Simultaneous determination of glucuronides of trimetozine in human urine by gas chromatography

A gas chromatographic method for the simultaneous determination of four glucuronides (metabolites) of trimetozine excreted in human urine is described. The methodinvolves pretreatment of the urine specimen [i.e. removal of interfering substances by solvent extraction, desalting on an ion-exchange (Amberlite XAD-2) column], and permethylation of glucuronides by reaction with methylsulfinyl carbanion and methyl iodide. The permethylated derivatives were submitted to gas chromatographic separation on an OV-17 column, and their structures were investigated by subsequent gas chromatographic—mass spectrometric analysis. The minimum detectable concentration of each glucuronide is 5 μg/ml when 1 ml of urine is used. The utility of the present method is successfully demonstrated by determining the urinary concentration of four glucuronides following oral administration of trimetozine to human subjects.     

Gas chromatographic method for the determination of urinary acetylpolyamines

A gas chromatographic method was developed for the determination of monoacetylputrescine, monoacetylcadaverine, N¹-acetylspermidine and N⁵-acetylspermidine in human urine. The amines were isolated from urine by silica gel column chromatography. 1, 10-Diaminodecane was used as internal standard. The amines were reacted with ethyl chloroformate in aqueous medium to four ethyloxycarbonyl derivatives prior to application to gas chromatography using a flame ionization detector. Separation and determination of the derivatives were carried out on a Uniport HP column (1.0 m) impregnated with 0.5% SP-1000 under temperature-programmed conditions. The monoacetylpolyamines could be measured accurately at the nanomole level. The method was used for the determination of the monoacetylpolyamines in urine of healthy volunteers. The values obtained were in the range of the published data.     

Quantitative analysis of terbutaline in serum and urine at therapeutic levels using gas chromatography—mass spectrometry

A simple and sensitive method for the determination of terbutaline in serum and urine has been developed. A mass spectrometer in the multiple ion detection mode was used as a gas chromatographic detector. Levels were monitored after oral and subcutaneous administration of the drug. The sensitivity is 1 ng/ml using 1 ml of serum.     

Determination of the cyanide metabolite 2-aminothiazoline-4-carboxylic acid in urine and plasma by gas chromatography-mass spectrometry

The cyanide metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) is a promising biomarker for cyanide exposure because of its stability and the limitations of direct determination of cyanide and more abundant cyanide metabolites. A simple, sensitive, and specific method based on derivatization and subsequent gas chromatography-mass spectrometry (GC-MS) analysis was developed for the identification and quantification of ATCA in synthetic urine and swine plasma. The urine and plasma samples were spiked with an internal standard (ATCA-d(2)), diluted, and acidified. The resulting solution was subjected to solid phase extraction on a mixed-mode cation exchange column. After elution and evaporation of the solvent, a silylating agent was used to derivatize the ATCA. Quantification of the derivatized ATCA was accomplished on a gas chromatograph with a mass selective detector. The current method produced a coefficient of variation of less than 6% (intra- and interassay) for two sets of quality control (QC) standards and a detection limit of 25 ng/ml. The applicability of the method was evaluated by determination of elevated levels of ATCA in human urine of smokers in relation to non-smokers for both males and females.     

Conformation and quantification of chloramphenicol in cow's urine, muscle and eggs by electron capture negative ion chemical ionization gas chromatography/mass spectrometry.

A method is described for the determination of residues of the antibiotic chloramphenicol in biological samples. The method is based on gas chromatography/negative ion chemical ionization mass spectrometry and uses (37Cl2)chloramphenicol as internal standard. Selective ion monitoring of four analyte-specific ions enables the determination of chloramphenicol levels in urine of 3 micrograms l-1 with a coefficient of variation of 8%. The limit of detection of the method is 0.1 p.p.b. for urine, muscle and egg.     

Determination of clenbuterol in urine as its cyclic boronate derivative by gas chromatography—mass spectrometry

A rapid and reliable gas chromatographic—mass spectrometric method for the determination of clenbuterol in urine is described. Penbutolol was used as internal standard. Four derivatization procedures have been tested, of which 1-butaneboronic acid gave the best results. The method includes extraction of the alkalinized urine (3 ml) with tert.-butyl methyl ether—n-butanol (9:1), derivatization with 1-butaneboronic acid (15 min at room temperature), and analysis in the selected-ion monitoring mode of the derivatives of clenbuterol at m/z 243, 327 and 342 and of penbutolol at m/z 342 and 357. The detection limit is 0.5 ng/ml and the recovery better than 90%.     

Human biomonitoring of pyrethrum and pyrethroid insecticides used indoors: determination of the metabolites E-cis/trans-chrysanthemumdicarboxylic acid in human urine by gas chromatography-mass spectrometry with negative chemical ionization

This work describes a gas chromatographic-mass spectrometric method employing negative chemical ionization (NCI) for the determination of E-cis/trans-chrysanthemumdicarboxylic acid (CDCA) in human urine used as a biomarker for the exposure to pyrethrum and/or certain pyrethroids in insecticide formulations applied indoors. Mixed-mode solid phase extraction was utilized for sample cleanup. Extraction recoveries ranged from 92 to 104% (2-9% R.S.D.). The acids were esterified with 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) allowing both their gas chromatographic separation and their sensitive mass spectrometric detection under NCI conditions. Detection limits of ca. 0.05 microg/l urine were achieved.     

Determination of pentacaine, trans-2-(1-pyrrolidinyl)cyclohexyl-3-pentyloxycarbanilate hydrochloride, in biological samples by gas chromatography/mass spectrometry

A quantitative and selective method has been developed for the determination of a novel local anaesthetic compound pentacaine, trans-2-(1-pyrrolidinyl)cyclohexyl-3-pentyloxycarbanilate hydrochloride, in biological samples. After ion pair extraction from 1 M HCl into 1,2-dichloroethane, pentacaine and a structurally related internal standard were derivatized to prevent thermal decomposition in the gas chromatograph. An on-column methylation technique with trimethylanilinium hydroxide was used. Determination was performed by gas chromatography/mass spectrometry (GC/MS) with selected ion monitoring. Interferences by endogenous lipophilic constituents were avoided by including an n-hexane wash before the ion pair extraction. This wash step did not reduce the drug recoveries. The method gave linear results over a concentration range of 5-100 ng ml-1 with a coefficient of variation less than 10% at 5 ng pentacaine ml-1. Specimens of plasma, whole blood, urine as well as in vitro preparations such as hepatic microsomes were successfully analysed.     

Determination of raloxifene and its glucuronides in human urine by liquid chromatography-tandem mass spectrometry assay

A selective, sensitive, accurate and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of raloxifene and its three glucuronides: raloxifene-6-β-glucuronide (M1), raloxifene-4'-β-glucuronide (M2), raloxifene-6,4'-diglucuronide (M3) in urine samples is presented in this paper. To our knowledge the developed analytical method is the first fully validated method capable of simultaneous determination of raloxifene and its glucuronides in real urine samples. Moreover, for the first time a method for determination of raloxifene diglucuronide in relevant biological samples was introduced. Metabolites were obtained by a bioconversion process of raloxifene to its glucuronides using the microorganism Streptomyces sp. and were used as standards for validation. Urine samples were introduced to a simple solid phase extraction prior to the analysis by LC-MS/MS. The method was linear in a wide range with high determination coefficient (r(2) > 0.997). The limits of quantification achieved were 1.01, 1.95, 2.83 and 4.69 nM for raloxifene, M1, M2 and M3, respectively. The recoveries were higher than 92.5%, the accuracy was within 100 ± 8.8% and the precision was better than 12% for all compounds. The developed method was successfully applied to the real urine samples and showed to be appropriate for use in further research of still not completely discovered raloxifene pharmacokinetics. Furthermore, the presented method could also serve for a potential application in anti-doping analysis.     

Application of the standard addition approach for the quantification of urinary benzene

Urinary benzene is used as biomarker of exposure to evaluate the uptake of this solvent both in non-occupationally exposed population and in benzene-exposed workers. The quantitative determination of benzene in urine is carried out in a three steps procedure: urine collection, sample analysis by head space/solid phase microextraction/gas chromatography/mass spectrometry and analyte quantification. The adopted quantification method influences the initial step, hence the whole procedure. Two quantification approaches were compared as regards precision and accuracy: the calibration curves and the standard addition method. Even if calibration curves obtained by using urine samples from different subjects were always linear, their slopes and intercepts showed noteworthy variations, attributable to the influence of the biological matrix on benzene recovery. The standard addition method showed to be more suitable for compensating matrix effects, and a three-point standard addition protocol was used to quantify benzene in urine samples of 11 benzene-exposed workers (smokers and non-smokers). Urine from occupationally exposed workers was collected before and after work-shift. Besides urinary benzene, the applicability of the method was verified by measuring the urinary concentration of the S-phenylmercapturic acid, a specific benzene metabolite, generally adopted as biomarker in biological monitoring procedures. A similar trend of concentration levels of both analytes measured in urine samples collected before work-shift with respect to the after work-shift ones was found, showing the actual applicability of the standard addition method for biological monitoring purposes.     

Determination of cyclophosphamide in urine by gas chromatography—mass spectrometry

A sensitive gas chromatographic method for the determination of cyclophosphamide in urine is presented. After liquid—liquid extraction with diethyl ether and derivatization with trifluoroacetic anhydride, cyclophosphamide was identified and quantified with mass spectrometry. The method is suitable for the determination of cyclophosphamide at concentrations of more than 0.25 ng/ml, which enables the uptake of cyclophosphamide during occupational activities, such as the preparation and administration of antineoplastic agents in hospitals, to be measured. Simple preparation makes the method appropriate for routine analysis.     

8-Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels in human urine do not depend on diet

In the present study, we used the method involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in human urine. The mean levels of 8-oxoGua and 8-oxodGuo in the urine samples of the subjects on unrestricted diet were respectively 1.87 nmol/kg 24 h (±0.90) and 0.83 nmol/kg 24h (±0.49), and in the case of the groups studied, they did not depend on the applied diet. The sum of the amounts of both compounds in urine can give information about the formation rate of 8-oxoGua in cellular DNA. It is also likely that the levels of modified nucleo-base/side in urine sample are reflective of the involvement of different repair pathways responsible for the removal of 8-oxodGuo from DNA, namely base excision repair (BER) and nucleotide excision repair (NER).     

Analysis of methadone and metabolites in biological fluids with gas chromatography—mass spectrometry

The analysis of methadone and its metabolites in biological fluids by gas chromatography—mass spectrometry is described with deuterated methadone and metabolites as internal standards. The method allowed the determination of 20 ng methadone in 0.5 ml of plasma or saliva. Mean saliva to plasma ratio of methadone for two patients was determined to be 0.51 ± 0.13. Methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine were measured by selected ion monitoring. Gas chromatography—mass spectrometry was found to have advantages over conventional gas chromatographic methods in terms of ratio analysis. 1,5-Dimethyl-3,3-diphenyl-2-pyrrolidone previously reported as a metabolite was shown to result primarily from the decomposition of EDDP free base.     

Stereospecific gas chromatographic/mass spectrometric assay of the chiral labetalol metabolite 3-amino-1-phenylbutane.

We previously identified 3-amino-1-phenylbutane (APB) as an oxidative N-dealkylated, metabolite of the antihypertensive agent labetalol. Labetalol has two asymmetric centers and is used clinically as a mixture of the four possible stereoisomers; APB has one asymmetric center. We now report an enantiospecific gas chromatographic/mass spectrometric assay for APB in urine. After adding the internal standard 1-methyl-2-phenoxyethylamine and alkalinizing, the urine samples were extracted with ether. The extracts were derivatized with the optically active acid chloride prepared from (S)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid. The derivatives were separated by capillary gas chromatography and detected by electron capture negative ion chemical ionization mass spectrometry with selected ion monitoring. The derivative of the R enantiomer eluted first, and the [M--32]- ions were monitored for both the drug and the internal standard. The method was linear in the 0.05-2.5 micrograms enantiomer-1 ml-1 range and had inter-assay and intra-assay coefficients of variation of less than 6%. The assay was used in the analysis of urine samples from a patient in labetalol therapy and no interference was found. Further studies are needed to elucidate the oxidative metabolism of labetalol and its stereochemical aspects.     

Analysis of benzene,toluene, ethylbenzene and m-xylene in biological samples from the general population

A method for the determination of benzene, toluene, ethylbenzene and xylene in blood and urine of people not occupationally exposed to solvents is described. The headspace technique combined with gas chromatography with a mass spectrometer detector is used. The sensitivity of recent mass spectrometers is good enough to furnish reliable results also in biological samples collected from the general population. No treatment for concentrating solvents present in the blood or urine is necessary. The main features of the method are easy preparation of biological samples, small volumes (7 ml), good repeatability and linearity in the range of interest. The limits of detection in blood were 16, 43, 22 and 52 ng/l for benzene, toluene, ethylbenzene and m-xylene respectively. Slightly greater sensitivity was found for urine samples. The results obtained in biological samples from 25 woodworkers not occupationally exposed to BTEX (15 non-smokers and 10 smokers) are comparable to those obtained by other investigators.     

A sensitive high-performance liquid chromatographic procedure with fluorometric detection for the analysis of decarboxylated S-adenosylmethionine and analogs in urine samples

A highly sensitive HPLC method for the determination of decarboxylated S-adenosylmethionine (dc-SAM) by fluorometric detection was developed. The reaction of dc-SAM and its analogs with chloroacetaldehyde leads to the corresponding 1,N6-etheno derivatives. These highly fluorescent derivatives were fully characterized through their proton nuclear magnetic resonance spectra and/or mass spectra. This derivatization procedure has been applied to the analysis of dc-SAM in rat and human urine. After a simple cation exchange column prepurification, the urine extracts were derivatized with chloroacetaldehyde and analyzed by reversed-phase HPLC with fluorometric detection. The method allowed the determination of subpicomole amounts of dc-SAM and was shown to be highly reproducible with the use of decarboxylated S-adenosylethionine as internal standard. The application of the method to the analysis of urine of rats treated with MDL 72175, a potent ornithine decarboxylase inhibitor, showed that the dc-SAM levels increased in a dose-related fashion.     

Determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in human urine

A method for simultaneous determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in urine is described. These compounds are metabolites of N-methyl-2-pyrrolidone, a powerful and widely used organic solvent. 5-Hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide were purified from urine by adsorption to a C₈ solid-phase extraction column and then elution by ethyl acetate–methanol (80:20). After evaporation, the samples were derivatised at 100°C for 1 h by bis(trimethylsilyl)trifluoroacetamide. Ethyl acetate was then added and the samples were analysed by gas chromatography–mass spectrometry in the electron impact mode. The extraction recovery for 5-hydroxy-N-methylpyrrolidone was about 80% while that for 2-hydroxy-N-methylsuccinimide was about 30%. The intra-day precision for 5-hydroxy-N-methylpyrrolidone was 2–4% and the between-day precision 4–21% (4 and 60 μg/ml). The intra-day precision for 2-hydroxy-N-methylsuccinimide was 4–8% and the between-day precision 6–7% (2 and 20 μg/ml). The detection limit was 0.2 μg/ml urine for both compounds. The method is applicable for analysis of urine samples from workers exposed to N-methyl-2-pyrrolidone.     

Improvement of ethyl glucuronide determination in human urine and serum samples by solid-phase extraction

An improved method for the determination of ethyl glucuronide (EtG) in human serum and urine was developed using solid-phase extraction (SPE) and gas chromatography (GC) with mass spectrometric detection (MS). EtG was isolated from serum and urine using aminopropyl SPE columns after deproteination with perchloric acid and hydrochloric acid, respectively. The chromatographic separation was performed on a DB 1701 fused-silica column. At a signal-to-noise ratio of 3:1, a quantification limit of 173 and 560 ng/ml and a detection limit of 37 and 168 ng/ml could be determined for serum and urine, respectively. This indicates high specificity and sensitivity of the described method. The mean absolute recovery was 85%, while intra- and inter-day precision of the assay were all less than 7.5%. The linearity of the calibration curves was satisfying as indicated by correlation coefficients of >0.993. The presented method provides the basis for determination and identification of EtG in human serum and urine samples in a low-concentration range for monitoring alcohol consumption during treatment for alcohol dependence and comorbid alcohol abuse of psychotherapy patients.     

Determination of Ro 19–6327 (Lazabemide) in human plasma and urine by gas chromatography-negative chemical ionization mass spectrometry

A sensitive and specific analytical method was developed for determination of Ro 19–6327 (Lazabemide) in human plasma and urine samples to provide pharmacokinetic data from clinical trials. The new method employs a simple liquid-liquid extraction to isolate the drug from biological samples. The extract is reacted to form the trifluoroacetyl derivative of Ro 19–6327 and then analyzed by gas chromatography-negative chemical ionization mass spectrometry (GC-NCIMS). The lower limit of quantitation of the assay is 0.05 ng/ml for plasma and 5.0 ng/ml for urine, based on 1-ml aliquots. No interferences from anticoagulants, collection devices, or endogenous constituents of plasma and urine were observed. Recovery (64.3%), inter-assay precision (<8% R.S.D.), and accuracy (>85%) of the method were considered acceptable. The assay proved reliable enough to be automated for unattended sample analysis of approximately 50 samples daily. In an additional series of tests, Ro 19–6327 was shown to be stable under conditions that might be encountered during the analysis of samples from clinical trials.     

Gas chromatographic-negative-ion chemical ionization mass spectrometric determination of a new dihydropyridine calcium antagonist (MPC-1304) and its metabolites in human plasma and urine

A gas chromatographic-negative-ion chemical ionization mass spectrometric method was developed for the determination of a new calcium antagonist, (±)-methyl 2-oxopropyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicar☐ylate, and its metabolites in plasma and urine. The sample was extracted with n-hexane-diethyl ether. The dried organic layer was subjected to acetylation: the aqueous layer was acidified and extracted with ethyl acetate, and after the ethyl acetate extract was dried the resulting residue was subjected to methylation. Aliquots of each reactant solution were injected into the gas chromatograph-mass spectrometer, equipped with a chemical ionization source and negative-ion monitoring mode, and analysed by the elected-ion monitoring method using deuterium-labelled internal standards. Detection was limited to 0.02–0.05 ng/ml of plasma and urine for each metabolite. A precise and sensitive assay for the determination of a new dihydropyridine calcium antagonist and its metabolites in plasma and urine was thus established.