Validated HPLC method for determination of amlodipine in human plasma and its application to pharmacokinetic studies

A rapid, simple and sensitive high-performance liquid chromatography (HPLC) method has been developed for quantification of amlodipine in plasma. The assay enables the measurement of amlodipine for therapeutic drug monitoring with a minimum detectable limit of 0.2 ng ml(-1). The method involves simple, one-step extraction procedure and analytical recovery was about 97%. The separation was performed on an analytical 125 x 4.6 mm i.d. Nucleosil C8 column. The wavelength was set at 239 nm. The mobile phase was a mixture of 0.01 M sodium dihydrogen phosphate buffer and acetonitrile (63:37, v/v) adjusted to pH 3.5 at a flow rate of 1.5 ml min(-1). The calibration curve was linear over the concentration range 0.5-16 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 10%.     

Simple high-performance liquid chromatographic determination of buspirone in human plasma

A simple, rapid and sensitive high-performance liquid chromatographic method for the determination of buspirone in plasma was developed. Buspirone was isolated from plasma using protein precipitation by acetonitrile and the recovery was complete. Citalopram was used as internal standard. The chromatographic conditions were as follows: analytical 125 x 4 mm, i.d. Nucleosil C18 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 1.0 ml min(-1), UV detection at 235 nm. The quantification limit for buspirone in plasma was 0.5 ng ml(-1).The calibration curve was linear over the concentration range 0.5-10 ng ml(-1). The inter- and intra-day assay coefficients of variation were found to be less than 8%. The present validated method was successfully used for bioequivalence studies of buspirone in human subjects.     

HPLC determination of omeprazole in human plasma using a monolithic column

A rapid and sensitive HPLC method using a monolithic column has been developed for quantification of omeprazole (CAS 73590-58-6) in plasma. The method was specific and sensitive with a quantification limit of 10 ng/ml. Sample preparation involves simple, one-step extraction procedure and analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 x 4.6 mm) column with an isocratic mobile phase consisting of 0.01 mol/l disodium hydrogen phosphate buffer-acetonitrile (73:27 v/v) adjusted to pH 7.1. The wavelength was set at 302 nm. The calibration curve was linear over the concentration range 20-1500 ng x ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 7%.     

Rapid determination of metformin in human plasma using ion-pair HPLC

A rapid, simple and sensitive ion-pair HPLC method has been developed for quantification of metformin in plasma. The assay enables the measurement of metformin for therapeutic drug monitoring with a minimum detectable limit of 20 ng/ml. The method involves simple, one-step extraction procedure and analytical recovery was complete. The separation was performed on an analytical 150 x 4.6 mm i.d. mubondapak C(18) column. The wavelength was set at 235 nm. The mobile phase was 40% acetonitrile, 0.01 M sodium dodecyl sulphate, 0.01 M sodium dihydrogen phosphate, and distilled water to 100%, adjusted to pH 5.1 at a flow rate of 1.5 ml/min. The calibration curve was linear over the concentration range 0.2-2.5 microg/ml. The coefficients of variation for inter-day and intra-day assay were within the range of clinical usefulness.     

Quantification of carvedilol in human plasma by liquid chromatography using fluorescence detection: application in pharmacokinetic studies

A simple, rapid and sensitive isocratic reversed-phase HPLC method with fluorescence detection using a monolithic column has been developed and validated for the determination of carvedilol in human plasma. The separation was performed on a Chromolith Performance (RP-18e, 100mm x 4.6mm) column with an isocratic mobile phase consisting of 0.01 M disodium hydrogen phosphate buffer-acetonitrile (40:60, v/v) adjusted to pH 3.5. The sample preparation involves protein precipitation procedure and analytical recovery was complete. Letrozole was used as internal standard. The assay enables the measurement of carvedilol for therapeutic drug monitoring with a minimum quantification limit (LOQ) of 1 ng ml(-1). The excitation and emission wavelengths were set at 240 and 340 nm, respectively. The calibration curve was linear over the concentration range 1-80 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 8.0%.     

Development an ion-pair liquid chromatographic method for determination of sotalol in plasma using a monolithic column

A rapid and sensitive ion-pair HPLC method using a monolithic column and fluorescence detection has been developed for quantification of sotalol in plasma. The assay enables the measurement of sotalol for therapeutic drug monitoring with a minimum quantification limit of 10 ng ml(-1). The analytical method involves simple, one-step protein precipitation and no extraction procedure is needed. Sample preparation is fast and the analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 mm x 4.6 mm) column at ambient temperature. The mobile phase was 10% acetonitrile, 0.001 M heptane sulfonic acid, 0.02 M sodium dihydrogen phosphate, and distilled water to 100%, adjusted to pH 5.5 at a flow rate of 1.8 ml/min. The excitation wavelength was set at 235 nm, emission at 300 nm. The calibration curve was linear over the concentration range 20-1500 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 7%. The method has been applied to the determination of sotalol in plasma from 12 subjects dosed with racemic sotalol.     

High performance liquid chromatography for routine monitoring of serum flecainide

We developed a simple, rapid, and selective assay method for determination of serum flecainide by using solid phase extraction and reversed phase high performance liquid chromatography (HPLC) equipped with ordinary octadecylsilyl silica (ODS) column and ultraviolet (UV) detector. Serum samples spiked with the internal standard were treated by a disposable C(18)-cartridge to extract flecainide. The flecainide and internal standard were separated on ODS column and were detected with an UV detector set at 298 nm. The mobile phase solvent consisting of 0.1 M 1-pentanesulfonic acid sodium salt, acetonitrile, and acetic acid (250:206:2.5 v/v) was used at the flow rate of 1.0 ml/min. The calibration curve for flecainide was linear at the concentration of 50-1500 ng/ml (r=0.9998). The recoveries of flecainide from serum samples were 92-98%. The coefficient of variations (CVs) for intra- and inter-day assay were 1.3-4.8 and 3.2-6.9%, respectively. The method could be applied to routine monitoring of serum flecainide in the patients with tachyarrhythmia.     

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.     

LC determination and pharmacokinetics of meloxicam

A simple and rapid HPLC assay method for the estimation of meloxicam in plasma was developed. The method totally eliminated the solvent extraction procedure. The plasma proteins were precipitated using perchloric acid (70%) and acetonitrile mixture (1:1 v/v) and the supernatant was directly injected to the HPLC system. The separation was achieved on a Lichrospher C18 5 micron (125x4.0 mm) analytical column with a mobile phase of sodium acetate buffer (pH 3.3, 170 mmol):acetonitrile (62:38 v/v) mixture. Detection was by UV detector at 355 nm. The retention time observed for meloxicam and piroxicam (internal standard) were at 6.0 and 4.0 min, respectively. The response was linear over a range of 50-1500 ng x ml(-1) in human plasma. The method was simple, specific, precise and accurate. The method was also used for the bioequivalence study of meloxicam formulation in healthy, human, Indian, male volunteers.     

A rapid high-performance liquid chromatographic method for the determination of pantoprazole in plasma using UV detection. Application in pharmacokinetic studies

A rapid, sensitive and reproducible HPLC method was developed and validated for the analysis of pantoprazole (CAS 102625-70-7) in human plasma. The separation was achieved on a monolithic silica column using acetonitrile-potassium dihydrogen phosphate buffer (25:75,v/v), pH 3.0, as the mobile phase at a flow rate of 1.5 ml min(-1). The wavelength was set at 290 nm. The assay enables the measurement of pantoprazole for therapeutic drug monitoring with a minimum quantification limit of 20 ng ml(-1). The method involves a simple protein precipitation procedure. Analyticil recovery was complete. The calibration curve was linear over the concentration range 20-3500 ng ml(-1) The coefficients of variation forthe inter-day and intra-day assay were found to be less than 7%.     

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.     

Determination of ABT-089 in human plasma by high performance liquid chromatography using in situ precolumn derivatization with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole

ABT-089 is a potent, selective neuronal cholinergic channel modulator with cognition enhancing activity in several animal paradigms. A simple and sensitive chromatographic method for the specific determination of ABT-089 in human plasma has been developed and validated. The method utilizes in situ precolumn fluorescence derivatization of the sample with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) prior to liquid-liquid extraction followed by reverse phase HPLC and fluorescence detection (lambda(ex) 495 nm, lambda(em) 533 nm). The described method significantly simplifies sample preparation. The derivatized product was separated from interference using a YMC ODS-AQ, 5 microm, 250x4.6 mm i.d. column using a mobile phase consisting of 30:5:65 (v:v:v) acetonitrile/methanol/aqueous buffer at a flow rate of 0.75 ml min(-1). The aqueous buffer consisted of 0.01 M tetramethylammonium perchlorate, 0.1% (v:v) trifluoroacetic acid, pH 3.0. An Alltech Absorbosphere CN, 5 microm, cartridge guard column was also used before the analytical column. Plasma samples were alkalinized with 0.1 M NaHCO3, 300 microl of a 1 mg ml(-1) ethanolic solution of NBD-F was added and the samples were heated in a water bath for 10 min at 50 degrees C. The samples were then extracted with tert-butylmethylether, evaporated to dryness and then reconstituted in mobile phase. For 1 ml of plasma, a limit of quantitation (LOQ) of 1.6 ng ml(-1) was obtained. The method was linear from 1.6 to 836 ng ml(-1). Inter and intra-day assay RSD (n = 6) were less than 9%. Accuracy determinations showed the quality control samples to range between 88-114% of the theoretical concentration.     

HPLC method for the determination of fluvoxamine in human plasma and urine for application to pharmacokinetic studies

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the assay of fluvoxamine in human plasma and urine. The method was based on reaction of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) forming orange colored product. The fluvoxamine-NQ derivative was separated by isocratic reversed-phase HPLC and detected at 450 nm. The chromatographic conditions were as follows: Phenomenex C(18) (250 mm x 4.6 mm i.d., 5 microm) column, mobile phase consisting of acetonitrile/water (80:20 v/v) at a flow rate of 1 ml/min. Tryptamine was selected as an internal standard. The assay was linear over the concentration range of 5-145 and 2-100 ng/ml for plasma and urine, respectively. The limits of detection (LOD) were 1.4 and 1 ng/ml for plasma and urine estimation at a signal-to-noise (S/N) ratio of 3. The limits of quantification (LOQ) were 5 and 2 ng/ml for plasma and urine, respectively. The extraction recoveries were found to be 96.66+/-0.69 and 96.73+/-2.17% for plasma and urine, respectively. The intra-day and inter-day standard deviations (S.D.) were less than 1. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This assay was demonstrated to be applicable for clinical pharmacokinetic studies.     

Simultaneous determination of amoxicillin and clavulanic acid in human plasma by isocratic reversed-phase HPLC using UV detection

A simple, rapid and sensitive isocratic reversed phase HPLC method with UV detection using internal standard has been developed and validated for simultaneous determination of amoxicillin and clavulanic acid in human plasma. The assay enables the measurement of amoxicillin and clavulanic acid for therapeutic drug monitoring with a minimum quantification limit of 15 and 30 ng ml(-1), respectively. The method involves simple, one-step extraction procedure and analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 mm x 4.6mm) column with an isocratic mobile phase consisting of 0.02 M disodium hydrogen phosphate buffer-methanol (96:4, v/v) adjusted to pH 3.0. The wavelength was set at 228 nm. The coefficients of variation for inter-day and intra-day assay were found to be less than 9.0%.     

Stereoselective determination of pyridoglutethimide enantiomers in serum with a chiral cellulose-based high-performance liquid chromatographic column using solid phase extraction and UV detection

A sensitive method for the separation and determination of R(+)- and S(-) enantiomers of pyridoglutehimide in serum by high performance liquid chromatography (HPLC) with UV detection was developed. The assay involves the use of a solid-phase extraction for serum sample clean-up prior to HPLC analysis using a C18 Bond-Elute column. Chromatographic resolution of the enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250 x 4.6 mm I.D.) under isocratic conditions using a mobile phase of 25:75 v/v acetonitrile-0.3 M aqueous sodium perchlorate (pH 6.2 adjusted with perchloric acid) at a flow rate of 0.8 ml/min. Recoveries for R(+)- and S(-)-pyridoglutethimide enantiomers were in the range 86-91% at 300-900 ng/ml level. Intra-day and inter-day precision calculated as %R.S.D. were in the ranges of 2.9-3.9 and 1.5-4.7% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percentage error were in the ranges of 1.9-3.3 and 1.5-3.9% for both enantiomers, respectively. Linear calibration curves in the concentration ranges of 100-1500 ng/ml for each enantiomer show correlation coefficient (r) of more than 0.9995. The limit of quantification (LOQ) of each enantiomer was 100 ng/ml using 1 ml of serum. The detection limit (LOD) for each enantiomer in serum using a UV detection set at 257 nm was 50 ng/ml (S/N = 2).     

Determination of saquinavir and ritonavir in human plasma by reversed-phase high-performance liquid chromatography and the analytical error function

Two simple and reproducible high-performance liquid chromatography methods with ultraviolet detection were developed and validated for the quantitation of two protease inhibitors, saquinavir and ritonavir, in human plasma. The same single liquid-liquid extraction procedure with ethyl acetate-hexane (50:50, v/v), reversed-phase column and mobile phase were used. The analyses were accomplished using a Luna C(18) column (150 mm x 4.6mm i.d.) with a C(18) guard column and, the mobile phase consisted of acetonitrile and 70 mM KH(2)PO(4) adjusted to pH 5 with 80 mM Na(2)HPO(4) (46:54, v/v). The wavelength was set at 240 nm for saquinavir and at 210 nm for ritonavir. The retention times were 6.4 min for saquinavir and 8.3 min for ritonavir. The methods were linear over the range of 100-2500 ng/ml for saquinavir and 200-2500 ng/ml for ritonavir. Intra and inter-day precision and accuracy were less than 10.2% for both drugs. Recovery were 90 and 87% for saquinavir and ritonavir, respectively. The drugs were stable at different relevant storage and working conditions. After the validation, their analytical error functions were established as standard deviation (S.D., ng/ml) = 4.84 + 7.14 x 10(-2)C (C is the theoretical concentration value) for saquinavir and S.D. (ng/ml) = 39.98 + 2.40 x 10(-5)C(2) for ritonavir.     

A rapid HPLC method for the determination of losartan in human plasma using a monolithic column

A rapid and simple high-performance liquid chromatography (HPLC) method using a monolithic column has been developed for quantification of losartan (CAS 12450-98-8) in plasma. The assay enables the measurement of losartan for therapeutic drug monitoring. The method involves a simple, one-step extraction procedure. The analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 x 4.6 mm) column with an isocratic mobile phase consisting of 0.01 mol/L disodium hydrogen phosphate buffer-acetonitrile (60:40 v/v) adjusted to pH 3.5. The wavelength was set at 254 nm. Thioridazine (CAS 50-52-2) was used as internal standard. Losartan, thioridazine and the EXP 3174, the active metabolite of losartan, appeared 3.4, 5.0 and 10.5 min post injection, respectively. The calibration curve was linear over the concentration range 5-300 ng mL(-1) with a minimum detectable limit of 2 ng mL(-1). The coefficients of variation for the inter-day and intra-day assay were found to be less than 8 %. The applicability of this method for pharmacokinetic studies in humans was demonstrated. The present assay is rapid, simple, precise, and accurate and is suitable for pharmacokinetic studies.     

A rapid reversed phase high performance liquid chromatographic method for determination of etoposide (VP-16) in human plasma

A rapid, simple and sensitive isocratic High Performance Liquid Chromatography (HPLC) method was developed to measure the concentration of etoposide in plasma samples with UV detection at 220 nm. The method uses a Bondapac C18 column at 60 degrees C. The mobile phase consists of Methanol: water (45:55 v/v) at a flow rate of 2.8 ml/min. Phenacetin was used as an internal standard. The plasma samples were extracted using ether with the organic layer evaporated under nitrogen. The residue was dissolved in 200 microl methanol with 20 microl injected into the HPLC column. The extraction method showed a recovery of 91.5+/-3% for etoposide. In this system, the retention time of phenacetin and etoposide were 3.3 and 4.4 min, respectively. The limit of detection of etoposide in plasma is 20 ng/ml and the limit of quantitation is 40 ng/ml. This analytical method has very good reproducibility (8.1% between-day variability at a concentration of 50 ng/ml). It is a fast, sensitive and economic method applicable for clinical and pharmacokinetic studies.     

Fully automated LC method for the determination of sotalol in human plasma using restricted access material with cation exchange properties for sample clean-up

A simple and rapid fully automated bio-analytical method for the liquid chromatographic (LC) determination of sotalol in human plasma has been described. The method is based on the use of a new kind of porous silica restricted access material (RAM) with cation exchange properties for sample clean-up. 100 microl of plasma samples were directly injected into the precolumn coupled on-line to a reversed-phase column (RP-Select B) by means of column switching system. The plasma matrix was washed out for 10 min using a washing liquid composed of 2 mM lithium perchlorate and methanol (97:3; v/v). By rotation of the switching valve, the analytes were then eluted in back-flush mode for 2 min and transferred to the analytical column by the LC mobile phase constituted of a mixture of methanol and 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM 1-octanesulphonic acid sodium salt (20:80; v/v). The flow-rate was 1.0 ml/min and sotalol was detected using fluorescence detection at 235 and 300 nm as excitation and emission wavelengths, respectively. The method was then validated using a new approach based on accuracy profile over a concentration range from 5 to 500 ng/ml. The limit of quantitation (LOQ) was 5 ng/ml and the total analysis time was 19 min.     

The measurement of propofol in human blood samples by liquid chromatography

A simple, sensitive, reliable and rapid column liquid chromatographic method was developed for the measurement of propofol, a new anesthetic agent, in human maternal and placental blood. The assay involved a single extraction of the drug and internal standard, thymol, from blood buffered with 0.1 M sodium dihydrogen phosphate buffer into cyclohexane. The organic extract, basified with tetramethylammonium hydroxide, in evaporation tube was evaporated to dryness at 30 degrees C under nitrogen. The residue was redissolved in 80 microliters acetonitrile and an aliquot (25-50 microliters) of the concentrate was injected into a C18 reversed-phase column (4 microns, Nova-Pak) linked to a C18 pre-column. The mobile phase consisted of 60% (v/v) acetonitrile in distilled water containing 1% v/v glacial acetic acid and was eluted at 2.5 ml min-1. The components of the column effluent was monitored by a fluorometric detector with excitation and emission wavelengths set at 276 nm and 310 nm, respectively. The method has been used to measure propofol concentrations in blood from maternal veins and placental arteries and veins during cesarean section.