Quantification of leukotriene B4 glucuronide in human urine

We have developed a method for measuring leukotriene B4 glucuronide, a marker of systemic leukotriene B4 biosynthesis, in human urine. This method involves the separation of two positional isomers of leukotriene B4 glucuronide by high-performance liquid chromatography, followed by hydrolysis with beta-glucuronidase and then leukotriene B4 quantification by enzyme immunoassay after purification by high-performance liquid chromatography. One of two positional isomers of leukotriene B4 glucuronide was predominantly present in urine. The concentration of the isomer increased in urine from aspirin-intolerant asthma patients after aspirin challenge. Urinary leukotriene E4 and leukotriene B4 glucuronide concentrations in 13 normal healthy adults were 94.6 pg/mg-creatinine (median) and 22.3 pg/mg-creatinine, respectively. Urinary LTE4 concentration increased during the first 3h after allergen inhalation in atopic patients. However, allergen-induced bronchoconstriction was not associated with an increased concentration of LTB4 glucuronide in urine. The method enabled us to precisely determine urinary leukotriene B4 glucuronide concentration.     

High-performance liquid chromatographic determination of the magnetic resonance imaging contrast agent Gadocoletate ion in human plasma, urine and faeces

Gadocoletate ion is a new paramagnetic intravascular contrast agent for magnetic resonance imaging (MRI). An high-performance liquid chromatographic method for assaying Gadocoletate ion in human plasma, urine and faecal samples is described. The analysis is based on the reversed-phase chromatographic separation of Gadocoletate ion from the endogenous components of the biological matrices and its detection during elution by ultraviolet light absorption at 200 nm. The selectivity of the method was satisfactory. The mean absolute recovery during the analytical sample preparation was greater than 87%. The precision, expressed as coefficient of variation (CV%) ranged from 0.29 to 5.90% and the accuracy, expressed as mean relative error (R.E.%) of the analytical method ranged from -3.7 to +7.1%. The detection limit in plasma and urine was 2.01 and 10.0 microg/mL (0.00203 and 0.0101 micromol/mL), respectively. The detection limit in homogenized faecal samples was 17.7 microg/g (0.0179 micromol/g). Stability studies were performed in human plasma and urine samples during the analytical cycle. Gadocoletate ion was shown to be stable in human plasma and in human urine when stored at about +4 degrees C for up 24 h, and after three freeze-thaw cycles. In addition, it was shown to be stable in samples of processed plasma and in diluted urine at about +4 degrees C for 48 h, and at room temperature for at least 24 h. As regards the long-term stability of Gadocoletate ion, the results of dedicated studies showed that Gadocoletate ion is stable in human plasma samples when stored at +4 degrees C for up to 30 days and at -80 degrees C for up to 90 days. Gadocoletate ion is stable in samples of human urine when stored at +4 degrees C for up to 30 days, and when stored at -20 degrees C and at -80 degrees C for up to 90 days. The method has been successfully validated in human plasma, urine and faeces and it has been shown to be precise, accurate and reliable.     

High-performance liquid chromatographic separation of acylcarnitines following derivatization with 4'-bromophenacyl trifluoromethanesulfonate

A high-performance liquid chromatographic method for the separation of acylcarnitines after derivatization with 4'-bromophenacyl trifluoromethanesulfonate is presented. Derivatization of acylcarnitines was achieved at room temperature within 10 min. Separation of the acylcarnitine 4'-bromophenacyl esters was accomplished by high-performance liquid chromatography using as the analytical column a Resolve-PAK 5-microns C18 radially compressed cartridge eluted with a tertiary gradient containing varying proportions of water, acetonitrile, tetrahydrofuran, triethylamine, potassium phosphate, and phosphoric acid. Acylcarnitine 4'-bromophenacyl esters were detected spectrophotometrically at 254 nm. Baseline separation was obtained for a standard mixture (5 nmol of each injected) containing carnitine, acetyl-, propionyl-, butyryl-, valeryl-, hexanoyl-, heptanoyl-, octanoyl-, nonanoyl-, decanoyl-, lauroyl-, myristroyl-, palmitoyl-, and stearoylcarnitine. Nearly complete separation was obtained for a standard mixture containing butyryl-, isobutyryl-, isovaleryl-, and 2-methylbutyrylcarnitine. The method was applied to a normal human urine and then to this same urine spiked with the acylcarnitine standards. Urinary acylcarnitine profiles from patients having propionic acidemia, isovaleric acidemia, and medium-chain acyl-CoA dehydrogenase deficiency were performed. Urinary isovalerylcarnitine was quantified in the patient with isovaleric acidemia using heptanoylcarnitine as an internal standard.     

Structural identification of sulfated tyrosine in human urine

A reliable HPLC method was used for the identification of positional isomerism and stereoisomerism of sulfated tyrosine residues in human urine. Upon separation of human urine by ion-pair HPLC on a reverse-phase column, p-tyrosine-O-sulfate (p-TyrS) was identified. Differentiation of the L and D forms was done by using a column with a chiral stationary phase. It was concluded that L-p-tyrosine (L-p-Tyr) which is the predominant tyrosine isomer in the human body, was sulfated and excreted in human urine as a normal constituent. The sulfated forms of D-p-Tyr and m-Tyr could not be detected under these analytical conditions.     

UHPLC method for the simultaneous determination of β-blockers, isoflavones and their metabolites in human urine

A rapid-resolution ultra high-performance liquid chromatography separation method (UHPLC) for the simultaneous determination of the following β-blockers: milrinone, sotalol, metoprolol, propranolol and carvedilol, and their metabolites: 5′-hydroxylphenyl-carvedilol, O-desmethylcarvedilol, 4-hydroxypropranolol, α-hydroxy-metoprolol, O-desmethyl-metoprolol; the following isoflavones: genistein, daidzein, glycitin, glycitein, puerarin and biochanin A; as well as their metabolites: dihydrogenistein, desmethylglycitein, 8-hydroxygenistein, daidzein-7,4′-diglucoside, 8-hydroxydaidzein, dihydrobiochanin A in human urine was optimized. The analysed compounds were extracted from human urine by means of solid phase extraction (SPE). The effective UHPLC separation of the examined compounds was applied on a Hypersil GOLD? (50 mm × 2.1 mm, 1.9 μm) column with a gradient mobile phase system and a UV detector. The complete separation of all analytes was achieved within 8.0 min. The method was validated for the determination of the aforementioned substances in human urine. The linear ranges, limits of detection (LOD) and limits of quantification (LOQ) for β-blockers, isoflavones and their metabolites were determined. The intra- and inter-day precision (%C.V.) was less than 4.48%, and the intra-day and inter-day accuracy was less than 4.74%. The tested SPE sorbent proved that appropriate absolute recoveries can be obtained for Oasis HLB (Waters). The mean recovery of the analytes, using the new SPE procedure, amounted from 70.14% to 99.85%. The present paper reports, for the first time, the method for the determination of β-blockers, isoflavones and their metabolites in human urine samples. The newly developed method was suitably validated and successfully applied for the analysis of the certain of the aforementioned analytes in human urine samples obtained from the patients suffering cardiovascular disease.     

Analysis of the antidiabetic drug acarbose by capillary electrophoresis

This study describes the derivatization of the pseudooligosaccharide acarbose and its main metabolite, component 2, with 7-aminonaphthalene-1,3-disulfonic acid (ANDS) in human urine. Their efficient separation was possible by means of capillary zone electrophoresis, using a capillary tube of fused-silica containing 100 mM triethylammonium phosphate buffer, pH 1.5. On column laser-induced fluorescence allowed the detection of the pseudooligosaccharides in human urine in the nanomolar range. With this method, acarbose and component 2 were quantified in human urine after application of 300 mg of acarbose.     

Quantitative measurement of sulforaphane, iberin and their mercapturic acid pathway metabolites in human plasma and urine using liquid chromatography-tandem electrospray ionisation mass spectrometry

A quantitative liquid chromatography positive ion electrospray tandem mass spectrometric method for the simultaneous determination of sulforaphane, iberin and their metabolites in human urine and plasma is described. The stability of the metabolites was determined in aqueous solution and in human plasma. Gradient liquid chromatographic separation was performed on a Zorbax SB-Aq 3.5 microm (100 x 2.1mm) column, using a mobile phase (flow rate 0.25 mL/min) consisting of ammonium acetate buffer at pH 4 and acetonitrile. Butyl thiocarbamoyl l-cysteine was used as internal standard. The assay was linear (r(2)>0.99) over the range of 0.03-300 microM in urine and 0.03-15 microM in plasma with intra- and inter-day assay precision (<10% CV) and accuracy (<20%). The lower limits of quantitation were in the range of 10-150 nmol/L. The method has been used to report, for the first time, individual quantitative measurement of each of the mercapturic acid pathway metabolites of sulforaphane and iberin in both human plasma and urine following a dietary study of broccoli consumption.     

Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry

A qualitative liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol-water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1-2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine.     

Amine metabolite profile of normal and uremic urine using gas chromatography—mass spectrometry

A method for the simultaneous analysis of phenolic amines and aliphatic amines in human urine is described. The amine metabolites in urine were extracted using Dowex 50W-X8 cationic resin, derivatized and analyzed by a gas chromatographic—mass spectrometric—computer system. The amine metabolites profile of 5 ml of urine was obtained with good gas chromatographic separation. The gas chromatographic method described here separates urinary phenolic amines, di- and polyamines and methylguanidine in a single chromatographic separation. The urinary levels of methylguanidine, putrescine, cadaverine, spermidine, p-tyramine, dopamine, and 3-methoxytyramine were quantitated by using a mass spectrometric technique. In uremic patients, only the urinary excretion of methylguanidine was increased in comparison with normal subjects, although the urinary excretion of other amines was decreased in uremic patients.     

Specific quantification of mouse and human keratin proteins by radioimmunoassay.

An accurate and sensitive method was developed for the complete separation of the native tetrapyrroles, such as bilirubin and its mono- and di-conjugates of glucuronic acid, glucose and xylose, by ion-pair reversed-phase high-pressure liquid chromatography. The application of this method was demonstrated by the analysis of bile pigments in human bile and urine, and the method also makes it possible to estimate very low UDP-glucuronyltransferase activity, such as is found in the human foetal and neonatal liver.     

Determination of fexofenadine in human plasma and urine by liquid chromatography-mass spectrometry

A sensitive method was developed to determine fexofenadine in human plasma and urine by HPLC-electrospray mass spectrometry with MDL 026042 as internal standard. Extraction was carried out on C18 solid-phase extraction cartridges. The mobile phases used for HPLC were: (A) 12 mM ammonium acetate in water and (B) acetonitrile. Chromatographic separation was achieved on a LUNA CN column (10 cm x 2.0 mm I.D., particle size 3 microm) using a linear gradient from 40% B to 60% B in 10 min. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions, m/z 502.3 for fexofenadine and m/z 530.3 for the internal standard. The limit of quantification achieved with this method was 0.5 ng/ml in plasma and 1.0 ng in 50 microl of urine. The method described was successfully applied to the determination of fexofenadine in human plasma and urine in pharmacokinetic studies.     

A novel clean-up method for urine analysis of low-molecular mass aldehydes by capillary electrophoresis with laser-induced fluorescence detection

A rapid clean-up method using ultra-filtration was developed to remove sample matrix in the determination of low-molecular mass aldehydes in human urine. The ensuing filtrate was derivatized with fluorescein 5-thiosemicarbazide and the labelled aldehydes determined by capillary zone electrophoresis with laser-induced fluorescence detection. Practical aspects related to the effect of the urine sample matrix on the label chemistry and the electrophoretic separation showed that the urine samples must be diluted 20-fold after their ultra-filtration. By using synthetic urine, linear ranges were established in the range of 15-5000 μg/l with limits of detection between 4.5 and 9 μg/l. The intra- and inter-assay precision (relative standard deviation, %) of the aldehydes ranged from 4.1% to 8.4% and 6.1%-9.6%, respectively, and the average specific uncertainty was 149±12 ng. The average recoveries performed on two levels by enriching synthetic urine samples ranged between 94% and 100%. Finally, the proposed method was applied to check low-molecular mass aldehydes in the human urine of a female volunteer to obtain information about the risk in her exposure to these chemicals in the workplace.     

Development of a LC/MS/MS method for the analysis of cannabinoids in human EDTA-plasma and urine after small doses of Cannabis sativa extracts

A novel high-performance liquid chromatographic separation method with tandem-mass spectrometry detection was developed for the simultaneous determination of Delta(9)-tetrahydrocannabinol (THC) and its major metabolites 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) as well as the components cannabidiol (CBD) and cannabinol (CBN) in human EDTA-plasma and urine. Run time was 25 min. Lower limit of quantification was 0.2 ng/ml. The coefficients of variation of all inter- and intra-assay determinations were between 1.3 and 15.5%. The method was successfully applied to the determination of cannabinoids in human plasma and human urine after administration of Delta(9)-tetrahydrocannabinol or Cannabis sativa extracts.     

Isolation and identification of S-adenosylmethionine from human urine

Procedures are described for the isolation and identification of adenosylmethionine from human urine. Previously described preliminary separative procedures using anion and cation exchange columns and an XAD-4 resin column have been extended to permit the separation of adenosylmethionine. The adenosylmethionine has been identified by conversion to methylthioadenosine followed by rechromatography of the latter compound with three different types of columns and elution systems. Mean adenosylmethionine values for urine were as follows: adults, 0.26; children, 0.36 nmole/mumole creatinine. Recovery of adenosylmethionine added to urine and determined by this separative procedure was 52%.     

N-acetyl[3H]leukotriene D4: a stable internal standard for automated BIO-FAST HPLC extraction and separation of LTE4 from human urine.

The BIO-FAST (Fully Automated Sample Treatment) HPLC can be used for the isolation and separation of leukotriene E4 (LTE4) from the urine of asthmatic patients. A chemically related leukotriene, N-acetyl[14,15-3H]leukotriene D4 (NAc[3H]LTD4), has been evaluated as an internal standard to allow full automation of the BIO-FAST method. NAcLTD4 is not a human metabolite, does not co-elute with endogenously produced LTs and is stable in native urine at 37 degrees C for at least 18 h. Recovery and stability studies were conducted by adding NAc[3H]LTD4 and [3H]LTE4 to the baseline urine of four asthmatic patients. Automated extraction of these four samples over 22 hours, using the BIO-FAST system, yielded recoveries of 80.5% (6.6 %CV, n = 12) and 72.4% (10.0 %CV, n = 12) for the NAc[3H]LTD4 and [3H]LTE4, respectively. The ratio of NAc[3H]LTD4 to [3H]LTE4 was 1.12 (6.3 %CV, n = 12) demonstrating the consistent relative extraction of these two leukotrienes.     

Sensitive high-performance liquid chromatographic method for the determination of 2-phenylethylamine in human urine

A new sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination of 2-phenylethylamine (PEA) in human urine. The analytical procedure involved a simple extraction of the analyte from urine, followed by precolumn derivatisation of the sample with o-phthalaldehyde. The HPLC separation was performed under isocratic conditions using an Erbasil S C₁₈ (250 × 4.0 mm I.D., particle size 3 μm) reversed-phase column. The limit of quantification was 0.5 ng of PEA/ml of urine. The method showed good linearity, accuracy and precision data in the concentration range 0.5–200 ng/ml of urine. The method was successfully applied to the determination of PEA urinary excretion in Parkinsonian patients after oral administration of the monoamine oxidase B (MAO-B) inhibitor, selegiline.     

Determination of the anti-platelet-activating factor BN-50727 and metabolites in human urine by high-performance liquid chromatography using solid-phase extraction

A sensitive and selective HPLC solid-phase extraction procedure was developed for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human urine. The procedure consisted in a double solid-phase extraction of the urine samples on cyanopropyl and silica cartridges, followed by an automated solid-phase extraction of the drug and metabolites on CBA cartridges and posterior elution on-line to the chromatographic system for its separation. The method allowed quantitation in the concentration range 10–2400 ng/ml urine for both BN-50727 and the main metabolite, the O-demethylated BN-50727 product. The limit of quantitation for both compounds was 10 ng/ml. The inter-assay precision of the method, expressed as relative standard deviation, ranged from 1.9 to 4.5% for BN-50727 and from 2.5 to 9.0% for the metabolite. The accuracy, expressed as relative error, ranged from −2.4 to 4.2% and from 0.2 to 6.2%, respectively. This paper describes the validation of the analytical methodology for the determination of BN-50727 in human urine and also for its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites in human urine after single-dose administration.     

Determination of ticarcillin epimers in plasma and urine with high-performance liquid chromatography

A high-performance liquid chromatogaphic method was developed for determining the concentrations of ticarcillin (TIPC) epimers in human plasma and urine. Samples were prepared for HPLC analysis with a solid-phase extraction method and the concentrations of TIPC epimers were determined using reversed-phase HPLC. The mobile phase was a mixture of 0.005 M phosphate buffer (pH 7.0) and methanol (12:1, v/v) with a flow-rate of 1.0 ml/min. TIPC epimers were detected at 254 nm. Baseline separation of the two epimers was observed for both plasma and urine samples with a detection limit of ca. 1 μg/ml with a S/N ratio of 3. No peaks interfering with either of the TIPC epimers were observed on the HPLC chromatograms for blank plasma and urine. The recovery was more than 80% for both plasma and urine samples. C.V. values for intra- and inter-day variabilities were 0.9–2.1 and 1.1–6.4%, respectively, at concentrations ranging between 5 and 200 μg/ml. The present method was used to determine the concentrations of TIPC epimers in plasma and urine following intravenous injection of TIPC to a human volunteer. It was found that both epimers were actively secreted into urine and that the secretion of TIPC was not stereoselective. Plasma protein binding was also measured, which revealed stereoselective binding of TIPC in human plasma.     

A rapid and accurate UPLC/MS/MS method for the determination of benzodiazepines in human urine

A rapid, sensitive, and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) assay method for simultaneous determination of 13 benzodiazepine compounds in human urine was developed and validated. Aliquots of 0.5 mL of urine specimens were used for the analysis and the benzodiazepines were extracted by single step methanol (containing 0.2% formic acid) precipitation and then separated on a BEH C18 (50 mm × 2.1 mm, 1.7 μm) analytical column with the temperature maintained at 45°C. The mobile phases consisted of methanol and water (both containing 0.2% formic acid) and the flow rate was 0.4 mL/min. The TQ detector, equipped with an electrospray ionization ion source, was set up with a positive mode. The acquisitions were performed in multiple-reaction monitoring (MRM) and the limit of quantification was 20 ng/mL for all of the 13 compounds. The low limits of detections (LODs) of the benzodiazepines in this method were between 0.5 and 2 ng/mL. The chromatographic separation time was 4 min and calibration curves in human urine were generated over the range of 20-2000 ng/mL. The method validation parameters such as accuracy, precision, carryover, recovery, stability, and specificity for all of the 13 compounds were within the acceptable range. This method is suitable for the high throughput screening of benzodiazepines in clinical laboratories.     

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.