Reversed-phase high-performance liquid chromatography method for the simultaneous quantitation of the lactone and carboxylate forms of the novel natural product anticancer agent 10-hydroxycamptothecin in biological fluids and tissues

Camptothecins are indole alkaloids isolated from a Chinese tree, Camptotheca acuminata, and have a wide spectrum of anticancer activity in vitro and in vivo. A novel camptothecin congener 10-hydroxycamptothecin (HCPT) has been shown to be more active and less toxic than camptothecin, and the lactone HCPT is believed to be responsible for its anticancer activity. In the present study, a reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection was developed and validated for the simulataneous analysis of HCPT for lactone form (I) and carboxylate form (II) in plasma, urine and feces and tissues. Biological samples were prepared by a liquid-liquid extraction method using ice-cold methanol-acetonitrile (1:1, v/v). This method was shown to be reproducible and reliable, with intra- and inter-day variations being less than 7%, and accuracy being 94.3%–102.7%. The limits of determination were 2 ng/ml, 2 ng/ml, 2 ng/g, and 10 ng/ml for HCPT forms I and II in rat plasma, urine, feces, and tissues, respectively. The assay was liner over the range 2–2000 ng/ml (r=0.999, P<0.001) with recoveries of greater than 90% for plasma and urine and approximately 70–80% for feces and tissues homogenates through the extraction procedure. This analytic procedure has been successfully applied to a pharmacokinetic study of HCPT in experimental animals and should be useful in the future human studies.     

Hydroxycamptothecin-loaded nanoparticles enhance target drug delivery and anticancer effect

Background  

Hydroxycamptothecin (HCPT) has been shown to have activity against a broad spectrum of cancers. In order to enhance its tissue-specific delivery and anticancer activity, we prepared HCPT-loaded nanoparticles made from poly(ethylene glycol)-poly(γ-benzyl-L-glutamate) (PEG-PBLG), and then studied their release characteristics, pharmacokinetic characteristics, and anticancer effects. PEG-PBLG nanoparticles incorporating HCPT were prepared by a dialysis method. Scanning electron microscopy (SEM) was used to observe the shape and diameter of the nanoparticles. The HCPT release characteristics in vitro were evaluated by ultraviolet spectrophotometry. A high-performance liquid chromatography (HPLC) detection method for determining HCPT in rabbit plasma was established. The pharmacokinetic parameters of HCPT/PEG-PBLG nanoparticles were compared with those of HCPT.     

Simultaneous determination of the carboxylate and lactone forms of 10-hydroxycamptothecin in human serum by restricted-access media high-performance liquid chromatography

A simple restricted-access media (RAM) HPLC method for simultaneous determination of the lactone and carboxylate forms of 10-hydroxycamptothecin (HCPT) in human serum was established. Using a RAM Hisep analytical column, serum samples were directly injected into the HPLC system. The eluted peaks of two forms of HCPT were monitored with a fluorescence detector. The separation was completed in 17 min. The linear range was 20-1000 ng/ml, intra-day and inter-day variations being less than 5%. The kinetic equation was introduced according to the analytical results. The equation shows that the course of the HCPT lactone form converting to carboxylate form in human serum at 4 degrees C is a first-order kinetic course. The concentration of each form at the moment of sampling was calculated by extrapolation.     

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 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.     

Quantitative analysis of sulpiride in body fluids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the analysis of sulpiride, N-ethyl-2-(2-methoxy-5-sulphonamido-benzamido-methyl)-pyrrolidine, in body fluids is described. A structurally related compound, N-ethyl-2-(2,4-dimethoxy-benzamido-methyl)-pyrrolidine, was used as internal standard.A fluorescence detector with excitation maximum at 299 nm and emission maximum at 342 nm was used for the quantitation. The detection limit was about 10 ng/ml in serum and cerebrospinal fluid and about 200 ng/ml in urine. The experimental error was 5–10% in the concentration range 25–100 ng/ml. Some preliminary data from a pharmacokinetic study in healthy volunteers are presented. The half-life for sulpiride in serum was about 8 h. Sulpiride was also measured in cerebrospinal fluid from five drug-treated psychotic patients.     

Solid-phase extraction-liquid chromatographic method for the determination and pharmacokinetic studies of albiflorin and paeoniflorin in rat serum after oral administration of Si-Wu decoction

A sensitive and rapid high-performance liquid chromatography (HPLC) method with solid-phase extraction (SPE) to simultaneously determine albiflorin and paeoniflorin in rat serum was described. Serum samples were pretreated with solid-phase extraction using Extract-Clean cartridges, and the extracts were analyzed by HPLC on a reversed-phase C(18) column and a mobile phase of acetonitrile-0.03% formic acid (17:83 (v/v)) with ultraviolet detection at 230 nm. Pentoxifylline was used as the internal standard (IS). The linear ranges of the calibration curves were 29-1450 ng/ml for albiflorin and 10-2000 ng/ml for paeoniflorin. The intra- and inter-day precisions (R.S.D.) were 相似文献   

Validation of HPLC analysis method of a novel antihypertensive agent MS23 in rat plasma

MS23 is a vasodilator with unique dual action pharmacological profile to inhibit type 4 PDE and antagonize L-type calcium channels. We validated an analytical protocol for MS23 in rat plasma using high performance liquid chromatography (HPLC). A C18 column and a phosphate/acetonitrite buffer were used for chromatographic separation. UV detection was performed at 307 nm. The calibration curve for MS23 was linear in the range from 50 to 10,000 ng/ml. The limit of quantification (LOQ) was 50 ng/ml. The results demonstrate that the method has linearity (R = 0.9989), specificity, and acceptable precision/accuracy. This method is simple, economic, and sufficient for in vivo pharmacokinetic studies on the compound.     

Column-switching high-performance liquid chromatographic method for the determination of a thymidylate synthase inhibitor, LY231514, an investigational agent for the treatment of solid tumors, in human plasma

A reversed-phase, column-switching high-performance liquid chromatographic (HPLC) method is described for the determination of a new thymidylate synthase inhibitor in human plasma. The compound and an internal standard are extracted from plasma using a Certify II solid-phase cartridge. Extracts are evaporated to dryness and the residue is reconstituted with mobile phase buffer. The analytes are separated from polar interferences and buffer salts originating from the elution step on a 4-mm YMC Basic pre-column. The fraction containing the analytes is further separated on a 25-cm YMC Basic column. The analytes are detected by their absorbance at 250 nm. The limit of quantitation is 10 ng/ml. The method is linear from 10 ng/ml to 80 μg/ml using three standard curve ranges. Validation studies for all three ranges show the method to be reproducible. The method has been successfully used to support pharmacokinetic studies.     

Pharmacokinetic conversion study of a new cyclosporine formulation in stable adult renal transplant recipients

Cyclosporine A (CyA) is a standard component of immunosuppressive regimens. It is a critical-dose drug for which a minor change in absorption can have important clinical consequences. The aim of the study was to compare the pharmacokinetics and safety of the new generic CyA formulation, Equoral capsules, after a switch from original formulation, Neoral capsules, in seventy stable adult renal transplant recipients. The extent and rate of pharmacokinetic parameters for bioequivalence were compared in a non-randomized, steady-state clinical study with fixed non-replicate study design. Pharmacokinetic analysis of CyA have shown that both the rate and extent of absorption of Equoral does not differ significantly from that of Neoral. At identical dosing, the new formulation was found to have geometric means of C(max) 717 ng/ml and AUCtau 3108 ng/ml.h, while corresponding results of comparator were 725 ng/ml and AUCtau 3039 ng/ml.h, respectively. The 90 % confidence intervals of C(max) and AUCtau were within 80- 125 % interval of the mean values. The results suggest that Equoral capsules can be used as an alternative treatment to Neoral capsules in CyA regimen.     

Sensitive determination of bisoprolol enantiomers in plasma and urine by high-performance liquid chromatography using fluorescence detection, and application to preliminary study in humans

A sensitive, stereoselective high-performance liquid chromatographic method with fluorescence detection for the measurement of bisoprolol enantiomers in human plasma and urine has been developed. Bisoprolol was extracted at alkaline pH with chloroform, followed by solid-phase extraction. The effluent was evaporated, and the reconstituted residue was chromatographed on a Chiralcel OD column with a mobile phase of hexane—2-propanol (10:0.9, v/v) containing 0.01% (v/v) diethylamine. Within the plasma and urine enantiomeric concentration ranges of 5–100 ng/ml and 25–1250 ng/ml, respectively, a linear relationship was obtained between the peak-height ratios and the corresponding concentrations. The limit of quantitation, defined as three times the baseline noise, was 2 ng/ml for each enantiomer in plasma. A preliminary pharmacokinetic study was undertaken in three healthy male volunteers following an oral dose of 5 mg of racemic bisoprolol. The results confirm that this assay is suitable for pharmacokinetic studies of bisoprolol enantiomers in humans following oral administration of the therapeutic dose.     

Determination of a new podophyllotoxin derivative, TOP-53, and its metabolite in rat plasma and urine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method was developed for the determination of a new podophyllotoxin derivative, TOP-53 (I), and TOP-53 glucoronide (II) as its major metabolite in rat plasma and urine. For the analysis of I, the sample was chromatographed on a reversed-phase C₁₈ column with electrochemical detection after consecutive two-step liquid-liquid extractions. Compound II was determined as I after enzymatic hydrolysis of II. This method was validated sufficiently with respect to specificity, accuracy, and precision. The limiits of quantitation for both I and II were 2 ng/ml in plasma and 10 ng/ml in urine. The method is thus useful for the pharmacokinetic study of I.     

Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.     

Effects of Gender and Phase of the Menstrual Cycle on the Kinetics of Ranitidine in Healthy Volunteers

The present study was undertaken to determine if differences exist in the pharmacokinetic parameters of oral ranitidine caused by gender and stage of the menstrual cycle. The study was performed in two steps, in the first a pharmacokinetic study was performed on 10 men (average age 35.5 yrs) and 10 women (average age 34.7 yrs) during the follicular phase, and in the second the pharmacokinetic study was performed only on the same women in their luteal phase. Subjects received a tablet dose of 300 mg ranitidine, and blood samples were drawn at several times after its ingestion. Plasma ranitidine concentration was determined by high performance liquid chromatography. Comparison of the pharmacokinetic parameters of women and men revealed statistically significant differences both in distribution volume (Vd) with values of 2.0 and 6.3 l/kg, Area Under Curve (AUC) with values of 7312.15 and 11471.94 ng/ml/h, and clearance (CLt) with values of 0.65 and 0.59 l/kg/h, respectively. Several pharmacokinetic parameters in women were different in the follicular compared to the luteal phase; for example, Vd was 2.0 and 5.6 l/kg, AUC was 7312.15 and 5195.83 ng/ml/h, and CLt was 0.65 and 0.97 l/kg/h, in the respective phases. Moreover, the maximum concentration (Cmax) was 1086 ng/ml in the follicular vs. 864 ng/ml in the luteal phase. The first study detected differences between men and women in several pharmacokinetic parameters, mainly those indicative of drug availability, for example, Vd, AUC, and CLt. Comparison of data obtained in the follicular phase with those obtained in the luteal phase revealed differences in most pharmacokinetic parameters, which is seemingly indicative of the characteristic physiological changes associated with the luteal phase that largely affect the kinetics and availability of drugs such as ranitidine. Although it has been postulated that hormonal fluctuation within the menstrual cycle phase is the primary cause of documented gender differences in the pharmacokinetics and pharmacodynamics of drugs, further study of related factors is required to fully understand how gender and menstrual cycle rhythms affect the pharmacokinetic process in their entirety.     

Rapid and sensitive determination of sertraline in human plasma using gas chromatography-mass spectrometry

A method for the determination of sertraline in human plasma using gas chromatography-mass spectrometry (GC-MS), with the selected ion-monitoring (SIM) mode, was described. The following was used in this study: (1) single liquid-liquid extraction at alkaline pH after deproteinization of plasma protein and (2) perfluoroacylation with HFBA, which has higher sensitivity (about 10-fold) compared with previous reported derivatization. The detection limit for the SIM of sertraline as an N-HFB derivative was 0.1 ng/ml, and its recovery was 80-85%. The linear response was obtained in the range of 0.2-10.0 ng/ml with a correlation coefficient of 0.999. The coefficient of variation (C.V.%) was less than 12.1% in the 1-30 ng/ml, and less than 18.2% at 0.2 ng/ml, and the accuracy was less than 10% at all of the concentration range. These findings indicate that this assay method has adequate precision and accuracy to determine the amount of sertraline in human plasma. After pharmacokinetics was performed with this assay method following oral administration of sertraline hydrochloride in man, moment analysis revealed that pharmacokinetic parameters for sertraline (Cmax, 10.3 ng/ml; Tmax, 8.0 h; T(1/2) 28.6 h) were similar to previously reported results. These results indicate that this simple and sensitive assay method is readily applicable to the pharmacokinetic studies of sertraline.     

Albendazole sulphoxide concentrations in plasma of endemic normals from a lymphatic filariasis endemic region using liquid chromatography

A simple and sensitive reversed-phase isocratic HPLC method for the determination of albendazole and its metabolites has been developed. The mobile phase consisting of acetonitrile-water-perchloric acid (70%) (30:110:0.06 (v/v/v)) was pumped at a flow rate of 0.80 ml/min on a 5 microm, reverse phase, Discovery RPamide C16 column with UV detection at 290 nm. The calibration graphs were linear in the range of 0.05- 1 microg/ml for albendazole, albendazole sulphoxide and albendazole sulphone. The limit of quantification was 50 ng/ml for albendazole, 25 ng/ml for albendazole sulphoxide and 30 ng/ml for albendazole sulphone. The within-day and day-to-day coefficient of variation averaged 4.98 and 6.95% for albendazole, 3.83 and 6.83% for albendazole sulphoxide and 3.44 and 5.51% for albendazole sulphone, respectively. The mean extraction recoveries of albendazole, albendazole sulphoxide and albendazole sulphone were 79.25, 93.03 and 88.78%, respectively. The method was applied to determine the plasma levels of albendazole sulphoxide in endemic normals administered with albendazole during pharmacokinetic studies.     

Determination of aloesin in rat plasma using a column-switching high-performance liquid chromatographic assay

A column-switching high-performance liquid chromatography (HPLC) method for the determination of aloesin in rat plasma using column-switching and ultraviolet (UV) absorbance detection is described. Plasma was directly injected onto the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The determination of aloesin was accurate and repeatable, with a limit of quantitation of 10 ng/ml in plasma. The standard calibration curve for aloesin was linear (r=0.998) over the concentration range of 10–1000 ng/ml in rat plasma. The intra- and inter-day assay variabilities of aloesin ranged from 1.0 to 4.7% and 1.1 to 8.8%, respectively. This highly sensitive and simple method has been successfully applied to a pharmacokinetic study after oral administration of aloesin to rats.     

Solid-phase extraction and determination of ranitidine in human plasma by a high-performance liquid chromatographic method utilizing midbore chromatography

An improved high-performance liquid chromatographic (HPLC) method utilizing solid-phase extraction (SPE) and midbore chromatography was developed for the determination of ranitidine in human plasma. A mobile phase of 20 mM K₂HPO₄-acetonitrile-triethylamine (87.9:12.0:0.1, v/v) pH 6.0 was used with a phenyl analytical column and ultraviolet detection (UV). The method demonstrated linearity from 25 to 1000 ng/ml in 500 μl of plasma with a detection limit of 10 ng/ml. The method was utilized in a pharmacokinetic study evaluating the effects of pancreatico-biliary secretions on ranitidine absorption.     

Quantitative determination of epinastine in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the quantitative determination of epinastine, a non-sedating histamine H₁ receptor antagonist, in rat plasma, was developed. A 100-μl volume of plasma sample was spiked with a solution of internal standard (diphenidol) and extracted with dichloromethane under alkaline conditions. The extract was applied onto the HPLC system and detected by ultraviolet absorption at a wavelength of 220 nm. The linearity of the calibration curve was preserved over the concentration range of 20--1000 ng/ml. Both intra-assay variation and relative error were less than 5% for the plasma sample containing 50 ng/ml or 1000 ng/ml of epinastine hydrochloride. The analytical method presented here should be useful for the investigation of the pharmacokinetic properties of epinastine, which is of clinical significance.     

Direct enantiospecific HPLC bioanalysis of (R,S)-atenolol on a chiral stationary phase

The simultaneous determination of the enantiomers of the β₁-selective adrenergic antagonist atenolol in human plasma and urine is described. After an alkaline preextraction atenolol is extracted from biological material at pH 12.3 using dichloromethane/propan-2-ol. The separation of the underivatized enantiomers is achieved by high-performance liquid chromatography on a chiral stationary phase (Chiralcel OD, cellulose tris-3, 5-dimethylphenylcarbamate, coated on silica gel) with fluorimetric detection. (?)-(S)-Pindolol is used as an internal standard. The detection limits of 5 ng/ml enantiomer in plasma and 50 ng/ml enantiomer in urine are sufficient for pharmacokinetic studies after therapeutic doses. © 1993 Wiley-Liss, Inc.     

Effects of gender and phase of the menstrual cycle on the kinetics of ranitidine in healthy volunteers

The present study was undertaken to determine if differences exist in the pharmacokinetic parameters of oral ranitidine caused by gender and stage of the menstrual cycle. The study was performed in two steps, in the first a pharmacokinetic study was performed on 10 men (average age 35.5 yrs) and 10 women (average age 34.7 yrs) during the follicular phase, and in the second the pharmacokinetic study was performed only on the same women in their luteal phase. Subjects received a tablet dose of 300 mg ranitidine, and blood samples were drawn at several times after its ingestion. Plasma ranitidine concentration was determined by high performance liquid chromatography. Comparison of the pharmacokinetic parameters of women and men revealed statistically significant differences both in distribution volume (Vd) with values of 2.0 and 6.3 l/kg, Area Under Curve (AUC) with values of 7312.15 and 11471.94 ng/ml/h, and clearance (CLt) with values of 0.65 and 0.59 l/kg/h, respectively. Several pharmacokinetic parameters in women were different in the follicular compared to the luteal phase; for example, Vd was 2.0 and 5.6 l/kg, AUC was 7312.15 and 5195.83 ng/ml/h, and CLt was 0.65 and 0.97 l/kg/h, in the respective phases. Moreover, the maximum concentration (Cmax) was 1086 ng/ml in the follicular vs. 864 ng/ml in the luteal phase. The first study detected differences between men and women in several pharmacokinetic parameters, mainly those indicative of drug availability, for example, Vd, AUC, and CLt. Comparison of data obtained in the follicular phase with those obtained in the luteal phase revealed differences in most pharmacokinetic parameters, which is seemingly indicative of the characteristic physiological changes associated with the luteal phase that largely affect the kinetics and availability of drugs such as ranitidine. Although it has been postulated that hormonal fluctuation within the menstrual cycle phase is the primary cause of documented gender differences in the pharmacokinetics and pharmacodynamics of drugs, further study of related factors is required to fully understand how gender and menstrual cycle rhythms affect the pharmacokinetic process in their entirety.