High-performance liquid chromatographic determination of angiotensin II receptor antagonists in human plasma and urine. I. DuP 532 (L-694,492)

A sensitive reversed-phase high-performance liquid chromatographic method with ultraviolet detection was developed for the analysis of a new angiotensin II receptor antagonist, DuP 532 (L-694,492), in human plasma and urine. The analyte and internal standard are extracted from plasma and urine at a pH between 3.3 to 3.6 by liquid-liquid extraction and analyzed on a C6 column with ultraviolet detection at 254 nm. The mobile phase is composed of acetonitrile and phosphate buffer at pH 2.5. The limits of quantification are 6 and 7.5 ng/ml for plasma and urine, respectively.     

Hyperhomocyst(e)inemia in renal transplant recipients with and without cyclosporine

A reversed-phase high-performance liquid chromatographic method for the determination of quercetin in human plasma following intravenous infusion is described. Quercetin in plasma was extracted with methanol-dimethyl sulphoxide (4:1 v/v) and separated on a C18 Hypersil-BDS column with 44% (v/v) methanol in 0.1 M ammonium acetate (pH 5.15) containing 0.27 mM EDTA as the mobile phase. The drug was detected specifically and sensitively at its absorption maximum of 375 nm, or electrochemically, with a detection limit of 80 ng/mL and 2 ng/mL, respectively.     

Simultaneous quantification of an anti-inflammatory compound (DuP 697) and a potential metabolite (X6882) in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method using fluorescence detection has been developed for the simultaneous analysis of low nanogram concentrations of an anti-inflammatory drug, 5-Bromo-2-(4-fluorophenyl)-3-[4-(methylsulfonyl)phenyl]thiophene (DuP 697), and a potential metabolite (X6882) in human plasma and of DuP 697 in human urine. This assay method used an EM Separations Lichrospher C18 endcapped column. The mobile phase was acetonitrile-water (75:25, v/v). The detection of DuP 697 and X6882 was by fluorescence at excitation and emission wavelengths of 256 and 419 nm, respectively. The chromatographic system could separate DuP 697 from X6882, the external standard (anthracene), and other endogenous substances present in human plasma. In human plasma the limits of quantification for DuP 697 and X6882 were 3 and 20 ng/ml, respectively; the limit of quantification for DuP 697 in human urine was 5 ng/ml. These compounds were shown to be stable in frozen (-20 degrees C) human plasma and urine for at least 9 weeks. The assay described has been used to characterize DuP 697 pharmacokinetics after oral administration in humans.     

Delivery of salbutamol to nonventilated preterm infants by metered-dose inhaler, jet nebulizer, and ultrasonic nebulizer

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 230 nm has been developed for the determination of paclitaxel in human plasma. Plasma samples were prepared by a selective one-step liquid-liquid extraction involving a mixture of acetonitrile-n-butyl chloride (1:4, v/v). Paclitaxel and the internal standard docetaxel were separated using a column packed with ODS-80A material, and a mobile phase consisting of water-methanol-tetrahydrofuran-ammonium hydroxide (37.5:60:2.5:0.1, v/v). The calibration graph for paclitaxel was linear in the range 10-500 ng/ml, with a lower limit of quantitation of 10 ng/ml, using 1 ml plasma samples. The extraction recoveries of spiked paclitaxel and docetaxel to drug-free human plasma were 89.6+/-8.52 and 93.7+/-5.0%, respectively. Validation data showed that the assay for paclitaxel is sensitive, selective, accurate and reproducible. The assay has been used in a single pharmacokinetic experiment in a patient to investigate the applicability of the method in vivo.     

Oral platelet aggregation inhibitor Ro 48-3657: determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 microl of plasma) and 25 ng/ml (50 microl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.     

Simple high-performance liquid chromatographic method for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of losartan and its E-3174 metabolite in human plasma, urine and dialysate. For plasma, a gradient mobile phase consisting of 25 mM potassium phosphate and acetonitrile pH 2.2 was used with a phenyl analytical column and fluorescence detection. For urine and dialysate, an isocratic mobile phase consisting of 25 mM potassium phosphate and acetonitrile (60:40, v/v) pH 2.2 was used. The method demonstrated linearity from 10 to 1000 ng/ml with a detection limit of 1 ng/ml for losartan and E-3174 using 10 microl of prepared plasma, urine or dialysate. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of losartan in patients with kidney failure undergoing continuous ambulatory peritoneal dialysis (CAPD).     

High-performance liquid chromatographic assay for a new fasciolicide agent, alphaBIOF10, in biological fluids

This paper describes a high-performance liquid chromatographic method with ultraviolet absorbance detection at 304 nm for the determination of 6-chloro-5-(1-naphthyloxy)-2-methylthio benzimidazole (alphaBIOF10) -- a new fasciolicide agent -- and its sulphoxide (SOalphaBIOF10), in plasma and urine. It requires 2 ml of biological fluid, an extraction using Sep-Pak cartridges, and methanol for drug elution. Analysis is performed on a microBondapak C18 (10 microm) column, using methanol-acetonitrile-water (40:30:30, v/v) as the mobile phase. Results showed that the assay is sensitive: 7.2 ng/ml for alphaBIOF10 and SOalphaBIOF10 in plasma and 3.6 ng/ml for both compounds in urine. The response was linear between 0.195 and 12.5 microg/ml. Maximum intra-day coefficient of variation was 5.3%. Recovery obtained was 97.8% for both alphaBIOF10 and SOalphaBIOF10. In urine, recovery was 99.6% and 93.1% for alphaBIOF10 and SOalphaBIOF10 respectively. The method was used to perform a preliminary pharmacokinetic study in two sheep and was found to be satisfactory.     

Anti-tumor gene therapy

A high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous measurement of pyrimethamine and sulphadoxine in human plasma. After an automated liquid-solid extraction on a C8 cartridge, the compounds are separated on a C18 column by isocratic elution; the mobile phase is methanol-acetonitrile-water (10:25:65, v/v/v) with triethylamine (1%) and adjusted to pH 5.6 with phosphoric acid. The eluent is monitored with an ultraviolet detector at 240 nm. The limit of quantification was 10 ng/ml for pyrimethamine and 22 microg/ml for sulphadoxine. No chromatographic interferences can be detected from endogenous compounds, other anti-malarial drugs or major drugs used for the treatment of children. Sulphadimethoxine is used as an internal standard. The method is accurate and precision is good with relative standard deviations lower than 6%. The chromatographic procedure takes 11 min. The method is comparatively rapid, simple, sensitive and can be used for therapeutic drug monitoring, clinical and pharmacokinetic studies.     

Determination of ticarcillin epimers in plasma and urine with high-performance liquid chromatography

A high-performance liquid chromatogaphic method was developed for determining the concentrations of ticarcillin (TIPC) epimers in human plasma and urine. Samples were prepared for HPLC analysis with a solid-phase extraction method and the concentrations of TIPC epimers were determined using reversed-phase HPLC. The mobile phase was a mixture of 0.005 M phosphate buffer (pH 7.0) and methanol (12:1, v/v) with a flow-rate of 1.0 ml/min. TIPC epimers were detected at 254 nm. Baseline separation of the two epimers was observed for both plasma and urine samples with a detection limit of ca. 1 microg/ml with a S/N ratio of 3. No peaks interfering with either of the TIPC epimers were observed on the HPLC chromatograms for blank plasma and urine. The recovery was more than 80% for both plasma and urine samples. C.V. values for intra- and inter-day variabilities were 0.9-2.1 and 1.1-6.4%, respectively, at concentrations ranging between 5 and 200 microg/ml. The present method was used to determine the concentrations of TIPC epimers in plasma and urine following intravenous injection of TIPC to a human volunteer. It was found that both epimers were actively secreted into urine and that the secretion of TIPC was not stereoselective. Plasma protein binding was also measured, which revealed stereoselective binding of TIPC in human plasma.     

High-performance liquid chromatographic determination of the enantiomers of the benzoquinolinone LY191704, a human type 1 5 alpha-reductase inhibitor, in plasma

A stereoselective reversed-phase liquid chromatographic method for the determination of compounds LY300502 and LY300503 (enantiomers of LY191704) in rat and dog plasma was developed. The assay involved extraction of the compounds using a strong cation-exchange solid-phase extraction column, from which the compounds are eluted with 1% of 1 M HCI in methanol. The enantiomers were separated on a Daicel Chiralcel OD-R column. The mobile phase consisted of water-acetonitrile-methanol (50:40:10, v/v) at a flow-rate of 0.3 ml/min. UV detection was achieved at 220 nm. The disposition of the enantiomers of LY191704 in rats and dogs was found to be stereoselective and species specific.     

Sensitive high-performance liquid chromatographic determination of cyclizine and its demethylated metabolite, norcyclizine, in biological fluids using coulometric detection

An accurate, sensitive, selective and reproducible high-performance liquid chromatographic method with coulometric detection for the determination of cyclizine and its inactive demethylated metabolite, norcyclizine, in biological fluids has been developed. The drugs were separated using a custom packed reversed-phase C18 analytical column and phosphate buffer (0.05 M, pH 3)-acetonitrile (7:3) as mobile phase. The dual electrode coulometric detector was operated in the "oxidative-screen" mode with the upstream electrode (detector 1) set at 0.55 V and the downstream electrode (detector 2) set at 0.90 V. Serum and urine samples were prepared for analysis by solid-phase extraction, followed by a simple phase-separation step. The limit of quantitation was 1 ng/ml for both cyclizine and norcyclizine in serum and urine.     

Determination of rifampicin and its main metabolite in plasma and urine in presence of pyrazinamide and isoniazid by HPLC method

A reversed phase HPLC method is described for the simultaneous estimation of rifampicin and its major metabolite desacetyl rifampicin, in the presence of isoniazid and pyrazinamide, in human plasma and urine. The assay involves simple liquid extraction of drug, metabolite and internal standard (rifapentine) from biological specimens and their subsequent separation on a C18 reversed phase column and single wavelength UV detection. In plasma as well as in urine samples, all the three compounds of interest eluted within 17 min. Using methanol-sodium phosphate buffer (pH 5.2; 0.01 M) (65:35, v/v) as mobile phase under isocratic conditions, it was established that isoniazid, pyrazinamide and ascorbic acid (added to prevent oxidative degradation of analytes) did not interfere with the analyte peaks. Recoveries (extraction efficiency) for drug were greater than 90% in both plasma and urine, whereas for metabolite the values were found to be 79 and 86% in plasma and urine, respectively. The plasma and urine methods were precise (total coefficient of variation ranged from 5 to 23%) and accurate (-7 to 5% of the nominal values) for both the analytes. Individual variance components, their estimates and their contribution to the total variance were also determined. Using the same method, unknown samples supplied by WHO were assayed and good correlations were obtained between the found and intended values. The method developed proved to be suitable for simultaneous estimation of rifampicin and desacetyl rifampicin in plasma and urine samples.     

Determinants of plasma anti-oxidant vitamin levels in a population at high risk for stomach cancer

A high-performance liquid chromatographic method was developed for the determination of a new antiulcer agent, YJA-20379-2, in human plasma and urine. The sample preparation was simple: 2.5-volume of acetonitrile was added to the biological sample to deproteinize. A 50-microliter aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase employed was methanol-0.1M S?rensen phosphate buffer of pH 7.0-H2O (75:2:25, v/v/v), and was run at a flow-rate of 1.0 ml/min. The column effluent was monitored by ultraviolet detector at 295 nm. The retention time for YJA-20379-2 was approximately 7.0 min. The detection limits for YJA-20379-2 in human plasma and urine were both 100 ng/ml. The coefficients of variation of the assay (within-day and between-day) were generally low (below 9.16%) for both the human plasma and urine. No interference from endogenous substances was found.     

Determination of ivabradine and its N-demethylated metabolite in human plasma and urine, and in rat and dog plasma by a validated high-performance liquid chromatographic method with fluorescence detection

A sensitive and selective high-performance liquid chromatographic method with native detection of fluorescence was developed and validated for the quantitation of ivabradine and its N-demethylated metabolite in plasma (rat, dog, human) and human urine. The procedure involves the use of an analogue as internal standard, solid-phase extraction on cyano cartridges, separation on a Nova-Pak C8 column and fluorescence detection. Calibration curves are linear in the concentration ranges from 0.5 to 100 ng/ml in plasma and 2.0 to 500 ng/ml in urine with a limit of quantitation set at 0.5 and 2.0 ng/ml in plasma and urine, respectively. The analysis of plasma and urine samples (spiked with the analytes at low, medium and high concentrations of the calibration range) demonstrates that both analytes can be measured with precision and accuracy within acceptable limits. Quality controls spiked with analyte concentrations up to 10000 ng/ml can also be analysed with excellent precision and accuracy after dilution of the samples. The parent drug and its metabolite are stable in plasma and urine after short-term storage (24 h at room temperature and after three freeze-thaw cycles) as well as after long-term storage at -20 degrees C (at least 6 months in animal plasma and 12 months in human plasma and urine). The method has been used to quantify both compounds in plasma and urine samples from clinical and non-clinical studies with ivabradine.     

Simple method for the routine determination of betaine and N,N-dimethylglycine in blood and urine

A simple and convenient method using commercially available derivatization reagents is described for the measurement of betaine and N,N-dimethylglycine (DMG) in blood and urine. Precolumn derivatization of plasma or urine is performed directly in acetonitrile without extraction with p-bromophenacyl bromide and crown ether as catalyst. The p-bromophenacyl ester derivatives are then separated by high-performance liquid chromatography, using an isocratic system of acetonitrile and water containing choline. Effluent was monitored at 254 nm. The limit of detection was 5 micromol/L for betaine and 2 micromol/L for DMG. Analytical recovery was >97% for both analytes. Total and within-day CVs were 2.0-4.4% and 0.9-2.2% for DMG. For betaine, the total and within-day CVs were 1.3-5.3% and 0.4-3.8%, respectively. The method is precise and cost-effective and has been used successfully to determine the concentrations of DMG and betaine in human plasma and urine.     

Highly sensitive high-performance liquid chromatographic determination method for a new erythromycin derivative, EM523, and its major metabolites in human plasma and urine using post-column tris(2,2'-bipyridine) ruthenium(III) chemiluminescence detection

A method for the simultaneous determination of de(N-methyl)-N-ethyl-8,9 -anhydroerythromycin A 6,9-hemiacetal (EM523, I) and its three metabolites in human plasma and urine has been developed using high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection. Plasma and urine samples spiked with erythromycin as an internal standard were extracted with a mixture of dichloromethane and diethyl ether under alkaline conditions. The organic layer was evaporated under a stream of nitrogen gas. The reconstituted sample was injected into an HPLC apparatus and separated on an ODS column using a gradient elution method. The eluate was reacted on-line with a mixture of tris(2,2'-bipyridine) ruthenium(II) and peroxodisulfate, and the generated CL intensity was detected. Optimization of the CL reaction conditions resulted in a sensitive and stable CL intensity for the determination of I and its metabolites. The recovery of each compound from human plasma and urine, and the sensitivity, linearity, accuracy and precision of the method were satisfactory. The lower limits of quantitation for each compound using 0.2 ml of plasma and 0.1 ml of urine were 1 and 10 ng/ml, respectively. This method has been used for the determination of 1 in samples from clinical trials.     

Determination of a new proton pump inhibitor, YH1885, in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new proton pump inhibitor, YH1885 (I), in human plasma and urine, and rat blood and tissue homogenate using fenticonazole as an internal standard. The sample preparation was simple: a 2.5 volume of acetonitrile was added to the biological sample to deproteinize it. A 50-microliter aliquot of the supernatant was injected onto a C8 reversed-phase column. The mobile phase employed was methanol-0.005 M tetrabutylammonium dihydrogenphosphate (77:23, v/v), and it was run at a flow-rate of 1.0 ml/min. The column effluent was monitored using an ultraviolet detector at 270 nm. The retention times for I and the internal standard were 9.0 and 10.3 min, respectively. The detection limits for I in human plasma and urine, and in rat tissue homogenate (including blood) were 50, 100 and 100 ng/ml, respectively. The coefficients of variation of the assay (within- day and between-day) were generally low (below 8.84%) for human plasma and urine, and for rat tissue homogenate. No interferences from endogenous substances were found.     

Accurate high-speed liquid handling of very small biological samples

An assay using reversed-phase high-performance liquid chromatography with ultraviolet detection, at 278 nm, was developed to measure diclofenac in human plasma and urine at concentrations suitable for biopharmaceutical studies. Indomethacin was used as internal standard and separation was performed at 40 degrees C on a C18 Spherisorb column with acetonitrile-0.1 M sodium acetate (35:65, v/v) (pH 6.3) as mobile phase. The sample preparation is simple and rapid (extractionless), and the total run time is less than 5 min. The retention time is 2.8 min for diclofenac and 3.6 min for indomethacin. The detection limit is 0.2 microgram/ml using a 20-microliters loop.     

Is plaque formation in the common carotid artery representative for plaque formation and luminal stenosis in other atherosclerotic peripheral arteries? A post mortem study

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate-acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15-2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

Inhibition of signal-regulated protein kinases by plant-derived hydrolysable tannins

A reversed-phase isocratic high-performance liquid chromatographic method has been developed for the determination of AG-331, a novel thymidylate synthase inhibitor, in human serum and urine. The method involves a solid-phase extraction from C18 cartridges without addition of an internal standard. The methanol eluent is evaporated under nitrogen at 40 degrees C, and reconstituted in mobile phase, acetonitrile-water (35:65, v/v) containing 25 mM ammonium phosphate. Separation of AG-331 was obtained on a C18 column at a flow-rate of 1 ml/min. Chromatographic signals were monitored by a photodiode-array detector at a primary wavelength of 457 nm with a bandwidth of 4.8 nm. Standard curves are linear in the range of 22-2175 ng/ml in plasma and 44-2175 ng/ml in urine, respectively. The extraction recovery ranged from 92.9-102.4%. Intra-day coefficient of variation was less than 9.5%, and inter-day coefficient of variation was less than 14.3% for an AG-331 concentration of 44 ng/ml. This method has been used to characterize the pharmacokinetics of AG-331 in cancer patients as part of ongoing Phase I trials.