High-performance liquid chromatographic analysis of nitrendipine in human plasma using ultraviolet detection and single-step solid-phase sample preparation

A high-performance liquid chromatographic (HPLC) method was developed for the determination of nitrendipine in human plasma using solid-phase extraction (SPE) and ultraviolet detection. A 30-microl aliquot of methanol (containing 2 microg/ml of the internal standard, nimodipine) was added to a 1-ml aliquot of biological sample. After vortex-mixing, the mixture was loaded on C(18) SPE cartridge which was conditioned with methanol and distilled water. After washing with distilled water, the SPE cartridge was eluted with 1-ml aliquot of diethyl ether. The organic phase was collected and evaporated under nitrogen gas. The residue was then reconstituted with a 100 microl aliquot of mobile phase, and a 50 microl aliquot was injected onto the C(18) reverse-phased column. The mobile phase, 10 mM phosphate buffer (pH 4.5):acetonitrile (50:50, v/v), was run at a flow rate of 1.2 ml/min. The column effluent was monitored using ultraviolet detector at 238 nm. The retention times for nitrendipine and the internal standard were approximately 10.1 and 12.6 min, respectively. The detection limit of nitrendipine in human plasma was 2.0 ng/ml. The coefficients of variation (CV) of the assay were below 16.5% for human plasma, and no interferences from endogenous substances were found. This specific, accurate and precise assay was useful in the study for the pharmacokinetic characteristics of nitrendipine.     

Validation of a method for the determination of zolpidem in human plasma using LC with fluorescence detection

A sensitive and selective high-performance liquid chromatography (HPLC) method was developed for the determination of zolpidem in human plasma. Zolpidem and the internal standard (trazodone) were extracted from human plasma that had been made basic. The basic sample was loaded onto a conditioned Bond Elut C18 cartridge, rinsed with water and eluted with methanol. Forty microliters were then injected onto the LC system. Separation was achieved on a C18 column (150 x 4.6 mm, 5 microm) with a mobile phase composed of acetonitrile:50 mM potassium phosphate monobasic at pH 6.0 (4:6, v/v). Detection was by fluorescence, with excitation at 254 nm and emission at 400 nm. The retention times of zolpidem and internal standard were approximately 4.7 and 5.3 min, respectively. The LC run time was 8 min. The assay was linear in concentration range 1-400 ng/ml for zolpidem in human plasma. The analysis of quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated excellent precision with relative standard deviations (RSD) of 3.7, 4.6, and 3.0%, respectively (n = 18). The method was accurate with all intraday (n = 6) and overall (n = 18) mean concentrations within 5.8% from nominal at all quality control sample concentrations. This method was also performed using a Gilson Aspec XL automated sample processor and autoinjector. The samples were manually fortified with internal standard and made basic. The aspec then performed the solid phase extraction and made injections of the samples onto the LC system. Using the automated procedure for analysis, quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated acceptable precision with RSD values of 9.0, 4.9, and 5.1%, respectively (n = 12). The method was accurate with all intracurve (n = 4) and overall (n = 12) mean values being less than 10.8% from nominal at all quality control sample concentrations.     

Validation of a method for the determination of (R)-warfarin and (S)-warfarin in human plasma using LC with UV detection

A sensitive and selective chiral high-performance liquid chromatography (HPLC) method was developed for the determination of (R)-warfarin and (S)-warfarin in human plasma. (R)- and (S)-warfarin and the internal standard (oxybenzone) were extracted from human plasma that had been made acidic with 1 N sulfuric acid into ethyl ether. The ethyl ether layer was removed and evaporated, and the residue was reconstituted in 200 microl of acetonitrile. A 50-microl aliquot was injected onto the HPLC system. Separation was achieved on a beta-cyclodextrin column (250 x 4.6 mm, 5 microm) with a mobile phase composed of acetonitrile:glacial acetic acid:triethylamine (1000:3:2.5, v/v/v). Detection was by ultraviolet absorbance at 320 nm. Late-eluting peaks were diverted from the analytical column by using a beta-cyclodextrin precolumn (50 x 4.6 mm, 5 microm) and a column switching device. The retention times of (R)- and (S)-warfarin and the internal standard were approximately 7.7, 6.9 and 4.0 min, respectively. The run time was 15 min. The assay was linear in concentration ranges of 12.5-2500 ng/ml for (R)- and (S)-warfarin in human plasma. The analysis of quality control samples for (R)- and (S)-warfarin (25.0, 400 and 2000 ng/ml) demonstrated excellent precision with relative standard deviations (R.S.D.) for (R)-warfarin of 10.9, 2.8, and 2.8%, respectively (n = 18), and for (S)-warfarin of 7.0, 2.4 and 2.6%, respectively (n = 18). The method was accurate with all overall (n = 18) mean concentrations being less than 6.0% from nominal at all quality control sample concentrations.     

Automatic determination of diltiazem and desacetyldiltiazem in human plasma using liquid-solid extraction on disposable cartridges coupled to HPLC--Part I: Optimization of the HPLC system and method validation.

A sensitive and automated method for the analysis of diltiazem and desacetyldiltiazem in plasma has been developed using liquid-solid extraction (LSE) on disposable extraction cartridges (DECs) in combination with HPLC. After isolation from plasma, the analytes are separated on a highly deactivated octyl silica column with a mobile phase of methanol-0.05 M phosphate buffer (pH 7.4) (62:38, v/v). The analytes are monitored photometrically at 238 nm. The complete preparation of the plasma sample as well as the injection of the final extract on to the analytical column are performed automatically by means of a sample processor equipped with a robotic arm to which is attached a needle dispensing the different liquids. The internal standard solution is first added to the plasma sample. The DEC is then conditioned successively with methanol and phosphate buffer (pH 7.4). A 1.0-ml volume of sample containing the internal standard solution is applied on an extraction cartridge filled with cyanopropyl silica (50 mg). After the DEC has been washed with the same buffer, the analytes are eluted with 0.16 ml of methanol. A 0.14-ml volume of buffer is then passed through the DEC and 0.25 ml of the final extract is injected onto the HPLC column. The absolute recoveries of the drugs are about 90% and the limit of detection for diltiazem is 0.8 ng ml-1. Relative standard deviations of 2.6% (within-day) and 3.7% (between-day) have been obtained for this compound at a plasma concentration of 50 ng ml-1.     

HPLC assay for albendazole and metabolites in human plasma for clinical pharmacokinetic studies

A sensitive and selective HPLC chromatography method using UV detection (295 nm) was developed for the determination of albendazole, albendazole sulfoxide (ABZSO), and albendazole sulfone (ABZSO2) in human plasma. Albendazole, ABZSO, ABZSO2, and the internal standard, oxibendazole, were extracted from human plasma by loading onto a conditioned C(18) SPE cartridge, rinsing with 15% methanol, and eluting with 90% methanol. Samples were evaporated under a stream of nitrogen, reconstituted with mobile phase, 1.25% triethylamine in water-methanol-acetonitrile (72:15:13, v/v/v) (pH* 3.1), and injected onto a Waters muBondapak Phenyl 3.9 x 300 mm HPLC column. Mobile phase flow rate was 1.0 ml/min. The retention times of albendazole, ABZSO, ABZSO2, and the internal standard were approximately 24.4, 7.9, 13.4, and 11.3 min, respectively. Total run time was 30 min. The assay was linear for concentration ranges in human plasma of 20-600 ng/ml for albendazole, 20-1000 ng/ml for ABZSO, and 20-300 ng/ml for ABZSO2. The analysis of quality control samples demonstrated excellent precision. Coefficients of variation for albendazole (20, 400, 600 ng/ml) were 6.7, 8.1 and 7.0%; ABZSO (20, 400, 800 ng/ml) were 6.0, 8.5 and 5.9%; ABZSO2 (20, 150, 300 ng/ml) were 3.1, 3.9 and 2.3%, respectively. The method appears to be robust and has been applied to a pharmacokinetic study of albendazole in healthy volunteers.     

Gas chromatographic assay of diethylcarbamazine in human plasma for application to clinical pharmacokinetic studies

A sensitive and selective gas chromatography method using flame ionization detection was developed for the determination of diethylcarbamazine (DEC) in human plasma. DEC and the internal standard, 1-diethylcarbamyl-4-ethyl piperazine HCl (E-DEC), were extracted from human plasma after loading onto a conditioned C(18) solid phase extraction cartridge, rinsed with water and eluted with methanol. After evaporation under a stream of nitrogen and reconstitution in methanol, 3 microl were injected onto the GC system. Separation was achieved on a A Heliflex(R) AT-35 capillary column (length 30 m, internal diameter 0.32 mm). Gas flow rates were: hydrogen, 35 ml/min; carrier gas (helium), 1.5 ml/min, make-up gas (helium), 25 ml/min; and air 420 ml/min. The retention times of DEC and internal standard were approximately 5.5 and 7.28 min, respectively. The GC run time was 22 min. The assay was linear in concentration range 100-2000 ng/ml for DEC in human plasma. The analysis of quality control samples for DEC (120, 1000, 2000 ng/ml) demonstrated excellent precision with coefficients of variation of 4.5,1.3, and 1.6%, respectively (n=6). The method was accurate with all intra-day (n=6) and inter-day (n=12) mean concentrations within 4.3% from nominal at all quality control sample concentrations. DEC was found to be stable after 3 freeze-thaw cycles, and with storage at -20 degrees C for 12 weeks. The method is currently being used for pharmacokinetic studies of DEC in healthy volunteers.     

Direct determination of verapamil in rat plasma by coupled column microbore-HPLC method

This report describes an automated coupled column microbore-high-performance liquid chromatography (HPLC) with fluorescence detection for direct determination of verapamil in small volume of rat plasma. We used HPLC system consisting of three columns such as precolumn, intermediate and analytical column and six-port switching valve and injected small volume of rat plasma to the system without sample preparation. An aliquot of sample was directly injected into Capcell Pak MF Ph precolumn for clean-up and enrichment, 35 mm Capcell Pak C18, intermediate column for concentration of compounds and 250 mm Capcell Pak C18 analytical column for separation of compounds and two mobile phases are used as mobile phase A (50mM ammonium phosphate, pH 4.5) and B (50mM ammonium phosphate:acetonitrile=70:30 v/v). Analysis of verapamil and internal standard, propranolol was performed with direct injection of 10 microl of rat plasma to the system and were eluted at 22 and 12 min, respectively, at a mobile phase flow rate of 0.5 (mobile phase A) and 0.15 ml/min (mobile phase B). The peaks of verapamil and internal standard were good shapes and well separated from any interfering endogenous peaks during a total run time of 25 min. The calibration curve for verapamil showed good linearity (r(2)=0.9997) over the concentration range of 0.01-2.50 microg/ml. The mean RSD (%) values of intra-day (n=5) and inter-day (n=5) variability of verapamil ranged from 1.96 to 9.06 and 0.62 to 3.08%, respectively. The LOD and LOQ were 0.01 and 0.025 microg/ml, respectively, for verapamil using 10 microl of rat plasma. An automated coupled column microbore-HPLC method was successfully applied to a pharmacokinetic study after intravenous injection of 3mg/kg of verapamil to the normal and dimethylnitrosamine (DMN)-induced hepatofibrotic rats.     

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.     

Automatic determination of diltiazem and desacetyldiltiazem in human plasma using liquid-solid extraction on disposable cartridges coupled to HPLC — Part I: Optimization of the HPLC system and method validation

A sensitive and automated method for the analysis of diltiazem and desacetyldiltiazem in plasma has been developed using liquid—solid extraction (LSE) on disposable extraction cartridges (DECs) in combination with HPLC. After isolation from plasma, the analytes are separated on a highly deactivated octyl silica column with a mobile phase of methanol—0.05 M phosphate buffer (pH 7.4) (62:38, v/v). The analytes are monitored photometrically at 238 nm. The complete preparation of the plasma sample as well as the injection of the final extract on to the analytical column are performed automatically by means of a sample processor equipped with a robotic arm to which is attached a needle dispensing the different liquids. The internal standard solution is first added to the plasma sample. The DEC is then conditioned successively with methanol and phosphate buffer (pH 7.4). A 1.0-ml volume of sample containing the internal standard solution is applied on an extraction cartridge filled with cyanopropyl silica (50 mg). After the DEC has been washed with the same buffer, the analytes are eluted with 0.16 ml of methanol. A 0.14-ml volume of buffer is then passed through the DEC and 0.25 ml of the final extract is injected onto the HPLC column. The absolute recoveries of the drugs are about 90% and the limit of detection for diltiazem is 0.8 ng ml⁻¹. Relative standard deviations of 2.6% (within-day) and 3.7% (between-day) have been obtained for this compound at a plasma concentration of 50 ng ml⁻¹.     

Determination of a new isoquinolinedione derivative, 7-anilino-5,8-isoquinolinedione,in plasma,urine and tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for the determination of a new isoquinolinedione derivative, 7-anilino-5,8-isoquinolinedione (IQO4), in rat plasma, urine, blood and tissue homogenates using diazepam as an internal standard. A 2 volume of acetonitrile was added to deproteinize the biological sample. A 50 microl aliquot of the supernatant was injected onto a C(18) reversed-phase column. The mobile phase, 0.05 M acetate buffer (pH 3):acetonitrile:methanol (40:40:20, v/v/v), was run at a flow rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector set at 298 nm. The retention times for IQO4 and the internal standard were approximately 5 and 7 min, respectively. The detection limits of IQO4 in rat plasma, urine and tissue homogenates (including blood) were 0.05, 0.1 and 0.1 microg/ml, respectively. The coefficients of variation of the assay were below 9.4% for rat plasma, urine and tissue homogenates. No interferences from endogenous substances were found.     

Determination of a new reversible proton pump inhibitor,DBM-819, in human plasma and urine,and rat tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for the determination of a new proton pump inhibitor, DBM-819, in human plasma and urine and rat tissue homogenates using KR-60461 as an internal standard. A 100-microl aliquot of acetonitrile (containing 0.5 microg/ml of the internal standard) and a 200-microl aliquot of 0.1 M Na(2)HPO(4) (adjusted pH 11 with 1 N NaOH) were added to a 100-microl aliquot of biological sample. After vortex-mixing, the mixture was extracted with 1 ml of ethylacetate. After centrifugation at 12000 x g for 3 min, the organic layer was collected and evaporated under nitrogen gas. The residue was then reconstituted with a 100-microl aliquot of mobile phase, and a 40-microl aliquot was injected onto the HPLC column. The mobile phase, 0.02 M phosphate buffer (pH 5): acetonitrile: methanol (46:44:10, v/v/v), was run at a flow rate of 0.5 ml/min and the column effluent was monitored by the fluorescence detector set at an excitation wavelength of 340 nm and an emission wavelength of 470 nm. The retention times for DBM-819 and the internal standard were approximately 10.5 and 12 min, respectively. The detection limits of DBM-819 in human plasma and urine, and rat tissue homogenates were 0.01, 0.02 and 0.02 (or 0.05) microg/ml. respectively. The coefficients of variation (CV) of the assay were below 11% for human plasma and urine, and rat tissue homogenates. No interferences from endogenous substances were found.     

Direct simultaneous determination of drug discovery compounds in monkey plasma using mixed-function column liquid chromatography/tandem mass spectrometry.

A direct injection method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for the simultaneous determination of two drug candidates in monkey plasma samples in support of pharmacodynamic studies. Each diluted monkey plasma sample containing internal standard was directly injected into a mixed-function column for sample cleanup, enrichment and chromatographic separation. The proteins and macromolecules first passed through the column while the drug molecules were retained on the bonded hydrophobic phase. The analytes retained on the column with an aqueous liquid mobile phase were then chemically eluted by switching to a strong organic mobile phase at a constant flow rate of 1.0 ml/min. The column effluent was also diverted from waste to mass spectrometer for analyte detection. Samples from two different analyte studies were assayed in one analytical procedure and the calibration curves were prepared using both analytes. The calibration curves were linear over the range of 5-2500 ng/ml for both analytes. The retention times for analytes and the internal standard were found to be consistent and no column deterioration was observed after 200 injections. The apparent on-column recoveries for the test compounds in monkey plasma samples were greater than 90% with 6% CV (N=5). The total analysis time was less than 5 min per sample.     

A liquid chromatography/tandem mass spectrometry assay to quantitate MS-275 in human plasma

A rapid, sensitive and selective method was developed and validated using LC/MS/MS for determination of MS-275 in human plasma. Sample preparation involved a single step liquid-liquid extraction by the addition of 0.2 ml of plasma with 5 ml acetonitrile/n-butyl-chloride. Separation of the compounds of interest, including the internal standard paclitaxel, was achieved on a Waters X-Terra C(18) (50 mm x 2.1mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile/ammonium acetate (pH 2.9; 2mM)(60:40, v/v) containing 0.1% formic acid and isocratic flow at 0.15 ml/min for 3 min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 0.5-100 ng/ml with values for the coefficient of determination of >0.99. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%). This method was subsequently used to measure concentrations of MS-275 in cancer patients receiving an oral weekly dose of 4 mg/m(2).     

Determination of a new phosphodiesterase V inhibitor,DA-8159, in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method using liquid-liquid extraction for sample preparation was developed for the determination of a new phosphodiesterase V inhibitor, DA-8159, in rat plasma and urine using sildenafil citrate as an internal standard. A 100 microl aliquot of 0.1 M Na(2)CO(3) (containing sildenafil citrate, 3 microg/ml as free sildenafil) and a 1 ml aliquot of ether were added to a 100 microl aliquot of biological samples (urine samples were diluted 20 times with distilled water). After vortex centrifugation at 9000 x g for 3 min, the ether layer was collected and dried under nitrogen gas. The residue was reconstituted with a 150 microl aliquot of the mobile phase, centrifuged, and a 100 microl aliquot of the supernatant was injected onto a reversed-phase column. The mobile phases, 20 mM KH(2)PO(4) (pH 4.7):acetonitrile (70:30, v/v for plasma and tissue samples, and 75:25, v/v for urine samples), were run at a flow rate of 1.0 ml/min. The column effluent was monitored by an ultraviolet detector set at 292 nm. The retention times for DA-8159 and the internal standard were approximately 10.7 and 9.1 min, respectively, in plasma and tissue samples and the corresponding values in urine samples were 47 and 33 min. The detection limits for DA-8159 in rat plasma and urine were 20 and 100 ng/ml, respectively. The coefficients of variation of the assay were generally low: below 10% for plasma and 9.9% for urine. No interferences from endogenous substances were found.     

Determination of the oral platelet aggregation inhibitor Sibrafiban in rat, dog, and human plasma utilising HPLC-column switching combined with turbo ion spray single quadrupole mass spectrometry

A sensitive and selective HPLC-column switching method with single quadrupole mass spectrometric detection was developed for the simultaneous determination of the oral platelet aggregation inhibitor Sibrafiban (double protected prodrug), its prodrug and the active metabolite in rat, dog, and human plasma. The three analytes together with their tri-deuterated internal standards were isolated from plasma by protein precipitation (0.5 M perchloric acid). The de-proteinated samples were injected onto a standard-bore trapping column (4.0 mm i.d., LC-ABZ) of an HPLC-column switching system. Polar plasma components were removed by flushing the trapping column with ammonium formate (pH 3.6; 5 mM). Enriched compounds (including the analytes of interest) were backflushed onto a narrow-bore analytical column (2.1 mm i.d., Inertsil ODS-2) and separated by gradient elution (formic acid/ methanol). The whole effluent (200 microl/min) from the analytical column was passed to the turbo ion spray interface without splitting. Selected ion monitoring (SIM) was used for mass spectrometric detection. The limit of quantification for all three analytes was 1 ng/ml, using a 250-microl specimen of plasma. The mean precision and inaccuracy for the three analytes in all species were < 6 and < 5%, respectively. The practicability of the new analytical method was demonstrated by the analysis of about 500 rat and dog plasma and about 14,000 human plasma samples. The new method represents a successful example for the application of LC single MS with ionspray ionisation to the analysis of small molecule drugs in biological matrices from toxicokinetic studies and large clinical trials.     

Development and validation of an automated method for the liquid chromatographic determination of sotalol in plasma using dialysis and trace enrichment on a cation-exchange pre-column as on-line sample preparation

A fully automated method for the determination of sotalol in human plasma was developed, involving dialysis through a cellulose acetate membrane, clean-up and enrichment of the dialysate on a strong cation-exchange pre-column and subsequent liquid chromatographic (LC) analysis with UV detection. All sample handling operations were carried out by means of an ASTED system. Before starting dialysis, the trace enrichment column (TEC) was conditioned. The plasma sample, to which the internal standard (atenolol) was automatically added, was then loaded in the donor channel and was kept static while the dialysis liquid, consisting of 0.017 M acetic acid, was passed through the acceptor channel in successive pulses. After each pulse, the dialysate was dispensed onto the TEC. When dialysis was discontinued, the analytes were eluted from the TEC by the LC mobile phase by rotation of a switching valve and transferred to the analytical column packed with octyl silica. The LC mobile phase was a mixture of methanol and pH 7.0 phosphate buffer containing 1-octanesulfonate at a concentration of 7.5×10⁻⁴ M (19:81; v/v). The UV detection was performed at 230 nm. The influence of several parameters of the dialysis and trace enrichment processes on analyte recovery and method selectivity was investigated. The method was then validated. The mean absolute recovery for sotalol was about 60%. The limit of quantitation was 25 ng/ml and R.S.D. for repeatability and intermediate precision obtained at a concentration level of 50 ng/ml were 4.3 and 5.8%, respectively.     

Quantitation of paclitaxel in micro-sample rat plasma by a sensitive reversed-phase HPLC assay

A sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of paclitaxel in micro-samples of rat plasma in order to study the mechanism of enhanced systemic exposure of paclitaxel co-administered with P-glycoprotein inhibitors. The assay involved solid-phase extraction procedures using 2'-methylpaclitaxel as the internal standard. Chromatographic separations were achieved using a ZORBAX ODS C18 column and mobile phase consisting of acetonitrile, methanol and ammonium acetate buffer (10 mM, pH 5.0) (48.5:16.5:35) pumped at 0.8 ml/min. The effluents were measured for UV absorption at 227 nm, with retention times of 8.5 and 11.0 min for paclitaxel and 2'-methylpaclitaxel, respectively. The chromatographic separation was excellent, with no endogenous interference. The standard curves showed a good linearity (r=0.9994) over the concentration ranges of 10-1,000 ng/ml. At 1,000 ng/ml, the absolute recoveries of paclitaxel and 2'-methylpaclitaxel are 89 and 90%, respectively. The intra- and inter-day variabilities of paclitaxel were both less than 15%. This validated method for the assay of paclitaxel in micro-sample rat plasma made it feasible to study the pharmacokinetics of the drug in a single rat.     

A rapid reversed phase high performance liquid chromatographic method for the determination of docetaxel (Taxotere®) in human plasma using a column switching technique

A rapid, simple and sensitive isocratic high performance liquid chromatography (HPLC) method was developed to measure the concentration of docetaxel in plasma samples with UV detection at 227 nm. The method uses a column switching technique with an Ultrasphere C₁₈ column (75×4.6 mm ID, 3μ, Altex, USA) as clean-up column and a CSC-nucleosil C₈ column (150×4.6 mm ID, 5μ, CSC, Montreal, Canada) as the analytical column. The mobile phase consisted of Phosphate buffer (30 mM, pH=3)-acetonitrile (47:53, v/v) with the flow rates of 1.1 and 1.3 ml min⁻¹ for clean-up and analytical columns, respectively. Paclitaxel was used as an internal standard. The plasma samples were extracted using a solid phase extraction method with Ammonium acetate (30 mM, pH=5)-acetonitrile (50:50, v/v) as final eluent. The extraction method showed a recovery of 92% for docetaxel. In this system, the retention times of docetaxel and Paclitaxel were 7.2 and 8.5 min, respectively. The detection limit of docetaxel in plasma is 2.5 ng ml⁻¹. This analytical method has a very good reproducibility (7.2% between-day variability at a concentration of 10 ng ml⁻¹). It is applicable in clinical and pharmacokinetic studies.     

HPLC with ultraviolet detection for the determination of chloroquine and desethylchloroquine in whole blood and finger-prick capillary blood dried on filter paper

A simple, sensitive, selective and reproducible method based on liquid chromatography was developed for the determination of chloroquine (CQ) and its active plasma metabolite desethylchloroquine (DECQ) in finger-pricked capillary blood spot onto filter paper (DBS) and whole blood samples. Both were separated from the internal standard quinine on a reversed phase C18 column, with the mobile phase consisting of a mixture of 1% diethylamine, acetonitrile and methanol (20:55:25, v:v:v) running at a flow rate of 1.0ml/min. Retention times of QN, DECQ and CQ were 4.5, 5.7 and 6.4min, respectively. Ultraviolet detection was at the wavelength 256nm. Sample preparation was done by extraction with hexane and tert-butyl methyl ether (1:1, v:v). Good precision and accuracy were obtained for both within-day repeatability and day-to-day reproducibility. Limit of quantification (LOQ) for both CQ and DECQ was accepted as 50ng/ml using 80μl DBS sample and 25ng/ml using 150μl whole blood sample. The mean recoveries for CQ, DECQ and internal standard for both whole blood and DBS were between 74 and 87%. The method was successfully applied for a pharmacokinetic study of CQ and DECQ in patients with Plasmodium vivax. Excellence correlation (r=0.997) was observed between the analysis of both CQ and DECQ in paired whole blood and DBS samples.     

Determination of actarit from human plasma for bioequivalence studies

An analytical method based on high-performance liquid chromatography with ultraviolet detection (245 nm) was developed for the determination of actarit in human plasma. Coumarin was used as an internal standard. Chromatographic separation was achieved with a C8 column using a mobile phase of methanol: 1% acetic acid (50-50, v/v) with a flow rate of 1.0 ml/min. The calibration curve was linear over the range of 0.1-4.0 μg/ml (r(2) > 0.99) and the lower limit of quantification was 0.1 μg/ml. The method was validated for sensitivity, accuracy, precision, recovery and stability. The method was used to determine the concentration-time profiles of actarit in the plasma following oral administration of 100 mg actarit tablets.