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.     

The dihydrouracil/uracil ratio in plasma, clinical and genetic analysis for screening of dihydropyrimidine dehydrogenase deficiency in colorectal cancer patients treated with 5-fluorouracil

A rapid and cost-effective reversed phase high performance liquid chromatography (HPLC) method for quantification of dihydrouracil to uracil ratio (UH2/U) in plasma has been developed and used to screen for dihydropyrimidine dehydrogenase (DPD) deficiency in nine patients treated with 5-fluorouracil (5-FU). This HPLC method is based on the use of a simultaneous UV detection at 205 and 268nm during the analysis run of the plasma extract and taking into account the particularity that UH2 shows no absorbance response at 268nm. The plasma UH2/U ratio values evaluated by the use of our HPLC assay were found to be highly correlated with the plasma 5-FU-half-life values and were significantly associated with the toxic side effects, whereas, data set provided from genetic analysis of the coding sequences of the DPD gene (DPYD) were found to be insufficient to explain all the cases of the 5-FU-related toxicity pattern. The proposed HPLC assay could be available for routine clinical use for DPD deficiency assessment in patients prior to 5-FU administration.     

Clonazepam serum levels in epileptic patients determined simply and rapidly by high-performance liquid chromatography using a solid-phase extraction column

We studied the use of high-performance liquid chromatography (HPLC), using a solid phase extraction column (Bond Elut cartridge column), for the simple, rapid and sensitive determination of serum clonazepam levels in epileptic patients. Extracted aliquots were analyzed by HPLC, using a reverse phase ODS column (mu-Bondapak C18). The analytical mean recovery of clonazepam added to the blank serum averaged 99.9%. The detection limit was as high as approximately 2 ng/ml in the serum. The reproducibilities were 2.3-8.6 CV % in the within-day assay and 6.5 CV % in the between-day assay, indicating that the analysis method was effective in the determination of clonazepam serum levels. Accordingly, we suggest that the present method, using a solid phase extraction column, may be useful for the routine monitoring of clonazepam serum levels in epileptic patients.     

High-performance liquid chromatographic method for the estimation of the novel investigational anti-cancer agent SR271425 and its metabolites in mouse plasma

A simple and reliable HPLC method was developed for the estimation of a new anti-cancer agent that belongs to the thioxanthone class, SR271425 in mouse plasma. SR271425, it's metabolites and internal standard (SR233377) were separated from plasma by liquid-liquid extraction using dichloromethane after quenching the plasma proteins with acetonitrile. Chromatography was performed on a reversed-phase C18 column using methanol-10 mM phosphate buffer, pH 3.5 (45:55) as mobile phase at a flow-rate of 0.8 ml/min for first 10 min and 1.4 ml/min for the next 15 min with UV-Vis detection at 264 nm and SR233377 as internal standard. The retention times of SR271425 and internal standard were 18.6 and 14.8 min, respectively. The limit of detection was 40 ng/ml and the limit of quantification was 78 ng/ml. This method was also able to detect the three metabolites of SR271425. The intra- and inter-day relative standard deviations were less than 13% at all concentrations. This analytical method was precise and reproducible for pharmacokinetics and metabolism studies of the drug in mice. SR271425 is proceeding to phase I clinical trials in 2001.     

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.     

High-performance liquid chromatographic method for sensitive determination of the alkylating agent CB1954 in human plasma.

A high-performance liquid chromatography (HPLC) method is described for the measurement of the weak alkylating agent CB1954 in human plasma. CB1954 can be used as an innocuous prodrug designed for activation by bacterial nitroreductases in strategies of gene-directed enzyme-prodrug therapy, and becomes activated to a potent bifunctional alkylating agent. The HPLC method involves precipitation and solvent extraction and uses Mitomycin C (MMC) as an internal standard, with a retention time for MMC of 5.85 +/- 0.015 min, and for CB1954 of 10.72 +/- 0.063 min. The limit of detection for CB1954 is 2.9 ng/ml, and this compares favourably with systems involving direct analysis of plasma (limit of detection 600 ng/ml, approximately). The method is now being used for pharmacokinetic measurements in plasma samples from cancer patients entering phase I clinical trials of CB1954. Results using serial plasma samples from one patient are presented. The patient was treated intravenously with CB1954 (6 mg/m2), and plasma clearance of the drug showed biphasic kinetics with alpha half-life 14.6 min, and beta half-life 170.5 min.     

Measurement of the novel antitumor agent 17-(allylamino)-17-demethoxygeldanamycin in human plasma by high-performance liquid chromatography

A sensitive HPLC assay has been developed to determine the concentration of 17-(allylamino)-17-demethoxygeldanamycin (AAG) in human plasma over the concentration range of 12.5 to 2,500 nM (7.33 to 1,465 ng/mL). After the addition of 1,000 nM geldanamycin as the internal standard, 1 mL samples of human plasma were subjected to solid-phase extraction, via Bond-Elut C18 cartridges, followed by analysis using an isocratic reversed-phase HPLC assay with UV detection. A Phenomenex Kingsorb, 3 micron, C18, 150x4.60 mm column and a Phenomenex Security Guard pre-column, C18 (ODS, Octadecyl), were used to achieve separation. AAG and GM were monitored at 334 and 308 nm, respectively, on a Hewlett-Packard 1050 Diode-Array Detector. The mobile phase, run at a flow-rate of 1 mL/min, was composed of 50% (v/v) 25 mM sodium phosphate (pH 3.00) with 10 mM triethylamine and 50% acetonitrile. HPLC effectively resolved AAG with retention times of 14.60 +/- 0.54 min and the internal standard geldanamycin at 10.72+/-0.38 min (n = 15). This assay was able to measure plasma concentrations of AAG, the lower limit of quantitation being 12.5 nM, at a starting dose of 10 mg/m2 infused intravenously over 1 h in a Phase I clinical trial in adult patients with solid tumors.     

Determination of 5-fluorouracil in microvolumes of human plasma by solvent extraction and high-performance liquid chromatography

In the present study, a new reversed-phase HPLC method has been developed and validated for the quantitative determination of 5-fluorouracil (5-FU) in human plasma using only 100-microl samples. The sample extraction and clean-up procedure involved a simple liquid-liquid extraction after addition of 5-chlorouracil (5-CU), used as internal standard, with 5 ml ethyl acetate. Chromatographic separations were performed on an Inertsil ODS-3 column (250x4.6 mm ID; 5 microM particle size), eluted with a mobile phase composed of acidified water (pH 2.0). The column effluent was monitored by UV absorption measurement at a wavelength of 266 nm. The calibration curves were constructed over a range of 0.20-50.0 microM and were fitted by weighted (1/x) linear regression analysis using the ratio of peak heights of 5-FU and 5-CU versus concentrations of the nominal standards. Extraction recoveries over the total range averaged 92 and 93% for 5-FU and 5-CU, respectively. The lower limit of quantitation was established at 0.20 microM (approximately 26 ng/ml), with within-run and between-run precisions of 4.2 and 7.0%, respectively, and an average accuracy of 109.3%. The within-run and between-run precisions at four tested concentrations analyzed in quintuplicate over a time period of four days were < 1.4 and < 4.4%, respectively. The accuracy at the tested concentrations ranged from 98.4 to 102.3%. Compared to previously described validated analytical methods for 5-FU, our present assay provides equivalent to superior sensitivity, using only microvolumes of sample.     

Development and validation of a bioanalytical assay for (E)-5-(2-bromovinyl)-2'deoxyuridine in plasma by capillary zone electrophoresis.

A capillary zone electrophoretic method for the quantification of (E)-5-(2-bromovinyl)-2'-deoxyuridine in plasma has been developed and validated. Separation was performed with a 25 mmol/l borate buffer, pH 9.0, after an initial rinsing step with sodium hydroxide. The rinsing step was necessary for reproducible analyses of aqueous samples and plasma extracts obtained by C18 solid-phase extraction after deproteination with perchloric acid. No interferences with plasma compounds were observed. The calibration graph was linear over the range of 30 to 3000 ng/ml using 5-fluorouracil as external standard. The limit of quantification was 24 ng/ml. The CZE method is fast, reproducible, linear and is therefore a good alternative for the already established HPLC methods.     

Determination of sulfadoxine in human blood plasma using packed-column supercritical fluid chromatography.

A sensitive, rapid, selective and reproducible method has been developed to measure plasma levels of sulfadoxine, 4-Amino-N-(5, 6-dimethoxy-4-pyrimidinyl) benzensulfonamide; in healthy, human volunteers using packed-column super-critical fluid chromatography. Omeprazole, 5-methoxy-2-[[(4-methoxy-3, 5-di-methyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole; was used as the internal standard (i.s.) at 15.0 microg/ml. The drug and the i.s. were extracted from plasma using dichloromethane. Separation of sulfadoxine and i.s. was done on a Nucleosil (250x4.6 mm) 10 microm, RP-C18 column with 7.4% (v/v) methanol-modified supercritical fluid carbon dioxide (2.5 ml/min) as the mobile phase. The column temperature was 40 degrees C and the outlet pressure was set at 8.83 MPa. The detection was done using a UV-Vis detector set at 265 nm. The limit of quantification was 0.50 microg/ml using 1 ml plasma specimen. The mean extraction recovery of the drug from plasma was found to be 94.9%. The SFC method was directly compared to a published HPLC/UV method. With respect to speed and use of organic solvents SFC was found to be superior; while in all other aspects the results were similar to the published technique. The method has been successfully used to estimate the sulfadoxine levels in healthy human volunteers from 0 to 240 h following an oral dose of 500 mg of sulfadoxine in combination with 25 mg of pyrimethamine.     

Development of a high-performance liquid chromatographic method to determine the concentration of karenitecin, a novel highly lipophilic camptothecin derivative, in human plasma and urine

Karenitecin is a novel, highly lipophilic camptothecin derivative with potent anticancer potential. We have developed a sensitive high-performance liquid chromatographic method for the determination of karenitecin concentration in human plasma and urine. Karenitecin was isolated from human plasma and urine using solid-phase extraction. Separation was achieved by gradient elution, using a water and acetonitrile mobile phase, on an ODS analytical column. Karenitecin was detected using fluorescence detection at excitation and emission wavelengths of 370 and 490 nm, respectively. Retention time for karenitecin was 16.2 +/- 0.5 min and 8.0 +/- 0.2 min for camptothecin, the internal standard. The karenitecin peak was baseline resolved, with the nearest peak at 3.1 min distance. Using normal volunteer plasma and urine from multiple individuals, as well as samples from the 50 patients analyzed to date, no interfering peaks were detected. Inter- and intra-day coefficients of variance were <4.4 and 7.1% for plasma and <4.9 and 11.6% for urine. Assay precision, based on an extracted karenitecin standard plasma sample of 2.5 ng/ml, was +4.46% with a mean accuracy of 92.4%. For extracted karenitecin standard urine samples of 2.5 ng/ml assay precision was +2.35% with a mean accuracy of 99.5%. The mean recovery of karenitecin, at plasma concentrations of 1.0 and 50 ng/ml, was 81.9 and 87.8% respectively. In urine, at concentrations of 1.5 and 50 ng/ml, the mean recoveries were 90.3 and 78.4% respectively. The lower limit of detection (LLD) for karenitecin was 0.5 ng/ml in plasma and 1.0 ng/ml in urine. The lower limit of quantification (LLQ) for karenitecin was 1 ng/ml and 1.5 ng/ml for plasma and urine, respectively. Stability studies indicate that when frozen at -70 degrees C, karenitecin is stable in human plasma for up to 3 months and in human urine for up to 1 month. This method is useful for the quantification of karenitecin in plasma and urine samples for clinical pharmacology studies in patients receiving this agent in clinical trials.     

Validated high-performance liquid chromatographic method for the determination of lamotrigine in human plasma

A high-performance liquid chromatographic (HPLC) procedure for lamotrigine was developed and validated. Lamotrigine (LTG) and an internal standard were extracted from plasma using liquid-liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 0.78-46.95 micromol/l, with a mean coefficient of correlation (r)> or =0.99923. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.58 micromol/l, respectively. Within- and between-run precision studies demonstrated C.V.<3% at all tested concentrations. LTG median recovery was 86.14%. Antiepileptic drugs tested did not interfere with the assay. The method showed to be appropriate for monitoring LTG in plasma samples.     

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 stavudine in human plasma and urine by high-performance liquid chromatography using a reduced sample volume

Sensitive high-performance liquid chromatographic assays have been developed for the quantification of stavudine (2',3'-didehydro-3'-deoxythymidine, d4T) in human plasma and urine. The methods are linear over the concentration ranges 0.025-25 and 2-150 microg/ml in plasma and urine, respectively. An aliquot of 200 microl of plasma was extracted with solid-phase extraction using Oasis cartridges, while urine samples were simply diluted 1/100 with HPLC water. The analytical column, mobile phase, instrumentation and chromatographic conditions are the same for both methods. The methods have been validated separately, and stability tests under various conditions have been performed. The detection limit is 12 ng/ml in plasma for a sample size of 200 microl. The bioanalytical assay has been used in a pharmacokinetic study of pregnant women and their newborns.     

Quantitative high-performance liquid chromatography-based detection method for calphostin C, a naturally occurring perylenequinone with potent antileukemic activity

Calphostin C is a potent inhibitor of protein kinase C and can induce Ca2+-dependent apoptosis in human ALL cells. Further development of calphostin C will require detailed pharmacodynamic studies in preclinical animal models. Therefore, we established a sensitive and accurate high-performance liquid chromatography (HPLC)-based quantitative detection method for the measurement of calphostin C levels in plasma. Extraction of calphostin C from plasma was performed by precipitation of plasma protein using acetonitrile and an aliquot of extracted supernatant was injected onto a Hewlett-Packard HPLC system constituting a 250x4 mm LiChrospher 100, RP-18 (5 microm) in conjunction with a 4x4 mm LiChrospher 100, RP-18 guard column (5 microm). The eluted compounds were detected by diode array detection set at a wavelength of 479 nm. Acetonitrile-water containing 0.1% trifluoroacetic acid and 0.1% triethylamine (70:30, v/v) was used as the mobile phase. The average extraction recovery from plasma was 97.3%. Good linearity (r>0.999) was observed throughout the concentration range of 0.05-40 microM for calphostin C in 50 microl of plasma. Intra- and inter-assay variabilities were less than 6% in plasma. The lowest detection limit of calphostin C in 50 microl plasma was 0.02 microM at a signal-to-noise ratio of approximately 3. The availability of this assay will now permit detailed pharmacodynamic and pharmacokinetic studies of calphostin C in vivo.     

Determination of 5-S-cysteinyldopa in plasma and urine using a fully automated solid-phase extraction--high-performance liquid chromatographic method for an improvement of specificity and sensitivity of this prognostic marker of malignant melanoma.

5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air-oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S-D-cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 microg l(-1) and 25 to 5000 microg l(-1) for plasma and urine samples, respectively, a limit of detection of 0.17 microg l(-1), intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons.     

Sensitive method for the determination of bisphenol-A in serum using two systems of high-performance liquid chromatography

The aim of this study was to establish an easy and accurate method for the determination of bisphenol-A (BPA) in the body liquid such as serum and urine. Two high-performance liquid chromatography (HPLC) systems, HPLC with electrochemical detector (ED), and HPLC with mass spectrometry (MS) using electrospray ionization (ESI) interface were used for the assay in the serum samples prepared with solid-phase extraction method. Water or EtOH at a concentration below 50% was suitable for the extraction of BPA from serum. The limit of detection of BPA was 0.2 ng ml(-1) for the HPLC-ED method and 0.1 ng ml(-1) for HPLC-MS. There was a good correlation between the data obtained by the two HPLC systems. BPA concentrations in healthy human serum were low (0-1.6 ng ml(-1)). From various commercial fetal bovine serum and sheep plasma, however, significant amounts of BPA were detected. Since no BPA was detected from sheep plasma immediately after collection, the high amounts of BPA were considered to be caused by the handling of blood during the preparation of the products after blood collection. In vitro study showed that the amount of BPA leached from polycarbonate tube into sheep plasma were 40 times larger than those into water and the leached amount of BPA depended on the temperature (37 degrees C>20 degrees C>5 degrees C).     

Development and validation of a sensitive solid-phase extraction and high-performance liquid chromatography assay for the novel antitumour agent CT2584 in human plasma

A HPLC assay and solid-phase extraction technique from human plasma has been developed and validated for the novel anticancer agent CT2584, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, which has recently completed a phase I trial at the Christie Hospital, Manchester under the auspices of the CRC phase I/II committee. Following addition of CT2576, 1-(11-octylamino-10-hydroxylundecyl)-3,7-dimethylxanthine, as internal standard, a solid-phase extraction cartridge (100 mg cyanopropyl) was used to isolate the drug CT2584 from human plasma. Analysis was performed by reversed-phase chromatography. CT2576 was used as internal standard at a concentration of 4 microg ml(-1) for the quantification of CT2584 from plasma for the duration of this work. The lower limit of quantification for the drug CT2584 in buffer using this assay was found to be 0.0122 microM (0.008 microg ml(-1)) and 0.048 microM (0.027 microg ml(-1)) when extracted from human plasma.     

Determination of lidocaine in plasma by direct solid-phase microextraction combined with gas chromatography

Direct-immersion solid-phase microextraction (SPME) has been used to extract the local anesthetic lidocaine from human plasma. A simplified model shows the relationship between the total amount of drug in plasma and the amount of drug extracted. The model takes into account that the drug participates between the fiber, sample and proteins. Therefore the model can also be used to obtain a good approximation of the drug-protein binding. Extraction yields of lidocaine in plasma are <1%, and the protein binding of lidocaine was found to be about 74% at pH 9.5. A SPME method has been developed for the determination of the total amount of lidocaine in plasma. The protein binding was reduced by acidification and, subsequently, the sample was deproteinized with trichloroacetic acid. With a 100-microm polydimethylsiloxane-coated fiber and addition of sodium chloride to the sample an extraction yield of about 12% at equilibrium (45 min) has been obtained. The relative standard deviation of this method is <10%. A linear range was found from 25 to 2000 ng ml(-1) lidocaine in plasma (r=0.998) with a detection limit of 5 ng ml(-1) in plasma. An extraction yield of about 80% could be obtained after an overnight extraction by use of a 65-microm polydimethylsiloxane-divinylbenzene-coated fiber. If an extraction time of 10 min is used with this fiber, the same yield is obtained as with the single-phase fiber in 45 min. However, the drawback of this mixed-phase fiber is its much shorter lifetime.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C18 reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 microg/ml for plasma, 1.6 microg/g for muscle tissue and 0.5 microg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.