Rapid High-performance Liquid Chromatographic Method Research Articles

Rapid high-performance liquid chromatography method to quantitate elaterinide in juice and reconstituted residues from a bitter mutant of hawkesbury watermelon.

A C(18) reversed-phase HPLC method has been developed to analyze the concentration of 2-O-beta-D-glycopyranosyl-cucurbitacin E (synonym, elaterinide) in juice or reconstituted residues of juice derived from a bitter mutant of Hawkesbury watermelon, Citrullus lanatus (Thunb.) Matsum. & Nakai. The method requires only two steps (homogenation and filtration) for sample preparation prior to column injection and, thus, is amenable to processing large numbers of test samples. Percent elaterinide in juice samples from whole fruit, processed in various ways, ranged from 0.0230 to 0.0808%. Error differences in percent elaterinide for repetitive analyses within the same lot of juice ranged from 1.2 to 5.6%.

Rapid quantification of delavirdine, a novel non-nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with fluorescence detection

Delavirdine is a novel non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1-infected patients. A simple and rapid high-performance liquid chromatographic method for the quantification of delavirdine in human plasma suitable for drug monitoring in patients is described. Sample pretreatment consists of protein precipitation with acetonitrile and subsequent evaporation of the extract to concentrate the analyte. The drug is separated from endogenous compounds by isocratic reversed-phase, high-performance liquid chromatography coupled with fluorescence detection. The optimal excitation and emission wavelengths are 300 and 425 nm, respectively. The method has been validated over the range of 50–50 000 ng/ml using only 200 μl of plasma samples. The assay is linear over this concentration range as indicated by the F-test for lack of fit. Within- and between-day precisions are less than 4.4% for all quality control samples. The lower limit of quantitation is 50 ng/ml. Recovery of delavirdine from human plasma is 93.8%. Delavirdine is stable under various conditions, for example 1 h at 60°C and one week at 4°C. This validated assay is suited for use in pharmacokinetic studies with delavirdine and can readily be implemented in the setting of a hospital laboratory for the monitoring of delavirdine concentrations.

Simultaneous determination of enrofloxacin and ciprofloxacin in animal biological fluids by high-performance liquid chromatography: Application in pharmacokinetic studies in pig and rabbit

A simple and rapid high-performance liquid chromatographic method for the simultaneous determination of enrofloxacin and ciprofloxacin has been developed in pig and rabbit samples. Solid-phase extraction was applied from samples on a C 18 cartridge using a mixture of methanol–hydrochloric acid (98:2, v/v). Analytical separation was performed on a C 18 column with UV detection at 277 nm under gradient conditions. The mobile phase was a mixture of orthophosphoric acid–triethylamine–acetonitrile. The method has been validated for both molecules in pig and rabbit plasma and adapted for rabbit tissue-cage fluid (TCF). The assay is specific and reproducible within the both drugs and mean recoveries for ciprofloxacin and enrofloxacin, respectively, were 92±6% and 90±5% for pig plasma over the range used. Mean recoveries for enrofloxacin were 108±9% and 102±7% for rabbit plasma and TCF, respectively, over the range used. The suitability of the assay for pharmacokinetic studies was determined by measuring enrofloxacin and ciprofloxacin concentrations either in pig plasma after administration of a single intravenous 5 mg/kg dose of enrofloxacin or in rabbit plasma and TCF during a 24 h infusion of enrofloxacin at a rate of 1.25 mg/kg per hour.

Therapeutic drug monitoring of risperidone using a new, rapid HPLC method: reappraisal of interindividual variability factors.

Because of the enormous gap between premarketing studies in physically healthy subjects and clinical practice in patients, the present study reconsidered interindividual variability factors affecting risperidone concentrations under routine therapeutic drug monitoring conditions. The study included 92 patients, 27% of whom were 70 years or older. The patients received risperidone orally (dose range, 0.5-11 mg per day) and had concentrations of risperidone and the active metabolite 9-hydroxyrisperidone measured at steady state by a new, rapid, and sensitive method of high-performance liquid chromatography (HPLC). After normalization to a dose of 4 mg/day, median concentrations were 2.9 ng/ml (80% range, 0.9-27.9 ng/ml) for the parent compound and 24.1 ng/ml (80% range, 12.0-57.6 ng/ml) for the metabolite. When considering linear regression models, age was identified as a major source of interindividual variability, with expected increases of 340% and 220% for concentrations of parent compound and metabolite, with age increasing from 20 to 80 years. Body weight provided an additional significant contribution to the variability of 9-hydroxyrisperidone concentration, a 20-kg higher body weight associated with a concentration decrease of 23%. Serotonin-specific reuptake inhibitor (SSRI) comedication (fluoxetine, two patients; citalopram, two patients; paroxetine, one patient; fluvoxamine, one patient) was significantly associated with 4.6-fold higher concentrations of parent compound, in keeping with an inhibitory action on CYP2D6 enzyme. Significantly higher concentrations of 9-hydroxy-risperidone (+ 29%) were also observed in the 17 patients with biperiden comedication. Therapeutic drug monitoring data, collected in patients representative of the population for which the drug was intended, allowed us to quantify the dose reduction needed in elderly patients and thus provided valuable information in addition to the one collected during premarketing studies performed with strict inclusion and exclusion criteria.

SIMULTANEOUS HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF NON-STEROIDAL ANTI-INFLAMMATORY OXICAMS IN PHARMACEUTICAL PREPARATIONS

A simple and rapid isocratic high-performance liquid chromatographic method has been developed for the simultaneous analysis of the non-steroidal anti-inflammatory drugs tenoxicam, piroxicam, meloxicam, and lornoxicam using isoxicam as an internal standard. The analyte was chromatographed using a Lichrosphere RP18 column, a Tris acetic acid buffer-tetrabutylammonium reagent-tetrahydrofuran-acetonitrile as the mobile phase, and UV detection at 360 nm. The separation obtained was very good. The method was applied to pharmaceutical formulations containing a single active ingredient and was shown to be linear, sensible, accurate, and reproducible. A very small modification of the mobile phase enabled the separation of tenoxicam, piroxicam, isoxicam, cinnoxicam,meloxicam, and lornoxicam with symmetrical and well resolved peaks.

Simultaneous determination of inulin and p-aminohippuric acid (PAH) in human plasma and urine by high-performance liquid chromatography.

Inulin and p-aminohippuric acid (PAH) clearances are used for the estimation of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). A simple and rapid high-performance liquid chromatography (HPLC) method with UV detection is described for the simultaneous determination of inulin and PAH in the same chromatogram in the plasma and urine of humans. Plasma and urine samples were hydrolyzed with perchloric acid (0.7%) in boiling water. The mobile phase consisted of 0.01 M potassium dihydrogenphosphate with 0.02 M tetramethylammonium chloride and o-phosphoric acid (pH 3)-acetonitrile (94:6, v/v), pumped at a rate of 1.2 ml min-1 on a C8 reversed-phase column. Tannic acid was used as the internal standard and UV detection at 285 nm was employed. The calibration curves were linear over the concentration range of 12.5-100 mg l-1 for inulin and 6.25-50 mg l-1 for PAH with determination coefficients greater than 0.997. The method is accurate (bias < 13%) and reproducible (intra- and inter-day relative standard deviation less than 11%), with a limit of quantitation of 12.5 mg l-1 and 6.25 mg l-1 for inulin and PAH, respectively. Analytical recoveries from urine and plasma were ranged from 81 to 108% for both compounds. This fully validated method, which allows the simultaneous determination of inulin and PAH clearances, is simple, rapid (total run time < 10 min) and requires only a 200 microliters plasma or urine sample.

Simultaneous Determination of Pyridoxine Hydrochloride and Doxylamine Succinate from Tablets by Ion Pair Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)

A new, simple, precise, and rapid ion pair reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed for the simultaneous determination of pyridoxine hydrochloride (PYR) and doxylamine succinate (DOX) in tablets. The stationary phase was a Microbondapak C18 column (10 μ, 300 mm × 3.9 mm i.d.). The mobile phase was water:methanol (60:40) containing 10 mM heptanesulfonic acid and 0.25% triethylamine and adjusted to pH 2.2 with orthophosphoric acid. Detection was carried out at 263 nm using an ultraviolet (UV) detector. The flow rate was 1.0 ml/min, and retention times were 3.65 min and 7.32 min for PYR and DOX, respectively. The linearity was obtained in the concentration range 0.5–500 μg/ml for PYR and DOX. Mean percentage recoveries were 100.20% and 101.20% for PYR and DOX, respectively.

Determination of thiamin in cooked sausages.

A reliable and rapid high-performance liquid chromatography (HPLC) method has been set up for the determination of total thiamin in difficult sample matrices such as cooked sausages. Different hydrolysis conditions and enzymes were tested to release the vitamin from its phosphate ester. The best data in the enzymatic digestion were obtained by incubating the samples with 6% clara-diastase at 50 degrees C for 3 h. After oxidation of thiamin to thiochrome, the sample extracts were purified by using a C(18) Sep-Pak cartridge. Final determination was performed by reversed-phase HPLC with fluorescence detector (excitation 360 nm, emission 430 nm), on a low-cost 25 cm x 4 mm i.d. Spherisorb C(8) cartridge using a mixture of 5 mM phosphate buffer pH 7.0 and acetonitrile (70:30, v/v) as mobile phase. Precision of the method was 1.5% (within a day) and 5. 2% (between days). The detection limit was 0.015 mg/100 g. All the recoveries from the different cooked sausages were better than 90% of thiamin hydrochloride added to samples of meats. In the samples analyzed, the mean value for thiamin was between 0.039 and 0.508 mg/100 g fresh weight.

Determination of piromidic acid residues in trout muscle tissue and in urine by liquid chromatography with post-column modification of pH and fluorimetric detection

A rapid high-performance liquid chromatographic method has been developed to determine piromidic acid in trout muscle tissue and in urine, in the presence of nalidixic, 7-hydroxymethylnalidixic, oxolinic and pipemidic acids and cinoxacin. A Nova-Pak C 18 column was used with acetonitrile–4·10 −4 M oxalic acid (40:60, v/v) as the mobile phase. A post-column change of pH was made with NaOH. Fluorimetric detection at 456 nm ( λ e x 275 nm) was used. The instrumental detection limit was 5.91 ng/ml, based on height of peak. Pretreatment of the urine samples was not necessary and fish samples were extracted with sodium hydroxide solutions and cleaned by means of an extraction with chloroform. Detection limit was 147 ng/ml for urine and 5.91 ng/g for trout muscle. Good separation without interference from any other components was obtained. Recovery was better than 87% in urine and better than 72% in trout muscle tissue.

Rapid and Sensitive Determination of Meropenem in Rat Plasma by High Performance Liquid Chromatography

A sensitive and rapid high-performance liquid chromato-graphic method using UV detection, has been developed for the analysis of meropenem in plasma. This involved a single liquid-solid extraction on Isolute C18-EC column in the presence of an internal standard (ceftazidime). Analysis was performed by isocratic elution with a mobile phase consisting of 0.01 M acetate buffer pH 4.8 containing 15% (v/v) methanol, with UV detection at 296 nm. The limit of quantitation of the assay was 0.125 mg/L. The method was applied to a pharmacokinetic study in rats receiving 100 mg/kg by IV route with a good accuracy (94–101%) and precision (less than 13%).

Determination of metformin in plasma by high-performance liquid chromatography after ultrafiltration

A rapid high-performance liquid chromatography (HPLC) method was developed for determination of metformin, an oral antidiabetic agent, in plasma. Sample preparation entailed a 30-min centrifugation of plasma through a micron filter with direct injection of the protein-free ultrafiltrate into an HPLC system consisting of a cation-exchange extraction column (7.5×4.6 mm), a column switching valve, and a cation-exchange analytical column (250×4.6 mm). The eluent was monitored at 232 nm. Metformin was well resolved at a retention time of about 5 min. There was less than 2% loss of metformin during ultrafiltration and good linearity was established from 0.10 to 40 mg/l of metformin hydrochloride. The lower limit of quantitation was about 0.05 mg/l, at which concentration the signal-to-noise was above 10. The intra- and inter-assay coefficients of variation at plasma concentrations of metformin hydrochloride between 0.25 and 25 mg/l were typically 0.8–1.4% and 3.5–6.4%, respectively. This method offers a rapid sample preparation time and achieves excellent sensitivity without resorting to extraction and evaporation techniques.

Rapid determination of nevirapine in human plasma by ion-pair reversed-phase high-performance liquid chromatography with ultraviolet detection

Nevirapine is a non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1-infected patients. A simple and rapid high-performance liquid chromatographic method for the quantification of nevirapine in human plasma is described. Sample pretreatment consists of protein precipitation with acetonitrile. The analyte is separated from endogenous compounds by isocratic reversed-phase, ion-pair, high-performance liquid chromatography with ultraviolet detection at 282 nm. The method has been validated over the range of 52–10 400 ng/ml using 250 μl of plasma. The assay was linear over this concentration range. Within- and between-day precisions were less than 4.5% for all quality control samples. The lower limit of quantitation was 52 ng/ml. Recovery of nevirapine from human plasma was 94.5%. This validated assay is suited for use in pharmacokinetic studies with nevirapine and can readily be used in a hospital laboratory for the monitoring of nevirapine concentrations.

Rapid analysis of ergovaline in ovine plasma using high-performance liquid chromatography with fluorimetric detection

A rapid high-performance liquid chromatographic method for the determination of the mycotoxin ergovaline in ovine plasma is described here. Ergotamine was used as an internal standard. A simple extraction procedure with diethyloxide was carried out, before chromatography on a C 8 column, with the excitation and emission wavelengths fixed at 250 and 420 nm respectively, on a fluorimetric detector. The method, which was found to be linear between 3.5 and 15 ng/ml, had good specificity, precision and accuracy. The limit of quantification and the limit of detection were 3.5 and 1.2 ng/ml, respectively. A preliminary application of the described assay to a plasma kinetic study, after intravenous administration of a single dose of ergovaline (17 μg/kg body mass) to four sheep, showed a very rapid decrease of the plasma ergovaline levels. The terminal half-life and the total clearance of the mycotoxin were found to be 23.6 min and 0.020 l/min kg −1 body mass, respectively.

Determination of lansoprazole in biological fluids and pharmaceutical dosage by HPLC.

A simple and rapid (extractionless) high-performance liquid chromatographic method with UV detection at 230 nm was developed for the determination of lansoprazole in biological fluids and pharmaceutical dosage. Niflumic acid was added as internal standard. The separation was performed at ambient temperature on a C18 Spherisorb column with acetonitrile + 0.1 M sodium acetate (40:60, v/v, pH 7) as mobile phase. The retention time was 5.2 min for niflumic acid and 6.7 min for lansoprazole. The detection limit was 20 ng/ml using a 100 microl loop. The method was successfully applied to a pharmacokinetic study of lansoprazole in humans.

Determination of fenbendazole, praziquantel and pyrantel pamoate in dog plasma by high-performance liquid chromatography

Simple and rapid high-performance liquid chromatographic methods were developed for the determination of fenbendazole, praziquantel and pyrantel pamoate in dog plasma. The combination of these drugs is the most powerful treatment against most types of worms. Blood plasma samples obtained in a pharmacokinetic trial were prepared using solid-phase extraction. Fenbendazole and praziquantel were analyzed simultaneously by reversed-phase high-performance liquid chromatography on an octadecyl-modified silica stationary phase employing acetonitrile–phosphate buffer (pH 3.0) eluent and ultraviolet detection at 220 nm. Pyrantel was analyzed separately on a base-deactivated reversed-phase column using methanol–tetrahydrofuran–ammonium acetate buffer (pH 4.6) eluent and ultraviolet detection at 317 nm. Average recoveries for fenbendazole, praziquantel and pyrantel pamoate were 76.8, 93.4 and 90.5%, respectively. Limits of quantitation were in the range of 15–25 ng/ml plasma.

Routine measurement of fluoxetine and norfluoxetine by high-performance liquid chromatography with ultraviolet detection in patients under concomitant treatment with tricyclic antidepressants.

A robust and rapid high-performance liquid chromatography (HPLC) method is described for therapeutic drug monitoring of fluoxetine and norfluoxetine in the presence of six frequently-used tricyclic antidepressants and their respective metabolites. Liquid-liquid extraction into n-hexane/acetonitrile is used with reextraction into hydrochloric acid for clean-up. The chromatographic separation is carried out on a CN column. The minimum detectable amount is 3 ng injected on column. In addition to qualitative and quantitative validation data for the assay method, results from patient samples are presented. It is concluded that for patients treated with fluoxetine, therapeutic drug monitoring is valuable for optimizing the therapy.

Rapid high-performance liquid chromatographic method of determining malondialdehyde for evaluation of rancidity in edible oils

A simple HPLC method for the determination of malondialdehyde (MDA) in oxidised vegetable oils was developed and the results were compared with those given by a spectrophotometric method. Vegetable oil was steam-distilled in a Kjeldahl distillation apparatus and the MDA was determined in the aqueous distillates by HPLC, using a µ-Bondapak C18 column, with a mobile phase of 1% acetic acid–acetonitrile (85 + 15, v/v). A total time of 2 min was necessary to assay each distillate and only MDA was detected. MDA can be determined at a level of 1.5 × 10–9 mol l–1.

Simultaneous Determination of Pseudoephidrine HC1 (PSE) and Terfanidine (TER) from Formulations by Reversed-Phase Ion Pair High-Performance Liquid Chromatography (RP-HPLC)

A new simple, precise, rapid, and selective reversed-phase ion pair high-performance liquid chromatography (RP-HPLC) method has been developed for the simultaneous determination of pseudoephidrine (PSE) and terfanidine (TER) from tablets using 60:15:25 acetontrile:methanol:water (v/v) containing 2.9 g sodium lauryl sulfate/liter, pH adjusted to 3.1 using phosphoric acid as a mobile phase and C18 Spherisorb ODS 2 (3 µm, 5 cm × 4.6 mm i.d.) as stationary phase. Detection was carried out using a UV detector at 254 nm. A constant flow of 1.0 ml/min was maintained throughout the analysis. Retention times for PSE and TER were 1.90 and 7.35 min, respectively. Linearity range and percentage recoveries for PSE and TER were 24–1200 and 12–600 µg/ml, and 100.01 and 100.4%, respectively.

Rapid isocratic HPLC analysis of beta-carotene in red peppers (Capsicum annuum L.) and food preparations

A simple, rapid high-performance liquid chromatography method has been devised in order to separate and quantify β-carotene present in red pepper (Capsicum annuum L.) fruits and food preparations. A reversed-phase isocratic non-aqueous system enables the separation of β-carotene within a few minutes, with detection at 450 nm. The selection of extraction solvents, mild saponification conditions, and chromatographic features, is evaluated and discussed. The method is proposed for rapid screening of large plant populations, plant selection, as well as for food products, and also for nutrition and quality control studies.

Quantitative High-performance Liquid Chromatographic Analysis of Famotidine in Pharmaceutical Dosage Forms

A rapid and specific high-performance liquid chromatographic (HPLC) method has been developed for quantitative analysis of famotidine in pharmaceutical dosage forms. The method was applied to medicines on the Turkish market. A LiChrospher RP-18 column, HP-1050 UV-detector and methanol—0·1 M aqueous ammonium acetate (30 : 70) as the solvent system (flow rate 1·0 mL min−1) were used. Quantitative determination of famotidine was carried out at 254 nm wavelength. The method was found to be rapid—under 5 min per assay—and was selective enough to allow the determination of famotidine in the presence of certain excipients.