Quantitative determination of epinastine in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the quantitative determination of epinastine, a non-sedating histamine H₁ receptor antagonist, in rat plasma, was developed. A 100-μl volume of plasma sample was spiked with a solution of internal standard (diphenidol) and extracted with dichloromethane under alkaline conditions. The extract was applied onto the HPLC system and detected by ultraviolet absorption at a wavelength of 220 nm. The linearity of the calibration curve was preserved over the concentration range of 20--1000 ng/ml. Both intra-assay variation and relative error were less than 5% for the plasma sample containing 50 ng/ml or 1000 ng/ml of epinastine hydrochloride. The analytical method presented here should be useful for the investigation of the pharmacokinetic properties of epinastine, which is of clinical significance.     

Rapid and simple method for the determination of zolpidem in human plasma by high-performance liquid chromatography

A simple and reproducible method for the determination of zolpidem in human plasma is presented. This method involves protein precipitation with methanol (2 ml of methanol are added to 0.5 ml of plasma) and reversed-phase chromatography with fluorescence detection (excitation wavelength 244 nm, emission wavelength 388 nm). The mobile phase consists of methanol–30 mM dihydrogen potassium phosphate–triethylamine (30:69:1). pH of the aqueous part of the mobile phase is 6.8. No internal standard is required. Limit of quantitation is 1.5 ng/ml and the calibration curve is linear up to 400 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy also does not exceed 9%. The assay is useful for pharmacokinetic studies.     

Free malondialdehyde determination in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for quantification of malondialdehyde (MDA) in human plasma. Deproteinized samples were injected onto a Waters carbohydrate analysis column which was eluted with 20% (v/v) 0.03 M Tris buffer, pH 7.4, in acetonitrile. Peak absorbancy was measured at 267 nm. In contrast to data already published, we did not detect any free MDA in normal human plasma. This suggests that the classical thiobarbituric acid test is not suitable for the determination of MDA in human plasma.     

A simple and rapid liquid chromatography method for simultaneous determination of zidovudine and nevirapine in plasma

We describe a simple, fast, isocratic, reversed-phase high performance liquid chromatographic method for simultaneous determination of plasma zidovudine and nevirapine with UV detection at 260 nm. The method involves liquid-liquid extraction with ethyl acetate and using 3-isobutyl 1-methyl xanthine as internal standard. The system requires a C(18) column (150 mm x 4.6 mm I.D.) and a mobile phase composed of potassium dihydrogen phosphate (15 mM; pH 7.5) and acetonitrile in the ratio of 80:20 (v/v). The assay was linear from 0.025 to 10.0 microg/ml for zidovudine and 0.05 to 10.0 microg/ml for nevirapine. The intra- and inter-day variations were less than 10% for both the drugs. The method was specific and sensitive enough to allow quantification of zidovudine and nevirapine in concentrations observed clinically. The average recoveries of zidovudine and nevirapine from plasma were 95 and 94%, respectively. The method was applied to a pharmacokinetic study in HIV-infected patients who were receiving antiretroviral treatment with zidovudine and nevirapine containing regimens. The method spans the blood concentration range of clinical interest. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring in patients taking a combination treatment of zidovudine and nevirapine.     

A simple and rapid purification of commercial trypsin and chymotrypsin by reverse-phase high-performance liquid chromatography

Commercial trypsin and chymotrypsin were further purified with respective recoveries of approximately 80 and 50% of the activity in a reverse-phase high-performance liquid chromatography system using acetonitrile in dilute trifluoroacetic acid at pH 2. The purified enzymes showed single enzymatic activities toward synthetic and protein substrates. The enzymes can be rapidly purified in amounts appropriate for structural analysis of proteins.     

A simple and rapid method of quantitative analysis of phosphoamino acids by high-performance liquid chromatography

A high-performance liquid chromatographic system for the separation of nonradiolabeled phosphoamino acids and orthophosphate by ion-pair reverse-phase chromatography has been developed. By the use of low-ionic-strength phthalate buffers at pH 6.3, the phosphoamino acids can be visualized by virtue of this uv-active eluant. The technique is sensitive to 200 pmol of phosphoamino acid and has been shown to be directly applicable to the analysis of isolated phosphoproteins.     

Rapid determination of citalopram in human plasma by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of citalopram in human plasma is presented. The sample preparation involved liquid–liquid extraction of citalopram with hexane–isoamyl alcohol (98:2 v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on a cyano column (45×4.6 mm, 5 μm particles), the mobile phase consisted of an acetonitrile–phosphate buffer, pH 6.0 (50:50, v/v). The run time was 2.6 min. The fluorimetric detector was set at an excitation wavelength of 236 nm and an emission wavelength of 306 nm. Verapamil was used as the internal standard. The limit of quantitation was 0.96 ng/ml using 1 ml of plasma. Within- and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 6%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Simultaneous determination of misonidazole and desmethylmisonidazole in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the simultaneous determination of misonidazole and desmethylmisonidazole in plasma is described. After plasma is deproteinized with methanol and the diluted supernatant is chromatographed on a C₁₈ reversed-phase column, both compounds are quantitated by means of an internal standard. The coefficients of variation of within-day and day-to-day precision are below 5.0% for misonidazole in the concentration range of 25–250 mg/l and below 6.1% for desmethylmisonidazole in the concentration range of 2.5–25.0 mg/l. Calibration curves are linear and an analytical recovery varying from 97.6 to 99.8% is obtained. The detection limits for misonidazole and desmethylmisonidazole in plasma are 1.4 mg/l and 0.7 mg/l, respectively.     

Rapid and simple micro-determination of carvedilol in rat plasma by high-performance liquid chromatography

We studied the use of high-performance liquid chromatography (HPLC) with spectrofluorometric detection, using a solid-phase extraction for a simple, rapid and sensitive determination of plasma carvedilol levels in rats. Extracted aliquots were analyzed by HPLC, using a reversed-phase octadecyl silica column. The analytical mean recovery of carvedilol added to the blank plasma was 94.2%. The detection limit was 3.6 ng/ml in the plasma. The reproducibilities (C.V.) were 2.7–7.5% for the within-day assay, and 2.6–7.4% for the between-day assay, indicating that the method was effective for the determination of carvedilol plasma levels.     

Enantiomeric determination of pantoprazole in human plasma by multidimensional high-performance liquid chromatography

Multidimensional HPLC is a powerful tool for the analysis of samples of a high degree of complexity. This work reports the use of multidimensional HPLC by coupling a RAM column with a chiral polysaccharide column to the analysis of Pantoprazole in human plasma by direct injection. The enantiomers from the plasma samples were separated with high resolution on a tris(3,5-dimethoxyphenylcarbamate) of amylose phase after clean-up by a RAM BSA octyl column. Water was used as solvent for the first 5 min in a flow-rate of 1.0 ml/min for the elution of the plasmatic proteins and then acetonitrile-water (35:65 v/v) for the transfer and analysis of pantoprazole enantiomers, which were detected by UV at 285 nm. Analysis time was 28 min with no time spent on sample preparation. A good linear relationship was obtained in the concentration range of 0.20 to 1.5 microg/ml for each enantiomer. Inter and intra-day precision and accuracy were determined by one low (0.24 microg/ml), one medium (0.70 microg/ml) and one high (1.3 microg/ml) plasma concentration and gave a C.V. varying from 1.80 to 8.43% and accuracy from 86 to 92%. Recoveries of pantoprazole enantiomers were in the range of 93.7-101.2%. The validated method was applied to the analysis of the plasma samples obtained from ten Brazilian volunteers who received an 80 mg oral dose of racemic pantoprazole and was able to quantify the enantiomers of pantoprazole in all clinical samples analyzed.     

Alternative method for determination of ceftazidime in plasma by high-performance liquid chromatography

A high-performance liquid chromatography procedure was developed to analyze ceftazidime concentrations in plasma. The procedure consisted of solid phase extraction followed by ion-pairing reverse-phase chromatography. An excellent linear relationship between ceftazidime peak height measurements and concentrations was demonstrated over the concentration range of 1–200 μg ml⁻¹. The advantage of this assay is the elimination of interference at the ceftazidime elution time that has been noted in previous studies and in our experience. Thus, this study describes an alternative, simple methodology that is clinically useful for analyzing ceftazidime in the research setting.     

Sensitive determination of nitrotyrosine in human plasma by isocratic high-performance liquid chromatography

A highly sensitive and simple isocratic high-performance liquid chromatography method was developed for determination of 3-nitrotyrosine in human plasma with precolumn derivatization with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole. The precision of the method was satisfactory (coefficient of variation 4.8%), and the detection limit was established at 0.1 pmol of 3-nitrotyrosine allowing the determination at the level of 6 pmol/ml in human plasma. The recoveries of 3-nitrotyrosine and α-methyltyrosine, an internal standard, were 89.3 +-7.1 and 85.7±7.6%, respectively. The 3-nitrotyrosine level was 31±6 pmol/ml (mean±S.D., n=9) in plasma from healthy volunteers. Since 3-nitrotyrosine is a stable product of peroxynitrite, an oxidant formed by a reaction of nitric oxide and superoxide radicals, the measurement of its plasma concentration may be useful as a marker of nitric oxide-dependent oxidative damage.     

Chiral determination of mirtazapine in human blood plasma by high-performance liquid chromatography

A method is described for the determination of the two enantiomers of mirtazapine in human blood plasma by high-performance liquid chromatography. Measurements were performed on drug free plasma spiked with mirtazapine and used to prepare and validate standard curves. Levels of enantiomers of mirtazapine were also measured in patients being treated for depression with racemic mirtazapine. Mirtazapine was separated from plasma by solid-phase extraction using CERTIFY columns. Chromatographic separation was achieved using a Chiralpak AD column and pre-column and compounds were detected by their absorption at 290 nm. Imipramine was used as an internal standard. The assay was validated for each analyte in the concentration range 10–100 ng/ml. The coefficient of variance was 16% and 5.5% for(+)-mirtazapine for 10 and 100 ng/ml control specimens respectively and 15% and 7.3% for mirtazapine for 10 and 100 ng/ml control specimens respectively. This assay is appropriate for use in the clinical range. The range of plasma mirtazapine concentrations from eleven patients taking daily doses of 30–45 mg of racemate was <5 to 69 ng/ml for (+)-mirtazapine and 13–88 ng/ml for (−)-mirtazapine for blood specimens collected 10–17.5 h after taking the dose.     

Alternative method for determination of pyrimethamine in plasma by high-performance liquid chromatography

A rapid, selective, sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative determination of pyrimethamine (PYR) in plasma is described. The procedure involved the two-step extraction of PYR and the internal standard, quinine (QN) with acetonitrile and dichloromethane at basic pH. Chromatographic separation consisted of the mobile phase (methanol-water containing 0.005 M octanesulfonic acid, 50:50, v/v) running through the column (Techopak-10 C₁₈) at a flow-rate of 1.6 ml/min. Detection was at UV wavelength of 240 nm. The mean recoveries of PYR and QN at a concentration range of 50 and 500 ng/ml were 98.9 and 89%, and 94.7 and 96% for PYR and QN. The within-day coefficients of variation were 2.1–5.1% for PYR and 5.9% for QN. The day-to-day coefficients of variation were 2.1–4.1% for PYR and 5% for QN. The minimum detectable concentrations for PYR and QN in plasma were 3 and 10 ng/ml. The method was found to be suitable for use in clinical pharmacokinetic study.     

Rapid determination of mycophenolic acid in plasma by reversed-phase high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure plasma concentrations of mycophenolic acid. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a Zorbax Eclipse C(8) reversed-phase column with a flow-rate of 1.5 ml/min, and monitoring the absorbance at 250 nm. Throughput was up to 100 samples in 24 h. Within the investigated concentration ranges of mycophenolic acid (0-100 mg/l), good linearity (r>0.99) was obtained. The method is sensitive (the limit of detection was about 20 microg/l) and precise (for 0.49 mg/l added to plasma, within-run C.V. was 2% and between-run was 4.2%; for 2.88 mg/l, within-run C.V. was 0.35% and between-run C.V. was 0.69%; for 24.38 mg/l, within-run C.V. was 0.77% and between-run C.V. was 3.1%). Analytical recoveries were 96% for 0.5 mg/l mycophenolic acid added to plasma, 100% for 12 mg/l and 102.5% for 24 mg/l.     

Rapid and sensitive determination of paclitaxel in mouse plasma by high-performance liquid chromatography

This report describes a rapid, simple and sensitive isocratic high-performance liquid chromatography with diode array UV detection for micro-sample analysis of paclitaxel in mouse plasma. The analysis utilized a Capcell-pak octadecyl analytical column and a mobile phase consisting of acetonitrile–0.1% phosphoric acid in deionized water (55:45, v/v). Paclitaxel and n-hexyl p-hydroxybenzoic acid (internal standard) were extracted from plasma by one-step extraction with tert.-butyl methyl ether. Peak purity was determined over a UV wavelength range of 200 to 400 nm. Paclitaxel and the internal standard were eluted at 3.4 min and 5.4 min, respectively, at a mobile phase flow-rate of 1.3 ml/min. No interfering peaks were observed and the total run time was 10 min. The standard curve was linear (r=0.9999) over the concentration range of 0.010–500 μg/ml. The extraction recovery was >90% for both paclitaxel and n-hexyl p-hydroxybenzoic acid. The intra- and inter-day assay variabilities of paclitaxel ranged from 0.4 to 2.2% and 0.6 to 7.8%, respectively. The LOD and LOQ were 5 and 10 ng/ml, respectively, for paclitaxel using a plasma sample volume of 100 μl. This highly sensitive and simple assay method was successfully applied to a pharmacokinetic study after i.v. administration of paclitaxel 20 mg/kg to mice.     

Simultaneous determination of oleuropein and its metabolites in plasma by high-performance liquid chromatography

A method based on high-performance liquid chromatography (HPLC) with a diode array detection system was developed and validated aiming at the simultaneous determination of oleuropein (OE) and its metabolites, hydroxytyrosol (HT) and tyrosol (T), in human plasma. These phenolic components are believed to play a vital role in the prevention of coronary artery disease and atherosclerosis. The proposed method includes a clean-up solid-phase extraction procedure (using a C(18) column) with high recovery efficiency (85-100%). The statistical evaluation of the method reveals good linearity, accuracy and reproducibility for all the compounds analyzed with RSD values less than 6.5%, while the detection limit is 50 ng/ml for both OE and T and 75 ng/ml for HT. This assay can be employed in bioavailability studies of olive oil phenolic compounds, thus assisting the evaluation of their pharmacological role.