Simple and sensitive determination of 5-fluorouracil in plasma by high-performance liquid chromatography. Application to clinical pharmacokinetic studies

5-Fluorouracil (5-FU) is an antineoplastic agent widely employed in the treatment of many types of cancer. Recent studies have proved the need for individual adjustment of 5-FU dosage based on pharmacokinetics. A simple and sensitive high-performance liquid chromatographic method for the determination of 5-FU in plasma and their preliminary clinical pharmacokinetics is described. After sample acidification with 20 microl of orthophosphoric acid (5%), the drug is extracted from plasma using n-propanol-diethyl ether (16:84). The organic layer is evaporated to dryness, the residue dissolved in 100 microl of mobile phase and 20 microl of this mixture is injected into a LiChrospher 100RP-18 (5 microm, 250 x 4.0 mm) analytical column. Mobile phase consisted of potassium dihydrogenphosphate (0.05 M, adjusted to pH 3). The limit of quantitation was 2 ng/ml. The method showed good precision: the within-day relative standard deviation (RSD) for 5-FU (10-20,000 ng/ml) was 3.75% (2.57-5.93); the between-day RSD for 5-FU, in the previously described range, was 5.74% (4.35-7.20). The method presented here is accurate, precise and sensitive and it has been successfully applied for 5-FU pharmacokinetic investigation and therapeutic drug monitoring.     

Simple and rapid determination of irinotecan and its metabolite SN-38 in plasma by high-performance liquid-chromatography: application to clinical pharmacokinetic studies

Irinotecan (CPT-11) is an anticancer agent widely employed in the treatment of colorectal carcinoma. A simple, rapid and sensitive high-performance liquid chromatographic method for the simultaneous determination of CPT-11 and its metabolite SN-38 in plasma, and their preliminary clinical pharmacokinetics are described. Both deproteinisation of plasma specimens (100 microl) and addition of the internal standard, camptothecin (CPT), are achieved by incorporating to samples 100 microl of a solution of CPT (1 microg/ml) in acetonitrile-1 mM orthophosphoric acid (90:10); 200 microl of this acidified acetonitrile solution, drug-free, is also added to accomplish complete deproteinisation: this procedure reduces sample preparation time to a minimum. After deproteinisation, samples are treated with potassium dihydrogenphosphate (0.1 M) and injected into a Nucleosil C18 (5 microm, 250 x 4.0 mm) column. Mobile phase consists of potassium dihydrogenphosphate (0.1 M)-acetonitrile (67:33), at a flow-rate of 1 ml/min. CPT-11, SN-38 and CPT are detected by fluorescence with excitation wavelength set at 228 nm and emission wavelengths of CPT-11, SN-38 and CPT fixed, respectively, at 450, 543 and 433 nm. The limits of quantitation for CPT-11 and SN-38 are 1.0 and 0.5 ng/ml, respectively. This method shows good precision: the within day relative standard deviation (RSD) for CPT-11 (1-10000 ng/ml) is 5.17% (range 2.15-8.27%) and for SN-38 (0.5-400 ng/ml) is 4.33% (1.32-7.78%); the between-day RSDs for CPT-11 and SN-38, in the previously described ranges, are 6.82% (5.03-10.8%) and 4.94% (2.09-9.30%), respectively. Using this assay, plasma pharmacokinetics of CPT-11, SN-38 and its glucuronidated form, SN-38G, have been determined in one patient receiving 200 mg/m2 of CPT-11 as a 90 min intravenous infusion. The peak plasma concentration of CPT-11 at the end of the infusion is 3800 ng/ml. Plasma decay is biphasic with a terminal half-life of 11.6 h. The volume of distribution at steady state (Vss) is 203 l/m2, and the total body clearance (Cl) is 14.8 l/h x m2. The maximum concentrations of SN-38 and SN-38G reach 28.9 and 151 ng/ml, respectively.     

Improved assay method for the determination of pyronaridine in plasma and whole blood by high-performance liquid chromatography for application to clinical pharmacokinetic studies

An improved high-performance liquid chromatography method using a diisopropyl-C14 reversed-phase column (Zorbax Bonus-RP column) and a liquid-liquid extraction technique with UV detection is presented for the analysis of pyronaridine in human whole blood and plasma. Tribasic phosphate buffer (50 mM, pH 10.3) and diethyl ether were used for liquid-liquid extraction. The mobile phase consists of acetonitrile-0.08 M potassium dihydrogen phosphate buffer (13:87, v/v) with the pH 2.8 adjusted by orthophosphoric acid. Amodiaquine was found to be a suitable internal standard for the method. The quantification limit with UV detection at 275 nm was 3 ng on-column for both plasma and blood samples. The method was applied to plasma and blood specimens from a rabbit after a single intramuscular dose of pyronaridine tetraphosphate (20 mg/kg as base). From this in vivo study, evidence was found that pyronaridine is concentrated in blood cells, with a blood:plasma ratio ranging from 4.9 to 17.8. We conclude that blood is the preferred matrix for clinical pharmacokinetic studies.     

Determination of 2-hydroxyflutamide in human plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

Flutamide is a potent antiandrogen used for the treatment of prostatic cancer. Flutamide undergoes extensive first-pass metabolism to the pharmacologically active metabolite 2-hydroxyflutamide. A simple, sensitive, precise, accurate and specific HPLC method, using carbamazepine as the internal standard, for the determination of 2-hydroxyflutamide in human plasma was developed and validated. After addition of the internal standard, the analytes were isolated from human plasma by liquid-liquid extraction. The method was linear in the 25 to 1,000 ng/ml concentration range (r>0.999). Recovery for 2-hydroxyflutamide was greater than 91.4% and for internal standard was 93.6%. The limit of quantitation was 25 ng/ml. Inter-batch precision, expressed as the relative standard deviation (RSD), ranged from 4.3 to 7.9%, and accuracy was better than 93.9%. Analysis of 2-hydroxyflutamide concentrations in plasma samples from 16 healthy volunteers following oral administration of 250 mg of flutamide provided the following pharmacokinetic data (mean+/-SD): Cmax, 776 +/- 400 ng/ml; AUC(0-infinity), 5,368 +/- 2,689 ng h/ml; AUC(0-t) 5,005 +/- 2,605 ng h/ml; Tmax 2.6 +/- 1.6 h; elimination half-life, 5.2 +/- 2.0 h.     

Simple method for determination of terbutaline plasma concentration by high-performance liquid chromatography

A method is described in which low nanomolar concentrations of terbutaline in plasma can be quantitated by use of a standard isocratic high-performance liquid chromatography system with electrochemical detection. Samples were prepared for injection by solid-phase extraction and preserved from degradation by addition of glutathione. Terbutaline and internal standard metaproterenol were resolved from plasma constituents on a single C(18) column by ion-pairing chromatography. The method is precise and accurate for measurement of freebase concentrations as low as 4.4 nmol/l (1 ng/ml).     

Simple and rapid high-performance liquid chromatography method for the determination of ofloxacin concentrations in plasma and urine

A high-performance liquid chromatographic method for the determination of ofloxacin in human plasma and urine was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C18 column and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was linear from 0.5 to 10.0 microg/ml. The relative standard deviation of intra- and inter-day assays was lower than 5%. The average recovery of ofloxacin from plasma was 93%. The method was evaluated in samples from healthy subjects whose drug levels were already measured by microbiological assay.     

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

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

Stereoselective determination of cisapride, a prokinetic agent, in human plasma by chiral high-performance liquid chromatography with ultraviolet detection: application to pharmacokinetic study

We have developed a simple, sensitive, specific and reproducible stereoselective high-performance liquid chromatography technique for analytical separation of cisapride enantiomers and measurement of cisapride enantiomers in human plasma. A chiral analytical column (ChiralCel OJ) was used with a mobile phase consisting of ethanol-hexane-diethylamine (35:64.5:0.5, v/v/v). This assay method was linear over a range of concentrations (5-125 ng/ml) of each enantiomer. The limit of quantification was 5 ng/ml in human plasma for both cisapride enantiomers, while the limit of detection was 1 ng/ml. Intra- and inter-day C.V.s did not exceed 15% for all concentrations except at 12.5 ng/ml for EII (+)-cisapride, which was approximately 20 and 19%, respectively. The clinical utility of the method was demonstrated in a pharmacokinetic study of normal volunteers who received a 20 mg single oral dose of racemic cisapride. The preliminary pharmacokinetic data obtained using the method we describe here provide evidence for the first time that cisapride exhibits stereoselective disposition.     

Sensitive stereospecific determination of acenocoumarol and phenprocoumon in plasma by high-performance liquid chromatography

We describe a normal-phase HPLC method for the stereospecific determination of R- and S-acenocoumarol and R- and S-phenprocoumon with S-warfarin as internal standard. The compounds were separated using a Whelk-O1 chiral stationary phase, detected by UV at 310 nm and quantified in the internal standard mode. Linearity was verified for acenocoumarol in the range of 15-2000 microg/l and for phenprocoumon from 15 to 2200 microg/l, respectively. The detection limits were 5 microg/l for all compounds. The recovery was >84% for R- and S-acenocoumarol and >74% for R- and S-phenprocoumon. The imprecision (C.V.) (50-1800 microg/l) for R- and S-acenocoumarol was <4.7% within-day and <7.8% between-day. For R- and S-phenprocoumon the respective values were <5.6% and <5.9%. The accuracy for all compounds was 96.5-110%.     

Quantitative determination of cefepime in plasma and vitreous fluid by high-performance liquid chromatography

An isocratic reversed-phase HPLC method was developed to determine cefepime levels in plasma and vitreous fluid. Cefepime and the internal standard cefadroxil were separated on a Shandon Hypersil BDS C18 column by using a mobile phase of 25 mM sodium dihydrogen phosphate monohydrate (pH 3) and methanol (87:13, v/v). Ultraviolet detection was carried out at 270 nm. The retention times were 4.80 min for cefepime and 7.70 min for cefadroxil. This fast procedure which involves an efficient protein precipitation step (addition of HClO4), allows a quantification limit of 2.52 microg ml(-1) and a detection limit of 0.83 microg ml(-1). Recoveries and absolute recoveries of cefepime from plasma were 96.13-99.44% and 94-102.5% respectively. The intra-day and inter-day reproducibilities were less than 2% for cefepime at 10, 30, 50 microg ml(-1) (n=10). The method was proved to be suitable for determining cefepime levels in human plasma and was modified to measure vitreous fluid samples.     

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

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

Determination of paroxetine in plasma by high-performance liquid chromatography for bioequivalence studies

A high-performance liquid chromatographic method is described for the determination of paroxetine in human plasma. Dibucaine was used as the internal standard. Paroxetine was isolated by solid phase extraction using a Bond-Elut C18 extraction column. Separation was obtained using a reversed-phase column under isocratic conditions with fluorescence detection. The sample volume was 500 microliters of plasma. The intra- and inter-assay accuracy and precision, determined as relative error and relative standard deviation, respectively, were less than 10%. The lower limit of quantitation, based on standards with acceptable relative error and relative standard deviation, was 10 ng ml-1. No endogenous compounds were found to interfere. The linearity was assessed in the range 5-100 ng ml-1. Stability of paroxetine during processing (autosampler) and in plasma was checked. This method proved suitable for bioequivalence studies following multiple doses in healthy volunteers.     

Automated solid-phase extraction method for the determination of atovaquone in plasma and whole blood by rapid high-performance liquid chromatography

A bioanalytical method for the determination of atovaquone in plasma and whole blood by solid-phase extraction and high-performance liquid chromatography has been developed and validated. A structurally similar internal standard was added and protein was precipitated from plasma and whole blood with acetonitrile before being loaded on to a C8 solid-phase extraction column. Atovaquone and internal standard were analysed by high-performance liquid chromatography on a C18 J'Sphere ODS-M80 (150x4.0 mm) column with mobile phase acetonitrile-phosphate buffer, 0.01 M, pH 7.0 (65:35, v/v) and UV detection at 277 nm. The intra-assay precisions for plasma and whole blood were 2.2% and 1.9% respectively at 12 microM and 6.0% and 5.6% respectively at 0.75 microM. The inter-assay precisions for plasma and whole blood were 1.4% and 2.1% respectively at 12 microM and 4.9% and 3.4% respectively at 0.75 microM. The lower limit of quantification in plasma and whole blood were 150 nM. The limit of detection in plasma and whole blood were 30 nM.     

Simple high-performance liquid chromatographic method for determination of alpha-tocopherol in human plasma.

A simple high-performance liquid chromatographic method using UV detection was developed for the determination of alpha-tocopherol in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile-tetrahydrofuran (3:2). The mobile phase comprised methanol-tetrahydrofuran (94:6) and analysis was run at a flow-rate of 1.5 ml/min with the detector operating at 292 nm. A Crestpak C18S (5 microm, 250 mm x 4.6 mm ID) was used for the chromatographic separation. The method had a mean recovery of 93%, while the within-day and between-day coefficients of variation and percentage errors were all less than 7%. The speed, specificity, sensitivity and reproducibility of this method make it particularly suitable for routine determination of alpha-tocopherol in human plasma. Moreover, only a small sample plasma volume (100 microl) is required for the analysis.     

Simple and sensitive high-performance liquid chromatography method for the determination of docetaxel in human plasma or urine

Several methods for quantification of docetaxel have been described mainly using HPLC. We have developed a new isocratic HPLC method that is as sensitive and simpler than previous methods, and applicable to use in clinical pharmacokinetic analysis. Plasma samples are spiked with paclitaxel as internal standard and extracted manually on activated cyanopropyl end-capped solid-phase extraction columns followed by isocratic reversed-phase HPLC and UV detection at 227 nm. Using this system, the retention times for docetaxel and paclitaxel are 8.5 min and 10.5 min, respectively, with good resolution and without any interference from endogenous plasma constituents or docetaxel metabolites at these retention times. The total run time needed is only 13 min. The lower limit of quantification is 5 ng/ml using 1 ml of plasma. The validated quantitation range of the method is 5-1000 ng/ml with RSDs < or = 10%, but plasma concentrations up to 5000 ng/ml can be accurately measured using smaller aliquots. This method is also suitable for the determination of docetaxel in urine samples under the same conditions. The method has been used to assess the pharmacokinetics of docetaxel during a phase I/II study of docetaxel in combination with epirubicin and cyclophosphamide in patients with advanced cancer.     

High-performance liquid chromatographic determination of tianeptine in plasma applied to pharmacokinetic studies

An improved analytical method for the quantitative measurement of tianeptine and its main metabolite MC5 in human plasma was designed. Extraction involved ion-paired liquid-liquid extraction of the compounds from 1.0 ml of human plasma adjusted to pH 7.0. HPLC separation was performed using a Nucleosil C18, 5 microm column (150x4.6 mm I.D.) and a mixture of acetonitrile and pH 3, 2.7 g l(-1) solution of sodium heptanesulfonate in distilled water (40:60, v/v) as mobile phase. UV detection was performed using a diode array detector in the 200-400 nm passband, and quantification of the analytes was made at 220 nm. For both tianeptine and MC5 metabolite, the limit of quantitation was 5 microg l(-1) and the calibration curves were linear from 5 to 500 microg l(-1). Intra- and inter-assay precision and accuracy fulfilled the international requirements. The recovery of tianeptine and its metabolite from plasma was, respectively, 71.5 and 74.3% at 20 microg l(-1), 71.2 and 70.8% at 400 microg l(-1). The selectivity of the method was checked by verifying the absence of chromatographic interference from pure solutions of the most commonly associated therapeutic drugs. This method, validated according to the criteria established by the Journal of Chromatography B, was applied to the determination of tianeptine and MC5-metabolite in human plasma in pharmacokinetic studies.     

Determination of ginsenoside Rg3 in plasma by solid-phase extraction and high-performance liquid chromatography for pharmacokinetic study.

A method using high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) is described for the determination of ginsenoside Rg3 in human plasma. A 2.5-ml volume of plasma was mixed with 2.5 ml 60% methanol aqueous solution, and centrifuged at 1100 g for 10 min, the supernatant fluid was further purified by SPE with 200 mg/5 ml 40 microns octadecyl silica and separation was obtained using a reversed-phase column under isocratic conditions with ultraviolet absorbance detection. The intra- and inter-day precision, determined as relative standard deviations, were less than 5.0%, and method recovery was more than 97%. The lower limit of quantitation, based on standards with acceptable RSDs, was 2.5 ng/ml. No endogenous compounds were found to interfere with analyte. A good linear relationship with a regression coefficient of 0.9999 in the range of 2.5 to 200 ng/ml was observed. This method has been demonstrated to be suitable for pharmacokinetic studies in humans. Method development for determination of drug with low UV absorption by SPE and HPLC is also discussed.     

Rapid and sensitive method for determination of nimesulide in human plasma by high-performance liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic method for determination of nimesulide in human plasma has been developed. The chromatographic system uses a reversed-phase C18 column with UV-Vis detection at 230 nm. Mobile-phase consisted of phosphate buffer (pH 5.5)-methanol-acetonitrile (50:20:30, v/v) at a flow-rate of 1.4 ml/min. Nimesulide was extracted in a single step into dichloromethane.The overall mean extraction recoveries were above 98% for both inter- and intra-assay reproducibility, with CVs from 0.3 to 1%. The calibration curve was linear in the concentration range of 0.05-5 microg/ml, and the lower limit of detection was 30 ng/ml. This simple and sensitive method allows for determination of the range of plasma concentrations that is observed after administration of clinically relevant doses of nimesulide.     

Simultaneous quantitative determination of cilostazol and its metabolites in human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cilostazol, a quinolinone derivative, and its known metabolites OPC-13015, OPC-13213, OPC-13217, OPC-13366, OPC-13269, OPC-13326 and OPC-13388 in human plasma was developed and validated. Cilostazol, its metabolites and two internal standards, OPC-3930 and OPC-13112, were extracted from human plasma by a combination of liquid-liquid and liquid-solid phase extractions, with combined organic solvents of n-butanol, methanol, chloroform, methyl-tert.-butyl ether, and a Sep-Pak silica column. The combined extract was then evaporated and the residue was reconstituted in ammonium acetate buffer (pH 6.5). The reconstituted solution was injected onto a HPLC system and was subjected to reversed-phase HPLC on a 5 microm ODS-80TM column to obtain quality chromatograph and good peak resolution. A gradient mobile phase with different percentages of acetonitrile in acetate buffer (pH 6.5) was used for the resolution of analytes. Cilostazol, its metabolites and the two internal standards were well separated at baseline from each other with resolution factor being 74 and 138. This HPLC method was demonstrated to be specific for all analytes of interest with no significant interference from the endogenous substances of human plasma. The lower limit of quantitation was 20 ng/ml for cilostazol and all metabolites. The method was validated initially for an extended linear range of 20-600 ng/ml for all metabolites and cilostazol, and has been revised later for a linear range of 20-1200 ng/ml for cilostazol and two major and active metabolites OPC-13015 and OPC-13213. The overall accuracy (relative recovery) of this method was established to be 98.5% to 104.9% for analytes with overall precision (CV) being 1.5% to 9.0%. The long-term stability of clinical plasma samples was established for at least one year at -20 degrees C. Two internal standards of OPC-3930 and OPC-13112 were evaluated and validated. However, the data indicated that there was no significant difference for all accuracy and precision obtained by using either OPC-3930 or OPC-13112. OPC-3930 was chosen as the internal standard for the analysis of plasma samples from clinical studies due to its shorter retention time. During the validation standard curves had correlation coefficients greater than or equal to 0.998 for cilostazol and the seven metabolites. These data clearly demonstrate the reliability and reproducibility of the method.