A new high-performance liquid chromatography （HPLC） method for the quantitative determination of sitagliptin in rat plasma was developed and validated for pharmacokinetics study. The plasma was spiked with the internal standard （hydrocortisone, IS）, treated with sodium hydroxide, and extracted with ethyl acetate. The extracted analyte was injected into an Agilent Zorbax Extend-C18 column （250 mm×4.6 mm, 4 μm） maintained at 30℃ and monitored at 267 nm. The mobile phase consisting of methanol-water （60：40, v/v, containing 10 mM Tris and 10 mM triethylamine） was titrated to pH 9.0 using 1 mol/L hydrochloric acid. The flow rate was 1.0 mL/min. The method showed high specificity. Calibration curves of the peak area ratio of each analyte/IS versus sitagliptin concentration were linear in the range of 0.75-100.0μg/mL （r〉0.9957）. The lower limit of quantification （LLOQ） was 0.75 μg/mL. The intra-day and inter-day coefficient of variation was lower than 10%. The accuracy （relative recovery） at three levels was 105.3% （0.75 μg/mL）, 99.8% （10.0 μg/mL） and 99.0% （100.0 μg/mL）. The extraction recovery was 81.5%, 82.4% and 84.5% at the concentrations of 0.75, 10.0 and 100.0 μg/mL, respectively. The short-term, long-term, freeze-thaw storage stability of plasma samples and the stability of standard solutions were satisfactory. This HPLC method is suitable for determining the concentration of sitagliptin in rat plasma and it was applied to determine the concentration-time profiles of sitagliptin in rat plasma following oral administration of sitagliptin.
An efficient and sensitive ion-pair HPLC-UV method using atenolol as internal standard （IS） was developed and validated for the determination of metformin in the plasma of diabetic rats. Plasma samples were deproteinated with 10% （v/v） perchloric acid. Separation was achieved on a UltimateTM AQ-C18 column （250 mm×4.6 mm, 5 μm） with a mobile phase （pH 5.05） composed of acetonitrile-water （31：69, v/v, containing 0.002 M sodium dodecyl sulfate, 0.0125 M potassium dihydrogen phosphate, 0.015 M triethylamine） at a flow rate of 1.0 mL/min. The calibration curve was linear （r〉0.994） between 7.5 and 4000 ng/mL. The lower limit of quantification （LLOQ） was 7.5 ng/mL. The precision was validated and the relative standard deviation was in the range of 1.87% to 15.70%; the accuracy was between 93.98%-106.89%. The mean recoveries were 95.40% and 95.31% for metformin and IS, respectively. The relative error （RE） of stability at different storage conditions was within ±9.00%. This method was used to determine the concentration-time profile of metformin in diabetic rat plasma following an oral administration of metformin at the dose of 10 mg/kg. Our results indicated that ion-pair HPLC-UV method using UltimateTM AQ-C18 column was effective for the pharmacokinetic studies of high polarity compounds like metformin.
A new HPLC-UV method was developed and validated for the quantitative determination of epidermal growth factor receptor inhibitor erlotinib in the plasma of tumor bearing BALB/c nude mice.Erlotinib and its internal standard l-（3-（（6,7-bis（2- methoxyethoxy）quinazolin-4-yl）amino）phenyl）ethanone were extracted from mice plasma samples using liquid-liquid extraction with a mixed solvent of methyl t-butyl ether and ethyl acetate（9：1,v/v）.Chromatographic separation was performed on a Luna C｜_（18）column（4.6 mm×250 mm,5μm）with acetonitrile：5 mM potassium phosphate buffer pH=5.2（41：59,v/v）as the mobile phase.UV detector was set at the wavelength of 345 nm,and the flow rate was 1.0 mL/min.The calibration curve was linear over the range of 20-10 000 ng/mL with acceptable intra-and inter-day precision and accuracy.The intra-day and inter-day precisions were within the range of 1.69%—5.66%,and the accuracies of intra-and inter-day assays were within the range of 105%—113%. The mean recoveries were 85.2%and 96.1%for erlotinib and IS,respectively.This method was successfully applied to a pharmacokinetic study in tumor bearing BALB/c nude mice following single oral administration at the dose of 12.5 mg/kg. The main pharmacokinetic parameters were as follows：C_（max）was 4.67μg/mL,T_（max）was 1.0 h,T_（1/2）was 2.78 h,and AUC_（0-24h）was 18.0μg/mL·h.
A simple, rapid and sensitive LC-MS/MS method was developed to quantify erlotinib and its active metabolite, OSI-420, simultaneously in BALB/c nude mice plasma. Erlotinib, OSI-420 and propranolol （internal standard） were extracted from nude mice plasma samples by liquid-liquid extraction. Separation was achieved on a reversed phase ClS column with a mobile phase of acetonitrile-water （35：65, v/v） containing 5 mM ammonium formate （pH = 3.0）. All compounds were monitored by mass spectrometry with electrospray positive ionization. The lower limit of quantification was 0.5 ng/mL for both erlotinib and OSI-420; accuracy was estimated by relative error, which was in the range from 0.07% to 8.00% for erlotinib and -2.83% to 6.67% for OSI-420; precision was validated by relative standard deviation, which was from 2.28% to 15.12% for erlotinib and from 1.96% to 11.50% for OSI-420. This method was applied to a pharmacokinetic study of BALB/c nude mice following oral administration of erlotinib at 12.5 mg/kg. A 2-compartment model was used to fit the pharmacokinetics of erlotinib and 1-compartment model for the pharmacokinetics of OSI-420. The ratio of the active metabolite to parent drug in mice was greater than previously reported in humans and probably reflects interspecies difference in the rate of conversion of erlotinib to OSI-420.
