Detection and quantification of glucuro- and sulfoconjugated metabolites in human urine following oral administration of xenobiotic 19-norsteroids

Recently, the endogenous origin of nandrolone (19-nortestosterone) and other 19-norsteroids has been a focus of research in the field of drug testing in sport. In the present study, we investigated metabolites conjugated to a glucuronic acid and to a sulfuric acid in urine following administration of four xenobiotic 19-norsteroids. Adult male volunteers administered a single oral dose (10 mg) of each of four 19-norsteroids. Urinary samples collected from 0 to 120 h were subjected to methanolysis and beta-glucuronidase hydrolysis and were derivatized by N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) before gas chromatography-mass spectrometry analysis. We confirmed that 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) were present in both glucuronide (g) and sulfate (s) conjugates and 19-norepiandrosterone (19-NEA) was excreted exclusively as a sulfate fraction in urine of all 19-norsteroids tested. The overall levels of the three metabolites can be ranked as follows: 19-NA(g+s)>19-NE(g+s)>19-NEA(s). The concentration profiles of these three metabolites in urine peaked between 2 to 12h post-administration and declined thereafter until approximately 72-96 h. 19-NA was most prominent throughout the first 24 h post-administration, except for a case in which an inverse relationship was found after 6h post-administration of nandrolone. Furthermore, we found that sulfate conjugates were present in both 19-NA and 19-NE metabolites in urine of all 19-norsteroids tested. The averaged total amounts of metabolites (i.e. 19-NA(s+g)+19-NE(s+g)+19-NEA(s)) excreted in urine were 38.6, 42.9, 48.3 and 21.6% for nandrolone, 19-nor-4-androsten-3,17-dione, 19-nor-4-androsten-3beta,17beta-diol and 19-nor-5-androstene-3beta,17beta-diol, respectively. Results from the excretion studies demonstrate significance of sulfate-conjugated metabolites on interpretation of misuse of the 19-norsteroids.     

Urinary excretion of 19-norandrosterone of endogenous origin in man: quantitative analysis by gas chromatography–mass spectrometry

A GC–MS method, using deuterium-labelled 19-noretiocholanolone as internal standard and following an extensive LC purification prior to selected ion monitoring of the bis(trimethylsilyl) ethers at ion masses m/z 405, 419, 420 and 421, allowed the quantitation of subnanogram amounts of 19-norandrosterone present in 10-ml urine samples at m/z 405. Thirty healthy men, free of anabolic androgen supply, delivered 24-h urine collections in 4 timed fractions. Accuracy was proven by the equation, relating added (0.05–1 ng/ml) to measured analyte, which had a slope not significantly different from 1. Precision (RSD) was 4% at a concentration of 0.4 ng/ml, and 14% at 0.04 ng/ml. Analytical recovery was 82%. The limit of quantitation was 0.02 ng/ml. The excretion ranges were 0.03–0.25 μg/24 h or 0.01–0.32 ng/ml in nonfractionated 24-h urine.Taking into account inter-individual variability and log-normal distribution, a threshold of 19-norandrosterone endogenous concentration of 2 ng/ml, calculated as the geometric mean plus 4 SD, was established. This value corresponds to the decision limit advised by sport authorities for declaring positive (anabolic) doping with nandrolone.     

Determination of ephedrines in urine by gas chromatography–mass spectrometry

A selective gas–liquid chromatographic method with mass spectrometry (GC–MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines — one derivative was formed for each ephedrine — were prepared and analyzed by GC–MS, after alkaline extraction of urine and evaporation of the organic phase, using d₃-ephedrine as internal standard. Calibration curves, with r²>0.98, ranged from 3.0 to 50 μg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.     

Comparison of sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.     

Determination of metabolites of pirimicarb in human urine by gas chromatography–mass spectrometry

The analytical method described permits the determination of 2-dimethylamino-5,6-dimethyl-4-hydroxypyrimidine (DDHP), 2-methylamino-5,6-dimethyl-4-hydroxypyrimidine (MDHP) and 2-amino-5,6-dimethyl-4-hydroxypyrimidine (ADHP) in human urine. These hydroxypyrimidines are metabolites of pirimicarb (2-dimethylamino-5,6-dimethylpyrimidin-4-yldimethylcarbamate) which is applied as insecticide. The analytes are extracted into a mixture of diethyl ether and acetonitrile. Pentafluorobenzyl bromide serves as derivatising reagent. The derivatives are analysed using capillary gas chromatography with mass selective detection. 2-Amino-4-hydroxy-6-methylpyrimidine and 4-hydroxy-6-trifluoromethylpyrimidine are used as internal standards. The detection limits are 0.5 μg/l (DDHP), 1 μg/l (MDHP) and 4 μg/l (ADHP), respectively. The method was used for analysing seven urine samples collected from workers who had applied pirimicarb. The three metabolites were found in every sample in concentrations up to 60 μg/l.     

Preliminary application of liquid chromatography–electrospray-ionization mass spectrometry to the detection of 5-methyltetrahydrofolic acid monoglutamate in human plasma

Liquid chromatography (LC) in direct combination with mass spectrometry (MS) has been shown to be a good analytical technique for the selective separation and detection of labile folate monoglutamates. Reversed-phase LC and electrospray-ionization MS conditions were developed and optimized for the separation and detection of 5-methyltetrahydrofolic acid, 5-formyl tetrahydrofolic acid, tetrahydrofolic acid, dihydrofolic acid and folic acid in aqueous samples. Representative and reproducible positive ion mass spectra were generated for each folate under mild MS conditions. The selective MS detection and identification of endogenous 5-methyltetrahydrofolic acid in human plasma was accomplished through the development of a straightforward C₁₈-based solid-phase extraction procedure. This procedure allows for the qualitative assessment of 5-methyltetrahydrofolic acid in plasma. Based upon an isotope-dilution internal standard calibration study with standards, the LC–MS limit of quantitation for 5M-THF was estimated to be 0.39 ng/ml.     

Determination of butorphanol in horse race urine by immunoassay and gas chromatography–mass spectrometry

An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC–MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC–MS system. The ELISA test (20 μl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC–MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC–MS method in the SIM mode using nalbuphine as internal standard included linearity studies (10–250 ng/ml), recovery (±100%), intra-assay (4.1–14.9%) and inter-assay (9.3–45.1%) precision, stability (10 days), limit of detection (10 ng/ml) and limit of quantitation (20 ng/ml).     

Application of solid-phase microextraction and gas chromatography–mass spectrometry for the determination of chlorophenols in urine

This study investigated the feasibility of applying solid-phase microextraction (SPME) combined with gas chromatography–mass spectrometry to analyze chlorophenols in urine. The SPME experimental procedures to extract chlorophenols in urine were optimized with a polar polyacrylate coated fiber at pH 1, extraction time for 50 min and desorption in GC injector at 290°C for 2 min. The linearity was obtained with a precision below 10% R.S.D. for the studied chlorophenols in a wide range from 0.1 to 100 μg/l. In addition, sample extraction by SPME was used to estimate the detection limits of chlorophenols in urine, with selected ion monitoring of GC–MS operated in the electron impact mode and negative chemical ionization mode. Detection limits were obtained at the low ng/l levels. The application of the methods to the determination of chlorophenols in real samples was tested by analyzing urine samples of sawmill workers. The chlorophenols were found in workers, the urinary concentration ranging from 0.02 μg/l (PCP) to 1.56 μg/l (2,4-DCP) depending on chlorophenols. The results show that trace chlorophenols have been detected with SPME–GC–MS in the workers of sawmill where chlorophenol-containing anti-stain agents had been previously used.     

Assay method for the carboxylic acid metabolite of clopidogrel in human plasma by gas chromatography–mass spectrometry

This paper describes a GC–MS method for the analysis of the carboxylic acid metabolite (SR26334, II) of methyl (+)-(S)--(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetate hydrogensulfate (clopidogrel, SR 25990, I) in plasma and serum. The analytical procedure involves a robotic liquid–liquid extraction with diethyl ether followed by a solid–liquid extraction on C₁₈ cartridges. The derivatization process was performed using n-ethyl diisopropylethylamine and -bromo-2,3,4,5,6-pentafluoro toluene. A structural analogue (III) of II, was used as internal standard. The 1/X²; weighted calibration curve obtained in the range 5–250 ng/ml was well described by a quadratic equation. The extraction efficiency was better than 48% over the range studied; for the internal standard it averaged 51% at 50 ng/ml. Precision ranged from 3.6 to 15.8%, and accuracy was between 92 and 114%. Dilution has no influence on the performance of the method which could then be used to quantitate plasma samples containing up to 25 000 ng/ml. The limit of quantification was 5 ng/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay methods for use in pharmacokinetic studies.     

Analysis of cyclophosphamide and five metabolites from human plasma using liquid chromatography–mass spectrometry and gas chromatography–nitrogen–phosphorus detection

An assay method for the quantification of cyclophosphamide (CY) and five metabolites from human plasma is presented. The procedure is adapted to the chemical properties of the compounds of interest: non-polar compounds are extracted into methylene chloride, concentrated and analyzed by GC–NPD after derivatization, and the remaining aqueous fraction is deproteinated with acetonitrile–methanol prior to separation via reversed-phase HPLC and detection using atmospheric pressure ionization (API)-MS. Standard curves are linear over the required range and reproducible over five months. Plasma concentration–time profiles of CY and metabolites from a patient receiving CY by intravenous infusion (60 mg/kg, once a day for two days) are presented.     

Simultaneous analysis of phenylglycols and phenylethanols in human urine by gas chromatography—mass spectrometry

A method is described for the determination of the neutral metabolites formed from catecholamines and various other structurally related phenylethylamines by using gas chromatography—chemical ionization—mass spectrometry. These metabolites (phenylglycols and phenylethanols) were extracted from urine specimens and converted to pentafluoropropionyl derivatives which were separated on either 3% OV-1, 3% SP-2250, or 3% QF-1 packed columns. Our results demonstrate the presence in human urine of p-hydroxyphenylglycol, a metabolite of octopamine. One patient excreted 13 and 91 μg/day of free and total (free + conjugated) p-hydroxyphenylglycol, respectively. Treatment with a monoamine oxidase inhibitor reduced the excretion of total p-hydroxyphenylglycol to 30% of baseline level.     

High-temperature solid-phase microextraction procedure for the detection of drugs by gas chromatography–mass spectrometry

High-temperature headspace solid-phase microextraction (SPME) with simultaneous (“in situ”) derivatisation (acetylation or silylation) is a new sample preparation technique for the screening of illicit drugs in urine and for the confirmation analysis in serum by GC–MS. After extraction of urine with a small portion of an organic solvent mixture (e.g., 2 ml of hexane–ethyl acetate) at pH 9, the organic layer is separated and evaporated to dryness in a small headspace vial. A SPME-fiber (e.g., polyacrylate) doped with acetic anhydride–pyridine (for acetylation) is exposed to the vapour phase for 10 min at 200°C in a blockheater. The SPME fiber is then injected into the GC–MS for thermal desorption and analysis. After addition of perchloric acid and extraction with n-hexane to remove lipids, the serum can be analysed after adjusting to pH 9 as described for urine. Very clean extracts are obtained. The various drugs investigated could be detected and identified in urine by the total ion current technique at the following concentrations: amphetamines (200 μg/l), barbiturates (500 μg/l), benzodiazepines (100 μg/l), benzoylecgonine (150 μg/l), methadone (100 μg/l) and opiates (200 μg/l). In serum all drugs could be detected by the selected ion monitoring technique within their therapeutic range. As compared to liquid–liquid extraction only small amounts of organic solvent are needed and larger amounts of the pertinent analytes could be transferred to the GC column. In contrast to solid-phase extraction (SPE), the SPME-fiber is reusable several times (as there is no contamination by endogenous compounds). The method is time-saving and can be mechanised by the use of a dedicated autosampler.     

Analysis of nitroaromatic compounds in urine by gas chromatography–mass spectrometry for the biological monitoring of explosives

Organic nitrocompounds are the most frequently used constituents of explosives and some of them have been evaluated to be highly toxic or even carcinogenic. Human contact with explosives may originate from a variety of sources, including occupational exposure during the production of ammunition as well as environmental exposure due to the contamination of soil and ground water reservoirs on former military production sites and training areas. This paper describes two gas chromatography–mass spectrometry–selected ion monitoring methods for the determination of twelve nitroaromatic compounds in urine (nitrobenzene, 1,2-dinitrobenzene, 1,3-dinitrobenzene, 1,3,5-trinitrobenzene, 2-nitrotoluene, 3-nitrotoluene, 4-nitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2,4,6-trinitrotoluene, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene). The analytes are detectable in the lowest μg/l range, with imprecisions of 3–22% within series and 5–29% between series, depending on the compound of interest. Both procedures are rapid and relatively easy to perform and, therefore, are advantageous for the screening of occupationally or environmentally exposed persons. We analysed urine samples obtained from nine workers from an ammunition dismantling workshop and from twelve control persons. 2,4,6-Trinitrotoluene was detected in six samples at concentrations between 4 and 43 μg/l. The main metabolites of 2,4,6-trinitrotoluene, 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene, were found in a concentration range from 143 to 16 832 μg/l and from 24 to 5787 μg/l, respectively. Nonconjugated aminodinitrotoluenes were present as varying percentages of the total amount. 2,4-Dinitrotoluene and 2,6-dinitrotoluene were found in two samples (2–9 μg/l). Nitroaromatics were not detectable in urine specimens from control persons.     

Determination of metabolites of pyrethroids in human urine using solid-phase extraction and gas chromatography–mass spectrometry

The described method permits the determination of the five most important metabolites of the pyrethroids permethrin, cypermethrin, deltamethrin, λ-cyhalothrin, fenvalerate, phenothrin and β-cyfluthrin in human urine in one run. The major urinary metabolites of these substances are cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl₂CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br₂CA), fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA). After acidic hydrolysis to release the conjugated carboxylic acid metabolites, the analytes were separated from the matrix by means of solid-phase extraction using a reversed-phase column. The components of the eluate were converted to their methyl esters and extracted in hexane. Separation and quantitative analysis of the pyrethroid metabolites was carried out by capillary gas chromatography and mass selective detection. 2-Phenoxybenzoic acid served as an internal standard. The detection limits lay between 0.3 and 0.5 μg per litre urine. The relative standard deviations of the within-series imprecision were between 1% and 6%. The relative recovery rates ranged between 90% and 98%. Using this method we determined the elimination of pyrethroid metabolites in 24-h urine samples from eight pest controllers after indoor application of permethrin. The detected concentrations ranged from 1 to 70 μg g⁻¹ creatinine.     

Determination of δC values of valine in protein hydrolysate by gas chromatography–combustion isotope ratio mass spectrometry

A gas chromatographic–combustion isotope ratio mass spectrometric (GC–C-IRMS) method for the determination of [1-¹³C]valine enrichments in protein hydrolysates is described. Using a quick derivatization method, δ¹³C values of the N-methoxycarbonyl methyl ester of valine can be determined from baseline separated GC peaks. Evaluation studies with respect to precision, accuracy, linearity, reduction capacity of the CuO combustion furnace and isotope dilution as a result of derivatization, showed that our GC–C-IRMS system allows robust measurement of enrichments of [1-¹³C]valine in the range 0 to 1.5 MPE (S.D.±0.01 MPE, n=3). Therefore this method is suited to determine fractional synthetic rates (FSRs) of proteins as low as one-tenth of the FSR of human albumin, in studies using a primed, continuous (6 h) infusion with [1-¹³C]valine plasma enrichments of approximately 15 MPE and an hourly sampling schedule.     

Analysis of benzphetamine and its metabolites in rat urine by liquid chromatography–electrospray ionization mass spectrometry

An analytical method to identify and determine benzphetamine (BMA) and its five metabolites in urine was developed by liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) using the solid-phase extraction column Bond Elut SCX. Deuterium-labeled compounds, used as internal standards, were separated chromatographically from each corresponding unlabeled compound in the alkaline mobile phase with an alkaline-resistant ODS column. This method was applied to the identification and determination of BMA and its metabolites in rat urine collected after oral administration of BMA. Under the selected ion monitoring mode, the limit of quantitation (signal-to-noise ratio 10) for BMA, N-benzylamphetamine (BAM), p-hydroxybenzphetamine (p-HBMA), p-hydroxy-N-benzylamphetamine (p-HBAM), methamphetamine (MA) and amphetamine (AM) was 700 pg, 300 pg, 500 pg, 1.4 ng, 6 ng and 10 ng in 1 ml of urine, respectively. This analytical method for p-HBMA, structurally closer to the unchanged drug of all the metabolites, was very sensitive, making this a viable metabolite for discriminating the ingestion of BMA longer than the parent drug or other metabolites in rat.     

Determination of lorazepam in plasma and urine as trimethylsilyl derivative using gas chromatography–tandem mass spectrometry

A procedure based on gas chromatography–tandem mass spectrometry for identification and quantitation of lorazepam in plasma and urine is presented. The analyte was extracted from biological fluids under alkaline conditions using solid-phase extraction with an Extrelut-1 column in the presence of oxazepam-d₅ as the internal standard. Both compounds were then converted to their trimethylsilyl derivatives and the reaction products were identified and quantitated by gas chromatography–tandem mass spectrometry using the product ions of the two compounds (m/z 341, 306 and 267 for lorazepam derivative and m/z 346, 309 and 271 for oxazepam-d₅ derivative) formed from the parent ions by collision-induced dissociation in the ion trap spectrometer. Limit of quantitation was 0.1 ng/ml. This method was validated for urine and plasma samples of individuals in treatment with the drug.     

Confirmation of a dopamine metabolite in parkinsonian brain tissue by gas chromatography—mass spectrometry

Gas chromatography—mass spectrometry was used to identify a dopamine metabolite isolated from the substantia nigra of parkinsonian brain tissue. Incubation of dopamine with monoamine oxidase B gave the same product which was identified as 3,4-dihydroxyphenylacetaldehyde. The structure of the compound was established by chemical synthesis, metastable ion measurement and high-resolution mass spectrometry.     

A three-phase liquid chromatographic method for δC analysis of amino acids from biological protein hydrolysates using liquid chromatography–isotope ratio mass spectrometry

We report a three-phase chromatographic method for the separation and analysis of δ¹³C values of underivatized amino acids from biological proteins (keratin, collagen, and casein) using liquid chromatography–isotope ratio mass spectrometry (LC–IRMS). Both precision and accuracy of δ¹³C values for standard amino acid mixtures over the range of approximately 8 to 1320 ng of carbon per amino acid on the column were assessed. The precision of δ¹³C values of amino acids was found to be better at higher concentrations, whereas accuracy improved at lower concentrations. The optimal performance for this method was achieved with between 80 and 660 ng of carbon of each amino acid on the column. At amino acid amounts lower than 20 ng of carbon on the column, precision and accuracy may become compromised. The application of this new three-phase chromatographic technique will allow the analysis of δ¹³C of amino acids to be carried out as a routine method and benefit fields of research such as biomedicine, forensics, ecology, nutrition, and palaeodiet reconstruction in archaeology.     

Speciation of ionic alkyllead compounds in human urine by gas chromatography–mass spectrometry after butylation through a Grignard reaction

One analytical procedure for the determination of ionic alkyllead in human urine has been studied. The system consists of the extraction of Me₃Pb⁺, Et₃Pb⁺ and Pb²⁺ at pH 9.0 with diethyldithiocarbamate to an organic phase. Then, the ionic compounds are butylated with BuMgCl and the final organic solution is analyzed by GC–MS–SIM. The elimination of both foam and gels in the extraction step and the general procedure for the urine are discussed. The recovery of compounds ranges from 105.1% for Me₃Pb⁺ to 97.2% for Et₃Pb⁺ using hexane as extracting agent and detection limits are 18.4 pg/ml of Me₃Pb⁺ and 19.2 pg/ml of Et₃Pb⁺ in urine. The speciation of ionic alkylleads in the urine of a petrol station worker showed a value of 27.9 pg/ml of Me₃Pb⁺ in urine and Et₃Pb⁺ was below the detection limit.