Anastrozole quantification in human plasma by high-performance liquid chromatography coupled to photospray tandem mass spectrometry applied to pharmacokinetic studies

A rapid, sensitive and specific method for quantifying the aromatase inhibitor (anastrozole) in human plasma using dexchlorpheniramine as the internal standard (I.S.) is described herein. The analyte and the I.S. were extracted from 200 microl of human plasma by liquid-liquid extraction using a mixture of diethyl ether:dichloromethane (70:30, v/v) solution. Extracts were removed and dried in the organic phase then reconstituted with 200 microl of acetonitrile:water (50:50; v/v). The extracts were analyzed by high performance liquid chromatography coupled with photospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed isocratically on a Genesis, C18 4 microm analytical column (100 mm x 2.1mm i.d.). The method had a chromatographic run time of 2.5 min and a linear calibration curve ranging from 0.05-10 ng ml(-1). The limit of quantification (LOQ) was 0.05 ng ml(-1). This HPLC-MS-MS procedure was used to assess pharmacokinetic studies.     

Quantitation of tamsulosin in human plasma by liquid chromatography-electrospray ionization mass spectrometry

A highly sensitive method for quantitation of tamsulosin in human plasma using 1-(2,6-dimethyl-3-hydroxylphenoxy)-2-(3,4-methoxyphenylethylamino)-propane hydrochloride as the internal standard (I.S.) was established using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). After alkalization with saturated sodium bicarbonate, plasma were extracted by ethyl acetate and separated by HPLC on a C18 reversed-phase column using a mobile phase of methanol-water-acetic acid-triethylamine (620:380:1.5:1.5, v/v). Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 228 for tamsulosin and m/z 222 for the I.S. Calibration curves, which were linear over the range 0.2-30 ng/ml, were analyzed contemporaneously with each batch of samples, along with low (0.5 ng/ml), medium (3 ng/ml) and high (30 ng/ml) quality control samples. The intra- and inter-assay variability ranged from 2.14 to 8.87% for the low, medium and high quality control samples. The extraction recovery of tamsulosin from plasma was in the range of 84.2-94.5%. The method has been used successfully to study tamsulosin pharmacokinetics in adult humans.     

Solid-phase extraction and high-performance liquid chromatographic determination of articaine and its metabolite articainic acid in human serum

A new method is described using solid-phase extraction (SPE) for preconcentration of articaine and the metabolite articainic acid and high-performance liquid chromatography (HPLC) for the determination of both compounds in human serum. Articaine and articainic acid were extracted in one step with SDB-RPS disk cartridges after precipitation of the serum proteins by perchloric acid. The HPLC separation was then performed on a reversed-phase C₈ column using phosphate buffer–acetonitrile (88:12, v/v). UV absorption at 274 nm was used for measuring the analytes with a low limit of quantitation of about 10 ng/ml, which is appropriate for pharmacokinetic studies of low dose submucosal injections of the local anaesthetic agent articaine hydrochloride in dentistry.     

Determination of glimepiride in human plasma using semi-microbore high performance liquid chromatography with column-switching

A fully automated semi-microbore high performance liquid chromatographic (HPLC) method with column-switching using UV detection was developed for the determination of glimepiride from human plasma samples. Plasma sample (900 microl) was deproteinated and extracted with ethanol and acetonitrile. The extract (70 microl) was directly injected into a Capcell Pak MF Ph-1 pre-column where the primary separation occurred to remove proteins and retain drugs using a mixture of acetonitrile and 10mM phosphate buffer (pH 2.18) (20:80, v/v). The analytes were transferred from the pre-column to an intermediate column using a switching valve and then subsequently separated on an analytical column and monitored with UV detection at 228 nm. Glimepiride was eluted with retention time 34.9 min without interference of endogenous substance from plasma. The limit of quantification (LOQ) was 10 ng/ml for glimepiride. The calibration curves were linear over the concentration range of 10-400 ng/ml (r(2) = 0.9997). Moreover, inter- and intra-day precisions of the method were less than 15% and accuracies were higher than 99%. The developed method was successfully applied for the quantification of glimepiride in human plasma and was used to support a human pharmacokinetic study following a single oral administration of 2 mg glimepiride.     

Simultaneous determination of lansoprazole enantiomers and their metabolites in plasma by liquid chromatography with solid-phase extraction

A simple and highly sensitive high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of lansoprazole enantiomers and their metabolites, 5-hydroxylansoprazole enantiomers and lansoprazole sulfone, in human plasma have been developed. Chromatographic separation was achieved with a Chiral CD-Ph column using a mobile phase of 0.5M NaClO(4)-acetonitrile-methanol (6:3:1 (v/v/v)). The analysis required only 100 microl of plasma and involved a solid-phase extraction with Oasis HLB cartridge, with a high extraction recovery (>94.1%) and good selectivity. The lower limit of quantification (LOQ) of this assay was 10 ng/ml for each enantiomer of both lansoprazole and 5-hydroxylansoprazole, and 5 ng/ml for lansoprazole sulfone. The coefficient of variation of inter- and intra-day assay was <8.0% and accuracy was within 8.4% for all analytes (concentration range 10-1000 ng/ml). The linearity of this assay was set between 10 and 1000 ng/ml (r2>0.999 of the regression line) for each of the five analytes. This method is applicable for accurate and simultaneous monitoring of the plasma levels of lansoprazole enantiomers and their metabolites in the renal transplant recipients.     

Determination of midazolam and its major metabolite 1'-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children

We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1'-hydroxymidazolam (1'-OHM) in a small volume (200 microl) of human plasma. Midazolam, 1'-OHM and 1'-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY Elite C18 (5 microm particle size; 100 mm x 2.1mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water-acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 microl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M+l]+ of parent drug and metabolite. Calibration curves in spiked plasma were linear (r2 > or = 0.99) from 15 to 600 ng/ml (MDZ) and 5-200 ng/ml (1'-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1'-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4+/-3.1% (n = 6) and 84.2+/-4.7% (n = 8), respectively; for 1'-OHM at 30 and 200 ng/ml the values were 89.9+/-7.2% (n = 6) and 86.9+/-5.6% (n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1'-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1'-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.).     

A rapid high performance liquid chromatographic determination of methyldopa in human serum with fluorescence detection and alumina extraction: application to a bioequivalence study

A simple and ultra rapid high performance liquid chromatographic (HPLC) method coupled with alumina extraction and fluorescence detection was described for determination of methyldopa in human serum. The drug and an internal standard were adsorbed onto alumina and eluted using acidic methanol. The eluate was directly injected onto ODS reverse phase column using a mixture of phosphate buffer (0.05 M) containing triethylamine (100 microl/l, v/v; pH 2.3) and methanol (92:8, v/v) at a flow rate of 2.1 ml/min as the mobile phase. The fluorescence detector excitation and emission wavelengths were set at 270 and 320 nm, respectively. No interference in the assay from any endogenous substances or other concurrently used drugs was observed and the retention times of I.S. and the drug were 1.7 and 2.4 min, respectively with total run time (injection to injection) of less than 3.5 min. The limit of quantification was evaluated to be 2 ng/ml. Validity of the method was studied and the method was precise and accurate with a linearity range from 20 ng/ml to 5000 ng/ml. This method has been used in a randomized crossover bioequivalence study of two different methyldopa preparations in 24 healthy volunteers.     

Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of donepezil, a centrally and selectively acting acetyleholinesterase inhibitor, in human plasma. After sample alkalinization with 0.5 ml of NaOH (0.1 M), the test compound was extracted from I ml of plasma using isopropanol-hexane (3:97, v/v). The organic phase was back-extracted with 75 microl of HCl (0.1 M) and 50 microl of the acid solution was injected into a C18 STR ODS-II analytical column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (6 M) and acetonitrile (59.5:0.5:40, v/v) and was delivered at a flow-rate of 1.0 ml/min at 40 degrees C. The peak was detected using a UV detector set at 315 nm, and the total time for a chromatographic separation was approximately 8 min. The method was validated for the concentration range 3-90 ng/ml. Mean recoveries were 89-98%. Intra- and inter-day relative standard deviations were less than 7.3 and 7.6%, respectively, at the concentrations ranging from 3 to 90 ng/ml. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.     

High-performance liquid chromatographic analysis of DA-7867, a new oxazolidinone,in human plasma and urine and in rat tissue homogenates

An HPLC method was developed for the determination of a new oxazolidinone, DA-7867 (I), in human plasma and urine and in rat tissue homogenates. To 100 microl of biological sample, 300 microl acetonitrile and 50 microl methanol containing 10 microg/ml DA-7858 (the internal standard) were added. After vortex-mixing and centrifugation, the supernatant was evaporated under a gentle stream of nitrogen. The residue was reconstituted in 100 microl of the mobile phase and a 50-microl aliquot was injected directly onto the reversed-phase (C(18)) column. The mobile phase, 20 mM KH2PO4:acetonitrile (75:25, v/v) was run at a flow rate of 1.5 ml/min and the column effluent was monitored by a UV detector set at 300 nm. The retention times of I and DA-7858 were approximately 6.5 and 8.7 min, respectively. The detection limits of I in human plasma and urine and in rat tissue homogenates were 20, 20, and 50 ng/ml, respectively.     

Determination of aesculin in rat plasma by high performance liquid chromatography method and its application to pharmacokinetics studies

A sensitive and reproducible high performance liquid chromatography method with UV detection was described for the determination of aesculin in rat plasma. After deproteinization by methanol using metronidazole as internal standard (I.S.), solutes were evaporated to dryness at 40 degrees C under a gentle stream of nitrogen. The residue was reconstituted in 100 microl of mobile phase and a volume of 20 microl was injected into the HPLC for analysis. Solutes were separated on a Diamonsil C18 column (250 mm x 4.6 mm i.d., 5 microm particle size, Dikma) protected by a ODS guard column (10 mm x 4.0 mm i.d., 5 microm particle size), using acetonitrile-0.1% triethylamine solution (adjusted to pH 3.0 using phosphoric acid) (10:90, v/v) as mobile phase (flow-rate 1.0 ml/min), and wavelength of the UV detector was set at 338 nm. No interference from any endogenous substances was observed during the elution of aesculin and internal standard (I.S., metronidazole). The retention times for I.S and aesculin were 10.4 and 12.4 min, respectively. The limit of quantification was evaluated to be 57.4 ng/ml and the limit of detection was 24.0 ng/ml. The method was used in the study of pharmacokinetics of aesculin after intraperitoneal injection (i.p.) administration in rats.     

Sensitive quantification of ranolazine in human plasma by liquid chromatography--tandem mass spectrometry with positive electrospray ionization

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with positive electrospray ionization (ESI) was developed for the quantification of ranolazine in human plasma. After liquid-liquid extraction of ranolazine and internal standard (ISTD) phenoprolamine from a 100 microl specimen of plasma, HPLC separation was achieved on a Nova-Pak C(18) column, using acetonitrile-water-formic acid-10% n-butylamine (70:30:0.5:0.08, v/v/v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 428.5-->m/z 279.1 for ranolazine and m/z 344.3-->m/z 165.1 for the internal standard, respectively. Linear calibration curves were obtained in the concentration range of 5-4000 ng/ml, with a lower limit of quantitation (LLOQ) of 5 ng/ml. The intra- and inter-day precision values were below 3.7% and accuracy was within +/-3.2% at all three quality control (QC) levels. This method was found suitable for the analysis of plasma samples collected during the phase I pharmacokinetic studies of ranolazine performed in 28 healthy volunteers after single oral doses from 200 mg to 800 mg.     

Improved liquid chromatographic method for mitoxantrone quantification in mouse plasma and tissues to study the pharmacokinetics of a liposome entrapped mitoxantrone formulation

A simple, rapid HPLC method for quantification of mitoxantrone in mouse plasma and tissue homogenates in the presence of a liposome entrapped mitoxantrone formulation (LEM-ETU) is described. Sample preparation is achieved by protein precipitation of 100 microl plasma or 200 microl tissue homogenate with an equal volume of methanol containing 0.5 M hydrochloric acid:acetonitrile (90:10, v/v). Ametantrone is used as the internal standard (i.s.). Mitoxantrone and i.s. are separated on a C18 reversed phase HPLC column, and quantified by their absorbance at 655 nm. In plasma, the standard curve is linear from 5 to 1000 ng/ml, and the precision (%CV) and accuracy (percentage of nominal concentration) are within 10%. In mouse tissue (heart, kidney, liver, lung, and spleen) homogenates (5%, w/v), the standard curve is linear from 25 to 2000 ng/ml, with acceptable precision and accuracy. The method was used to successfully quantify mitoxantrone in mouse plasma and tissue samples to support a pharmacokinetic study of LEM-ETU in mice.     

A simple HPLC method for the determination of bifendate: application to a pharmacokinetic study of bifendate liposome

A rapid, sensitive and simple high-performance liquid chromatographic (HPLC) method with ultraviolet detector (UV) has been developed for the determination of bifendate in 100 microl plasma of rats. Sample preparation was carried out by deproteinization with 100 microl of acetonitrile. A 20 microl of supernatant was directly injected into the HPLC system with methanol-double distilled water (65/35, v/v) as the mobile phase at a flow rate of 1.0 ml/min. Separation was performed with a microBondapak C(18) column at 30 degrees C. The peak was detected at 278 nm. The calibration curve was linear (r(2)=0.9989) in the concentration range of 0.028-2.80 microg/ml in plasma. The intra- and inter-day variation coefficients were not more than 6.55% and 6.07%, respectively. The limit of detection was 5 ng/ml. The mean recoveries of bifendate were ranged from 94.53% to 99.36% in plasma. The present method has been successfully applied to the pharmacokinetic study of bifendate liposome in rats.     

Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative

Asymmetric dimethylarginine (ADMA; N(G),N(G)-dimethyl-L-arginine) is the most important endogenous inhibitor of nitric oxide synthase and a potential risk factor for cardiovascular diseases. This article describes a gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate quantification of ADMA in human plasma or serum and urine using de novo synthesized [2H(3)]-methyl ester ADMA (d(3)Me-ADMA) as the internal standard. Aliquots (100 microl) of plasma/serum ultrafiltrate or native urine and of aqueous solutions of synthetic ADMA (1 microM for plasma and serum; 20 microM for urine) are evaporated to dryness. The residue from plasma/serum ultrafiltrate or urine is treated with a 100 microl aliquot of 2M HCl in methanol, whereas the residue of the ADMA solution is treated with a 100 microl aliquot of 2M HCl in tetradeuterated methanol. Methyl esters are prepared by heating for 60 min at 80 degrees C. After cooling to room temperature, the plasma or urine sample is combined with the d(3)Me-ADMA sample, the mixture is evaporated to dryness, the residue treated with a solution of pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v) and the sample is incubated for 30 min at 65 degrees C. Solvent and reagents are evaporated under a stream of nitrogen gas, the residue is treated with a 200 microl aliquot of 0.4M borate buffer, pH 8.5, and toluene (0.2 ml for plasma, 1 ml for urine). Reaction products are extracted by vortexing for 1 min, the toluene phase is decanted, and a 1 microl aliquot is injected into the GC-tandem MS instrument. Quantitation is performed by selected reaction monitoring (SRM) of the common product ion at m/z 378 which is produced by collision-induced dissociation of the ions at m/z 634 for endogenous ADMA and m/z 637 for d(3)Me-ADMA. In plasma and urine of healthy humans ADMA was measured at concentrations of 0.39+/-0.06 microM (n=12) and 3.4+/-1.1 micromol/mmol creatinine (n=9), respectively. The limits of detection and quantitation of the method are approximately 10 amol and 320 pM of d(3)Me-ADMA, respectively.     

High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4-700 ng/ml of quercetin in plasma and 20-1000 ng/ml of quercetin in urine. The lower limit of quantification was approximately 7 ng/ml of quercetin in plasma and approximately 35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was approximately 0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.     

High performance liquid chromatographic determination of topiramate in human serum using UV detection

Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquid-liquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264 nm. A mixture of phosphate buffer (0.05M) containing triethylamine (1 ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5 ml/min was used as mobile phase. No interference was found with endogenous substances. Validity of the method was studied and the method was precise and accurate with a linearity range from 40 ng/ml to 40 microg/ml. The limit of quantification was 40 ng/ml of serum. The correlation coefficient between HPLC methods using fluorescence and UV detections was studied and found to be 0.992.     

Development and validation of a simple liquid chromatographic method with ultraviolet detection for the determination of imatinib in biological samples

The aim of this study was to develop a rapid and sensitive HPLC method with UV detection for the estimation of imatinib from the plasma of patients with chronic myeloid leukemia (CML). The robustness of the method was checked by conducting first dose pharmacokinetics on blood samples from four patients who had been administered Gleevec (100 mg) in an oral dose. Samples were prepared in a simple and single step by precipitating the plasma proteins with methanol and injecting 50 microl aliquot from supernatant was subjected for analysis. Assay was conducted using a C8 column (250 mm x 4.6 mm, 5 microm particle size) under isocratic elution with 0.02 M potassium dihydrogen phosphate-acetonitrile (7:3, v/v) at a flow rate of 1 ml/min and detected using photodiode array at 265 nm. Calibration plots in spiked plasma were linear in a concentration range of 0.05-25 microg/ml. The inter and intra-day variation of standard curve was <4% (R.S.D.). This method could be a simple and quick method for the estimation of imatinib from the patient's plasma.     

Sensitive liquid chromatography assay with ultraviolet detection for a new phosphodiesterase V inhibitor, DA-8159, in human plasma and urine

A sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet absorption detection (292 nm) was developed and validated for the determination of the new phosphodiesterase V inhibitor, DA-8159 (DA), in human plasma and urine. A single step liquid-liquid extraction procedure using ethyl ether was performed to recover DA and the internal standard (sildenafil citrate) from 1.0 ml of biological matrices combined with 200 microl of 0.1M sodium carbonate buffer. A Capcell Pak C18 UG120 column (150 mm x 4.6 mm I.D., 5 microm) was used as a stationary phase and the mobile phase consisted of 30% acetonitrile and 70% 20mM potassium phosphate buffer (pH 4.5) at a flow rate of 1.0 ml/min. The lower limit for quantification was 5 ng/ml for plasma and 10 ng/ml for urine samples. Within- and between-run accuracy and precision were < or =15 and < or =10%, respectively, in both plasma and urine samples. The recovery of DA from human plasma and urine was greater than 70%. Separate stability studies showed that DA is stable under the conditions of analysis. This validated assay was used for the pharmacokinetic analysis of DA during a phase I, rising dose study.     

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.     

Rapid and sensitive high-performance liquid chromatographic determination of four cephalosporin antibiotics in pharmaceuticals and body fluids

A rapid, accurate and sensitive method has been developed and validated for the quantitative simultaneous determination of four cephalosporins, cephalexin and cefadroxil (first-generation), cefaclor (second-generation) and cefataxim (third-generation), in pharmaceuticals as well as in human blood serum and urine. A Spherisorb ODS-2 250 x 4-mm, 5-microm analytical column was used with an eluting system consisting of a mixture of acetate buffer (pH 4.0)-CH(3)OH 78-22% (v/v) at a flow-rate 1.2 ml/min. Detection was performed with a variable wavelength UV-Vis detector at 265 nm resulting in limit of detection of 0.2 ng for cefadroxil and cephalexin, but only 0.1 ng for cefotaxime and cefaclor per 20-microl injection. Hydrochlorothiazide (HCT) (6-chloro-3,4-dihydro-7 sulfanyl-2H-1,2,4-benzothiadiazine-1-1-dioxide) was used as internal standard at a concentration of 2 ng/microl. A rectilinear relationship was observed up to 8, 5, 12 and 35 ng/microl for cefadroxil, cefotaxime, cefaclor, cephalexin, respectively. Analysis time was less than 7 min. The statistical evaluation of the method was examined by means of within-day repeatability (n=8) and day-to-day precision (n=9) and was found to be satisfactory with high accuracy and precision. The method was applied to the determination of the cephalosporins in commercial pharmaceuticals and in biological fluids: human blood serum after solid-phase extraction and urine simply after filtration and dilution. Recovery of analytes in spiked samples was in the range from 76.3 to 112.0%, over the range of 1-8 ng/microl.