Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS.     

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS.     

The determination of organophosphonate nerve agent metabolites in human urine by hydrophilic interaction liquid chromatography tandem mass spectrometry

A sensitive, robust isotope dilution LC/MS/MS method is presented for the quantitative analysis of human urine for the alkyl methylphosphonic acid metabolites of five organophosphorus nerve agents (VX, rVX or VR, GB or Sarin, GD or Soman, and GF or Cyclosarin). The selective sample preparation method employs non-bonded silica solid-phase extraction and is partially automated. While working with a mobile phase composition that enhances the electrospray ionization process, the hydrophilic interaction chromatography method results in a 5-min injection-to-injection cycle time, excellent peak shapes and adequate retention (k'=3.1). These factors lead to limits of detection for these metabolites as low as 30 pg/mL in a 1-mL sample of human urine. The quality control data (15 and 75 ng/mL) demonstrate accurate (-0.5 to +3.4%) and precise (coefficients of variation of 2.1-3.6%) quantitative results over the clinically relevant urine concentration range of 1-200 ng/mL for a validation set of 20 standard and quality control sets prepared by five analysts over 54 days. The selectivity of the method is demonstrated for a 100-individual reference range study, as well as the analysis of relevant biological samples. The combined sample preparation and analysis portions of this method have a throughput of 288 samples per day.     

Analysis of amphetamines and metabolites in urine with ultra performance liquid chromatography tandem mass spectrometry

A simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and fully validated for the quantitative determination of seven amphetamines and metabolites in urine. The method was validated for selectivity, linearity, LOQ, LOD, imprecision, bias, analyte and processed sample stability, matrix effect, recovery, carryover and dilution integrity. A classic liquid–liquid extraction with ethyl acetate was used as sample preparation procedure. The compounds were separated on an Acquity UPLC HSS C₁₈ column in 6.8 min. The linear dynamic range was established from 25 to 500 ng/mL. The limit of quantification was fixed to the lowest calibrator level and the limit of detection ranged from 0.125 to 2.5 ng/mL. The method presented an excellent intra- and inter-assay imprecision and bias (<10.7%) at each measured concentration of two external quality controls (QC) and three “in house” QC. No matrix effects were observed and good recoveries (>70%) were obtained for all the compounds. No carryover was observed after the analysis of high concentrated samples (8000 ng/mL). The method was subsequently applied to authentic samples.     

Investigation of piceid metabolites in rat by liquid chromatography tandem mass spectrometry

There is considerable evidence that stilbenes provide health benefits. Trans-piceid is one of the major stilbenoid compounds in red wine and other plants. The purpose of this study is to investigate the metabolism of piceid in rats, including its conversion product by intestinal microflora in vitro and urinary metabolites. A HPLC-MS/MS method with electrospray ionization (ESI), negative ion mode and collision induced dissociation (CID), was used to elucidate the structures of the major metabolites of piceid. Three metabolites resveratrol, dihydropiceid and dihydroresveratrol were detected after incubating with gut microbiota for 5h. Four urinary metabolites of piceid were identified as resveratrol, dihydroresveratrol monosulfate, piceid monosulfate and piceid monoglucuronide.     

Simultaneous determination of six dialkylphosphates in urine by liquid chromatography tandem mass spectrometry

Dialkylphosphates (DAP) are urinary markers of the exposure to organophosphates pesticides. The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of the following DAP: dimethylphosphate (DMP), dimethythiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP). Dibutylphosphate (DBP) was used as internal standard. This method was based on a liquid-liquid extraction procedure, a chromatographic separation using an Inertsil ODS3 C18 column and mass spectrometric detection in the negative ion, multiple reaction monitoring (MRM) mode, following two ion transitions per compound. It yielded a limit of quantification of 2 microg/L for the six compounds and intra-assay coefficients of variation (CV%) lower than 20%. This method was applied to the analysis of urines samples from a small cohort of non-exposed volunteers. At least one of the six DAP was detected in each sample. This result confirmed the feasibility of a LC-MS/MS procedure for monitoring the general population exposure to some frequently employed organophosphate pesticides.     

Global analysis of metabolites in rat and human urine based on gas chromatography/time-of-flight mass spectrometry

Sediment in urine may contain low-molecular-weight compounds that should be included in the analysis. To date, no systematic investigation has addressed this issue. We investigated three primary factors that influence the extraction efficiency of metabolites during preparation of urine samples for metabolomic research: centrifugation, pH, and extraction solvents. Obtained with the use of gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) technique and principal component analysis (PCA), our results indicate that (1) conventional centrifugation causes an apparent loss of some metabolites, indicating that urine samples for metabolomic research should not be centrifuged before procedures are undertaken to recover the metabolites; (2) pH adjustment has a large impact on the recovery of metabolites and is therefore not encouraged; (3) with design of experiment analysis, methanol and water yield the optimal extraction efficiency. Differences between rat and human urine were observed and are discussed. Ninety-nine metabolites identified in rat and human urine are presented. An efficient protocol is proposed for the pretreatment of urine samples.     

Quantification of dinor, dihydro metabolites of F2-isoprostanes in urine by liquid chromatography/tandem mass spectrometry

The F2-isoprostanes (F2-IsoP) are a series of prostaglandin (PG)-F2-like compounds that are produced by free-radical-mediated oxidation of arachidonic acid. One F2-IsoP with potent biological activity is 15-F2t-IsoP and increased levels of 15-F(2t)-IsoP have been measured in several diseases. The major urinary metabolite of 15-F2t-IsoP (8-iso-PGF(2alpha)) is 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M). Previously, we developed a stable isotope dilution gas chromatography/negative chemical ionization/mass spectrometry (MS) assay for 15-F2t-IsoP-M, which, while highly sensitive, required time-consuming derivatization and thin-layer chromatography purification. We now report the development of a more rapid high-performance liquid chromatography method coupled to electrospray ionization-tandem mass spectrometry (LC/MS/MS) to analyze all of the dinor,dihydro metabolites of the F2-IsoP isomers (F2-IsoP-M). The precision of this assay was +/-5.0% and the accuracy 80%. The assay remained linear over a range of 1-100 ng injected onto the LC column. Levels of F2-IsoP-M determined by the LC/MS/MS assay method significantly correlated with levels of 15-F2t-IsoP-M determined by the GC/MS assay (R = 0.77y = 67.2x-0.5). The levels of F2-IsoP-M detected in spot urines from 40 normal subjects were 38.1+/-19.1 ng/mg creatinine (mean+/-SD). This method provides an accurate and rapid assay to assess oxidative status in vivo.     

Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated frrm normal human urine was acheived by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urin.     

Quantitative analysis of cocaine and its metabolites in whole blood and urine by high-performance liquid chromatography coupled with tandem mass spectrometry

AIM: In forensic toxicology it is important to have specific and sensitive analysis for quantification of illicit drugs in biological matrices. This paper describes a quantitative method for determination of cocaine and its major metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and urine by liquid chromatography coupled with tandem mass spectrometry LC/MS/MS. METHOD: The sample pre-treatment (0.20 g) consisted of acid precipitation, followed by centrifugation and solid phase extraction of supernatant using mixed mode sorbent columns (SPEC MP1 Ansys Diag. Inc.). Chromatographic separation was performed at 30 degrees C on a reverse phase Zorbax C18 column with a gradient system consisting of formic acid, water and acetonitrile. The analysis was performed by positive electrospray ionisation with a triple quadropole mass spectrometer operating in multiple reaction monitoring (MRM) mode. Two MRM transitions of each analyte were established and identification criteria were set up based on the retention time and the ion ratio. The quantification was performed using deuterated internal analytes of cocaine, benzoylecgonine and ecgonine methyl ester. The calibration curves of extracted standards were linear over a working range of 0.001-2.00 mg/kg whole blood for all analytes. The limit of quantification was 0.008 mg/kg; the interday precision (measured by relative standard deviation-%RSD) was less than 10% and the accuracy (BIAS) less than 12% for all analytes in whole blood. Urine samples were estimated semi-quantitatively at a cut-off level of 0.15 mg/kg with an interday precision of 15%. CONCLUSION: A liquid chromatography mass spectrometric (LC/MS/MS) method has been developed for confirmation and quantification of cocaine and its metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and semi-quantitative in urine. The method is specific and sensitive and offers thereby an excellent alternative to other methods such as GC-MS that involves derivatisation.     

Determination of norgestimate and its metabolites in human serum using high-performance liquid chromatography with tandem mass spectrometric detection

A rapid and reliable analytical method is described for the simultaneous determination of a synthetic progestin norgestimate (NGM), and its metabolites, 17-deacetylnorgestimate (17-DA-NGM), 3-ketonorgestimate (3-keto-NGM) and norgestrel (NGL) in human serum using reversed phase high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. The assay was linear over the concentration ranges of 0.1–5.0 ng/ml for 17-DA-NGM and NGL and 0.5–5.0 ng/ml for NGM and 3-keto-NGM. The inter-assay reproducibility was consistently less than 10%. The overall recovery of the analytes ranged from 72 to 92%. Serum profiles following oral administration of norgestimate to female volunteers are presented.     

Determination of azatadine in human plasma by liquid chromatography/tandem mass spectrometry

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 μL of the resulting solution was injected onto the LC-MS/MS system. A 4.6 mm × 150 mm, I.D. 5 μm, Agilent TC-C(18) column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2m/z for azatadine, 383.3 → 337.3m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93-11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83-105.07% of the nominal values.     

Detection and mass spectrometric characterization of novel long-term dehydrochloromethyltestosterone metabolites in human urine

The biotransformation of dehydrochloromethyltestosterone (DHCMT, 4-chloro-17β-hydroxy,17α-methylandrosta-1,4-dien-3-one) in man was studied with the aim to discover long-term metabolites valuable for the antidoping analysis. Having applied a high performance liquid chromatography for the fractionation of urinary extract obtained from the pool of several DHCMT positive urines, about 50 metabolites were found. Most of these metabolites were included in the GC-MS/MS screening method, which was subsequently applied to analyze the post-administration and routine doping control samples. As a result of this study, 6 new long-term metabolites were identified tentatively characterized using GC-MS and GC-MS/MS as 4-chloro-17α-methyl-5β-androstan-3α,16,17β-triol (M1), 4-chloro-18-nor-17β-hydroxymethyl,17α-methyl-5β-androsta-1,13-dien-3α-ol (M2), 4-chloro-18-nor-17β-hydroxymethyl,17α-methyl-5β-androst-13-en-3α-ol (M3), its epimer 4-chloro-18-nor-17α-hydroxymethyl,17β-methyl-5β-androst-13-en-3α-ol, 4-chloro-18-nor-17β-hydroxymethyl,17α-methylandrosta-4,13-dien-3α-ol (M4) and its epimer 4-chloro-18-nor-17α-hydroxymethyl,17β-methylandrosta-4,13-dien-3α-ol. The most long-term metabolite M3 was shown to be superior in the majority of cases to the other known DHCMT metabolites, such as 4-chloro-18-nor-17β-hydroxymethyl,17α-methylandrosta-1,4,13-trien-3-one and 4-chloro-3α,6β,17β-trihydroxy-17α-methyl-5β-androst-1-en-16-one.     

Lewisite metabolites detection in urine by liquid chromatography-tandem mass spectrometry

A sensitive and simple method for the quantification and for the detection of 2-chlorovinylarsonous (CVAA) and 2-chlorovinylarsonic (CVAOA) acids was developed. CVAA and CVOA are important biological markers in human and rat urine specific to lewisite (chlorovinylarsonous chloride compounds) exposure. The developed assay was based on the use of solid-phase extraction (SPE) followed by liquid-chromatography coupled to electrospray ionization (negative ion-mode) low-energy collision dissociation-tandem mass spectrometry (ESI-CID-MS/MS). The method demonstrated linearity over at least three orders of magnitude and had a detection limit (LOD) of 0.5 ng/ml for CVAA and 3 ng/ml for CVAOA. The relative standard deviations for the quality control samples ranged from 6 to 11%. Application of this procedure was demonstrated in the lewisite animals exposure model. Rats were exposed intravenously by no lethal doses of lewisite and markers levels in urine samples were analyzed for 21 days post-exposure.     

Screening of eltanolone metabolites in dog urine by anion-exchange/reversed-phase liquid chromatography and mass spectrometry

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated ¹⁴C-labelled eltanolone (5β-Pregnan-3α-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of ¹⁴C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC–ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography–mass spectrometry with an electron impact ion source (GC–MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).     

Simultaneous quantitative determination of olmesartan and hydrochlorothiazide in human plasma and urine by liquid chromatography coupled to tandem mass spectrometry

A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the simultaneous determination of olmesartan (OLM) and hydrochlorothiazide (HCTZ) in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the analytes from biological matrices followed by injection of the extracts onto a C₁₈ column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using negative electrospray ionization (ESI). The method was validated over the concentration range of 1.00–1000 ng/mL and 5.00–5000 ng/mL for OLM in human plasma and urine as well as 0.500–200 ng/mL and 25.0–25,000 ng/mL for HCTZ in human plasma and urine, respectively. Inter- and intra-run precision of OLM and HCTZ were less than 15% and the accuracy was within 85–115% for both plasma and urine. The average extraction recoveries were 96.6% and 92.7% for OLM, and 87.2% and 72.1% for HCTZ in human plasma and urine, respectively. The linearity, recovery, matrix effect and stability were validated for OLM/HCTZ in human plasma and urine.     

Determination of succinylacetone in dried blood spots and liquid urine as a dansylhydrazone by liquid chromatography tandem mass spectrometry

Succinylacetone (SA) is a specific marker for the inherited metabolic disease, hepatorenal tyrosinemia. We developed a stable-isotope dilution liquid chromatography tandem mass spectrometry for the determination of SA in dried blood spots (DBS) and liquid urine using a (13)C(4)-SA as internal standard. SA was extracted, converted to the butyl ester and derivatized with dansylhydrazine (Dns-H). Calibration curves in DBS and urine calibrators were linear up to 100 and 30 microM, respectively. At a signal-to-noise ratio of 3, the limits of detection in DBS and urine were 0.2 and 0.005 microM, respectively. Total run time was 5 min. Intra- and inter-assay precision expressed as coefficient of variation were better than 9.1% with more than 96% recovery. The method was applied retrospectively and prospectively for the diagnosis of hepatorenal tyrosinemia and for follow-up of patients under treatment.     

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.     

Quantitative urine amino acid analysis using liquid chromatography tandem mass spectrometry and aTRAQ reagents

Ion-exchange chromatography (IEC) is the most widely used method for amino acid analysis in physiological fluids because it provides excellent separation and reproducibility, with minimal sample preparation. The disadvantage, however, is the long analysis time needed to chromatographically resolve all the amino acids. To overcome this limitation, we evaluated a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method, which utilizes aTRAQ reagents, for amino acid analysis in urine. aTRAQ reagents tag the primary and secondary amino groups of amino acids. Internal standards for each amino acid are also labeled with a modified aTRAQ tag and are used for quantification. Separation and identification of the amino acids is achieved by liquid chromatography tandem mass spectrometry using retention times and mass transitions, unique to each amino acid, as identifiers. The run time, injection-to-injection, is 25 min, with all amino acids eluting within the first 12 min. This method has a limit of quantification (LOQ) of 1 μmol/L, and is linear up to 1000 μmol/L for most amino acids. The Coefficient of Variation (CV) was less than 20% for all amino acids throughout the linear range. Method comparison demonstrated concordance between IEC and LC-MS/MS and clinical performance was assessed by analysis of samples from patients with known conditions affecting urinary amino acid excretion. Reference intervals established for this method were also concordant with reference intervals obtained with IEC. Overall, aTRAQ reagents used in conjunction with LC-MS/MS should be considered a comparable alternative to IEC. The most attractive features of this methodology are the decreased run time and increased specificity.