High-performance liquid chromatographic determination of pyridostigmine bromide, nicotine, and their metabolites in rat plasma and urine

This study reports on the development of a rapid and simple method for the determination of the antinerve agent drug pyridostigmine bromide (3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) (PB), its metabolite N-methyl-3-hydroxypyridinium bromide, nicotine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidine), and its metabolites nornicotine (2-(3-pyridyl)pyrrolidine) and cotinine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidone) in rat plasma and urine. The compounds are extracted and eluted by methanol and acetonitrile using C18 Sep-Pak cartridges and separated using high-performance liquid chromatography by a gradient of methanol, acetonitrile, and water (pH 3.2) at a flow rate of 0.8 mL/min in a period of 14 min. UV detection was at 260 nm for nicotine and its metabolites and at 280 nm for PB and its metabolite. The limits of detection ranged between 20 and 70 ng/mL, and the limits of quantitation were 50-100 ng/mL. The average percent recovery of five spiked plasma samples were 85.7 +/- 7.3%, 80.4 +/- 5.8%, 78.9 +/- 5.4%, 76.7 +/- 6.4%, and 79.7 +/- 5.7% and for urine were 85.9 +/- 5.9%, 75.5 +/- 6.9%, 82.6 +/- 7.9%, 73.6 +/- 5.9%, and 77.7 +/- 6.3% for nicotine, nornicotine, cotinine, PB, and N-methyl-3-hydroxypyridinium bromide, respectively. The calibration curves for standard solutions of the compounds of peak areas and concentration are linear for a range between 100 and 1,000 ng/mL. This method is applied in order to analyze the previously mentioned chemicals and metabolites following their oral administration in rats.     

High-performance liquid chromatographic determination of lansoprazole and its metabolites in human serum and urine.

A simple and sensitive high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous determination of lansoprazole and its metabolites in human serum and urine. The analytes in serum or urine were extracted with diethyl ether-dichloromethane (7:3, v/v) followed by evaporation, dissolution and injection into a reversed-phase column. The recoveries of authentic analytes added to serum at 0.05-2 micrograms/ml or to urine at 1-20 micrograms/ml were greater than 88%, with the coefficients of variation less than 7.1%. The minimum determinable concentrations of all analytes were 5 ng/ml in serum and 50 ng/ml in urine. The method was successfully applied to a pharmacokinetic study of lansoprazole in human.     

High-performance liquid chromatographic method for the determination of mangiferin in rat plasma and urine

A reversed-phase high-performance liquid chromatography assay for mangiferin in rat plasma and urine was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) containing 3% acetic acid at a flow rate of 1 mL/min. Detection was at 257 and 365 nm for mangiferin in plasma and urine, respectively. The limit of quantitation (LOQ) of mangiferin was 0.6 microg/mL in plasma, and 0.48 microg/mL in urine. The standard curve was linear from 0.6 to 24 microg/mL in plasma, and 0.48 to 24 microg/mL in urine, both intra- and inter-day precision of the mangiferin were determined and their RSD did not exceed 10%. The method provides a technique for rapid analysis of mangiferin in rat plasma and urine, which can be used in pharmacokinetic studies.     

High-performance liquid chromatographic determination of the polar metabolites of nifedipine in plasma, blood and urine

A relatively simple reversed-phase high-performance liquid chromatographic method for the determination of the polar metabolites of nifedipine in biological fluids is described. After conversion of 2-hydroxymethyl-6-methyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid 5-methyl ester (IV) into 5,7-dihydro-2-methyl-4-(2-nitrophenyl)-5-oxofuro[3,4-b] pyridine-3-carboxylic acid methyl ester (V) by heating under acidic conditions, V was extracted with n-pentane-dichloromethane (7:3) and analysed on a C18 column with ultraviolet detection. Subsequently, 2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid monomethyl ester (III) was extracted with chloroform and analysed on the same system. Limits of determination in blood were 0.1 microgram/ml for III and 0.05 microgram/ml for IV and V; these limits were two to ten times higher for urine. This inter-assay variability was always less than 7.5%.     

High-performance liquid chromatographic determination of vinca-alkaloids in plasma and urine

A liquid chromatographic method is described for separating and determining vinblastine, vincristine and vindesine in plasma and urine. The drugs are extracted from the biological material using an ion-pair extraction, with sodium octylsulphate as counter-ion at pH 3. The extracts are injected on a reversed-phase system with a cyano column as stationary phase and a mobile phase composed of acetonitrile-phosphate buffer, pH 3 (65:35, vol. %). Stability studies are carried out for stock solutions of the drugs in water at different temperatures and pH values. The stability of these compounds in plasma is also investigated in the presence of an antioxidant. The method is applied to determine drug levels of vindesine and vinblastine in preliminary pharmacokinetic studies, using vincristine as the internal standard.     

High-performance liquid chromatographic determination of the stereoisomeric metabolites of ibuprofen

A stereospecific reversed-phase high-performance liquid chromatographic (HPLC) method has been developed to simultaneously quantitate the stereoisomers of the two major metabolites of ibuprofen: hydroxyibuprofen and carboxyibuprofen. The metabolites were derivatized with S-(alpha)-methylbenzylamine to form diastereomeric amides which were separated and quantified on a C8 column. The validity of the stereoselective assay was confirmed by comparison with a non-stereoselective HPLC method. The stereoselective assay was applied to the quantification of all the stereoisomeric ibuprofen metabolites in urine from human volunteers dosed with racemic ibuprofen or the individual enantiomers of ibuprofen. Significant substrate and product stereo-selectivities were observed in the formation of carboxyibuprofen.     

High-performance liquid chromatographic determination of hippuric acid in human urine

A method is described for the determination of urinary hippuric acid by high-performance liquid chromatography. The method used ethyl acetate extraction for partial clean up of the urine. The separation was carried out on a reversed-phase column using 20% methanol in 0.01 M aqueous potassium phosphate containing 0.5% acetic acid as a mobile phase. The column effluent was monitored with a UV detector at 254 nm. Hippuric acid was separated from other normal urine constituents in less than 10 min. Metabolites of xylene and styrene did not interfere with the assay. Analytical recoveries from urine were excellent and peak height and concentration were linearly related.     

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.     

High-performance liquid chromatographic determination of nicotinamide and its metabolites in human and murine plasma and urine.

A high-performance liquid chromatographic method is described which enables the determination of nicotinamide and eight of its possible metabolites in human and murine plasma and urine, using ion-pairing on a base-deactivated reversed-phase column. Calibration curves were linear up to 2 mumol/ml for nicotinamide and 200 nmol/ml for the metabolites; both the intra- and inter-assay relative standard deviations ranged between 1 and 8%. In murine plasma, the N-oxide was the major nicotinamide metabolite, but in man, formation of 1-methylnicotinamide and the 2- and 4-pyridones was also significant. In urine, nicotinuric acid was seen in the mouse, but no nicotinic acid metabolites were seen in man.     

High-performance liquid chromatographic determination of 4,4'-methylenedianiline in human urine

A method is described for the determination of urinary 4,4'-methylenedianiline (MDA) by high-performance liquid chromatography (HPLC). MDA was extracted from hydrolyzed urine using C18 solid-phase extraction columns. The extract was analyzed by reversed-phase HPLC with electrochemical detection at a cell potential of 0.8 V. The method was very sensitive (detection limit 2.5 micrograms/l) and quantitation using 4,4'-ethylenedianiline as an internal standard correlated well with results by gas chromatography-mas spectrometry. Run-to-run precision (n = 25) averaged 8.9%. In analysis of more than 160 potentially exposed workers, MDA was detected in less than 20% of the urines and concentrations ranged up to 210 micrograms MDA per g of creatinine.