Gas chromatograph-mass spectrometric method for the determination of carvedilol and its metabolites in human urine

A sensitive and efficient method was developed for the determination of carvedilol and its metabolites in human urine by gas chromatography-mass spectrometry (GC-MS). Urine samples were hydrolyzed with beta-glucuronidase/arylsulfatase (from Helix pomatia) and the target compounds were extracted with liquid-liquid extraction. The extracts were completely derivatized with MSTFA and MBTFA and analyzed by GC-MS using an Ultra-2 column. The linearity of the assay ranges were 0.75-75 ngmL(-1) for carvedilol and o-desmethyl carvedilol (o-DMC), and 3.0-75 ngmL(-1) for 4-hydroxyphenyl carvedilol (4-HPC) and 5-hydroxyphenyl carvedilol (5-HPC). The absolute recovery of carvedilol and its metabolites added to a blank urine sample was 80.1-97.8%. The limits of detection (LOD) and quantitation (LOQ) of carvedilol and o-DMC were 0.30 and 0.75 ngmL(-1), and its of 4-HPC and 5-HPC were 0.75 and 3.0 ngmL(-1), respectively. The reproducibilities were 1.86-11.5% for the intra-day assay, and 0.70-1.71% for the inter-day assay precision and the degree of inaccuracy was -3.0 to 3.9% at the concentration of 75 ngmL(-1). The proposed GC-MS method was effective for the determination of carvedilol and its three metabolites in human urine.     

Determination of urinary trans,trans-muconic acid by gas chromatography-mass spectrometry

A sensitive and specific method for the determination of trans,trans-muconic acid (t,t-MA) in urine is described. After clean-up on an anion-exchange cartridge, t,t-MA was derivatized with BF₃-methanol to the dimethyl ester and analyzed by gas chromatography-mass spectrometry (GC-MS), with 2-bromohexanoic acid as an internal standard. The limit of detection was 0.01 mg/l, the coefficient of variation for duplicate analysis in a series of urine samples (n = 50) was 2.6% and the recovery rate ranged from 93.3 to 106.3%. The between-day and within-day precision for the analysis were 7.4 and 14.6%, respectively. The method was applied to the determination of t,t-MA in urine samples from smokers and non-smokers. The mean concentration of t,t-MA in urine of 10 smokers was 0.09 ± 0.04 mg/g creatinine and was significantly (p = 0.012) higher than that found in urine of 10 non-smokers (0.05 ± 0.02 mg/g creatinine). In contrast to the results obtained with the commonly used high-performance liquid chromatographic ultraviolet detection (HPLC-UV) methods, no interference between t,t-MA and other urinary compounds was found. This GC-MS method is both specific and sensitive for biomonitoring of low environmental benzene exposure.     

Determination of bisphenol A in river water and body fluid samples by stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography-mass spectrometry

A new method, based on stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) is described for the determination of trace amounts of bisphenol A (BPA) in river water, urine, plasma, and saliva samples. The derivatization conditions with acetic acid anhydride and the SBSE conditions such as sample volumes and extraction time are investigated. Then, the stir bar is subjected to TD followed by GC-MS. The detection limits of BPA in river water, urine, plasma, and saliva samples are 1-5, 20, 100, and 20pgml(-1) (ppt), respectively. Calibration for BPA was shown to be linear with a correlation coefficient of >0.99. The average recoveries of BPA in all samples are higher than 95% (R.S.D. < 10%) with correction using an added surrogate standard, 13C12-bisphenol A. This simple, accurate, sensitive, and selective analytical method may be applicable to the determination of trace amounts of BPA in liquid samples.     

Sensitive determination of alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine using gas chromatography-mass spectrometry

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography-mass spectrometry (GC-MS). AMT and 5MeO-DIPT were extracted using an Extrelut column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC-MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.     

Direct extract derivatization for determination of amino acids in human urine by gas chromatography and mass spectrometry

The purpose of this study was to develop a simple and accurate analytical method to determine amino acids in urine samples. The developed method involves the employment of an extract derivatization technique together with gas chromatography-mass spectrometry (GC-MS). Urine samples (300 microl) and an internal standard (10 microl) were placed in a screw tube. Ethylchloroformate (50 microl), methanol-pyridine (500 microl, 4:1, v/v) and chloroform (1 ml) were added to the tube. The organic layer (1 microl) was injected to a GC-MS system. In this proposed method, the amino acids in urine were derivatized during an extraction, and the analytes were then injected to GC-MS without an evaporation of the organic solvent extracted. Sample preparation was only required for ca. 5 min. The 15 amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, tryptophan, valine) quantitatively determined in this proposed method. However, threonine, serine, asparagine, glutamine, arginine were not derivatized using any tested derivatizing reagent. The calibration curves showed linearity in the range of 1.0-300 microg/ml for each amino acid in urine. The correlation coefficients of the calibration curves of the tested amino acids were from 0.966 to 0.998. The limit of detection in urine was 0.5 microg/ml except for aspartic acid. This proposed method demonstrated substantial accuracy for detection of normal levels. This proposed method was limited for the determination of 15 amino acids in urine. However, the sample preparation was simple and rapid, and this method is suitable for a routine analysis of amino acids in urine.     

Miniaturized hollow fiber assisted liquid-phase microextraction with in situ derivatization and gas chromatography-mass spectrometry for analysis of bisphenol A in human urine sample

A new method that involves miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS) is described for the determination of trace amounts of bisphenol A (BPA) in human urine samples. The detection limit and the quantification limit of BPA in human urine sample are 0.02 and 0.1 ng ml(-1) (ppb), respectively. The calibration curve for BPA is linear with a correlation coefficient of >0.999 in the range of 0.1-50 ng ml(-1). The average recoveries of BPA in human urine samples spiked with 1 and 5 ng ml(-1) BPA are 101.0 (R.S.D.: 6.7%) and 98.8 (R.S.D.: 1.8%), respectively, with correction using the added surrogate standard, bisphenol A-(13)C12. This simple, accurate, sensitive and selective analytical method can be applicable to the determination of trace amounts of BPA in human urine samples.     

Simultaneous determination of 2-naphthol and 1-hydroxy pyrene in urine by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometric (GC-MS) method was developed for the determination of 2-naphthol (2-NAP) and 1-hydroxypyrene (1-HOP) in human urine. Extraction from urine after the enzyme hydrolysis with β-glucuronidase/arylsulfatase was achieved with a liquid extraction using 5 mL of pentane. After addition of 50 μL of N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBDMSTFA) to prevent the loss of 2-NAP during drying, the extract was completely dried and derivatized with MTBDMSTFA for 30 min at 60 °C. The accuracies were in the range of 96-109% at a concentration of 0.5, 10 and 25 μg/L and their precisions were less than 15%. Method detection limits of 2-NAP and 1-HOP were 0.07 and 0.01 μg/L, respectively. This method was used to analyze twenty urine samples, and they were found in the concentration range <0.07-13.7 μg/L (2-NAP) and <0.01-0.88 μg/L (1-HOP). The concentrations of 2-NAP and 1-HOP were well correlated to those of naphthalene and pyrene in blood, respectively.     

Hollow-fiber-supported liquid phase microextraction with in situ derivatization and gas chromatography-mass spectrometry for determination of chlorophenols in human urine samples

A simple and highly sensitive method that involves hollow-fiber-supported liquid phase microextraction (HF-LPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS) was developed for the determination of chlorophenols (CPs) such as 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TrCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) in human urine samples. Human urine samples were enzymatically de-conjugated with beta-glucuronidase and sulfatase. After de-conjugation, HF-LPME with in situ derivatization was performed. After extraction, 2mul of extract was carefully withdrawn into a syringe and injected into the GC-MS system. The limits of detection (S/N=3) and quantification (S/N>10) of CPs in the human urine samples are 0.1-0.2ngml(-1) and 0.5-1ngml(-1), respectively. The calibration curve for CPs is linear with a correlation coefficient of >0.99 in the range of 0.5-500ngml(-1) for DCP and TrCP, and of 1-500ngml(-1) for TeCP and PCP, respectively. The average recoveries of CPs (n=6) in human urine samples are 81.0-104.0% (R.S.D.: 1.9-6.6%) with correction using added surrogate standards. When the proposed method was applied to human urine samples, CPs were detected at sub-ngml(-1) level.     

Determination of menthol in plasma and urine of rats and humans by headspace solid phase microextraction and gas chromatography--mass spectrometry

A method for the determination of menthol and menthol glucuronide (M-G) after enzymatic hydrolysis in plasma and urine of rats and humans was developed using headspace solid phase microextraction and gas chromatography-mass spectrometry in the selected ion monitoring mode (HS-SPME/GC-MS). The assay linearity for plasma ranged from 5 to 1000 ng/ml. The limit of quantification (LOQ) in plasma was 5 ng/ml. The intra- and inter-day precision for menthol and M-G were < or = 18.1% R.S.D. at the LOQ and < or = 4.0% at higher concentrations. Menthol and M-G were determined in rat and human plasma and urine after administration of menthol.     

Identification and quantitative determination of 3-chloro-2-hydroxypropylmercapturic acid and α-chlorohydrin in urine of rats treated with epichlorohydrin

Epichlorohydrin (ECH) is used in many industrial processes. Different toxic effects of ECH were found in rodents. The metabolism of ECH was investigated before in rats using [¹⁴C]ECH. The aim of this investigation was the development of non-radioactive quantitative analytical methods for measuring two urinary metabolites of ECH, namely 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) and α-chlorohydrin (α-CH). The identity of CHPMA and α-CH excreted in urine of rats treated with 5 to 35 mg/kg ECH was confirmed by GC-MS. The quantitative analysis of CHPMA, involving ethyl acetate extraction from acidified urine and subsequent methylation and analysis by gas chromatography-flame photometric detection (GC-FPD), showed a method limit of detection of 2 μg/ml. The analysis of α-CH, based on ethyl acetate extraction and subsequent analysis by GC-ECD, showed a method limit of detection of 2 μg/ml. CHPMA and α-CH derivatives could be determined quantitatively down to concentrations of 0.5 and 0.4 μg/ml urine, respectively, by selected-ion monitoring GC-MS under EI conditions. Cumulative urinary excretion of CHPMA and α-CH by rats treated with ECH were found to be 31 ± 10 and 1.4 ± 0.6% (n = 13) of the ECH dose, respectively. For CHPMA, the dose-excretion relationship suggested partially saturated ECH metabolism. For α-CH, the dose-excretion relationship was linear. With fractionated urine collection it was found that approximately 74 and 84% of the total cumulative excretion of CHPMA and α-CH, respectively, took place within the first 6 h after administration of ECH. From these investigations it is concluded that the GC-FPD and GC-ECD based methods developed are sufficiently sensitive to measure urinary excretion of CHPMA and α-CH in urine from rats administered 5 to 35 mg/kg ECH. It is anticipated that the analysis of CHPMA and α-CH based on GC-MS may be sufficiently sensitive to investigate urinary excretion from humans occupationally exposed to ECH.     

Monitoring for occupational exposure to 4,4′-methylenebis(2-chloroaniline) by gas chromatographic-mass spectrometric analysis of haemoglobin adducts, blood, plasma and urine

The feasibility of using plasma, blood and haemoglobin adducts for monitoring occupational exposure to the suspected human carcinogen 4,4′-methylenebis(2-chloroaniline) (MOCA) was investigated. A method utilising capillary gas chromatography-negative-ion chemical-ionisation mass spectrometry (GC-MS) for the determination of pentafluoropropionyl (PFP) derivatives of MOCA, released by alkaline hydrolysis from protein adducts and conjugates, was both sensitive and selective. When selected ion monitoring was used, sub-femtomole amounts of PFP-MOCA could be measured. The detection limit for haemoglobin adducts of MOCA was below 10 fmol/g Hb, well below the levels found for occupationally exposed individuals. Capillary GC with electron-capture detection also had the required sensitivity for the determination of MOCA in blood and urine of five individuals who were exposed to MOCA during the manufacture of polyurethane elastomers were determined by the GC-MS method. The MOCA concentrations for the various blood fractions and urine were within the following ranges: haemoglobin adducts, 0.73–43.3 pmol MOCA/g Hb; plasma alkaline hydrolysate, 0.05–22.0 nmol/l; whole blood, 0.13–17.4nmol/l; urine, 4.5–2390 nmol/l. Because the products of MOCA in the blood reflect metabolic activation of MOCA and integrate exposure over a period of weeks, the use of blood samples for monitoring exposure to MOCA offers advantages over the currently used urinary MOCA measurements.     

Determination of dichloromethane, trichloroethylene and perchloroethylene in urine samples by headspace solid phase microextraction gas chromatography-mass spectrometry

A method for the determination of volatile chlorinated hydrocarbons, namely dichloromethane (DCM), trichloroethylene (TCE), and perchloroethylene (PCE), in urine samples was developed using headspace solid phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS). HS-SPME was performed using a 75 microm Carboxen-polydimethylsiloxane fiber. Factors, which affect the HS-SPME process, such as adsorption and desorption times, stirring, salting-out effect, and temperature of sampling have been evaluated and optimized. The highest extraction efficiency was obtained when sampling was performed at room temperature (22 degrees C), from samples saturated with salt and under agitation. Linearity of the HS-SPME-GC-MS method was established over four orders of magnitude and the limit of detection was 0.005 microg/l for all the compounds. Precision, calculated as %R.S.D. at three different concentration levels, was within 1-8% for all intra- and inter-day determinations. The method was applied to the quantitative determination of TCE and PCE in human urine samples from exposed (TCE, n=5; median, 9.32 microg/l and PCE, n=39; median, 0.58 microg/l) and non-exposed individuals (n=120; median concentrations, 0.64, 0.22 and 0.11 microg/l for DCM, TCE and PCE, respectively. In addition, two cases of acute accidental exposure to DCM are reported, and the elimination kinetics in blood and urine was followed up. The calculated half-lives of urinary and blood DCM were, respectively, 7.5 and 8.1 h for one subject and 3.8 and 4.3 h for the other.     

Simultaneous determination of 5-hydroxyindoles and catechols from urine using polymer monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection

To make analytes amenable for fluorescence (FL) detection, polymer monolith microextraction (PMME) coupled to high-performance liquid chromatography with FL detection was developed for the simultaneous determination of catechols and 5-hydroxyindoleamines (5-HIAs) from urine samples. In this method, a two-step pre-column derivatization method was employed to derivatize the analytes and a poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolithic capillary column was used as the extraction medium for PMME. The conditions for the derivatization and subsequent extraction of 5-HIAs and catechols derivatives were optimized. Using our optimum conditions, the detection limit of the target analytes were 0.11–21 nM. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations less than 12% and a recovery of higher than 82%. In this study, we show how our proposed method can be used as a rapid sensitive technique for the determination of catechols and 5-HIAs from urine samples.     

Improved solid-phase extraction and HPLC measurement of torasemide and its important metabolites

Torasemide is a "loop type" diuretic drug. For pharmacokinetic studies sensitive analytic methods are essential for authentic qualitative and quantitative information. A robust, selective and sensitive HPLC method is described for the simultaneous determination of torasemide, its major metabolite M5 and its active metabolites M1 and M3 and an internal standard within 17 min. Solid-phase extraction with C(2)-cartridges was used for the clean-up of plasma samples. The chromatographic separation was carried out on a CN-column with a mobile phase consisting of perchloric acid (0.02 M, pH 2.5)/acetonitrile (90/10, v/v)). The calibration range used reached from 20 to 1000 ng/ml for all analytes. Coefficients of variation were less than 10% at every calibration point for each analyte. Plasma concentrations in samples obtained from volunteers in the course of a clinical study could be reliably measured with this method. Median maximum concentrations in plasma after a 10mg oral dose during a 24h study interval were located at 1h for torasemide, 1h for M1 and 2h for M5. Concentrations between 2226 and <20 ng/ml for torasemide, between 159 and <20 ng/ml for M1 and between 420 and <20 ng/ml for M5 were observed.     

Quantitative determination of tulobuterol and its metabolites in human urine by mass fragmentography

A method is described for the simple and simultaneous determination of tulobuterol and its metabolites in human urine by gas chromatography-mass spectrometry. Quantification was achieved by single-ion monitoring at m/e 86 derived from trimethylsilyl-tulobuterol and its metabolites using a column packed with a mixed phase, 2% OV-1–2% QF-1 (1 : 1, w/w). The detection limits were estimated to be 2 ng/ml in urine for tulobuterol and 5 ng/ml for metabolites, respectively.     

Gas chromatographic method with mass-selective detection for the determination of 2-isopropoxyphenol in human urine

Human metabolism of the insecticide propoxur yields 2-isopropoxyphenol (IPP) which is excreted conjugated in urine. In this publication a sensitive and selective analytical method is described which permits the determination of IPP as a suitable parameter for biomonitoring. The clean-up of the hydrolysed urine samples consisted of steam distillation and solid-phase extraction using a reversed-phase column. IPP and the internal standard 2-ethoxyphenol were converted to their pentafluorobenzyl ethers. Excess of the derivatisation reagent was removed using deactivated silica gel. Separation and quantitative analysis was carried out by capillary gas chromatography and mass selective detection. Coefficients of variation were below 5% for concentrations from 6 to 300 μg/l. The detection limit was 0.5 μg/l. The method was checked by analysing six urine samples from pest controllers after indoor application of propoxur. The IPP concentrations ranged from 45 to 306 μg/g creatinine. IPP was not detected in urine specimens from 10 non-exposed persons. The sensitivity of the developed method permits the detection of latent exposure to propoxur.     

Simultaneous determination of barbiturates in human biological fluids by direct immersion solid-phase microextraction and gas chromatography-mass spectrometry

Simultaneous determination of seven barbiturates in human whole blood and urine by combining direct immersion solid-phase microextraction (DI-SPME) with gas chromatography-mass spectrometry (GC-MS) is presented. The main parameters affecting the DI-SPME process, such as SPME fibers, salt additives, pHs, extraction temperatures and immersion times were optimized for simultaneous determination of the drugs. The extraction efficiencies were 0.0180-0.988 and 0.0156-2.76% for whole blood and urine, respectively. The regression equations of the drugs showed excellent linearity for both samples; the correlation coefficients (r(2)) were 0.994-0.999. The detection limits for whole blood were 0.05-1 microg x ml(-1), and those for urine 0.01-0.6 microg x ml(-1). Actual quantitation could be made for pentobarbital in whole blood and urine obtained from volunteers, who had been orally administered a therapeutic dose of the drug. The DI-SPME/GC-MS procedure for barbiturates established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology.     

Determination of urinary triclosan by stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry

We have developed an analytical method for the determination of urinary 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan), which utilizes stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Human urine sample is de-conjugated by treatment with beta-glucuronidase and sulfatase. A stir bar coated with polydimethylsiloxane (PDMS) is added to the urine sample in a vial and the sample is stirred for 60 min at room temperature (25 degrees C). Then, the PDMS stir bar is subjected to TD-GC-MS. The detection limit of triclosan is 0.05 ng mL(-1). The method shows linearity over the calibration range (0.1-10 ng mL(-1)) and the correlation coefficient (r) is higher than 0.993 for triclosan standard solution. The average recoveries of triclosan in human urine sample are 102.8-113.1% (RSD: 2.4-6.7%). This simple, sensitive, and selective analytical method may be used in the determination of trace amounts of triclosan in human urine samples.     

Determination of urinary 5-hydroxytryptophol glucuronide by liquid chromatography-mass spectrometry

5-Hydroxytryptophol glucuronide (GTOL) is the major excretion form of 5-hydroxytryptophol (5-HTOL), a minor serotonin metabolite under normal conditions. Because the concentration of 5-HTOL is markedly increased following consumption of alcohol, measurement of 5-HTOL is used as a sensitive biomarker for detection of recent alcohol intake. This study describes the development and evaluation of a liquid chromatography-electrospray ionization mass spectrometry (LC-MS) procedure for direct quantification of GTOL in human urine. Deuterium labelled GTOL (GTOL-(2)H(4)) was used as internal standard. GTOL was isolated from urine by solid-phase extraction on a C(18) cartridge prior to injection onto a gradient eluted Hypurity C(18) reversed-phase HPLC column. The detection limit of the method was 2.0 nmol/L and the measuring range 6-8500 nmol/L. The intra- and inter-assay coefficients of variation were <3.5% (n=10) and <6.0% (n=9), respectively. The new LC-MS method was highly correlated with an established GC-MS method for urinary 5-HTOL (r(2)=0.99, n=70; mean 5-HTOL/GTOL ratio=1.10). This is the first direct assay for quantification of GTOL in urine. The method is suitable for routine application.     

Stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography--mass spectrometry for measurement of phenolic xenoestrogens in human urine samples

A high-sensitivity analytical method that uses stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) for the simultaneous measurement of trace amounts of phenolic xenoestrogens (PXs), such as 2,4-dichlorophenol (DCP), 4-tert-butylphenol (BP), 4-tert-octylphenol (OP), 4-nonylphenol technical isomers (NP), pentachlorophenol (PCP) and bisphenol A (BPA), in human urine samples was developed. The urine sample (1 ml) was de-conjugated by adding beta-glucuronidase and sulfatase. Then, protein precipitation was performed by the addition of acetonitrile. After centrifugation, the supernatant was diluted with purified water and subjected to SBSE with in situ derivatization and TD-GC-MS. The detection limits of DCP, BP, OP, NP, PCP and BPA in the urine samples were 20, 10, 10, 50, 20 and 20 pg ml-1 (ppt), respectively. The calibration curves for PXs were linear and had correlation coefficients higher than 0.99. The average recoveries of those analytes in the urine samples were higher than 95% (RSD: <10%, n=6) with correction using the added surrogate standards. This simple, accurate, sensitive and selective method can be used in the determination of PXs in human urine samples.