High performance liquid chromatography determination of cyanide in urine by pre-column fluorescence derivatization

A method for the determination of cyanide in human urine has been developed. The method is based on the reaction of cyanide with 2,3-naphthalenedialdehyde and taurine to give a fluorescent product for reversed-phase HPLC separation and fluorometric detection. After centrifugation followed by dilution of urine samples, the specimens could be analysed directly by this method. The recovery of cyanide added to urine at concentration levels of 50-1000 pmol/mL was 85-96%. The detection limit of cyanide was 30 pmol/mL in urine. The method was successfully applied to the analysis of urine from smokers and nonsmokers. The mean concentrations of cyanide were found to be 215 pmol/mL for the former and 84 pmol/mL for the latter.     

Simple, rapid and sensitive determination of plasma taurine by high-performance liquid chromatography using pre-column derivative formation with fluorescamine.

A simple, rapid and sensitive method for the determination of plasma taurine by high-performance liquid chromatography in the isocratic mode has been developed. The deproteinized plasma was treated with fluorescamine. These derivatives were separated on a LiChrospher 100 RP-8 column within 15 min. The detection limit for taurine was 0.2 microM. The plasma taurine contents of yellowtail fish, Seriola quinqueradiata, beef cattle, dairy cows and chickens were determined to be 125 +/- 54, 5.6 +/- 1.4, 2.2 +/- 0.7 and 20.0 +/- 9.6 micrograms/ml, respectively.     

Determination of 15N enrichment of taurine in cat urine by high resolution fast-atom bombardment mass spectrometry.

A method is described for measuring the stable isotopic enrichment of taurine in cat urine samples by high resolution fast-atom bombardment mass spectrometry, after 15N labelled taurine was given to cats for the purpose of investigating taurine metabolism. The 15N enrichment of taurine was measured after hydrolysis and purification of taurine by anion/cation exchange chromatography. The isotopic ratio of taurine was determined by measuring the [M+H]+ ion peaks in the spectra of the unlabelled and labelled compounds under multiple ion scan conditions. The overall standard deviation of the measurement is better than 4%. This method requires no derivation and uses only 500 microL of urine samples.     

High-performance liquid chromatographic determination of formate as benzimidazole in biological samples

Formate was determined as benzimidazole by high-performance liquid chromatography after reaction with o-phenylenediamine at 130 degrees C for 2 h in 1 M perchloric acid. The useful concentration range was 1.6-40 mumol/l and the determination limit was 20 pmol. The recoveries from rat liver homogenate and human urine were 90.3 +/- 2.9 and 89.4 +/- 2.5%, respectively. Using this method, the activity of formaldehyde dehydrogenase in biological samples could be measured, and also the formate concentration in the liver and urine of rats to which methanol had been administered.     

Quantification of urinary N-acetyl-S- (propionamide)cysteine using an on-line clean-up system coupled with liquid chromatography/tandem mass spectrometry

Acrylamide has been reported to be present in high-temperature processed foods and normal processed food intake could lead to significant acrylamide exposure. Acrylamide in vivo can be conjugated with glutathione in the presence of glutathione transferase. This conjugation product is further metabolized and excreted as N-acetyl-S-(propionamide)cysteine (NASPC) in the urine. NASPC could be considered a biomarker for acrylamide exposure. The objective of this study was to develop a highly specific, rapid and sensitive method to quantify urinary NASPC, serving as a biomarker for acrylamide exposure assessment. Isotope-labeled [13C3]NASPC was successfully synthesized and used as an internal standard. This urine mixture was directly analyzed using a newly developed liquid chromatographic/tandem mass spectrometric method coupled with an on-line clean-up system. The detection limit for this method was estimated as < 5 microg l(-1)(0.4 pmol) on-column. The method was applied to measure the urinary level of NASPC in 70 apparently health subjects. The results showed that the NASPC urinary level was highly associated with smoking. Smokers had a significantly higher urinary NASPC level (135 +/- 88 microg g(-1) creatinine) than non-smokers (76 +/- 30 microg g(-1) creatinine). A highly sensitive and selective LC/MS/MS isotope dilution method was successfully established. With an on-line clean-up system, this system is capable of routine high-throughput analysis and accurate quantitation of NASPC in urine. This could be a useful tool for health surveillance for acrylamide exposure in a population for future study.     

HPLC with fluorescence detection of urinary phenol, cresols and xylenols using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a fluorescence labeling reagent.

A simple and sensitive HPLC method for the determination of phenolic compounds, i.e., phenol (Phe), cresols (Cres) and xylenols (Xyls), was developed. After a pre-column fluorescence derivatization of these compounds with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) at 60 degrees C for 30 min, 11 DIB derivatives were successfully separated within 50 min with an ODS column using CH3CN-H2O-CH3OH (25 + 22 + 53, v/v) as the eluent. The detection limits of DIB derivatives at a signal-to-noise ratio of 3 ranged from 0.15 to 1.09 microM (0.2-1.6 pmol per 20 microliters). The precision of the proposed method for both within- and between-day assays of free and total phenol related compounds was satisfactory (RSD < 9.5%). By the proposed method, Phe and p-Cre could be detected in normal urine samples, and the calculated concentrations of free Phe and p-Cre in unhydrolysed urine samples were 1.5 +/- 1.3 and 23.9 +/- 24.3 microM and those of total Phe and p-Cre in hydrolysed urine samples were 87.3 +/- 81.2 and 200.7 +/- 195.4 microM (n = 21), respectively.     

Quantitative determination of taurine in real samples by high-performance anion-exchange chromatography with integrated pulsed amperometric detection

As taurine is a very important compound involved in a large number of metabolic processes, it is naturally present in the mammal tissues and is often deliberately added in some foods as a fortifying component. A detailed knowledge of taurine metabolic roles in biological systems can be obtained only if a sensitive, reliable and rapid analytical method is available. This article describes the successful application of high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-IPAD) for taurine determination in egg white and yolk samples, as well extracts of human serum and urine. Applications are shown for determination of taurine in soft drinks and pharmaceutical preparations where the taurine content was evaluated by standard additions. These results were achieved without prior derivatization of taurine.     

High-performance liquid chromatographic determination of dopamine sulfoconjugates in urine after L-dopa administration

A procedure was developed for the separation and determination of dopamine-3-O-sulfate (DM-3-S) and dopamine-4-O-sulfate (DM-4-S) in the urine of subjects administered L-DOPA. The method consists of sample preparation using cation- and anion-exchange resins followed by determination of the sulfates by high-performance liquid chromatography. The addition recoveries were 96 +/- 2.9% (S.D.) for DM-3-S and 93 +/- 3.0% (S.D.) for DM-4-S. Twenty samples could be measured per day. When every 2-h urine specimen from normal subjects was analyzed after L-DOPA administration (0.5 g), the maximum excretion of each sulfate was observed in the second 2-h specimen. For the first 6 h 7.5 +/- 1.5% (S.D.) of the administered L-DOPA was excreted as DM-O-sulfates. During this time, the ratio of DM-4-S to the DM-O-sulfates was 11.7 +/- 0.58% (S.D.).     

Quantitative on-chip determination of taurine in energy and sports drinks

A new method for the quantitative determination of taurine in beverages by microchip electrophoresis was developed. A rapid and simple sample preparation procedure, only including two dilution steps and the addition of the fluorogenic labeling reagent NBD-Cl (4-chloro-7-nitrobenzofurazan), is applied. Using a home-built wavelength-resolved fluorescence detector, the separation and determination of the taurine derivative could be achieved in only 12 s, while the additional spectral information was utilized to ensure peak purity. Spanning from 0.1 to 50 mmol L(-1), the linear dynamic range of the applied method was adapted to the apparent contents in common taurine containing beverages. The smallest detectable amount of the taurine derivative actually injected into the separation channel was as low as 60 amol. The method was successfully validated by an independent liquid chromatographic method.     

Use of an automated chromium reduction system for hydrogen isotope ratio analysis of physiological fluids applied to doubly labeled water analysis

The doubly labeled water method is commonly used to measure total energy expenditure in free-living subjects. The method, however, requires accurate and precise deuterium abundance determinations, which can be laborious. The aim of this study was to evaluate a fully automated, high-throughput, chromium reduction technique for the measurement of deuterium abundances in physiological fluids. The chromium technique was compared with an off-line zinc bomb reduction technique and also subjected to test-retest analysis. Analysis of international water standards demonstrated that the chromium technique was accurate and had a within-day precision of <1 per thousand. Addition of organic matter to water samples demonstrated that the technique was sensitive to interference at levels between 2 and 5 g l(-1). Physiological samples could be analyzed without this interference, plasma by 10000 Da exclusion filtration, saliva by sedimentation and urine by decolorizing with carbon black. Chromium reduction of urine specimens from doubly labeled water studies indicated no bias relative to zinc reduction with a mean difference in calculated energy expenditure of -0.2 +/- 3.9%. Blinded reanalysis of urine specimens from a second doubly labeled water study demonstrated a test-retest coefficient of variation of 4%. The chromium reduction method was found to be a rapid, accurate and precise method for the analysis of urine specimens from doubly labeled water.     

Mass fragmentographic determination of prostaglandin F2 alpha in human and rabbit urine

Analysis of prostaglandin F2 alpha (PGF2 alpha) in urine is a useful indicator of renal prostaglandin synthesis. A mass fragmentographic method for PGF2 alpha analysis in human urine was developed using [3,3,4,4-2H4]PGF2 alpha as an internal standard and carrier. PGF2 alpha was extracted from urine (20 ml) with chloroform, purified by preparative thin-layer chromatography and converted to the methyl ester trimethylsilyl ether before analysis by gas chromatography--mass spectrometry. The specificity of the urine analysis was demonstrated by retention time and the use of two pairs of fragments m/e 494/498 and 513/517 with the same results. The coefficient of variation for duplicate analysis averaged 12.6%, n = 17. Urine from recumbent women contained 4.9 +/- 2.6 (S.D.) ng/ml or 4.1 +/- 1.0 ng PGF2 alpha per mg creatinine (n = 10) with little diurnal variation. Male urine contained 5.0 +/- 2.7 (S.D.) ng/ml or 3.7 +/- 2.1 ng/mg creatinine (n = 10). Similar concentrations were found in boys and in girls. These observations indicate that urinary PGF2 alpha originates from the kidneys with little contribution from the male accessory sexual glands. This method can also be applied to analysis of PGF2 alpha in rabbit urine.     

Spectrofluorometric determination of ketorolac tromethamine via its oxidation with cerium(IV) in pharmaceutical preparations and biological fluids

A simple and sensitive fluorometric method for determination of ketorolac tromethamine was studied. The method depends on oxidation of the drug with cerium(IV) and subsequent monitoring of the fluorescence of the induced cerium(III) at lambda(em) 365 nm after excitation at 255 nm. Different variables affecting the reaction conditions, such as the concentrations of cerium(IV), sulfuric acid concentration, reaction time, and temperature, were carefully studied and optimized. Under the optimum conditions, a linear relationship was found between the relative fluorescence intensity and the concentration of the investigated drug in the range of 0.1-0.8 microg/mL. No interferences could be observed from the excipients commonly present in dosage forms. The proposed method was successfully applied to the analysis of the investigated drug in its pure form, pharmaceutical preparations, and biological fluids with good accuracy and precision. The recoveries for pharmaceutical formulations ranged from 99.8-101.0 +/- 0.6% for tablets, 98.5-101.0 +/- 1.0% for ampoules, and 99.0-100.5 +/- 0.7% for eye drops. The results obtained by the proposed method were satisfactory compared with those obtained by the official method. The recoveries for biological fluids were 99.1-100.4 +/- 0.7 and 99.0-100.0 +/- 0.5% for plasma and urine, respectively.     

稀土对大鼠尿液成分影响的核磁共振研究

目前,稀土生物效应的研究已进入了分子和细胞水平,但在活体动物内研究稀土的作用情况还不多见。稀土化合物随食物进入动物体内后,会对动物体内的器官、组织、细胞产生一定影响,将导致其体液容量、分布、电解质浓度等方面的变化。现代NMR技术是目前药理毒理学研究的有效手段之一,它具有简便、全面、快捷、非破坏性等优点,可用于对体液样品的检测和研究。本文采用现代核磁共振技术研究了稀土化合物硝酸镧灌胃给药后对Wistar大鼠尿液中代谢产物的影响,为阐明稀土化合物的毒理学机制和稀土的进一步开发利用提供理论依据。     

Rapid and sensitive quantification of urinary N7-methylguanine by isotope-dilution liquid chromatography/electrospray ionization tandem mass spectrometry with on-line solid-phase extraction

A rapid, highly specific and sensitive isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) method coupled with an on-line solid-phase extraction (SPE) system was developed to measure N7-methylguanine (N7-MeG) in urine. 15N5-Labeled N7-MeG was synthesized to serve as an internal standard, and an on-line SPE cartridge was used for on-line sample cleanup and enrichment. The urine sample can be directly analyzed within 15 min without prior sample purification. The detection limit for this method was estimated as 8.0 pg/mL (4.8 pmol) on-column. This method was further applied to study exposure to methylating agents arising from cigarette smoke. Sixty-seven volunteers were recruited, including 32 regular smokers and 35 nonsmokers. Urinary cotinine, a major metabolite of nicotine, was also determined using an isotope-dilution LC/MS/MS method. The results showed that urinary levels of N7-MeG observed in smokers (4215 +/- 1739 ng/mg creatinine) were significantly (P < 0.01) higher than those in nonsmokers (3035 +/- 720 ng/mg creatinine). It was further noted that the urinary level of N7-MeG was found to be correlated with that of cotinine for smokers, implying that cigarette smoking resulted in increased DNA methylation, followed by depurination and excretion of N7-MeG in urine. As a result of the on-line extraction system, this method is capable of routine high-throughput analysis and accurate quantitation of N7-MeG, and could be a useful tool for health surveillance of methylating agent exposure.     

Identification and quantification of apolipoprotein D in normal human urine

Apolipoprotein D has been identified in normal human urine, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with monospecific antibodies. Urinary apolipoprotein D appeared as a main 33,000 u protein together with a minor fraction corresponding to its partially deglycosylated species of lower molecular mass. No high molecular mass forms of apolipoprotein D naturally occurring in plasma could be detected. The apolipoprotein D mean +/- SD concentration assayed with rocket immunoelectrophoresis, in urine samples from nine apparently healthy normal men, was 1.4 +/- 1.0 mg/L (range: 0.2-3.0 mg/L). Among the plasma apolipoproteins, apolipoprotein D behaves uniquely as regards its excretion in urine; the other apolipoproteins belonging to the A, B, D and E groups, although of low molecular masses, are present, at most, in trace amounts in normal urine.     

Method for measuring endogenous 3-O-methyldopa in urine and plasma.

The present report describes a method using column liquid chromatography with electrochemical detection for assaying concentrations of 3-O-methyldopa in urine and plasma. The technique combines a one-step sample preparation scheme with post-column flow-through electrodes in series, allowing adequate chromatographic separation of 3-O-methyldopa from other endogenous substances in urine. The validity of the method was confirmed by markedly decreased urinary 3-O-methyldopa levels after administration of an inhibitor of catechol-O-methyltransferase to rats, radioactivity in chromatographic fractions corresponding to 3-O-methyldopa in urine of rats undergoing infusion of [3H]-L-DOPA, and correlations between excretion rates of 3-O-methyldopa and catechols in humans. In healthy humans, urinary excretion of 3-O-methyldopa averaged 974 +/- 707 (S.D.) nmol per day, and plasma levels of 3-O-methyldopa averaged 89 +/- 32 nmol/l. The method should be useful in studies about the metabolism of endogenous and exogenous DOPA.     

Determination of cadmium and lead in urine and other biological samples by graphite-furnace atomic-absorption spectrometry

A method for determining cadmium and lead in urine and other biological samples by graphite-furnace atomic-absorption spectrometry is reported. Samples were analysed after wet or dry ashing and without extraction or matrix-modification techniques, in laminar-flow clean-room; negligible blank contributions were found. Matrix interference effects were observed only for lead and were resolved by the method of standard additions. Five NBS biological reference materials were used as internal quality-control standards. The urinary levels for non-exposed volunteers ranged from 0.16 +/- 0.01 to 1.65 +/- 0.20 and from 6 +/- 1 to 31 +/- 6 ng/ml for cadmium and lead, respectively; this corresponds to 0.15 +/- 0.02 to 2.01 +/- 0.16 and 7 +/- 1 to 31 +/- 3 mug/day. The average relative standard deviation for 60 urine samples was 10% for cadmium and 13% for lead.     

A rapid gas chromatographic method for the determination of chlorophenoxyisobutyric acid in plasma and urine.

A rapid gas chromatographic method is described for the determination of chlorophenoxyisobutyric acid (the active metabolite of clofibrate) in plasma and urine. The assay involves an extraction into toluene and back-extraction of the chlorophenoxyisobutyric acid and the internal standard (2-naphthoic acid) into the methylating reagent (trimethylanilinium hydroxide). Concentrations of 1 mug/ml in plasma and urine can easily be measured; the precision of the method is 3.3 +/- 0.7% for plasma and 2.7 +/- 0.4% for urine. There is no interference from endogenous compounds or from drugs commonly prescribed together with clofibrate.     

Quantitative determination of taurine and related biomarkers in urine by liquid chromatography–tandem mass spectrometry

Current urinary bladder cancer diagnosis is commonly based on a biopsy obtained during cystoscopy. This invasive method causes discomfort and pain in patients. Recently, taurine and several other compounds such as L-phenylalanine and hippuric acid in urine were found to be indicators of bladder cancer. However, because of a lack of sensitive and accurate analytical techniques, it is impossible to detect these compounds in urine at low levels. In this study, using liquid chromatography–tandem mass spectrometry (LC-MS/MS), a noninvasive method was developed to separate and detect these compounds in urine. ¹⁵N₂-L-glutamine was used as the internal standard, and creatinine acted as an indicator for urine dilution. A phenyl-hexyl column was used for the separation at an isocratic condition of 0.2% formic acid in water and 0.2% formic acid in methanol. Analytes were detected in multiple-reaction monitoring with positive ionization mode. The limit of detection range is 0.18–6 nM and the limit of quantitation ranges from 0.6 to 17.6 nM. The parameters affecting separation and quantification were also investigated and optimized. Proper clinical validation of these biomarkers can be done using this reliable, fast, and simple method. Furthermore, with simple modifications, this method could be applied to other physiological fluids and other types of diseases.     

High-performance liquid chromatographic analysis of isoxicam in human plasma and urine

A sensitive, selective, and rapid high-performance liquid chromatographic procedure was developed for the determination of isoxicam in human plasma and urine. Acidified plasma or urine were extracted with toluene. Portions of the organic extract were evaporated to dryness, the residue dissolved in tetrahydrofuran (plasma) or acetonitrile (urine) and chromatographed on a mu Bondapak C18 column preceded by a 4-5 cm X 2 mm I.D. column packed with Corasil C18. Quantitation was obtained by UV spectrometry at 320 nm. Linearity in plasma ranged from 0.2 to 10 micrograms/ml. Recoveries from plasma samples seeded with 1.8, 4 and 8 micrograms/ml isoxicam were 1.86 +/- 0.077, 4.10 +/- 0.107 and 8.43 +/- 0.154 micrograms/ml with relative standard deviations of 3.3%, 2.5% and 5.4%, respectively. The linearity in urine ranged from 0.125 to 2 micrograms/ml. The precision of the method was 3.3-9.0% relative standard deviation over the linear range.