Rapid high-performance liquid chromatographic method for determination of adefovir in plasma using UV detection: application to pharmacokinetic studies

A rapid, sensitive and reproducible HPLC method was developed and validated for the analysis of adefovir (CAS 106941-25-7) in human plasma. The separation was achieved on a monolithic silica column (Chromolith Performance RP-18e, 100 x 4.6 mm) using acetonitrile-ammonium dihydrogen phosphate buffer (6:94, v/v), pH 5.2, as the mobile phase at a flow rate of 1.5 ml min(-1). The wavelength was set at 260 nm. The assay enables the measurement of adefovir for therapeutic drug monitoring with a minimum quantification limit of 1 ng ml(-1). The method involves a simple protein precipitation procedure. Analytical recovery was complete. The calibration curve was linear over the concentration range 1-40 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 5%. The method was applied to the determination of adefovir in plasma from 12 subjects dosed with adefovir 2 x 10 mg tablets and pharmacokinetic parameters were evaluated.     

A validated high-performance liquid chromatographic method for the determination of glibenclamide in human plasma and its application to pharmacokinetic studies

Glibenclamide is a potent second generation oral sulfonylurea antidiabetic agent widely used for the treatment of type II diabetes melitus. A rapid, sensitive, precise, accurate and specific HPLC assay for the determination of glibenclamide in human plasma was developed and validated. After addition of flufenamic acid as internal standard, the analytes were isolated from human plasma by liquid-liquid extraction. The method was linear in the 10-400 ng/ml concentration range (r > 0.999). Recovery for glibenclamide was greater than 91.5% and for internal standard was 93.5%. Within-day and between-day precision, expressed as the relative standard deviation (RSD%), ranged from 1.4 to 5.9% and 5.8 to 6.6%, respectively. Assay accuracy was better than 93.4%. The assay was used to estimate the pharmacokinetics of glibenclamide after oral administration of a 5 mg tablet of glibenclamide to 18 healthy volunteers.     

A rapid and sensitive high-performance liquid chromatographic method for the determination of diclofenac sodium in serum and its use in pharmacokinetic studies

A rapid, and sensitive high-performance liquid chromatographic method has been developed for the determination of diclofenac sodium in serum using flufenamic acid as the internal standard. Serum protein was precipitated with acetonitrile. The drugs were eluted from a 5 microns C-8 reversed-phase column at ambient temperature with a mobile phase consisting of acetonitrile-water (50:50% v/v) adjusted to pH 3.3 with glacial acetic acid, at a flow rate of 2 mL min-1 with UV detection at 280 nm. Each analysis required no longer than 10 min. Quantitation was achieved by the measurement of the peak-height ratio and the relative and absolute recoveries varied from 90 to 98%. Detection limits for diclofenac sodium in serum is 25 ng mL-1. Intraday coefficients of variation (CV) ranged from 2.47 to 4.61% and interday CVs from 3.52 to 7% at three different concentrations. Preliminary stability tests showed that diclofenac sodium is stable for at least 2 weeks in serum after freezing. The method is applied for the determination of the pharmacokinetic parameters of diclofenac after administration of two formulations (enteric-coated tablet and slow-release tablet), to a healthy male volunteer.     

A high-performance liquid chromatographic method for determination of praziquantel in plasma

A simple, sensitive, selective and reproducible method based on a reversed-phase chromatography was developed for the determination of praziquantel in human plasma. Praziquantel was separated from the internal standard (diazepam) on a Luna C18 column (250 mm x 4.6mm, 5 microm particle size), with retention times of 4.8 and 6.2 min, respectively. Ultraviolet detection was set at 21 7 nm. The mobile phase consisted of acetonitrile and distilled water (70:30, v/v), running through the column at a flow rate of 1.0 ml/min. The chromatographic analysis was operated at 25 degrees C. Sample preparation (1 ml plasma) was done by a single step liquid-liquid extraction with the mixture of methyl-tert-butylether and dichloromethane at the ratio of 2:1 (v/v). Calibration curves in plasma at the concentrations 0, 50, 100, 200, 400, 800 and 1600 ng/ml were all linear with correlation coefficients better than 0.999. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was below 15% (relative standard deviation: R.S.D.). Good accuracy was observed for both the intra-day or inter-day assays, as indicated by the minimal deviation of mean values found with measured samples from that of the theoretical values (below +/-15%). Limit of quantification (LOQ) was accepted as 5 ng using 1 ml samples. The mean recovery for praziquantel and the internal standard were greater than 90% for both praziquantel and internal standard. The method was free from interference from the commonly used antibiotic and antiparasitic drugs. The method appears to be robust and has been applied to a pharmacokinetic study of praziquantel in three healthy Thai volunteers following a single oral dose of 40 mg/kg body weight praziquantel.     

A rapid liquid chromatographic method for the determination of lamotrigine in plasma

A rapid, sensitive and simple high-performance liquid chromatographic (HPLC) method for the determination of lamotrigine in plasma is described. The drug was extracted from 100 μl of plasma with chloroform:isopropanol (95:5% v/v) in the presence of 100 μl of phosphate buffer (10 mM). The extract was evaporated and the residue was reconstituted with mobile phase and injected onto the HPLC system. The drug and the internal standard (chloramphenicol) were eluted from a Symmetry C₁₈ stainless steel column at ambient temperature with a mobile phase consisting of 0.01 M potassium phosphate–acetonitrile–methanol (70:20:10% v/v/v), adjusted to pH 6.7, at a flow rate of 1.3 ml min⁻¹ and the detector was monitored at 214 nm. Quantitation was achieved by measurement of the peak-area ratio of the drug to the internal standard and the lower limit of detection for lamotrigine in plasma was 20 ng ml⁻¹. The intraday precision ranged from 3.34 to 6.12% coefficient of variation (CV) and the interday precision ranged from 2.15 to 8.34% CV. The absolute and relative recoveries of lamotrigine ranged from 86.93 to 90.71% and from 95.18 to 107.13%, respectively. The method was applied in studying the pharmacokinetics of lamotrigine administered orally to rabbits. This reliable micro-method would have application in pharmacokinetic studies of lamotrigine where only small sample sizes are available, e.g. paediatric patients.     

Sensitive high-performance liquid chromatographic method for determination of captopril in plasma.

A rapid, simple and sensitive high-performance liquid chromatographic method for the determination of captopril in plasma has been developed. Captopril is derivatized with a new reagent, 2-bromo-2'-acetonaphthone to form a product that showed ultraviolet-absorbing properties. For plasma samples, the protein was removed with 6% perchloric acid and the derivatized captopril was extracted with diethyl ether. The chromatographic separation was performed on an analytical mu bondapak NH2 column (300 x 3.9 mm, i.d.) with an isocratic mobile phase consisting of n-hexane-2-propanol-methanol-acetic acid (68:15:15:2). Using ultraviolet detection at 246 nm, the quantification limit for captopril in plasma was 10 ng/ml. The calibration curve was linear over the concentration range 12.5-500 ng/ml. The average recovery was 95% for plasma. The inter-day and intra-day assay coefficients of variation were found to be less than 12%.     

Improved high-performance liquid chromatographic method for the determination of indomethacin in plasma.

Indomethacin is an inhibitor of prostaglandin synthesis and has several uses, including the ability to encourage closure of a patent ductus in premature neonates. A rapid, accurate, and sensitive high-performance liquid chromatographic (HPLC) method is described for measuring plasma concentrations of indomethacin. An acidic buffer (citrate, pH 3.0) was employed to alter the pH of the aqueous phase prior to extraction to minimise interference interference from endogenous compounds. Extraction of indomethacin from plasma was optimally achieved with petroleum ether (boiling fraction of 40-60 degrees C):dichloromethane (50:50). However, separation of indomethacin from plasma proteins was attempted unsuccessfully using acetonitrile precipitation; severe band broadening occurred due to injected sample solvent problems. The absolute recoveries for indomethacin and internal standard (mefenamic acid) were over 90% (n = 3). Precision was expressed as the coefficient of variation (n = 4), which was less than or equal to 8% at each of six indomethacin concentrations in the range 50-2,000 ng/ml. The limit of quantitation of indomethacin in plasma was 50 ng/ml (0.5 ml of plasma injected). We report an HPLC method for the quantitation of indomethacin in plasma that may have widespread applicability for routine drug assay laboratories.     

A rapid liquid chromatographic method for the determination of metoclopramide in human plasma

A new sensitive analytical method is described for the measurement of metoclopramide in 1 ml plasma samples. The extraction step is followed by simple back-extraction and direct injection into the high-performance chromatographic (HPLC) column, with ultraviolet absorbance detection at 275 nm and reverse phase chromatography. The limit of detection for metoclopramide was 3 ng/ml and standard curves were linear over a concentration range of 5 to 1,000 ng/ml. The lowest quantifiable concentration of 5 ng/ml could be determined with a coefficient of variation of 6.5%. The method compares favourably with HPLC methods already described for metoclopramide and gives rapid and reproducible results in subjects receiving the drug.     

A fully automated high-performance liquid chromatographic method for the determination of indomethacin in plasma

A fully automated method is described, which enables the determination of indomethacin in plasma by reversed-phase HPLC following on-line sample enrichment and clean-up on a short pre-column.

The plasma sample is introduced directly into the column switching system. The pre-column, filled with a pellicular bonded phase, is first washed with phosphate buffer, pH 7.4. The compounds retained on the pre-column are then eluted in the fore-flush mode and separated on an octadecylsilica column with a methanolic phosphate buffer (pH 7.4) mobile phase. Indomethacin is determined spectrophotometrically at 254 or 260 nm.

The effect of changes in the pH and flow rate of the washing eluent are studied. Under the conditions selected, memory effects can be avoided, the absolute recovery of the drug is 70% and the limit of detection 10 ng ml⁻¹ for a 100 μl injection of plasma. At a concentration of 100 ng ml⁻¹, the relative standard deviations (RSD) are 1.7% (within-day) and 3.5% (between-day), respectively.     

A simple high-performance liquid chromatographic method for the determination of acyclovir in human plasma and application to a pharmacokinetic study

A rapid, simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the measurement of acyclovir (CAS 59277-89-3) concentrations in human plasma and its use in bioavailability studies is evaluated. The method was linear in the concentration range of 0.05-4.0 microg/ml. The lower limit of quantification (LLOQ) was 0.05 microg/ml in 0.5 ml plasma sample. The intra- and inter-day relative standard deviations across three validation runs over the entire concentration range were less than 8.2%. This method was successfully applied for the evaluation of pharmacokinetic profiles of acyclovir capsule in 19 healthy volunteers. The main pharmacokinetic parameters obtained were: AUC(o-t) 6.50 +/- 1.47 and 7.13 +/- 1.44 microg x h/ml, AUC(0-infinity) 6.77 +/- 1.48 and 7.41 +/- 1.49 microg x h/ml, C(max) 2.27 +/- 0.57 and 2.27 +/- 0.62 microg/ml, t(1/2) 2.96 +/- 0.41 and 2.88 +/- 0.33 h, t(max) 0.8 +/- 0.3 and 1.0 +/- 0.5 h for test and reference formulations, respectively. No statistical differences were observed for C(max) and the area under the plasma concentration--time curve for acyclovir. 90% confidence limits calculated for C(max) and AUC from zero to infinity (AUC(0-infinity)) of acyclovir were included in the bioequivalence range (0.8-1.25 for AUC).     

A simple and rapid HPLC method for the determination of atorvastatin in human plasma with UV detection and its application to pharmacokinetic studies

A rapid and sensitive high-performance liquid chromatographic method for the determination of atorvastatin (CAS 134523-00-5) in plasma was developed in this study. Atorvastatin was isolated from plasma using protein precipitation by acetonitrile. Diltiazem (CAS 33286-22-5) was used as internal standard. The chromatographic conditions were as follows: analytical 125 x 4 mm (i.d.) Nucleosil C8 column (5 microm particle size), mobile phase consisting of sodium dihydrogen phosphate buffer-acetonitrile (60:40, v/v) adjusted to pH 5.5 at a flow rate of 1.5 ml/min, UV detection at 245 nm. The detection limit for atorvastatin in plasma was 1 ng ml(-1). The calibration curve was linear over the concentration range 20-800 ng ml(-1). The recovery was complete. The inter-day and intra-day assay coefficients of variation were found to be less than 7%. The present validated method was successfully used for pharmacokinetic studies of atorvastatin in human subjects.     

Development and validation of a high-performance liquid chromatographic method for determination of pinocembrin in rat plasma: Application to pharmacokinetic study

A sensitive and specific reversed-phase high-performance liquid chromatography with ultraviolet detection (RP-UV-HPLC) method has been developed and validated for the identification and quantification of pinocembrin in rat plasma using chrysin as the internal standard. Following protein precipitation with acetonitrile, the analytes were separated by the mobile phase 0.01 M ammonium acetate (pH 4.0)–methanol (35:65, v/v) with an Agilent TC-C18 column (5 μm, 4.6 mm × 150 mm) at a flow rate of 1 ml/min, column temperature 40 °C and detection wavelength 290 nm. A good linear relationship was obtained in the concentration range studied (0.07–133.33 μg/ml, r = 0.9995). The lowest limit of quantification (LLOQ) was 66.7 ng/ml and the lowest limit of detection (LLOD) was 25 ng/ml. Average recoveries ranged from 93.9 to 97.8% in plasma at the concentrations of 0.33 and 33.33 μg/ml. Intra- and inter-batch relative standard deviations were 0.15–2.03 and 1.18–9.96%, respectively. This method was successfully applied to the pharmacokinetic studies in rats after intravenous administration of pinocembrin.     

Determination of methyldopa in plasma using high-performance liquid chromatography with electrochemical detection. Application to pharmacokinetic/bioavailability studies

The problem of quantitatively measuring methyldopa (a-methyldopa) in biological matrices after applying therapeutic doses to humans is still challenging. Numerous methods have been published but most of them require a tedious, time-consuming sample preparation, are not specific enough or lack the necessary sensitivity. As the basis of conclusive human pharmacokinetic and bioavailability/bioequivalence studies is a validated analytical method, which is reliable, selective, sensitive and able to proceed hundreds or even thousands of samples in a limited time, an assay to fulfill these needs was developed. The present method employs a hiph-performance liquid chromatographic system consisting of a pump, an ODS column, an autosampler and an electrochemical detector. The assay is sensitive down to 50 ng/ml plasma, the calibration curves are linear in a range of 50-2000 ng/ml, the chromatographic peaks are well resolved and the precision and accuracy are excellent. The assay has been successfully used for the determination of very low methyldopa plasma levels during several clinical studies.     

A simple and rapid method for the estimation of quinine using reversed-phase high-performance liquid chromatography with UV detection

A method for measurement of quinine by high-performance liquid chromatography with detection by absorbance at 240 nm is described. The method is sensitive and free from analytical interferences from other drugs, including the quinine diastereoisomer quinidine. Data obtained by analysis of serum from a patient who took a quinine overdose are reported, and the results are compared to those obtained with a spectrofluorometric method.     

Rapid high-performance liquid chromatographic determination of bleomycin A2 in plasma.

A rapid and specific method for the determination of bleomycin A2 is described. A 50-microliter aliquot of 20% trichloroacetic acid was added to 200 microliter of plasma. The sample was vortexed and centrifuged, and 50 microliter of the clear supernate was injected into a liquid chromatograph equipped with a microparticulate reversed-phase column and a fixed wavelength detector. Elution was carried out using methanol-acetonitrile-0.0085 M heptanesulfonic acid-acetic acid. A linear calibration curve was found in the 0.05-5 microgram/ml range with an estimated precision of +/-6% (CV). Preliminary pharmacokinetic data in the rabbit also are reported.     

A simple high-performance liquid chromatographic method for detection of hydroxyzine in human plasma after overdose

INTRODUCTION: Hydroxyzine, a piperazine H1-receptor antagonist, is effective in generalised anxiety disorder. For toxicological purposes, a simple reversed-phase high-performance liquid chromatographic assay was developed for the detection of hydroxyzine in human plasma. METHODS: A liquid-liquid procedure was used to extract the drug from plasma in the presence of an internal standard (clothiapine). The analysis was performed on a Spherisorb S5 C(8) analytical column with UV detection. RESULTS: A linear response was observed over the concentration range 20-1500 ng/ml. A good accuracy (bias<7.3%) was achieved for all quality controls, with intraday and interday variation coefficients inferior to 8.5%. The limit of detection was 10 ng/ml, without interference of endogenous components. DISCUSSION: This rapid method (run time<13 min) is currently used for poison management involving hydroxyzine.