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.     

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.     

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC–MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1000 μg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1±13.5 μg/l for 2-ethylhexanoic acid, 104.8± 80.6 μg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2± 389.5 μg/l for 2-ethyl-3-oxohexanoic acid.     

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).     

Quantification of 3,4-methylenedioxymetamphetamine and its metabolites in plasma and urine by gas chromatography with nitrogen–phosphorus detection

A gas chromatographic method with nitrogen–phosphorus detection involving a solid–liquid extraction phase was developed and validated for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in plasma. A modification of this method was validated for the analysis of MDMA, MDA, 4-hydroxy-3-methoxymethamphetamine (HMMA) and, 4-hydroxy-3-methoxyamphetamine (HMA) in urine. Under the analytical conditions described, the limits of detection in plasma and urine were less than 1.6 μg/l and 47 μg/l, respectively, for all the compounds studied. Good linearity was observed in the concentration range evaluated in plasma (5–400 μg/l) and urine (100–2000 μg/l) for all compounds tested. The recoveries obtained from plasma were 85.1% and 91.6% for MDMA and MDA, respectively. Urine recoveries were higher than 90% for MDMA and MDA, 74% for HMMA, and 64% for HMA. Methods have been successfully used in the assessment of plasma and urine concentrations of MDMA and its main metabolites in samples from clinical studies in healthy volunteers.     

High-performance liquid chromatographic–tandem mass spectrometric method for the determination of ethionamide in human plasma, bronchoalveolar lavage fluid and alveolar cells

We have developed and validated an accurate, sensitive, and rapid high-performance liquid chromatographic–tandem mass spectrometric method (HPLC–MS–MS) for the determination of ethionamide in plasma, bronchoalveolar fluid (BAL) and alveolar cells (AC). The retention times for ethionamide, clemastine fumarate (internal standard for plasma), promethazine (internal standard for plasma) and propranolol (internal standard for BAL and AC) were approximately 2.62, 1.21, 2.14, and 2.22 min, respectively, with a total run time of 3.2 min. Ethionamide detection for plasma was carried out on a PE Sciex API III (Perkin-Elmer, Foster City, CA, USA). BAL and cell pellets and some plasma specimens were analyzed on a Micromass Quattro LC (Micromass Co., Manchester, UK). The detection limits for ethionamide were 0.05 μg/ml for plasma, and 0.005 μg/ml for BAL supernatants and alveolar cell suspensions.     

Development, validation and application of assays to quantify metrifonate and 2,2-dichlorovinyl dimethylphosphate in human body fluids

Gas chromatographic procedures [GC with electron-capture detection (ECD) and GC–MS] for the quantitative analysis of metrifonate and its active metabolite 2,2-dichlorovinyl dimethylphosphate (DDVP) in human blood and urine were developed, validated, and applied to the analysis of clinical study samples. Analysis of metrifonate involved extraction of acidified blood with ethyl acetate followed by solid-phase clean-up of the organic extract. Acidified urine was extracted with dichloromethane and the residue of evaporated organic phase was reconstituted in toluene. ECD and diethyl analogue of metrifonate internal standard (I.S.) were used for quantitation of metrifonate. The metrifonate lower limit of quantitation (LOQ) was 10.0 μg/l. The DDVP metabolite was chromatographed separately after cyclohexane extraction of acidified blood and urine using d₆-DDVP I.S. and MS detection. The LOQ of DDVP was 1 μg/l. Stability studies have confirmed that the matrix should be acidified prior to storage at −20°C or −80°C to inhibit chemical and enzymatic degradation of the analytes and to avoid overestimation of DDVP concentrations. Metrifonate was found to be stable in acidified human blood after 20 months of storage at −20°C and after 23 months of storage at −80°C. Under these conditions DDVP was found to be stable after 12 months of storage. Both assay procedures were cross-validated by different world-wide laboratories and found to be accurate and robust during analyses of clinical study samples.     

Identification of a flunixin metabolite in camel by gas chromatography–mass spectrometry

A flunixin metabolite, a hydroxylated product, has been identified in camel urine and plasma samples using gas chromatography–mass spectrometry (GC–MS) and GC–MS–MS in the electron impact and chemical ionization modes. Its major fragmentation pattern has been verified by GC–MS–MS in daughter ion and parent ion scan modes. The method could detect flunixin and its metabolite in camel urine after a single intravenous dose of 2.2 mg of flunixin/kg body weight for 96 and 48 h, respectively, which increases the reliability of antidoping control analysis.     

Headspace solid-phase microextraction and gas chromatographic–mass spectrometric screening for volatile hydrocarbons in blood

Optimization for headspace solid-phase microextraction (SPME) was studied with a view to performing gas chromatographic–mass spectrometric (GC–MS) screening of volatile hydrocarbons (VHCs) in blood. Twenty hydrocarbons comprising aliphatic hydrocarbons ranging from n-hexane to n-tridecane, and aromatic hydrocarbons ranging from benzene to trimethylbenzenes were used in this study. This method can be used for examining a burned body to ascertain whether the victim had been alive or not when the burning incident took place. n-Hexane, n-heptane and benzene, the main indicators of gasoline components, were found as detectable peaks through the use of cryogenic oven trapping upon SPME injection into a GC–MS instrument. The optimal screening procedure was performed as follows. The analytes in the headspace of 0.2 g of blood mixed with 0.8 ml of water plus 0.2 μg of toluene-d₈ at −5°C were adsorbed to a 100-μm polydimethylsiloxane (PDMS) fiber for 30 min, and measured using the full-mass-scanning GC–MS method. The lower detection limits of all the compounds were 0.01 μg per 1 g of blood. Linearities (r²) within the range 0.01 to 4 μg per 1 g of blood were only obtained for the aromatic hydrocarbons at between 0.9638 (pseudocumene) and 0.9994 (toluene), but not for aliphatic hydrocarbons at between 0.9392 (n-tridecane) and 0.9935 (n-hexane). The coefficients of variation at 0.2 μg/g were less than 8.6% (n-undecane). In conclusion, this method is feasible for the screening of volatile hydrocarbons from blood in forensic medicine.     

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.     

Qualitative and quantitative analysis of some synthetic, chemically acting laxatives in urine by gas chromatography—mass spectrometry

A method for the qualitative and quantitative simultaneous analysis of dioxyanthraquinone, desacetyl-Bisacodyl, phenolphthalein and Oxyphenisatin in human urine using gas chromatography—mass spectrometry (GC—MS) has been developed. The compounds were extracted from urine at pH 7.5 with diethyl ether using Extrelut extraction columns, followed by evaporation and trimethylsilylation.The method used electron beam ionization GC—MS employing a computer-controlled multiple-ion detector (mass fragmentography). The recovery from urine for the various compounds was between 80% and 100%. The detection limit for these compounds was in the range 0.01–0.05 μg/ml of urine.The method proved to be suitable for measuring urine concentrations for at least four days after administration of a single oral low therapeutic dose of the laxatives to sixteen healthy volunteers.     

Gas chromatographic–mass spectrometric determination of 4-nonylphenols and 4-tert-octylphenol in biological samples

A simple and rapid method is described for the GC–MS determination of 4-nonylphenols (NOs) and 4-tert-octylphenol (OC) in biological samples. The NOs and OC in the sample are extracted with acetonitrile and the lipid in the sample extract is eliminated by partitioning between hexane and acetonitrile. After Florisil PR column clean-up, the sample extract is analyzed by GC–MS in the selected ion monitoring (SIM) mode. Average recoveries in pale chub (fish) and corbicula (shellfish) are 86.0 and 93.4% for NOs, and 95.8 and 96.4% for OC, respectively, spiked at the levels of 1.0 μg of NOs and 0.1 μg of OC per 5 g of fish and shellfish samples. The detection limits are 20 ng/g for NOs and 2 ng/g for OC.     

Determination of urinary 18β-glycyrrhetinic acid by gas chromatography and its clinical application in man

A sensitive and quantitative gas chromatographic assay for the determination of 18β-glycyrrhetinic acid (18β-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18β-GA from urine two Sep-Pak C₁₈ extractions, hydrolysis with Helix pomatia and three liquid–liquid extractions were performed, using 18α-glycyrrhetinic acid (18α-GA) as internal standard. Both 18β-GA and internal standard were converted into their pentafluorobenzyl-ester/trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 μg/l with a urine volume of 10 ml. A detection limit of 3 μg/l was obtained by performing GC–MS. The GC method was used to monitor the urinary excretion of 18β-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption.     

Simple analysis of local anaesthetics in human blood using headspace solid-phase microextraction and gas chromatography–mass spectrometry–electron impact ionization selected ion monitoring

A simple method for analysis of five local anaesthetics in blood was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry–electron impact ionization selected ion monitoring (GC–MS–EI-SIM). Deuterated lidocaine (d₁₀-lidocaine) was synthesized and used as a desirable internal standard (I.S.). A vial containing a blood sample, 5 M sodium hydroxide and d₁₀-lidocaine (I.S.) was heated at 120°C. The extraction fiber of the SPME system was exposed for 45 min in the headspace of the vial. The compounds adsorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC–MS system. The calibration curves showed linearity in the range of 0.1–20 μg/g for lidocaine and mepivacaine, 0.5–20 μg/g for bupivacaine and 1–20 μg/g for prilocaine in blood. No interfering substances were found, and the time for analysis was 65 min for one sample. In addition, this proposed method was applied to a medico–legal case where the cause of death was suspected to be acute local anaesthetics poisoning. Mepivacaine was detected in the left and right heart blood samples of the victim at concentrations of 18.6 and 15.8 μg/g, respectively.     

Determination of zearalenone and its metabolites α- and β-zearalenol in beer samples by high-performance liquid chromatography–tandem mass spectrometry

A fast, robust and sensitive LC–MS–MS method for the determination of zearalenone (ZON) and its metabolites α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) in beer samples is described. Sample preparation was performed by direct RP-18 solid-phase extraction of undiluted beer samples followed by selective determination of analytes by LC–MS–MS applying an atmospheric pressure chemical ionization (APCI) interface. Using the negative ion mode limits of determination of 0.03–0.06 μg l⁻¹ beer and limits of quantification of 0.07–0.15 μg l⁻¹ beer were achieved, which was distinctly more sensitive than in the positive ion mode. Twenty-three beer samples from different countries, produced from different grains and under different brewing conditions, were investigated by this method, but only in one sample could β-ZOL and ZON be detected. Independently of the type of beer, relative standard deviations between 2.1% and 3.3%, a linear working range of 0.15 μg l⁻¹ to 500 μg l⁻¹ beer and recovery rates around 100% could be achieved when zearalanone (ZAN) was used as internal standard.     

Simultaneous determination of eight lipid peroxidation degradation products in urine of rats treated with carbon tetrachloride using gas chromatography with electron-capture detection

One of the major processes that occur as a result of radical-induced oxidative stress is lipid peroxidation (LPO). Degradation of lipid peroxides results in various products, including a variety of carbonyl compounds. In the present study eight different lipid degradation products, i.e., formaldehyde, acetaldehyde, acetone, propanal, butanal, pentanal, hexanal and malondialdehyde were identified and measured simultaneously and quantitatively in rat urine after derivatization with O-(2,3,4,5,6-pentafluorbenzyl)hydroxylamine hydrochloride, extraction with heptane and using gas chromatography–electron-capture detection (GC–ECD). The identity of the respective oximes in urine was confirmed by gas chromatography–negative ion chemical ionization mass spectrometry (GC–NCI-MS). Simultaneously measured standard curves were linear for all oxime-products and the detection limits were between 39.0±5.3 (n=9) and 500±23 (n=9) fmol per μl injected sample. Recoveries of all products from urine or water were 73.0±5.2% and higher. In urine of CCl₄-treated rats an increase in all eight lipid degradation products in urine was found 24 h following exposure. ACON showed the most distinct increase, followed by PROPA, BUTA and MDA. It is concluded that the rapid, selective and sensitive analytical method based on GC–ECD presented here is well suited for routine measurement of eight different lipid degradation products. These products appear to be useful as non-invasive biomarkers for in vivo oxidative stress induced in rats by CCl₄.     

Simultaneous determination of local anesthetics including ester-type anesthetics in human plasma and urine by gas chromatography–mass spectrometry with solid-phase extraction

The present study describes the simultaneous determination of seven different kinds of local anesthetics and one metabolite by GC–MS with solid-state extraction: Mepivacaine, propitocaine, lidocaine, procaine (an ester-type local anesthetics), cocaine, tetracaine (an ester-type local anesthetics), dibucaine (Dib) and monoethylglycinexylidide (a metabolite of lidocaine) were clearly separated from each other and simultaneously determined by GC–MS using a DB-1 open tubular column. Their recoveries ranged from 73–95% at the target concentrations of 1.00, 10.0 and 100 μg/ml in plasma, urine and water. Coefficients of variation of the recoveries ranged from 2.3–13.1% at these concentrations. The quantitation limits of the method were approximately 100 ng/ml for monoethylglycinexylidide, propitocaine, procaine, cocaine, tetracaine and dibucaine, and 50 ng/ml for lidocaine and mepivacaine. This method was applied to specimens of patients who had been treated with drip infusion of lidocaine, and revealed that simultaneous determination of lidocaine and monoethylglycinexylidide in the blood and urine was possible.     

Determination of pyrazinamide and its main metabolites in rat urine by high-performance liquid chromatography

A new high-performance liquid chromatograhic procedure for simultaneous determination of pyrazinamide (PZA) and its three metabolites 5-hydroxypyrazinamide (5-OH-PZA), pyrazinoic acid (PA), and 5-hydroxypyrazinoic acid (5-OH-PA), in rat urine was developed. 5-OH-PZA and 5-OH-PA standards were obtained by enzymatic synthesis (xanthine oxidase) and checked by HPLC and GC–MS. Chromatographic separation was achieved in 0.01 M KH₂PO₄ (pH 5.2), circulating at 0.9 ml/min, on a C₁₈ silica column, at 22°C. The limits of detection were 300 μg/l for PZA, 125 μg/l for PA, 90 μg/l for 5-OH-PZA and 70 μg/l for 5-OH-PA. Good linearity (r²>0.99) was observed within the calibration ranges studied: 0.375–7.50 mg/l for PZA, 0.416–3.33 mg/l for PA, 0.830–6.64 mg/l for 5-OH-PZA and 2.83–22.6 mg/l for 5-OHPA. Accuracy was always lower than ±10.8%. Precision was in the range 0.33–5.7%. The method will constitute a useful tool for studies on the influence of drug interactions in tuberculosis treatment.     

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography–mass spectrometry

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC–MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into the GC–MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 μg ml⁻¹ for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.     

Routine analysis of amphetamine class drugs as their naphthaquinone derivatives in human urine by high-performance liquid chromatography

We describe a simple HPLC method which is suitable for the routine confirmation of immunoassay positive amphetamine urine samples. The precolumn derivisation method employing sodium naphthaquinone-4-sulphonate was found to have adequate sensitivity, selectivity and precision for the measurement of amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxyethylamphetamine (MDEA) at 500 μg/l cutoff level for confirmatory analysis of amphetamines in urine. The specificity of the method is enhanced by detecting the peaks at two different wavelengths. The ratios of the peak heights measured at the two wavelengths were different for each of the 5 amphetamines analysed. There was no interference from other phenylethylamine analogues that are commonly found in “over the counter” preparations. The HPLC method is compared to a commercial TLC system for detecting amphetamines in urine of drug abusers attending drug rehabilitation programmes. The HPLC confirmatory method described is a viable alternative to GC or to the more complex and costly GC–MS techniques for confirming amphetamine, methamphetamine, MDMA, MDA and MDEA in urine of drug abusers especially when used in a clinical care setting