Determination of metabolic profile of anti-malarial trioxane CDRI 99/411 in rat liver microsomes using HPLC

CDRI 99/411 is a potent 1,2,4‐trioxane anti‐malarial candidate compound of the Central Drug Research Institute, India. This study aimed to conduct comprehensive in vitro metabolic investigations of CDRI 99/411 to corroborate its preclinical investigations. Preliminary in vitro metabolic investigations were performed to assess the metabolic stability [in vitro half‐life (t_1/2) and in vitro hepatic intrinsic clearance (Cl_int)] of CDRI 99/411 in male Sprague–Dawley rat and human liver microsomes using validated high‐performance liquid chromatography with photodiode array detector. The observed in vitro t_1/2 of the compound in rat and human liver microsomes was 13 min with in vitro Cl_int 130.7 ± 25.0 μL/min/mg and 19 min with in vitro Cl_int 89.3 ± 17.40 μL/min/mg. These observations suggested moderate metabolic degradation and in vitro Cl_int with insignificant difference (p > 0.05) in the metabolic stability profile in rat and human. Hence, in vitro metabolic investigations were performed with rat liver microsomes. It was observed that CDRI 99/411 exhibited sigmoidal kinetics. At nonlinear regression (r ≥ 0.99) EC₅₀ and Hill slope values were 17 µm and 1.50, respectively. The metabolism of CDRI 99/411 was primarily mediated by CYP3A2 and was inferred by CYP reaction phenotyping with known potent inhibitors. Two metabolites of CDRI 99/411 were detected which were undetectable on incubation with 1‐aminobenzotriazole and ketoconazole. Copyright © 2011 John Wiley & Sons, Ltd.     

An HPLC method for the determination and pharmacokinetic study of lehmannine in rat plasma

A high-performance liquid chromatographic (HPLC) method for determining lehmannine (LMN) in rat plasma was developed for application in the pharmacokinetics study. The plasma was deproteinized with acetonitrile that contained an internal standard and was separated from the aqueous layer by adding sodium chloride. The HPLC assay was carried out using a VP-ODS column at 40 degrees C. The mobile phase was acetonitrile-0.02 mol/L ammonium acetate buffer-triethylamine (35:65:0.04, v/v/v). The flow rate was 1.0 mL/min. The detection wavelength was set at 220 nm. The method was used to determine the concentration-time profiles of LMN in the plasma following oral administration or bolus injection of LMN aqueous solution. The pharmacokinetic parameters of LMN were calculated for the first time by Drug and Statistics 1.0 program.     

Determination of the active metabolites of sibutramine in rat serum using column-switching HPLC

A simple and direct analysis using column-switching HPLC method was developed and validated for the quantification of active metabolites of sibutramine, N-mono-desmethyl metabolite (metabolite 1, M1) and N-di-desmethyl metabolite (metabolite 2, M2) in the serum of rats administered sibutramine HCl (5.0 mg/kg, p.o.). Rat serum was directly injected onto the precolumn without sample prepreparation step following dilution with mobile phase A, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (8.3:4.5:87.2 by volume). After the endogenous serum components were eluted to waste, the system was switched and the analytes were eluted to the trap column. Active metabolites M1 and M2 were then back-flushed to the analytical column for separation with mobile phase B, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (35.8:19.2:45 by volume) and detected at 223 nm. The calibration curves of active metabolites M1 and M2 were linear in the range of 0.1-1.0 microg/mL and 0.15-1.8 microg/mL. This method was fully validated and shown to be specific, accurate (10.4-10.7% error), and precise (1.97-8.79% CV). This simple and rapid analytical method using column-switching appears to be useful for the pharmacokinetic study of active metabolites (M1 and M2) of sibutramine.     

Determination of erythromycin A in rat plasma and urine by high-performance liquid chromatography with chemiluminescence detection using Tris(2,2'-bipyridine)ruthenium(II)

A simple and sensitive method was developed for the determination of erythromycin A (EA), decladinosyl erythromycin A (dClEA) and erythromycin B (EB) in rat plasma and urine by high-performance liquid chromatography with electrogenerated chemiluminescence detection using Tris(2,2'-bipyridine)ruthenium(II). The recovery rates of EA, dClEA and EB were 97, 94 and 85% from rat plasma and 89, 83 and 93% from rat urine, respectively. The calibration curves were linear over the concentration ranges 0.05-5 microg/mL for plasma and 0.5-50 microg/mL for urine. The precision and accuracy for all analytes in rat plasma were < or =9.0 and -6.3-7.2%, and those in urine were < or =9.4% and -6.1-7.6%, respectively. This method proved to be a powerful tool for determination of EA, dClEA and EB concentrations in samples from rats.     

Determination of cnidilin and its two metabolites in rat bile and stool after oral administration by HPLC/electrospray ionization tandem mass spectrometry

A simple, fast and sensitive method for the simultaneous determination of cnidilin and its two metabolites (M1 and M2) in rat bile and stool using HPLC coupled with electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MS/MS) has been developed. The sample pretreatment was simple, because methanol was the only additive used for dilution of bile and ultrasound of stool. Pimpinellin was used as internal standard (IS). The separation was performed on a reverse phase C₁₈ column with gradient elution consisting of 0.5‰ aqueous formic acid and methanol (containing 0.5‰ formic acid). The detection was in the multiple‐reaction monitoring mode within 7 min. All the analytes were in accordance with the requirement of the validation of the method in vivo (linearity, precision, accuracy, limit of detection and limit of quantification). After oral administrating 24 mg/kg of the prototype drug cnidilin, M1 and M2 were determined in bile within 36 h, and in stool within 60 h. Cnidilin in bile was completely excreted in 24 h, and the main excretive amount of cnidilin was 80% in the first 6 h, but the drug recovery in bile within 24 h was <1.95%. In stool, the main excretive amount of cnidilin was 95.8% in the first 24 h, and the drug recovery within 48 h was lower than 1.48%. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of HPLC with fluorescent detection using NBD‐F for the quantification of colistin sulfate in rat plasma and its pharmacokinetic applications

Colistin sulfate, composed of a mixture of colistin A sulfate (CLA) and colistin B sulfate (CLB), is available for treating life‐threatening infections caused by multidrug‐resistant Gram‐negative bacteria. In this study, the CLA and CLB were quantified separately. Colistin sulfate was extracted from rat plasma with a solid‐phase extraction C₁₈ cartridge and reacted with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), and the fluorescent derivatives were subjected to reversed‐phase high‐performance liquid chromatography analysis and used to investigate the pharmacokinetics of CLA and CLB in rat plasma. The recovery rates of CLA and CLB were 41.2 ± 4.4 and 45.5 ± 3.1%, respectively. The recovery rate calculated from the total area of CLA and CLB was 43.9 ± 3.6%. When 2 mm NBD‐F and 10 mm boric acid buffer (pH 9.5) were added to colistin sulfate, the highest recovery rate was obtained. The best heating time was 5 min at 60°C. The lower limits of quantification for CLA, CLB and the total area of CLA and CLB were 0.05, 0.05 and 0.1 μg/mL; the coefficients of variations were 13.5, 14.5 and 14.1%, respectively. This method was found to have acceptable linearity, precision and accuracy, and has been successfully applied to a pharmacokinetic study in rat plasma.     

Simultaneous determination of D- and L-serine in rat brain microdialysis sample using a column-switching HPLC with fluorimetric detection

Both D- and L-serine in rat brain microdialysis sample were simultaneously determined by pre-column fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), separation of the derivatives on ODS column, TSKgel ODS-80TsQA, followed by Pirkle type chiral columns, Sumichiral OA-2500 (S), which gave a sufficient enantiomeric separation of NBD-D-serine and NBD-L-serine, and fluorimetric detection at a wavelength of 540 nm with an excitation wavelength of 470 nm. The peaks of NBD-D-serine and NBD-L-serine in the rat brain microdialysis sample were clearly found, and the validation study showed satisfactory results; the precision and accuracy were within 5.14 and 109%, respectively. Using the proposed HPLC method, the time-course profile of D-serine concentration in rat prefrontal cortex following intraperitoneal administration of D-serine was investigated. As a consequence, D-serine appeared to be rapidly distributed in the brain, and then decreased gradually with time in the extracellular fluid of the rat prefrontal cortex. The proposed HPLC method will be useful for in vivo studies on D-serine, which acts as a coagonist for N-methyl-D-aspartate receptor, to the extracellular fluid of rat brain.     

Determination of rifampicin in rat plasma by modified large‐volume direct injection RAM‐HPLC and its application to a pharmcokinetic study

A direct large volume injection high‐performance liquid chromatography (HPLC) method with homemade restricted‐access media (RAM) pre‐column and combined with a column‐switching valve was established and developed for determination rifampicin (RIP) in rat plasma. The rat plasma samples (100 μL) were injected directly onto pre‐column, where RIP was retained and pre‐concentrated, while proteins were washed to waste using a methanol–water (5:95) as the mobile phase at a flow rate of 1 mL/min. Then, by rotation of the switching valve at 5 min, the RIP were eluted from the pre‐column and transferred to an Luna C₁₈ analytical column by the chromatographic mobile phase consisting of methanol–acetonitrile–10 mm ammonium format (60:5:35) at a flow rate of 1 mL/min. The total analytical run time was 15 min with UV detection wavelength at 254 nm. Carbamazepine was used as the internal standard. Excellent linear correlation (r = 0.9993) was obtained in the range of 0.25–8 µg/mL for rat plasma. The intra‐day and inter‐day precisions of RIP were all <5.0%. The recoveries were in the range of from 99.98–113.66% for plasma. This on‐line RAM‐HPLC method was successfully applied to the pharmacokinetic study of RIP in rat plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of the putative cancer chemopreventive flavone tricin in plasma and tissues of mice by HPLC with UV--visible detection

AHPLC method developed and validated for the determination of tricin in human plasma published previously was cross-validated to allow measurement of the flavone tricin in plasma and tissues of mice. Blank samples of plasma, liver or intestinal mucosa were spiked with tricin at 0.5--4.0, 1.0--8.0 and 5.0--40 microg/mL, respectively. These tricin concentration ranges covered the tricin levels achieved in the mouse tissues in the dose-escalating experiments. Analysis afforded linear calibration curves with regression coefficients of >0.99. Endogenous compounds did not interfere with tricin detection when the detection wavelength was set at 355 nm, the maximum absorbance of tricin. Accuracy and precision were <15% for all concentrations in all matrices except for the precision at the lower limit of quantification (0.5 microg/mL) in mouse plasma, which was 18.4%. Consumption of diet mixed with tricin at 0.05, 0.2 or 0.5% for one week furnished steady-state levels in plasma, liver and small intestine in the 1--3 x 10(-7), 4--22 x 10(-7) and 3--46 x 10(-5) m ranges, respectively.     

高效液相色谱-共振瑞利散射光谱法测定大鼠血清中的奈替米星

建立了高效液相色谱-共振瑞利散射光谱联用测定大鼠血清中奈替米星的方法.采用的色谱条件为:以C18柱为分离柱,以甲醇(含0.22%三氟乙酸)-20 mmol/L醋酸钠水溶液(8∶92,v/v)为流动相进行等度洗脱.以滂胺天蓝为分子探针,奈替米星在柱后与滂胺天蓝结合生成离子缔合物,产生强烈的共振瑞利散射信号,荧光检测器在激...     

Development and validation of a RP‐HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of tofacitinib in rat plasma. The bioanalytical procedure involves extraction of tofacitinib and itraconazole (internal standard, IS) from rat plasma with a simple liquid–liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1.0 mL/min and C₁₈ column maintained at 40 ± 1 °C. The eluate was monitored using an UV detector set at 287 nm. Tofacitinib and IS eluted at 6.5 and 8.3 min, respectively and the total run time was 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 182–5035 ng/mL (r² = 0.995). The intra‐ and inter‐day precisions were in the range of 1.41–11.2 and 3.66–8.81%, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Validated RP‐HPLC/UV method for the quantitation of abiraterone in rat plasma and its application to a pharmacokinetic study in rats

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of abiraterone (ART) in rat plasma. The analytical procedure involves extraction of ART and diclofenac (internal standard, IS) from rat plasma with a simple liquid–liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system with a Betasil C₁₈ column maintained at ambient room temperature and an isocratic mobile phase [acetonitrile–water–10 mm potassium dihydrogen phosphate (pH 3.0), 55:5:40, v/v/v] at a flow rate of 1.00 mL/min with a total run time of 10 min. The eluate was monitored using an UV detector set at 255 nm. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 93.4–3251 ng/mL (r² = 0.997). The intra‐ and inter‐day precisions were 0.56–4.98 and 3.03–7.18, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study of ART in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of an RP‐HPLC method for the quantitation of odanacatib in rat and human plasma and its application to a pharmacokinetic study

A simple, specific, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of odanacatib in rat and human plasma. The bioanalytical procedure involves extraction of odanacatib and itraconazole (internal standard, IS) from a 200 μL plasma aliquot with simple liquid–liquid extraction process. Chromatographic separation was achieved on a Symmetry Shield RP₁₈ using an isocratic mobile phase at a flow rate of 0.7 mL/min. The UV detection wave length was 268 nm. Odanacatib and IS eluted at 5.5 and 8.6 min, respectively with a total run time of 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 50.9–2037 ng/mL (r² = 0.994). The intra‐ and inter‐day precisions were in the range of 2.06–5.11 and 5.84–13.1%, respectively, in rat plasma and 2.38–7.90 and 6.39–10.2%, respectively, in human plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of a reversed‐phase HPLC method for the determination of γ‐tocotrienol in rat and human plasma

γ‐Tocotrienol (γ‐T3) is a member of the vitamin E family. Recently, γ‐T3 has attracted the attention of the scientific community due to its potent anticancer activity and other therapeutic benefits. The objective of this study was to develop and validate a simple and practical reversed‐phase HPLC method with satisfactory sensitivity for the routine quantification of γ‐T3 in rat and human plasma. The separation of γ‐T3 from the plasma components was achieved with a C₁₈ reversed‐phase column with an isocratic elution using a mixture of methanol, ethanol and acetonitrile (85:7.5:7.5, v/v/v) with a UV detection at 295 nm. γ‐T3 was extracted from rat and human plasma by liquid–liquid extraction with an average recovery of 60%. The method proved linear in the range 100–5000 ng/mL. The inter‐day precision ranged from 5.8 to 12.8% and the accuracy ranged from 92.4 to 108.5%, while the intra‐day precision ranged from 0.7 to 7.9% in both rat and human plasma. This data confirm that the developed method has a satisfactory sensitivity, accuracy and precision for the quantification of γ‐T3 in plasma. To assess its applicability the method was successfully applied to the quantitative analysis for pharmacokinetic studies of γ‐T3 in rats administered a 10 mg/kg single oral dose. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of an RP‐HPLC method for the quantitation of Orteronel (TAK‐700), a CYP17A1 enzyme inhibitor,in rat plasma and its application to a pharmacokinetic study

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography assay method has been developed and validated for the estimation of Orteronel in rat plasma. The bioanalytical procedure involves extraction of Orteronel and phenacetin (internal standard) from rat plasma with a simple liquid–liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1 mL/min and a C₁₈ column maintained at ambient room temperature. The eluate was monitored using a photodiode array detector set at 242. Orteronel and internal standard eluted at 4.8 and 6.2 min, respectively and the total run time was 9 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 100–3149 ng/mL (r² = 0.995). The intra‐ and inter‐day precisions were in the ranges of 0.31–7.87 and 3.97–6.35, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study of Orteronel in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of memantine in rat plasma by HPLC-fluorescence method and its application to study of the pharmacokinetic interaction between memantine and methazolamide

A sensitive high-performance liquid chromatographic method with fluorescence detection was developed to determine memantine (MT) in rat plasma. The method consists of pre-column labeling of MT with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) and a clean-up step with solid-phase extraction. A good separation of DIB-MT was achieved within 12 min on an octadecylsilica (ODS) column (150 × 4.6 mm i.d.; 5 μm) with a mobile phase of acetonitrile-water (70:30, v/v). The calibration curve prepared with fluoxetine as an internal standard showed good linearity in the range of 10-400 ng/mL (r = .999). The limits of detection and quantitation at signal-to-noise ratios of 3 and 10 were 2.0 and 6.6 ng/mL, respectively. The method was shown to be reliable with precisions of <5% for intra-day and <9% for inter-day as relative standard deviation. The fluorescence property and reaction yield of authentic DIB-MT were also examined. The proposed method was successfully applied to study the pharmacokinetic interaction between MT and methazolamide.     

A comparative pharmacokinetic study of three flavonoids and three anthraquinones in normal and gastrointestinal motility disorders rat plasma after the oral administration of Wei‐Chang‐Shu tablet using high‐performance liquid chromatography–tandem mass spectrometry

A simple, fast and reliable high‐performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification and pharmacokinetic study of three flavonoids (liquiritigenin, isoliquiritigenin and formononetin) and three anthraquinones (emodin, rhein and aloe‐emodin), which are the bioactive ingredients of Wei‐Chang‐Shu tablet found in rat plasma. After extraction by liquid–liquid extraction with ethyl acetate, chromatographic separation was achieved on an Agilent Zorbax SB‐C₁₈ column (4.6 × 150 mm, 5 μm) at a flow rate of 1 mL/min by gradient elution using 0.1% aqueous acetic acid and acetonitrile. The detection was performed using a triple quadrupole mass spectrometer equipped with electrospray ionization source in the negative ionization and selected reaction monitoring mode. Method validation was performed in terms of specificity, carryover, linearity (r > 0.99), intra−/inter‐day precision (1.0–10.1%), accuracy (relative error, <7.6%), stability (0.6–13.2%), extract recovery (74.9–91.9%) and matrix effect (89.1–109%). The lower limits of quantification of the six analytes varied from 0.92 to 10.4 ng/mL. The validated method was successfully applied to compare the pharmacokinetic properties of Wei‐Chang‐Shu tablet in normal rats and in rats with gastrointestinal motility disorders. The results indicated that there were obvious differences in the pharmacokinetic behavior between normal and model rats. This study will be helpful in the clinical application of Wei‐Chang‐Shu tablet.