraction and derivatization. The calibration ranges were 0.01–5 µg/mL for glucuronides of androsterone and 11β‐OHAn‐3G, and 5–500 ng/mL for glucuronides of 11‐ketoAn, DHEA, testosterone, epitestosterone and DHT. The linearity of the method was 0.992–0.998, and the limits‐of‐detection at a signal‐to‐noise ratio of 3 were 5–10 ng/mL. The coefficients of variation were in the range of 4.0–9.0% for intra‐day assay and 4.1–9.8% for inter‐day assay. The proposed method may be applicable to metabolic profiling in both quantitative and qualitative analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Quantitative gas chromatographic/mass spectrometric analysis of morphine glucuronides in human plasma by negative ion chemical ionization mass spectrometry

A sensitive and specific method for the determination of morphine glucuronides in human plasma is presented. Morphine glucuronides, namely morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G), were extracted from plasma by solid-phase extraction on C(18) cartridges at pH 9.3 and derivatized to their pentafluorobenzyl ester trimethylsilyl ether derivatives. The compounds were measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further purification. Using this detection mode, a diagnostic useful fragment ion at m/z 748 was obtained at high relative abundance for both target compounds. [(2)H(3)]-labeled morphine glucuronides were used as internal standards. Calibration graphs were calculated by polynomial fit within a range of 10-1280 and 15-1920 nmol l(-1) for the 6- and 3-glucuronide, respectively. At the limit of quantitation (LOQ), the inter-assay precision was 2.21% (M3G) and 2.23% (M6G) and the GC/MS assay variability was 1.8% (M3G) and 0.9% (M6G). The accuracy at the LOQ showed deviations of +4.92% (M3G) and +1.5% (M6G). The sample recovery after solid-phase extraction was 84.7% for both M3G and M6G. The method is rugged, rapid and robust and has been applied to the batch analysis of morphine glucuronides during pharmacokinetic profiling of the drugs.     

Designer drug 2,4,5-trimethoxyamphetamine (TMA-2): studies on its metabolism and toxicological detection in rat urine using gas chromatographic/mass spectrometric techniques

Studies are described on the metabolism and the toxicological detection of the amphetamine-derived designer drug 2,4,5-trimethoxyamphetamine (TMA-2) in rat urine using gas chromatographic/mass spectrometric (GC/MS) techniques. The identified metabolites indicated that TMA-2 was metabolized by oxidative deamination to the corresponding ketone followed by reduction to the corresponding alcohol, O-demethylation followed by oxidative deamination, and finally O,O-bis-demethylation. All metabolites carrying hydroxy groups were found to be partly excreted in urine as glucuronides and/or sulfates. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction, and microwave-assisted acetylation allowed the detection, in rat urine, of an intake of TMA-2 that corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure in human urine should be suitable as proof of an intake of TMA-2.