Determination of methimazole in urine by liquid chromatography

A liquid chromatography methodology is developed and validated for detection and quantification of methimazole in urine. The approach is based on derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate, reversed-phase high-performance liquid chromatography (HPLC) separation of so formed methimazole 2-S-quinolinium derivative from other urine matrix components, followed by detection and quantification with the use of ultraviolet–visible detector. Neither extraction, nor preconcentration of the sample are necessary. The methimazole standards added to normal urine before derivatization step show that the response of the detector, set at 345 nm, is linear within the concentration range studied, that is, from 0.25 to 50 mg/l urine. The relative standard deviation values for precision and recovery within the calibration range were from 1.8 to 5.0% and from 95.7 to 103.3%, respectively. Lower limits of detection and quantitation were 0.15 and 0.25 mg/l urine, respectively.     

A simplified assay of furosemide in plasma and urine by high-pressure liquid chromatography.

A simplified high-pressure liquid chromatograhic method for determination of furosemide in plasma and urine has been developed using a fluorometric detector directly coupled to the column effluent. The method includes an ether extraction from acidified biologic samples. The mobile phase used for chromatography on a reversed-phase column (C15 hydrocarbon permanently bonded to silica particles) is sufficiently acidic to induce fluorescence of furosemide. The methylester of furosemide is employed as an internal standard. The sensitivity is 0.1 and 0.25 microgram per ml plasma and urine, respectively. The applicability to pharmacokinetic studies of furosemide is shown.     

Determination of cetirizine in human urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of the histamine H1-receptor antagonist cetirizine in human urine was developed. Cetirizine and the internal standard are extracted from acidified (pH 5) urine (0.5 ml) into chloroform and the organic layer is evaporated to dryness. The residue is chromatographed on a Spherisorb 5ODS-2 column using Pic A (5 mM aqueous tetrabutylammonium phosphate)-methanol-tetrahydrofuran (33:65:2, v/v) as the mobile phase with ultraviolet detection (230 nm). The calibration graph is linear from 0.1 to 10 micrograms/ml and using 0.5 ml of urine the detection limit is 20 ng/ml. The within-run relative standard deviation is less than 6% and the accuracy is within 10% of the theoretical value at concentrations between 0.1 and 10 micrograms/ml in urine. There is a good correlation (r = 0.99606) with a previously described capillary gas chromatographic assay.     

Determination of sodium phenobarbital in animal chow by high-pressure liquid chromatography

An analytical procedure is described for determining residues of sodium phenobarbital in animal chow at levels as low as 0.14 ppm. The methanol extract is subjected to a liquid-liquid cleanup at pH 13 and 1, further cleaned up on a silica gel column and assayed by high-pressure liquid chromatography by using an ultraviolet absorption detector at 210 nm. Data concerning extraction efficiency, partition values and stability of the chemical in animal chow are also presented.     

Determination of the carotenoid phytoene in blood by high-pressure liquid chromatography

A sensitive and specific high-pressure liquid chromatographic assay was developed for the determination of phytoene in blood with an overall recovery of 86 ± 6.0% and a limit of detection of 50–100 ng per ml of blood. This method provides for rapid and simple quantitation of phytoene using 1 ml or less of blood.

The assay was used in the determination of phytoene blood levels in the dog following intravenous and oral administration of 10-mg/kg doses.     

Determination of sparfloxacin in serum and urine by high-performance liquid chromatography.

A specific and sensitive analytical method for the determination of sparfloxacin in serum and urine is described. Serum proteins are removed by precipitation with acetonitrile after the addition of ofloxacin as an internal standard. The supernatant solvent is evaporated in a vacuum concentrator and the dry residue is redissolved in the mobile phase. Separation is performed on a cation-exchange column (Nucleosil 100 5SA, 125 x 4.0 mm I.D., 5 microns particle size) protected by a guard column (Perisorb RP-18, 30 x 4.0 mm I.D., 30-40 microns particle diameter). The mobile phase consisted of 750 ml of acetonitrile and 250 ml of 100 mmol/l phosphoric acid (v/v) to which sodium hydroxide had been added. The final concentration of sodium was 23 mmol/l and the pH was 3.82. Sparfloxacin and ofloxacin were determined by spectrofluorimetry (excitation wavelength 295 nm; emission wavelength 525 nm). The flow-rate was 1.5 ml/min and the retention times were 4.7 (sparfloxacin) and 8.0 (ofloxacin) min. Validation of the method yielded the following results for serum: detection limit 0.05 mg/l; precision between series 10.4-3.6%; recovery 99.5-100.0%; comparison with a microbiological assay c(bioassay) = 1.035c(HPLC) - 0.06. The test organism was Bacillus subtilis ATCC 6633. For urine the results were: detection limit 0.5 mg/l; precision between series 7.8-5.0%; recovery 97.0-97.8%; method comparison c(bioassay) = 1.092c(HPLC) - 1.09. No interferences were observed in human volunteers. The method can also be applied to stool samples.     

Determination of 6-N-trimethyllysine in urine by high-performance liquid chromatography

A method for determination of 6-N-trimethyllysine in urine is described. Trimethyllysine and the chemically analogous 6-N-triethyllysine internal standard were isolated from aqueous samples by microcolumn ion-exclusion chromatography. The specimens were derivatized by reaction with 1-fluoro-2,4-dinitrobenzene and reaction byproducts extracted by organic solvents. The trimethyllysine and internal standard derivatives were separated easily from other sample constituents by reversed-phase paired-ion high-performance liquid chromatography with spectrophotometric detection at 405 nm. Standard curves were linear over a sample concentration range of 10-150 nmol/ml; the detection limit corresponded with 0.1 nmol trimethyllysine injected into the chromatograph. The procedure was used for determination of trimethyllysine in human urine.     

Determination of vitamin E in different biological samples by high-pressure liquid chromatography

Summary The different methods for the determination of vitamin E in all types of biological materials are reviewed. The following subjects are dealt with: isolation of the vitamin from biological samples, foodstuffs and pharmaceuticals; instrumental aspects (injection, reversed-phase HPLC, straight-phase HPLC); standardization; detection limits.

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Bestimmung von Vitamin E in verschiedenen biologischen Proben durch HPLC

Zusammenfassung Die Übersicht behandelt folgende Aspekte: Isolierung des Vitamins aus biologischem Material, Lebensmitteln und Pharmazeutica; instrumentelle Probleme (Injektion, reversed-phase und straight-phase HPLC); Standardisierung; Nachweisgrenzen.

     

Determination of picotamide in human plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of picotamide in human plasma and urine is described. After addition of an internal standard (bamifylline), the plasma and urine samples were subjected to liquid-liquid extraction and clean-up procedures. The final extracts were evaporated to dryness and the resulting residues were reconstituted in 100 microliters of methanol-water (50:50, v/v) and chromatographed on a LiChrosorb RP-SELECT B reversed-phase column coupled to an ultraviolet detector monitored at 230 nm. Chromatographic analysis takes about 10 min per sample. The assay was linear over a wide range and has a limit of detection of 0.005 and 0.1 micrograms/ml in plasma and urine, respectively. It was selective for picotamide, accurate and robust and thus suitable for routine assays after therapeutic doses of picotamide.     

Analysis of warfarin in plasma by high-pressure liquid chromatography.

An improved high-pressure liquid chromatography method for the estimation of warfarin in plasma was developed. Plasma was acidified and extracted with ethylene dichloride spiked with methylated warfarin [3-(alpha-acetonylbenzyl)-4-methoxy-coumarin] as internal standard. The residue, redissolved in dioxane, was chromatographed on a reversed-phase column using a mobile phase of 40% dioxane in water (pH 4.2) on a high-pressure liquid chromatograph fitted with an UV absorbance detector. Recoveries from extraction, quantitated using tracer amounts of [14C]warfarin and methylated [14C]warfarin were 92.2 +/- 3.16% and 82.33 +/- 1.03%, respectively. The standard curve was linear between o.625 and 5.0 microng/ml. Detection was sensitive to approximately 0.5 microng/ml and specific without the inter ference of normal plasma constituents and warfarin metabolites.     

Determination of pirlindole in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the analysis of pirlindole [2,3,3a,4,5,6-hexahydro-8-methyl-1H-pyrazino(3,2,1-jk)carbazole hydrochloride], a new antidepressive drug. The drug was extracted from plasma into dichloromethane, and the analysis was carried out on a reversed-phase column, the effluent being monitored by fluorescence detection. The method is selective and sensitive (limit of detection 1-2 ng/ml plasma). Urine analysis was done by direct injection of the diluted sample. The method was applied to the analysis of plasma and urine samples of eight healthy male volunteers who received a 75-mg oral dose of a tablet formulation of pirlindole. The method was also applied to a study in three beagle dogs which received pirlindole (1 mg/kg) by infusion (0.1 mg/kg/min) and orally (10 mg/kg) to estimate the absolute bioavailability of the drug.     

Determination of thiosulfate in human urine by high performance liquid chromatography

Thiosulfate is a sulfate analogue with a thiosulfur substituent and is found in human samples. Its concentration in urine is increased in some diseases and after exposure to hydrogen sulfide gas. We have developed a sensitive, simple and cheap method for thiosulfate determination in urine. The method is based on precolumn derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate followed by reversed-phase liquid chromatography separation and ultraviolet detection of 1-methyl-2-thioquinolone at 375 nm. The calibration curve for thiosulfate was linear in the tested range 0.5-50 μmol L⁻¹ with correlation coefficient better than 0.999. The analytical recovery and relative standard deviation values for precision within the calibration range were from 90.1% to 104.2% and from 2.39% to 5.59%, respectively. The lower limit of detection and quantitation were 0.3 and 0.5 μmol L⁻¹, respectively. The mean (range) concentration of thiosulfate normalized against creatinine for apparently healthy seven women and six men was 2.21 (1.45-2.77) and 2.51 (1.36-4.89) mmol mol⁻¹ creatinine, respectively. We monitored thiosulfate in urine samples from one volunteer for 24 h. The urinary excretion of thiosulfate was 21.4 μmol per 24 h. This method can be used for routine clinical monitoring thiosulfate in urine. Cysteine and cysteinylglycine can be measured concurrently, if needed.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10 micrometer muBondapak phenyl column with an eluting solvent of water--methanol--1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(D-(-)-alpha-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 +/- 6.3% (S.D.) in the concentration ranges of 0.1-20 microgram per 0.2 ml of plasma with a limit of detection equivalent to 0.5 microgram/ml plasma. The urine assay was validated over a concentration range of 0.025-5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 microgram/ml) using a 0.1-ml urine specimen per assay. The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Determination of the fatty-acid composition of soybean oil by high-pressure liquid chromatography

A method for determining the fatty-acid composition of soybean oil by using high-pressure liquid chromatography (HPLC) is discussed and compared with the determination using gas chromatography. The oil is saponified and an aliquot is treated directly to form the p-bromophenacyl esters of the fatty acids, which are separated by gradient-elution HPLC. It is shown that glycerol does not interfere with the esterification, thus obviating the solvent extraction previously used to isolate fatty acids from biological samples.