Urinary excretion of anthocyanins in humans after cranberry juice ingestion

Cranberry, which is rich in polyphenols, including anthocyanins and proanthocyanidins, has been found to have various effects beneficial to human health, including prevention of urinary tract infections. These effects have been associated with polyphenols in the fruit. We investigated the excretion of anthocyanins in human urine after ingestion of cranberry juice. Eleven healthy volunteers consumed 200 ml of cranberry juice containing 650.8 microg total anthocyanins. Urine samples were collected within 24 h before and after consumption. Six of 12 anthocyanins identified in cranberry were quantified in human urine by HPLC coupled with electrospray ionization and tandem mass spectrometry (HPLC-ESI-MS-MS). Among these, peonidin 3-O-galactoside, the second most plentiful anthocyanin in the juice, was found most abundantly in urine within 24 h, corresponding to 41.5 nmol (56.1% of total anthocyanins). The urinary levels of anthocyanins reached a maximum between 3 and 6 h after ingestion, and the recovery of total anthocyanins in the urine over 24 h was estimated to be 5.0% of the amount consumed. This study found high absorption and excretion of cranberry anthocyanins in human urine.     

Method for the determination of five toxicologically relevant arsenic species in human urine by liquid chromatography-hydride generation atomic absorption spectrometry

An analytical method for the simultaneous quantitation of arseneous acid (As(III)), arsenic acid (As(V)), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and trimethylarsine oxide (TMAO) in human urine by coupling of high-performance liquid chromatography with hydride generation atomic absorption spectrometry (HPLC/HG-AAS) via a flow-injection interface is presented. After arsenic species separation by anion-exchange displacement chromatography the compounds are on-line reduced to their corresponding hydrides and detected by atomic absorption spectrometry. Detection limits range from 1.1 (TMAO) to 2.6 microg/L (As(V)). The method has been applied to determine arsenic species in the urine of a volunteer before and after consumption of seafood as well as to analyse certified reference urine samples for their arsenic species content.     

Automated high-performance liquid chromatographic method for the determination of nedocromil sodium in human urine using bimodal column switching

An automated HPLC method is described for the determination of nedocromil sodium in human urine. An HPLC autosampler is used to inject urine samples onto a short reversed-phase column. This column acts as a concentration column and performs a preliminary extraction. The concentration column is automatically backflushed onto an ion-exchange column where final separation of nedocromil sodium from urine constituents occurs. Recovery, accuracy, precision, sensitivity and specificity were investigated. The method has been applied to urine samples from clinical studies, and the results were compared to those obtained using a radioimmunoassay developed previously.     

Column-switching high-performance liquid chromatographic assay for determination of apigenin and acacetin in human urine with ultraviolet absorbance detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of apigenin and the 4′-methylated derivative acacetin in human urine using column-switching and ultraviolet (UV) absorbance detection. Urine samples were enzymatically hydrolysed and solid-phase extracted prior to injection onto the HPLC system. Prior to elution of apigenin and the internal standard, 5,7,8-trihydroxyflavone, from the first column used for sample clean-up, the six-port valve was switched to the second column for analysis with UV detection. Detection of apigenin was precise and reproducible, with a limit of quantification of 10 ng ml⁻¹ urine. Detection and quantification of acacetin was linear down to 70 ng ml⁻¹ urine. The method has been successfully applied to determine the level of apigenin in 100 human urine samples from an intervention study with parsley.     

Highly sensitive high-performance liquid chromatographic determination method for a new erythromycin derivative, EM523, and its major metabolites in human plasma and urine using post-column tris(2,2′-bipyridine) ruthenium(III) chemiluminescence detection

A method for the simultaneous determination of de(N-methyl)-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal (EM523, I) and its three metabolites in human plasma and urine has been developed using high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection. Plasma and urine samples spiked with erythromycin as an internal standard were extracted with a mixture of dichloromethane and diethyl ether under alkaline conditions. The ortanic layer was evaporated under a stream of nitrogen gas. The reconstituted sample was injected into an HPLC apparatus and separated on an ODS column using a gradient elution method. The elute was reacted on-line with a mixture of tris(2,2′-bipyridine) ruthenium(II) and peroxodisulfate, and the generated CL intensity was detected. Optimization of the CL reaction conditions resulted in a sensitive and stable CL intensity for the determination of I and its metabolites. The recovery of each compound from human plasma and urine, and the sensitivity, linearity, accuracy and precision of the method were satisfactory. The lower limits of quantitation for each compound using 0.2 ml of plasma and 0.1 ml of urine were 1 and 00 ng/ml, respectively. This method has been used for the determination of I in samples from clinical trials.     

High-performance liquid chromatographic determination of PEG 600 in human urine

Polyethylene glycols (PEGs) are non-ionic, water-soluble synthetic polymers which have been widely used for many applications. Since they are of very low toxicity and are readily excreted in urine, PEGs in the molecular weight range 400–6000 have been used extensively in the study of intestinal physiology in man. A high-performance liquid chromatographic (HPLC) method has been developed for the determination of PEG 600 in human urine, which includes a pre-column derivatisation step. The dibenzoate derivatives of PEG 600 can be quantitatively prepared, and this, coupled with ultraviolet detection at 230 nm, has greatly improved the limit of detection for the determination of PEGs by HPLC. A suitable extraction procedure has also been developed which enabled PEG levels in urine to be monitored with much greater sensitivity than any previously reported method.     

A sensitive high-performance liquid chromatographic procedure with fluorometric detection for the analysis of decarboxylated S-adenosylmethionine and analogs in urine samples

A highly sensitive HPLC method for the determination of decarboxylated S-adenosylmethionine (dc-SAM) by fluorometric detection was developed. The reaction of dc-SAM and its analogs with chloroacetaldehyde leads to the corresponding 1,N6-etheno derivatives. These highly fluorescent derivatives were fully characterized through their proton nuclear magnetic resonance spectra and/or mass spectra. This derivatization procedure has been applied to the analysis of dc-SAM in rat and human urine. After a simple cation exchange column prepurification, the urine extracts were derivatized with chloroacetaldehyde and analyzed by reversed-phase HPLC with fluorometric detection. The method allowed the determination of subpicomole amounts of dc-SAM and was shown to be highly reproducible with the use of decarboxylated S-adenosylethionine as internal standard. The application of the method to the analysis of urine of rats treated with MDL 72175, a potent ornithine decarboxylase inhibitor, showed that the dc-SAM levels increased in a dose-related fashion.     

Antioxidant capacity of human blood plasma and human urine: simultaneous evaluation of the ORAC index and ascorbic acid concentration employing pyrogallol red as probe

The oxygen radical absorbance capacity (ORAC) methodology has been employed to estimate the antioxidant capacity of human blood plasma and human urine using pyrogallol red (ORAC-PGR) as target molecule. Uric acid, reduced glutathione, human serum albumin, and ascorbic acid (ASC) inhibited the consumption of pyrogallol red, but only ASC generated an induction time. Human blood plasma and human urine protected efficiently pyrogallol red. In these assays, both biological fluids generated neat induction times that were removed by ascorbate oxidase. From these results, ORAC-PGR method could be proposed as a simple alternative to evaluate an ORAC index and, simultaneously, to estimate the concentration of ascorbic acid in human blood plasma or human urine.     

Simultaneous quantification of tiloronoxim and tilorone in human urine by liquid chromatography-tandem mass spectrometry

A simple, sensitive and specific HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the simultaneous quantification of tiloronoxim and its major active metabolite, tilorone, in human urine. The analytes, together with metoprolol, which was employed as an internal standard (IS), were extracted with a mixture solvent of chloroform/ethyl ether (1/2, v/v). The chromatographic separation was performed on a narrow-bore reversed phase HPLC column with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). The API 3,000 mass spectrometer was equipped with a TurboIonSpray interface and was operated on positive-ion, multiple reaction-monitoring (MRM) mode. The mass transitions monitored were m/z 426.3-->100.0, m/z 411.3-->100.0 and m/z 268.3-->116.1 for tiloronoxim, tilorone and the IS, respectively. The assay exhibited a linear dynamic range of 1-100 ng/ml for both tiloronoxim and tilorone based on the analysis of 0.2 ml aliquots of urine. The lower limit of quantification was 1 ng/ml for both compounds. Acceptable precision and accuracies were obtained for concentrations over the standard curve ranges. Run time of 8 min for each injection made it possible to analyze a high throughput of urine samples. The assay has been successfully used to analyze human urine samples from healthy volunteers.     

Determination of grepafloxacin in plasma and urine by a simple and rapid high-performance liquid chromatographic method

A rapid, specific, sensitive and economical method has been developed and validated for the determination of grepafloxacin in human plasma and urine. The assay consisted of reversed-phase HPLC with UV detection. Plasma proteins were removed by a fast and efficient procedure that has eliminated the need for costly extraction and evaporation. For the urine samples, the only required sample preparation was dilution. Separation was achieved on a reversed-phase TSK gel column with an isocratic mobile system. The method had a quantification limit of 0.05 μg/ml in plasma and 0.5 μg/ml in urine. The coefficients of variation (C.V.) were less than 4% for within- and between-day analyses. The method was successfully applied to a pharmacokinetic study, and was proved to be simple, fast and reproducible.     

Validation of an HPLC/MS/MS method with isotopic dilution for quantitative determination of trans,trans-muconic acid in urine samples of workers exposed to low benzene concentrations

Urinary trans,trans-muconic acid (t,t-MA), a biomarker of benzene exposure, is usually determined by HPLC methods with detection by either UV or, more recently, electrospray tandem mass spectrometry. However, not all these methods have been fully validated for quantitative analysis. This paper presents an HPLC/MS/MS method for reliable quantitative determination of t,t-MA that uses a commercial deuterium-labeled isotope as internal standard; the matrix effect has been evaluated and LOD is 0.22 microg/L. We used this method to test 200 urine samples, 175 of them collected at end-of-shift from workers in an oil refinery.     

High-performance thin-layer chromatographic determination of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in urine and comparison with high-performance liquid chromatography

The consumption of N-ethyl-3,4-methylenedioxyamphetamine (MDE, 1), an analogue of ecstasy, can be detected by direct in situ HPTLC-FTIR measurement of the main metabolite N-ethyl-4-hydroxy-3-methoxyamphetamine (HME, 2). HME (2) can, like the other important metabolite 3,4-methylenedioxyamphetamine (MDA, 3) and unchanged MDE (1), be determined quantitatively in urine by HPTLC-UV after two-step automatic development. The results have been compared with those obtained using an HPLC method. The differences were not generally significant. Small deviations were attributable to the different sample preparation methods necessary. The working range for the HPTLC method was between 0.1 and 8.2 μg/ml and for the HPLC method between 0.2 and 60.0 μg/ml. The method standard deviations were 2.66–4.91% (HPTLC) and 0.48–3.67% (HPLC).     

Quantification of pravastatin in human plasma and urine after solid phase extraction using high performance liquid chromatography with ultraviolet detection

A high performance liquid chromatography (HPLC) method for the estimation of pravastatin in human plasma and urine samples has been developed. The preparation of the samples was performed by automated solid phase extraction using clonazepam as internal standard. The compounds were separated by isocratic reversed-phase HPLC (C(18)) and detected at 239 nm. The method was linear up to concentrations of 200 ng/ml in plasma and 2000 ng/ml in urine. The intra-assay variability for pravastatin in plasma ranged from 0.9% to 3.5% and from 2.5% to 5.3% in urine. The inter-assay variability ranged from 9.1% to 10.2% in plasma and from 3.9% to 7.5% in urine. The validated limits of quantification were 1.9 ng/ml for plasma and 125 ng/ml for urine estimation. These method characteristics allowed the determination of the pharmacokinetic parameters of pravastatin after administration of therapeutic doses.     

Urine sex steroid profile in healthy volunteers during isolation in a pressurized compartment

In order to study the time course of the parameters of urine sex steroid profile and its potential boundary changes, the quantitative determination of a number of endogenous steroids and their metabolites in healthy human urine has been carried out by the gas chromatography method using mass-selective detection. The samples were obtained from six volunteers under the conditions of total monitoring of vital activity factors affecting urine steroid profile (diet, water consumption, physical activity, temperature and air composition, day-night rhythm, and the psychoemotional state) in an experimental study using a pressurized compartment. The healthy human profile parameters of urine steroids were found, which were affected by conditions of controlled vital activity in a pressurized compartment. Parameters of the individual and group variability of the parameters of steroid profile and their dependence on experimental factors, salt consumption mode, and autonomous vital activity, were found.     

Purification of human epidermal growth factor (urogastrone) from urine

Human epidermal growth factor has been isolated from a concentrated chromatographic eluate of human urine. The purification method utilizes six chromatographic steps including adsorption to aminoethylcellulose (AE-11), gel filtration on Sephadex G-50, carboxymethylcellulose (CM-52) chromatography, ion-exchange HPLC and reverse-phase HPLC. The final product appears homogeneous and identical to pure gamma-urogastrone when analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC using two eluent systems. The yield of the method described above allowed the development of a sensitive radioimmunoassay system for this growth factor.     

High-performance liquid chromatographic methods for the determination of a new carbapenem antibiotic, L-749,345, in human plasma and urine

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of a new carbapenem antibiotic assay using ultraviolet detection has been developed for a new carbapenem antibiotic L-749,345 in human plasma and urine. A plasma sample is centrifuged and then injected onto an extraction column using 25 mM phosphate buffer, pH 6.5. After 3 min, using a column-switching valve, the analyte is back-flushed with 10.5% methanol–phosphate buffer for 3 min onto a Hypersil 5 μm C₁₈ BDS 100×4.6 mm analytical column and then detected by absorbance at 300 nm. The sample preparation and HPLC conditions for the urine assay are similar, except for a longer analytical column 150×4.6 mm. The plasma assay is specific and linear from 0.125 to 50 μg/ml; the urine assay is linear from 1.25 to 100 μg/ml.     

Detection of cystathionine and lanthionine ketimines in human urine

A recently developed HPLC procedure for the determination of cystathionine ketimine (CK) and lanthionine ketimine (LK) has been applied to the detection of these compounds in human urine. The assay has taken advantage of the selective production of an absorbance at 380 nm, not seen with other amino acids, when the two ketimines are reacted with phenylisothiocyanate. Coelution with authentic phenylthiohydantoin derivatives of CK and LK and the identical absorption spectra establish the identity of the compounds found in the urine with the synthetic products. Quantitation of the two ketimines by HPLC indicates that the excretion of CK and LK is respectively 606 micrograms and 84 micrograms per g of creatinine as mean values of 10 healthy subjects of both sexes, 20-40 years old, in the early morning voided urine.     

Simultaneous analysis of several non-steroidal anti-inflammatory drugs in human urine by high-performance liquid chromatography with normal solid-phase extraction

A practical and reproducible high-performance liquid chromatographic method using normal solid-phase extraction has been developed for the simultaneous analysis of twelve non-steroidal anti-inflammatory drugs (NSAIDs) in human urine. A urine specimen mixed with acetate buffer pH 5.0 was purified by solid-phase extraction on a Sep-Pak Silica cartridge. The analyte was chromatographed by a reversed-phase Inertsil ODS-2 column using a phosphate buffer-acetonitrile at pH 5.0 as the mobile phase, and the effluent from the column was monitored at 230 or 320 nm. Absolute recoveries were greater than 73% for all of the twelve NSAIDs. The present method enabled simple manipulation and isocratic HPLC with UV analysis as well as high sensivity of 0.005 μg/ml for naproxen, and 0.05 μg/ml for sulindac, piroxicam, loxoprofen, ketoprofen, felbinac, fenbufen, flurbiprofen, diclofenac, ibuprofen and mefanamic acid as the quantitation limit in human urine using indomethacin as an internal standard.     

Sensitive analytical method for the novel H1-receptor antagonist HSR-609 in human plasma and urine by high-performance liquid chromatography with pre-column fluorescent derivatization

A simple and sensitive method for quantitation of HSR-609 (I) in human plasma and urine was developed using HPLC with the fluorescence labelling reagent 4-(N,N-dimethylaminosulfonyl)-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ). Compound I was extracted from human plasma and urine, and derivatized by reaction with DBD-PZ in the presence of Mukaiyama reagent A, an equimolar solution of 2,2′-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP) in acetonitrile. The reaction mixture was cleaned up by liquid-liquid extraction following the derivatization. The conjugate was analyzed by ion-pair HPLC with fluorometric detection. The quantitation limits for I were 0.5 ng/ml in plasma and 5 ng/ml in urine. Using this method, plasma concentration and urinary excretion of I were studied after oral administration of I to human volunteers.