Human bioavailability of olive oil secoiridoids: screening of metabolites in plasma and urine using UPLC coupled with high resolution mass spectrometry
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One of the metabolites of ochratoxin A (OA) is 4R-4-hydroxyochratoxin A (4-OH-OA), and the ratio of 4-OH-OA to OA excreted in urine can be linked to the carcinogenic potential of this compound. As further support to the hypothesis that OA can be involved in Balkan endemic nephropathy and the associated urinary system tumours, it was decided to investigate the presence of these two compounds in the urine of affected populations. A sensitive method is described for the determination of the compounds at the 10 ng l–1 level. It involves extraction, two purification steps by column chromatography and high-performance liquid chromatography (HPLC), an analysis by HPLC and a confirmatory test by HPLC after derivatisation.
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A sensitive and reliable high-pressure liquid chromatography (HPLC) assay for miloxacin and its two principal metabolites, 5,8-dihydro-8-oxo-2H-1,3-dioxolo[4,5-g]quinoline-7-carboxylic acid (M-1) and 1,4-dihydro-1,6-dimethoxy-7-hydroxy-4-oxoquinoline-3-carboxylic acid (M-2), in human serum and urine was developed. A strong anion-exchange Zipax SAX column using a mobile phase of 0.01 M citric acid solution containing 0.03 M sodium nitrate with pH 5.0 was used to achieve separation of the three compounds. The retention times of miloxacin, M-1, and M-2 were 3.8, 9.3, and 5.9 min, respectively. Serum and urine concentrations of these compounds as low as 10 ng/ml were measured. When results from the HPLC assay were compared with those from the microbiological assay of serum and urine samples from human subjects receiving miloxacin orally, the correlation coefficients were 0.94 for the serum and 0.99 for the urine. The HPLC assay method presents an alternative to the microbiological assay and permits future pharmacokinetic investigations of miloxacin.
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Conjugate
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A reproducible high performance liquid chromatography (HPLC) method was developed for analysis of uric acid in canine serum and urine. The method consists of precipitating serum proteins with phosphotungstic acid prior to HPLC analysis. Urine is analyzed after dilution with buffer. Chromatography is performed on a reversed-phase C-18 column with UV detection at 292 nm. Sensitivity of the method will allow reproducible measurement of uric acid at concentrations of 0.05 mg/dl in serum and 0.1 mg/dl in urine. The HPLC method has been used to quantify hundreds of canine serum and urine samples. The method is superior to UV absorption or colorimetric methods because its lower limit of detection allows measurement of uric acid at concentrations found in canine serum and urine.
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