Determination of gatifloxacin in human serum and urine by high-performance liquid chromatography with ultraviolet detection

A simple and sensitive HPLC method for the determination of gatifloxacin concentrations in human serum and urine was developed and validated. Serum proteins were removed by ultrafiltration through a filtering device after adding a displacing agent. Urine samples were diluted with mobile phase prior to injection. Separation was achieved with a C18 reverse-phase column and gatifloxacin concentrations were determined using ultraviolet detection. The quantitation limits of the assay were 100 ng/ml in serum and 1.0 microg/ml in urine. The assay method was successfully applied to a pharmacokinetic study of gatifloxacin in healthy volunteers.     

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 μg/ml and using 0.5 ml of urine the detection limit is 20 ng/ml. The within-run relative standard deviation is <6% and the accuracy is within 10% of the theoretical value at concentrations between 0.1 and 10 μg/ml in urine. There is a good correlation (r = 0.99606) with a previously described capillary gas chromatographic assay.     

Determination of oxalic acid in urine by high-performance liquid chromatography

A new method for determination of oxalic acid in urine is described. The method encloses sample purification prior to the treatment by High Performance Liquid Chromatography (HPLC). The purification step consists in the passage of acidified urine through Sep-pak C18 cartridge (Waters), followed by the precipitation of the oxalic acid eluted with CaCl2, new dilution of the calcium oxalate precipitate, oxalic acid extraction with diethyl ether and total dryness of the sample. The losses of oxalic acid during this process are evaluated by the addition of oxalic acid (U-14C) before the precipitation step. The dried samples are redissolved in mobile phase (o-H3PO4, 0.05 M) and injected into a HPLC chromatograph, with reversed phase column (Lichrosorb RP-8, Merck). Oxalate peak is detected spectrophotometrically at 220 nm, with a retention time of 3.20 minutes. The method shows a mean recovery value of 98.25%, with an intra-run and between-run values of 5.13 and 8.06 respectively. The oxalic acid measured in urine by this method is 35.52 +/- 9.42 mg/24 h in normal subjects.     

Determination of epitestosterone and testosterone in urine by high-performance liquid chromatography

This paper describes an alternative HPLC method for the determination of testosterone and epitestosterone, which is simple, rapid, selective, sensitive and reproducible. Samples were prepared using a previous enzymatic hydrolysis with liquid-liquid extraction. The determination was carried out on a Hypersil BDS-C₁₈ reversed-phase column with gradient elution and UV absorbance detection (240 nm). The limits of quantification (signal-to-noise ratio = 6) were 20 ng/ml for testosterone and 30 ng/ml for epitestosterone.     

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-μm μBondapak 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-(-)-α-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 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/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 μg/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 monoacetyldiamines and polyamines in urine by high-performance liquid chromatography

A procedure is described for the determination of monoacetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine in human urine. The procedure is based on the high-performance liquid chromatographic separation of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivatives of these amines using two different chromatographic modes. Monoacetyl-1,6-diaminohexane was used as an internal standard. The amines were extracted from urine using a silica gel cartridge. The dansyl monoacetylpolyamines were separated from the mixture of dansyl derivatives of urinary amines on a bonded-phase CN column using a programmed solvent gradient elution. The dansyl acetylpolyamines were rechromatographed on a silica gel column.This chromatographic procedure was used for the determination of the concentration of N¹-acetylspermidine, N⁸-acetylspermidine and monoacetylputrescine in the urine of healthy volunteers and cancer patients.     

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

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (Cipralan ^TM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile---phosphate buffer (0.015 mol/1, pH 6.0) (80:20). A 10-μ ion-exchange (sulfonate) column was used with acetonitrile—phosphate buffer (0.015 mol/1, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard.The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10–1000 ng/ml and 50–5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.     

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

A rapid, reliable and specific reversed-phase high-performance liquid chromatographic procedure is described for the determination of diphenylpyraline hydrochloride at nanogram concentrations in plasma and urine. After extraction of the drug with n-pentane-2-propanol (50:1) from alkalinized samples, the organic extract was evaporated to dryness, reconstituted with methanol and chromatographed using a 5-μm Asahipak ODP-50 C₁₈ column with UV detection at 254 nm. The elution time for diphenylpyraline was 7.9 min. The overall recovery of diphenylpyraline from spiked plasma and urine samples at concentrations ranging from 53 to 740 ng/ml were 94.65% and 92.29%, respectively. Linearity and precision data for plasma and urine standards after extraction were acceptable. The limit of detection was 15 ng/ml for both plasma and urine samples at 0.002 AUFS.     

Determination of methotrexate in serum by high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method with ultraviolet detection was developed to quantitate methotrexate in serum-based calibrators, controls and patient samples. Sample clean-up was achieved with C₁₈ Sep-Pak Classic cartridges. The chromatographic separation was accomplished on a 5-μm Ultrasphere ODS Beckman column. 8-Chlorotheophylline was used as an internal standard. The method was validated by recovery, linearity, accuracy and precision studies. Two standard curves were constructed to cover the high and the low ends of the calibrator range (0.05–1.0 μmol/l). Response was found linear over the whole range of the calibrator set with a correlation coefficient of 0.999 and 1.00 for the low-level and the high-level curves, respectively. Accuracy varied from 12% at the lowest level to 1.2% at the highest level. The precision study showed a C.V. of 14.4% at the lowest level and 3.3% at the highest level.     

Determination of some benzodiazepines and metabolites in serum,urine and saliva by high-performance liquid chromatography

The performance of a number of liquid—solid systems, consisting of mixtures of buffers (0.05 M) and methanol as mobile phase and methyl-silica as stationary phase, were investigated with respect to their use in the separation of 1,4-benzodiazepines by reversed-phase high-performance liquid chromatography with UV detection at 254 nm. Phase system selectivities and column efficiencies were determined. A nomogram is presented from which the chromatographic parameters can be calculated.A complete separation of nine benzodiazepines within 12 min has been achieved, using methyl-silica as the stationary phase and 50% methanol as the eluent.The results were applied to the development of a method for the determination of therapeutic levels of diazepam and its metabolites in human serum, urine and saliva. The first step in the analysis, the extraction of diazepam and its metabolites from serum and urine, was also investigated and good recoveries were achieved. A low detection limit (0.2 ng) and high precision were obtained. The concentrations of diazepam and its metabolites in human serum, urine and saliva were determined after both single and multiple oral doses of diazepam (and oxazepam).     

Determination of iodide in serum and urine by ion-pair reversed-phase high-performance liquid chromatography with coulometric detection

An HPLC method is described for the determination of iodide in serum and urine using ion-pair chromatography with coulometric detection. After adding hexadecyltrimethylammonium chloride, the ions pairs formed with the iodide in the sample are extracted using an organic solvent. The solvent is then evaporated and the dry residue obtained is mixed with an appropriate volume of mobile phase so as to concentrate the sample prior to injection into the chromatograph. For a sample of 0.5 ml of serum, the method features a limit of detection (signal-to-noise ratio of 3) of 0.2 μgl⁻¹, sufficient to be applied in paediatric assays for the diagnosis of both iodide deficiency and excess.     

Determination of aromatic metabolites in ruminant urine by high-performance liquid chromatography

A method based on reversed-phase HPLC is reported for the separation and quantification of various urinary aromatic metabolites: hippuric, phenylaceturic, salicyluric, benzoic, phenylacetic, salicylic. 3-phenylpropionic and cinnamic acids and several phenols in ruminant urine. In this method, a Nova-Pak C₁₈ (4 μm) 150×3.9 mm I.D. column, two solvents [A: 15°b methanol in 20 mM acetic acid (pH 3.3); B: methanol] in a gradient mode at a flow-rate of 0.8 ml/min, and UV detection at 210 nm were used. Quantification of the total (free and conjugated) benzoic, phenylacetic and salicylic acids present in urine was achieved by hydrolysis of the samples in 3 M HCl at 100°C for 24 h prior to HPLC analysis. The lowest detection concentration was 50 μmol/I. This method is useful for scanning the profile of aromatic metabolites in urine of ruminants, which provides information on the diets the animals receive.     

Determination of serum retinol by reversed-phase high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic method was developed for the determination of serum levels of retinol in humans. A direct serum injection technique after deproteinisation was used to avoid lengthy pretreatment steps which can result in degradation of retinol during analysis. The column used was CLC-ODS, the mobile phase was acetonitrile-water and detection wavelength was 328 nm. Deterioration in column performance was not observed even after injection of 300 samples. The lower detection limit was 10 μg/l. On analyzing a serum pool six times, a C.V of 0.7% was obtained. The method is quantitative, reproducible, rapid and highly accurate for routine analysis.