Determination of a new naphthoquinone derivative, NQ12, in human plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the determination of a new phospholipase A2 inhibitor, NQ12, in human plasma and urine. The sample preparation was simple: 2 volumes of acetonitrile were added to the biological samples to deproteinize it. A 50-microl aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase employed was 0.05M acetate buffer (pH 3) : acetonitrile : methanol (30:45:25, v/v/v) and run at a flow rate of 1.5 ml/min. The column effluent was monitored by a UV detector set at 298 nm. The retention times for NQ12 and the internal standard were approximately 5.5 and 7.0 min, respectively. The detection limits for NQ12 in human plasma and urine were all 20 ng/ml. The coefficients of variation of the assay (within-day and between-day) were generally low (below 11.8%) for human plasma and urine. No interferences from endogenous substances were found.     

Simultaneous determination of the HIV protease inhibitors indinavir, amprenavir, saquinavir, ritonavir and nelfinavir in human plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the simultaneous quantitative determination of five HIV protease inhibitors (i.e. indinavir, amprenavir, saquinavir, ritonavir and nelfinavir) in human plasma is described. An aliquot of 500 microl plasma was extracted with 0.5 ml of 0.1 M NH4OH and 5 ml of methyl tert.-butyl ether. After evaporating, the residue was dissolved in eluent mixture of acetonitrile and 50 mM KH2PO4 adjusted to pH 5.6 with 50 mM Na2HPO4 (43:57, v/v). Subsequently, the eluent was washed with hexane. Chromatography was performed using a C18 reversed-phase column. Ultraviolet detection at 215 nm was used. Linearity of the method was obtained in the concentration range of 0.05-20 microg ml(-1) for all five protease inhibitors. Our method is now in use to analyse plasma samples from patients treated with co-administration of HIV protease inhibitors.     

Sensitive determination of ephedrine and norephedrine in human plasma samples using derivatization with 9-fluorenylmethyl chloroformate and liquid chromatography

A high-performance liquid chromatography procedure for the determination of ephedrine and norephedrine using fluorimetric detection in plasma samples is described. A double liquid-liquid extraction was performed, followed by derivatization with 9-fluorenylmethyl chloroformate. The extracts were chromatographed with a 5-microm C18 (150x4.6 mm I.D.) column using a mobile phase composed of acetonitrile and water (52:48; v/v). The excitation and emission wavelengths were respectively 264 nm and 313 nm. Calibration curves were linear over the range 0 to 300 ng/ml for each analyte. The specificity of the method was demonstrated with several FMOC-reacting drugs. The limits of quantification are similar to those obtained with the reference method: 2 ng/ml for ephedrine and 5 ng/ml for norephedrine. This method has been successfully applied to the determination of ephedrine and norephedrine plasma levels after administration of low doses of ephedrine to healthy subjects.     

High-performance liquid chromatographic analysis of a new neuroprotective agent for ischemia-reperfusion damage, KR-31378.

A high-performance liquid chromatographic method was developed for the determination of a neuroprotective agent for ischemia-reperfusion damage, KR-31378, in human plasma and urine and in rat tissue homogenates. The method involved deproteinization of the the biological samples with 0.5 volumes of saturated Ba(OH)2, 0.5 volumes of 0.04 M ZnSO4 and 1 volume of acetonitrile. A 80-microl aliqout of the supernatant was injected onto a reversed-phase C18 column. The mobile phase, 50 mM triethylamine acetate : acetonitrile : tetrahydrofuran (65:30:5, v/v/v), was run at a flow rate of 1.0 ml/min. The column effluent was mornitored by a ultraviolet detector set at 310 nm. The retention time of KR-31378 was approximately 6.5 min. The detection limits of KR-31378 in human plasma and urine and rat tissue homogenates were 0.2, 0.5 and 0.5 microg/ml, respectively. The coefficients of variation (within-day and between-day) were below 13.6% for human plasma and urine and rat homogenates. No interferences from endogenous substances were found.     

Determination of the cephalosporin antibiotic cephradine in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic method, for the determination of cephradine in human plasma samples has been developed and validated. Cephradine and cephaloridine (internal standard) were extracted from human plasma by perchloric acid protein precipitation followed by centrifugation. Aliquots of the extracts were analysed by reversed-phase high-performance liquid chromatography (HPLC) utilising a polymeric reversed-phase PLRP-S column, followed by ultraviolet detection at 260 nm. The method has a working dynamic range from 0.2 to 30.0 microg/ml from 200 microl human plasma. The precision of the method at 0.2 microg/ml was 4.9% (intra-assay) and negligible (inter-assay) as calculated by one-way analysis of variance and the accuracy of the method at 0.2 microg/ml was -4.1% in terms of percentage bias. This method has been successfully applied to clinical studies including an oral bioequivalence study comparing the pharmacokinetics of 500 mg tablets of Kefdrin with 500 mg tablets of Velosef in healthy human volunteers.     

Determination of cyclosporin A in human and mouse plasma by reversed-phase high-performance liquid chromatography.

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 227 nm has been validated for the determination of cyclosporin A in human and mouse plasma. The cyclosporin D analog PSC 833 was used as internal standard. Plasma samples were pretreated by liquid-liquid extraction with diethyl ether. A good chromatographic separation between cyclosporin A, the internal standard and two potentially interfering endogenous peaks was achieved using a stainless steel column packed with 5 microm Nova-Pak phenyl material operated at 72 degrees C, and a mobile phase consisting of acetonitrile-methanol-water (20:52:28, v/v/v). The calibration curve for cyclosporin A in human plasma was linear over the tested concentration range of 0.11 to 5.34 microM. Murine plasma samples (200 microl) were diluted up to a total volume of 500 microl with blank human plasma and the concentrations were read from the calibration curve prepared in human plasma. The lower limit of quantitation was 0.11 microM using 500 microl of human plasma and 0.28 microM using 200 microl of mouse plasma. The validation data showed that the assay is sensitive, selective and reproducible for determination of cyclosporin A. The applicability was demonstrated in a pharmacokinetic experiment where mice received oral cyclosporin A.     

Rapid determination of the anti-cancer drug chlorambucil (Leukeran) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A bioanalytical method for the determination of the anticancer drug chlorambucil (Leukeran) and its phenyl acetic acid mustard metabolite in human serum and plasma is described. Automated solid-phase extraction of the analytes is carried out with C18 sorbent packed in a 96 well format microtitre plate using a robotic sample processor. The extracts are analysed by isocratic reversed-phase liquid chromatography using pneumatically and thermally assisted electrospray ionisation (TurboIonspray) with selected reaction monitoring. The method is specific and sensitive, with a range of 4-800 ng/ml in human serum and plasma for both parent drug and metabolite (sample volume 200 microl). The method is accurate and precise with intra-assay and inter-assay precision (C.V.) of <15% and bias <15% for both analytes. The automated extraction procedure is significantly faster than manual sample pre-treatment methods, a batch of 96 samples is extracted in 50 min allowing for faster sample turnaround. The method has been used to provide pharmacokinetic support to biocomparability studies of Leukeran following single doses of oral tablet formulations.     

Determination of a new asiatic acid derivative, AS 2-006A in rat plasma and urine, and human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new wound healing agent, AS 2-006A (ethoxymethyl 2-oxo-3, 23-O-isopropylideneasiatate), in rat plasma and urine, and human plasma. The sample preparation was simple: 2 volumes of acetonitrile were added to the biological samples to deproteinize it. A 50-microl aliquot of the supernatant was injected onto the reversed-phase column. The mobile phase employed was acetonitrile : H2O (9:1, v/v) and run at a flow rate of 1.1 ml/min. The column effluent was monitored by a UV detector set at 205 nm. The retention time for AS 2-006A was approximately 29.5 min. The detection limits for AS 2-006A in rat and human plasma were both 1 microg/ml, and in rat urine was 2 microg/ml. The coefficients of variation of the assay (within-day and between-day) were generally low (below 10.8%) for rat plasma and urine, and human plasma. No interferences from endogenous substances were found.     

Comparison of versions 1.0 AND 1.5 of the UltraSensitive AMPLICOR HIV-1 MONITOR test for subjects with low viral load

We compared the performance of two UltraSensitive AMPLICOR HIV-1 MONITOR kits (version 1.5 [v1.5] versus v1.0) by retesting 404 plasma samples with low viral loads (<3,000 copies/ml) with both kits. With 292 samples that initially had <50 copies/ml by the v1.0 kit, the v1.5 assay was more sensitive than the v1.0 assay for samples with human immunodeficiency virus type 1 RNA near the 50-copy/ml cutoff (P = 0.0146). Median numbers of copies per milliliter were similar for 112 samples with 50 to 3,000 copies/ml with no difference in sensitivity with a 200-copy/ml cutoff.     

Liquid chromatographic determination of ketoconazole, a potent inhibitor of CYP3A4-mediated metabolism

A high-performance liquid chromatographic assay with UV detection has been developed for the determination of ketoconazole in human plasma. Quantitative extraction was achieved by a single solvent extraction involving a mixture of acetonitrile-n-butyl chloride (1:4, v/v). Ketoconazole and the internal standard (clotrimazole) were separated on a column packed with Inertsil ODS-80A material and a mobile phase composed of water-acetonitrile-tetrahydrofuran-ammonium hydroxide-triethylamine (45:50.2:2.5:0.1:0.1, v/v). The column effluent was monitored at a wavelength of 206 nm with a detector range set at 0.5. The calibration graph was linear in the range of 20-2000 ng/ml, with a lower limit of quantitation of 20.0 ng/ml. The extraction recoveries for ketoconazole and clotrimazole in human plasma were 93+/-9.7% and 83+/-10.0%, respectively. The developed method has been successfully applied to a clinical study to examine the pharmacokinetics of ketoconazole in a cancer patient.     

Simultaneous determination of the HIV-protease inhibitors indinavir, nelfinavir, saquinavir and ritonavir in human plasma by reversed-phase high-performance liquid chromatography.

A sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of the four licensed HIV-protease inhibitors indinavir, nelfinavir, saquinavir and ritonavir. An aliquot of 500 microl plasma, spiked with internal standard, was extracted with 0.5 ml 0.1 M NH4OH and 5 ml methyl tert.-butyl ether. After evaporating, the residue was dissolved in eluent consisting of acetonitrile-50 mM phosphate buffer, pH 5.63 (40:60, v/v). Subsequently, the eluent was washed with hexane. Chromatography was performed using a C18 reversed-phase column and gradient elution with a linear increase of acetonitrile from 36 to 66%. Ultraviolet detection at 215 nm was used. Linearity of the method was obtained in the concentration range of 45-30 000 ng/ml for all four analytes. The method was validated extensively and stability tests under various conditions were performed. The assay is now in use to analyse plasma samples from patients treated with (combinations of) HIV-protease inhibitors.     

Sensitive high-performance liquid chromatographic method for the determination of the lactone form and the lactone plus hydroxy-acid forms of the new camptothecin derivative DX-8951 in human plasma using fluorescence detection

A sensitive quantitation of the lactone form and the lactone plus hydroxy-acid forms of DX-8951, a camptothecin derivative, in human plasma has been investigated by high-performance liquid chromatography (HPLC). This assay method consisted of two analytical procedures. In Procedure I, the lactone form was collected by the stepwise separation on a C18 cartridge. In Procedure II, the lactone plus hydroxy-acid forms were collected using another batch of the plasma sample by co-elution of the two forms from a C18 cartridge with acidic solution. The hydroxy-acid form of DX-8951 was quantitated from the difference of the lactone plus hydroxy-acid forms and the lactone form. Thereafter, these pre-treated samples were assayed by HPLC under the same HPLC conditions with a spectrofluorometer and a reverse-phase ODS column. The mobile phase was acetonitrile/0.05 M potassium dihydrogen phosphate (pH 3) (18:82, v/v) at a flow-rate of 1.0 ml/min. For the assay of the lactone form and the lactone plus hydroxy-acid forms of DX-8951 in plasma, analytical method were validated over the range 0.2-50 ng/ml.     

High-performance liquid chromatographic analysis of nitrite and nitrate in human plasma as S-nitroso-N-acetylcysteine with ultraviolet absorbance detection

A rapid HPLC method with UV absorbance detection at 333 nm for the measurement of nitrite and nitrate in ultrafiltrate samples of human plasma is described. The method is based on hydrochloric acid-catalyzed conversion of nitrite by N-acetyl-L-cysteine to S-nitroso-N-acetyl-L-cysteine and isocratic elution using 10 mM NaH2PO4 in acetonitrile-water, pH 2.0 (15:85, v/v). The limit of detection of the method is 50 nM nitrite. The method was validated by gas chromatography-mass spectrometry.     

Determination of a new oral iron chelator, ICL670, and its iron complex in plasma by high-performance liquid chromatography and ultraviolet detection

ICL670 is a representative of a new class of orally active tridentate selective iron chelators. Two molecules of ICL670 are required to form a complete hexacoordinate chelate Fe-[ICL670]2 with one ferric iron. A simple and rapid HPLC-UV method for the separate determination of ICL670 and Fe-[ICL670]2 in the plasma of iron-overloaded patients is described. Plasma samples were prepared as rapidly as possible, the tubes being kept at 4 degrees C. Plasma proteins were precipitated with methanol. The supernatant was diluted with water and placed on the refrigerated sample rack of an autosampler before injection. The chromatographic separations were achieved on an Alltima C18 column using 0.05 M Na2HPO4 and 0.01 M tetrabutylammonium hydrogen sulfate-acetonitrile-methanol (41:9:50, v/v/v) as mobile phase. The analytes were detected at 295 nm. Calibration and quality control samples were prepared in normal human plasma. The mean accuracy (n=6) over the entire investigated concentration range 0.25-20 microg/ml ranged from 91 to 109% with a coefficient of variation (C.V.) from 4 to 8% for ICL670, and from 95 to 105% with a C.V. from 2 to 20% for the iron complex. The dissociation of the complex during analysis was shown to be marginal. The iron removal from plasma of iron-overloaded patients by free ICL670 during analysis was low. The in vitro iron transfer from the iron pools of iron-overloaded plasma onto ICL670 was shown to be a slow process.     

Determination of the enaminone DM5, an anti-epileptic agent, in mouse plasma and brain tissue by high-performance liquid chromatography with ultraviolet detection

Enaminone derivatives of the 4-carbomethoxy-5-methylcyclohexane-1,3-dione series represent a new and potentially active series of compounds for the treatment of Epilepsy. Enaminone esters have been previously evaluated as compounds with potent oral anticonvulsant activity similar to class 1 anticonvulsants phenytoin, carbamazepine, and lamotrigine. DM5, a member of this class with -Cl in the para-substituted position, has been assessed to have the most potent pharmacological activity (ED50) in both the mouse and rat. A selective and specific high-performance liquid chromatography method was developed to quantitate DM5 in plasma and brain tissue in mice. Reverse phase chromatography with ultraviolet (lambda = 307 nm) detection was utilized to quantitate eluate. A C18 analytical column was used and the mobile phase consisted of acetonitrile and 0.05 M NaH2PO4 buffer (60:40; v/v). Liquid-liquid extraction with ether was used to extract the DM5 from plasma or brain homogenates. DM5 and carbamazepine (internal standard) eluted at approximately 6.0 and 9.0 min without any interfering peaks. The calibration curves were found to be linear (r > or = 0.9999) in the range of 0.1-5.0 microg/ml or microg/g. Intra-run precision's were in all in the range of 90%. The absolute recovery of the analyte in brain and plasma samples was < or = 90%. The valid method accurately quantified DM5 in plasma and brain tissue samples collected from a pharmacokinetic study consisting of an intravenous bolus in the tail vein of wild type and genetically altered mice.     

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.     

Determination of a novel calmodulin antagonist, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1- (4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, DY-9760e, in human plasma using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of a novel calmodulin antagonist, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-( 4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, DY-9760e and its major metabolite, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyi ndazole, DY-9836 in human plasma has been developed. DY-9760e, DY-9836 and the internal standard (I.S.) were extracted from plasma by means of an Isolute C18 (EC) column. The extracts were chromatographed on a reversed-phase TSK-gel ODS-80Ts column using 0.1 M acetate buffer (pH 5)-CH3CN (65:35, v/v) as the mobile phase at a flow-rate of 1.0 ml/min. Fluorescence detection at an excitation wavelength of 303 nm and an emission wavelength of 347 nm resulted in a limit of quantitation of 1.000 ng/ml for plasma. The method showed satisfactory sensitivity, precision, accuracy, recovery and selectivity. Stability studies showed that DY-9760 and DY-9836 were stable in plasma up to at least eight weeks at -80 degrees C.     

High-performance liquid chromatographic determination of vinorelbine in human plasma and blood: application to a pharmacokinetic study

A sensitive and specific high-performance liquid chromatographic method with fluorescence detection (excitation wavelength: 280 nm; emission wavelength: 360 nm) was developed and validated for the determination of vinorelbine in plasma and blood samples. The sample pretreatment procedure involved two liquid-liquid extraction steps. Vinblastine served as the internal standard. The system uses a Spherisorb cyano analytical column (250x4.6 mm I.D.) packed with 5 microm diameter particles as the stationary phase and a mobile phase of acetonitrile-80 mM ammonium acetate (50:50, v/v) adjusted to pH 2.5 with hydrochloric acid. The assay showed linearity from 1 to 100 ng/ml in plasma and from 2.5 to 100 ng/ml in blood. The limits of quantitation were 1 ng/ml and 2.5 ng/ml, respectively. Precision expressed as RSD was in the range 3.9 to 20% (limit of quantitation). Accuracy ranged from 92 to 120%. Extraction recoveries from plasma and blood averaged 101 and 75%, respectively. This method was used to follow the time course of the concentration of vinorelbine in human plasma and blood samples after a 10-min infusion period of 20 mg/m2 of this drug in patients with metastatic cancer.