Systematic approach to the determination of cephalosporins in biological fluids by reversed-phase liquid chromatography

The chromatographic behaviour of some cephalosporins as a function of pH and ionic strength of the mobile phase was studied on 10-microns LiChrosorb RP-18. Acidic cephalosporins were retained longest in their neutral form with an acidic eluent. Amphoteric cephalosporins were retained longest in their protonated form with an acidic eluent of low ionic strength. Cefotiam was retained longer with an alkaline mobile phase. LiChrosorb RP-18, Nucleosil C18 and muBondapak C18 gave rise to different selectivities when an acidic eluent, methanol-water (25:75) containing 0.2% of 1.8 M H2SO4 was used. This may be related to interactions with residual silanol groups. The studied cephalosporins (with the exception of cefotiam and cefsulodin) were separated from compounds present in biological fluids on 5-microns LiChrosorb RP-18 using the mobile phase 0.2% of 1.8 M H2SO4 in a mixture of methanol and water with various methanol contents. The determination of cefotiam in biological fluids was performed with an alkaline mobile phase. The preparation of the sample was simple and rapid: precipitation of plasma proteins or dilution of urine. The method was applied to the determination of ceftizoxime in human plasma and urine. Concentrations down to 0.2 micrograms/ml of plasma and 25 micrograms/ml of urine could be determined with good reproducibility and accuracy.     

Automation of sample pretreatment for liquid chromatography

The purpose of this review is to discuss the strategic problems of automating sample preparation (SP) for high performance liquid chromatography (HPLC). There is a general feeling that SP is the bottleneck of many HPLC procedures. Despite numerous reports of successful automation of SP, there are still many laboratories using manual or semiautomated SP procedures. This calls for a reevaluation of the present situation.     

Recent advances in sample introduction for supercritical fluid chromatography

New strategies for sample introduction in supercritical fluid chromatography are reviewed. Both open tubular and packed column systems are examined as the injection demands of each column type are addressed. In addition to advances made in solvent injection methods, supercritical fluid extraction (SFE) is discussed as a solventless injection technique for supercritical fluid chromatography.     

Direct determination of tocopherols in paprika and paprika oleoresin by liquid chromatography

A method for the determination of tocopherols and tocopherol esters, quantified as tocopherol acetate, in paprika and paprika oleoresin is reported. Paprika samples were extracted with ethyl acetate and aliquots of the extracts were directly injected into a liquid chromatograph. Reverse phase high-performance liquid chromatography with spectrophotometric detection at 280 nm was used. Gradient elution was applied, allowing the determination of tocopherols and tocopherol esters in the presence of carotenoids. The method does not need previous separation steps, so is useful for the routine determination of vitamin E in commercial samples.     

Simultaneous determination of retinol and tocopherols by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method to determine retinol and all four tocopherols (alpha-, beta-, gamma- and delta-) simultaneously was established using a reversed-phase column (YMC-PACK A-302 S-5 120A ODS). The HPLC conditions were mobile phase 65% isopropanol, sample solvent 99.5% methanol and temperature 30 degrees C. Retinol and tocopherols were measured in rat liver.     

Recent advances in the determination of enantiomeric drugs and their metabolites in biological fluids by capillary electrophoresis-mediated microanalysis

This review summarizes the recent developments in the application of electromigration techniques for the enantioselective analysis of drugs and metabolites in biological fluids. During the period covered by this review, it has been observed an increase in the use of negatively charged chiral selectors, particularly sulfobutyl ether-beta-cyclodextrin and sulfated-beta-cyclodextrin, and the combination of two different chiral selectors in the running buffer to obtain the resolution of drugs and their metabolites. Low detection limits as required for pharmacokinetic studies were obtained by using concentration techniques, including sample stacking procedures, and more sensitive detection systems, such as laser-induced fluorescence and mass spectrometry. Finally, the major points are discussed that can be considered to obtain reliable methods for enantioselective bioanalysis.     

Determination of disulfiram and metabolites from biological fluids by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of disulfiram, diethyldithiocarbamate, diethyldithiocarbamate methyl ester, carbon disulfide, and diethylamine from a single sample of plasma or urine. The analytical procedure is based on a quantitative stepwise extraction of disulfiram and diethyldithiocarbamate methyl ester, or the conversion of diethyldithiocarbamic acid, carbon disulfide and diethylamine to diethyldithiocarbamate methyl ester for chromatographical determination. The procedure is specific, precise and simple. The application of the analytical methods developed for the determination of disulfiram and the various metabolites in plasma from mice given disulfiram intraperitoneally or humans given Antabuse orally is illustrated.     

Corticosteroid analysis in biological fluids by high-performance liquid chromatography

A sensitive, specific, and reproducible high-performance liquid chromatographic assay for the simultaneous determination of prednisone, prednisolone and cortisol in biological fluids was developed with dexamethasone as the internal standard. Samples are extracted with methylene chloride, washed with sodium hydroxide and then water, and chromatographed on a microparticulate silica gel column with UV detection at 254 nm. Sensitivity was greater than 15 ng for all four steroids. Specificity was supported by use of dual wave-length UV detection and/or radioimmunoassay. The assay has been applied in pharmacokinetic studies and a typical plasma concentration--time profile for the three steroids is presented for one subject who received 50 mg of prednisone.     

Simple reversed-phase liquid chromatography method for determination of tocopherols in edible plant oils

A simple and rapid reversed-phase high-performance liquid chromatography method for determination of alpha-, (beta + gamma), and delta-tocopherols in edible plant oils has been developed. Oils are diluted in 2-propanol and injected directly onto Symmetry C18 column. Methanol and acetonitrile (1:1) are used as a mobile phase. Tocopherols are detected using fluorescence detector set at excitation and emission wavelength 295 nm and 325 nm, respectively. The method is precise (R.S.D. not higher than 2.24%) and sensitive-detection limits (DL) are 8 ng/ml for gamma- and delta-tocopherols and 28 ng/ml for alpha-tocopherol; quantification limits (QL) were calculated as three times higher than DL.     

Determination of trimethoprim in biological fluids by high-performance liquid chromatography

A rapid, sensitive, and specific high-performance liquid chromatographic assay was developed for the determination of trimethoprim in blood, plasma, and urine using normalphase (adsorption) chromatography on a microparticulate silica column and UV monitoring at 280 nm. Trimethoprim is selectively extracted from the biological sample matrix at alkaline pH with chloroform, providng nearly quantitative extraction (greater than 95%) and a sensitivity limit of 0.01 to 0.02 microgram/ml blood or plasma, without interference from sulfonamides.     

Determination of bepridil in biological fluids by high-performance liquid chromatography

A rapid, selective and sensitive assay has been developed for the determination of the anti-anginal drug, bepridil, in biological samples. The lowest concentration of bepridil which can be measured accurately and precisely in a 2-ml plasma or urine sample is 10 ng/ml. The standard curve is linear in the concentration range 10-2000 ng/ml. Accuracy and precision of the assay, expressed as relative deviation and coefficient of variation (inter-run) are less than 6.5% at all concentrations in the linear range. No interfering peaks are observed. Using an automatic injector and a laboratory computer system, 48 samples are analyzed routinely in an 8-h day.     

Simultaneous determination of uric acid and creatinine in biological fluids by column-switching liquid chromatography with ultraviolet detection

A column-switching liquid chromatographic method for the simultaneous determination of uric acid and creatinine in human serum and urine was developed. Creatinine and uric acid were separated by size-exclusion chromatography on a hydrophilic gel column (C1) and creatinine eluted from Cl was separated from proteins by filtration through a longer hydrophilic gel column (C2). The creatinine fraction eluted from C2 was transferred to a weakly acidic cation-exchange column (C3) and then to a strongly acidic cation-exchange column (C4). Uric acid eluted from Cl after creatinine was transferred to an anion-exchange column (C5) and then to a hydrophilic gel column (C6). The mobile phase was a mixed buffer of pH 5.1 (propionic acid-succinic acid-NaOH, 60:15:60 mmol/1 in water). Diluted serum and urine could be injected onto C1, and Cl was backflushed after the transfer of uric acid from Cl to C5.

Creatinine and uric acid in the eluate were determined by measuring their ultraviolet absorption at 234 and 290 nm, respectively. The recovery of uric acid and creatinine added to diluted serum (20-fold dilution, concentration 20 and 5 μmol/1, respectively) was 98.9±0.56% and 100.9±1.29%, respectively. The recovery of uric acid and creatinine added to diluted urine (100-fold dilution, concentration 50 and 100 μmol/l, respectively) was 99.4±0.72% and 98.7±1.45%, respectively (mean±R.S.D., n=6).