Determination of haloacetic acids in human urine by headspace gas chromatography–mass spectrometry

Haloacetic acids (HAAs) are water disinfection byproducts (DBPs) formed by the reaction of chlorine oxidizing compounds with natural organic matter in water containing bromine. HAAs are second to trihalomethanes as the most commonly detected DBPs in surface drinking water and swimming pools. After oral exposure (drinking, showering, bathing and swimming), HAAs are rapidly absorbed from the gastrointestinal tract and excreted in urine. Typical methods used to determine these compounds in urine (mainly from rodents) only deal with one or two HAAs and their sensitivity is inadequate to determine HAA levels in human urine, even those manual sample preparation protocols which are complex, costly, and neither handy nor amenable to automation. In the present communication, we report on a sensitive and straightforward method to determine the nine HAAs in human urine using static headspace (HS) coupled with GC–MS. Important parameters controlling derivatisation and HS extraction were optimised to obtain the highest sensitivity: 120 μl of dimethylsulphate and 100 μl of tetrabutylammonium hydrogen sulphate (derivatisation regents) were selected, along with an excess of Na₂SO₄ (6 g per 12 ml of urine), an oven temperature of 70 °C and an equilibration time of 20 min. The method developed renders an efficient tool for the precise and sensitive determination of the nine HAAs in human urine (RSDs ranging from 6 to 11%, whereas LODs ranged from 0.01 to 0.1 μg/l). The method was applied in the determination of HAAs in urine from swimmers in an indoor swimming pool, as well as in that of non-swimmers. HAAs were not detected in the urine samples from non-swimmers and those of volunteers before their swims; therefore, the concentrations found after exposure were directly related to the swimming activity. The amounts of MCAA, DCAA and TCAA excreted from all swimmers are related to the highest levels in the swimming pool water.     

Determination of scopolamine in human serum and microdialysis samples by liquid chromatography–tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC–MS–MS) method with a rapid and simple sample preparation was developed for the determination of scopolamine in biological fluids. Scopolamine and the internal standard atropine in serum samples were extracted and cleaned up by using an automated solid phase extraction method. Microdialysis samples were directly injected into the LC–MS system. The mass spectrometer was operated in the multi reaction monitoring mode. A good linear response over the range of 20 pg/ml to 5 ng/ml was demonstrated. The accuracy for added scopolamine ranged from 95.0 to 104.0%. The lower limit of quantification was 20 pg/ml. This method is suitable for pharmacokinetic studies.     

Assay of bromhexine in human plasma by capillary gas—liquid chromatography with nitrogen-selective detection and selected ion monitoring

A specific, sensitive method for the determination of bromhexine in human plasma is described. It comprises a selective extraction procedure and a specific determination with capillary gas—liquid chromatography and nitrogen-selective flame ionization detection. The detection limit of the assay is about 0.5 ng/ml. The specificity of the assay was checked by gas chromatography—mass spectrometry. The method is applied to the pharmacokinetics of bromhexine in humans.     

Determination of domperidone in human serum and human breast milk by high-performance liquid chromatography–electrospray mass spectrometry

A sensitive and selective method for the determination of domperidone in human breast milk and serum has been developed. The same method may be successfully applied to both matrices to a lower limit of quantitation of 0.5 ng/ml. Samples are processed by a liquid–liquid extraction, and analyzed by LC–ESI-MS in positive ion mode. There was no interference, on the domperidone quantitation, from over 30 drugs. Samples from patients, at various times post-dose, were analyzed and a large number showed significant levels of domperidone in the breast milk as well as in the serum.     

Micro-determination of clonazepam in plasma or serum by electron-capture gas—liquid chromatography

A rapid method is described for the electron-capture gas chromatographic determination of clonazepam in plasma or serum using methyl-clonazepam as an internal standard. The analysis is performed isothermally on the silicone stationary phase SP-2510DA (Supelco). With this liquid phase, gas chromatographic properties are comparable to methods involving acid hydrolysis or derivatisation. A short pre-column containing another phase is added to enhance resolution. The method involves a single extraction, requires 100 μl of sample and has a detection limit of 3 nmol/l. Response is linear at concentrations from 5–900 nmol/l and thus clonazepam analysis both during therapy and after overdosage is possible. Plasma and serum clonazepam levels are interchangeable.     

Determination of Δ9-tetrahydrocannabinol from human saliva by tandem immunoaffinity chromatography-high-performance liquid chromatography

Smoking or ingestion of cannabis causes cognitive, perceptual and behavioural changes, which are responsible for impaired performance in driving motor vehicles. In this paper a novel liquid chromatographic assay for the selective quantification of Δ⁹-tetrahydrocannabinol, the major indicator of a present cannabis intoxication in saliva, is described. The method involves a column-switching procedure and requires an extremely simple pre-treatment of the sample. Deproteinized saliva was directly injected into the chromatographic system. The clean-up and enrichment procedure was performed in an immunoaffinity column, followed by the transfer of the antigens to an octylsilica analytical column. The immunoaffinity sorbent was obtained by covalent immobilization of specific antibodies on epoxy-activated silica. The mobile phase consisted of methanol-aqueous 0.15 mol/1 NaCl solution (elution programmed) and the analyte was detected by measuring the UV absorption at 220 nm. Using an injection volume of 4.5 ml (dilution 3:2, v/v) the limit of quantification was 20 ng/ml, at a signal-to-noise ratio of 5. Recoveries were estimated to be in the range of 70%. Both intra- and inter-day coefficients of variation were below 5%     

Micro-determination of plasma diphenylhydantoin by gas—liquid chromatography

A selective, sensitive and precise gas—liquid chromatographic method for the determination of diphenylhydantoin in micro samples of blood plasma is described. After a double extraction with chloroform containing an analogue of diphenylhydantoin as an internal standard, the drug and standard are N,N-dimethylated in alkaline aqueous solution with methyl iodide followed by extraction into acetone. The methylated derivatives are separated gas chromatographically and measured using a flame-ionization detector. The lowest concentration of diphenylhydantoin in plasma which can be measured in a 100-μl sample is 1 μg/ml, which is well below the normal therapeutic concentration of 10–20 μg/ml in plasma. The methylated derivatives of diphenylhydantoin and the internal standard have been identified by their proton magnetic resonance spectra and mass spectra.     

Determination of ginsenoside Rg3 in human plasma and urine by high performance liquid chromatography–tandem mass spectrometry

Here we report a method capable of quantifying ginsenoside Rg3 in human plasma and urine. The method was validated over linear range of 2.5–1000.0 ng mL⁻¹ for plasma and 2.0–20.0 ng mL⁻¹ for urine using ginsenoside Rg1 as I.S. Compounds were extracted with ethyl acetate and analyzed by HPLC/MS/MS (API-4000 system equipped with ESI⁻ interface and a C₁₈ column). The inter- and intra-day precision and accuracy of QC samples were ≤8.5% relative error and were ≤14.4% relative standard deviation for plasma; were ≤5.6% and ≤13.3% for urine. The Rg3 was stable after 24 h at room temperature, 3 freeze/thaw cycles and 131 days at −30 °C. This method has been applied to pharmacokinetic study of ginsenoside Rg3 in human.     

Determination of very-long-chain fatty acids in serum by gas chromatography–nitrogen–phosphorus detection following cyanomethylation

A sensitive method for very-long-chain fatty acid analysis was developed by gas chromatography–nitrogen–phosphorus detection by using cyanomethyl derivatization. Bromoacetonitrile as alkylating reagent was used to improve nitrogen phosphorus detection detectability of compounds containing non-nitrogen. The carboxyl group of very-long-chain fatty acid was alkylated to cyanomethyl esters. Reaction conditions were 40 min at 60°C under potassium carbonate base. Heptacosanoic acid was used as an internal standard and hexane was used as a solvent of extraction. The extraction yield was 82.8% or more, relative standard deviation of the precision test was 8.3 % or more and the result of linearity test showed a good correlation coefficient of r²=0.999 in the range of 0.1–50 μg/ml. The quantification limits were 10 ng/ml when 0.5 ml of serum was used. The present method proved simple, rapid, inexpensive and resistant to contaminants. When it was applied to serum samples taken from patients with X-linked adrenoleukodystrophy which is a hereditary X-linked disorder characterized by progressive demyelination and adrenal insufficiency during childhood, relative increase of the concentration of hexacosanoic acid and the concentration ratios of hexacosanoic, lignoceric to behenic acid was observed in comparison with control samples.     

Quantitative analysis of oxepinac in human plasma,urine and saliva by gas chromatography—mass fragmentography

A sensitive and specific method is described for the quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e]oxepin-3-acetic acid (oxepinac) in human plasma, urine and saliva. Oxepinac and internal standard are extracted from acidified plasma, urine or saliva, converted to the corresponding n-propyl esters and analysed by gas chromatography—mass fragmentography using selected ion monitoring. The method is accurate and precise over the range 100 μg/ml to 1.0 ng/ml. The method has been applied to the analysis of plasma, urine and saliva from healthy volunteers receiving therapeutic doses of oxepinac.     

Enatiomeric determination of tramadol and O-desmethyltramadol in human urine by gas chromatography–mass spectrometry

A GC–MS assay for stereoselective determination of tramadol and its pharmacologically active phase I metabolite O-desmethyltramadol in human urine was developed. Nefopam was used as internal standard. The method involves a simple solid phase extraction with chiral analysis by gas chromatography–electron ionization mass spectrometry using m/z 263; 58, 249; 58, and 179; 58 for the determination of concentration of tramadol, O-desmethyltramadol and internal standard, respectively. Chromatography was performed on a Rt-βDEXcst column containing alkylated beta-cyclodextrins as a chiral selector. The calibration curves were linear in the concentration range 0.1–20 μg/mL (R² ≥ 0.998). Intra-day accuracies ranged between 97.2–104.9%, 96.1–103.2%, and 97.3–102.8% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged between 95.2–105.7%, 99.1–105.2%, and 96.5–101.2% at the lower, intermediate, and high concentration for all analytes, respectively. This method was successfully used to determine the concentration of enantiomers of T and ODT in a pharmacogenetic study.     

Identification of flavanone metabolites in rat urine by combined gas–liquid chromatography and mass spectrometry

1. The metabolism of flavanone in the rat was studied after oral or intraperitoneal administration of the compound. Flavone and flav-3-ene together with five other unidentified minor metabolites were excreted in the urine. 2. The formation of flavanone metabolites was not suppressed by the administration of high doses of the antibacterial compounds aureomycin and phthaloylsulphathiazole. 3. No aromatic acids that could be attributed to ring cleavage of flavanone were detected. 4. Administration of 100 or 200mg of flavanone daily per rat caused some deaths during the 7-14-day period. 5. The application of combined gas-liquid chromatography/mass spectrometry and proton nuclear-magnetic-resonance spectroscopy to the separation and identification of the flavanone metabolites is described. 6. Measurement of the two major flavanone metabolites was carried out by gas-liquid chromatography.     

Assay of HA-966 in rat plasma by capillary gas—liquid chromatography with nitrogen-selective detection

A gas—liquid chromatographic method for the determination of the γ-aminobutyric acid-like drug 1-hydroxy-3-aminopyrrolidone-2 (HA-966) in plasma is described. HA-966 was converted into its diacetyl derivative Ac₂HA-966 with acetic anhydride. This compound could be suitably eluted from a capillary OV-17 support-coated open tubular column. A sensitive detection method was achieved by making use of nitrogen—phosphorus-selective flame ionization.     

Determination of β-blockers in pharmaceutical and human urine by capillary electrophoresis with electrochemiluminescence detection and studies on the pharmacokinetics

A novel method for simultaneous determination of atenolol, metoprolol and esmolol was proposed by capillary electrophoresis (CE) separation and electrochemiluminescence (ECL) detection. Poly-β-cyclodextrin (Poly-β-CD) was used as an additive in the running buffer to improve the separation of three analytes. The conditions for CE separation, ECL detection and effect of Poly-β-CD were investigated in detail. The three β-blockers with very similar structures were well separated and detected under the optimum conditions. The linear ranges of the standard solution for atenolol and esmolol were 2.5-125 μmol/L with a detection limit (S/N=3) of 0.5 μmol/L, and for metoprolol was 0.5-25 μmol/L with a detection limit of 0.1 μmol/L. For three β-blockers from spiked aqueous and urine samples, the accuracy and precision including intraday and interday experiments were performed by calculating the recovery, the relative standard deviations of the ECL intensity and the migration time, respectively. The developed method was applied to the determination of metoprolol content in commercial pharmaceutical, and the analytical results are in good agreement with the nominal value with recoveries in the range of 98.7-105%. The proposed method was also applied to the monitoring of pharmacokinetics for metoprolol in human body.     

Determination of terbutaline enantiomers in human urine by coupled achiral–chiral high-performance liquid chromatography with fluorescence detection

A coupled achiral–chiral high-performance liquid chromatographic system with fluorescence detection at excitation/emission wavelengths of 276/306 nm has been developed for the determination of the enantiomers of terbutaline, (S)-(+)-terbutaline and (R)-(−)-terbutaline in urine. Urine samples were prepared by solid-phase extraction with Sep-pak silica, followed by HPLC. The terbutaline was preseparated from the interfering components in urine on Phenomenex silica column and the terbutaline enantiomers and betaxolol were resolved and determined on a Sumichiral OA-4900 chiral stationary phase. The two columns were connected by a switching valve equipped with silica precolumn. The precolumn was used to concentrate the terbutaline in the eluent from the achiral column before back flushing onto the chiral phase. For each enantiomer the assay was linear between 1 and 250 ng/ml (R²=0.9999) and the detection limit was 0.3 ng/ml. The intra-day variation was between 4.6 and 11.6% in relation to the measured concentration and the inter-day variation was 4.3–11.0%. It has been applied to the determination of (S)-(+)-terbutaline and (R)-(−)-terbutaline in urine from a healthy volunteer dosed with racemic terbutaline sulfate.     

Determination of the quinidine analog, 7′-trifluoromethyldihydrocinchonidine-2HCl in plasma and urine by high-performance liquid chromatography

A rapid and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiarrhythmic quinidine analog, 7′-trifluoromethyldihydrocinchonidine-2HCl ([I]-2HCl) in plasma and urine. The overall recovery of [I] from plasma was 86 ± 9% with a sensitivity limit of detection of 0.2 μg/ml.The assay involves extraction of [I] into benzene-methylene chloride (9:1) from plasma or urine made alkaline with 0.1 N sodium hydroxide (pH 13) and saturated sodium chloride, the residue of which is dissolved in methylene chloride, an aliquot of which is analyzed by HPLC using adsorption chromatography on silica gel with UV detection at 254 nm. The mobile phase composed of methylene chloride-methanol-conc. ammonium hydroxide (95.5:4:0.5) yields baseline resolution of quinidine used as the internal (reference) standard, compound [I] and dihydroquinidine, a common contaminant in quinidine.The assay was applied to the analysis of plasma and urine samples taken from a dog administered a single 20 mg/kg dose via intravenous and oral routes. The stability of [I] in human plasma for up to 37 days of storage at ?17°C was also demonstrated.