Determination And Pharmacokinetic Study Of Medicine In Biological Samples By High Performance Liquid Chromatography-tandem Mass Spectrometry

ObjectiveTo develop and validate rapid, sensitive and specific methods for the determination and pharmacokinetics study of bivalirudin in human plasma and metformin and rosiglitazone in human urine by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).Methods(1) Determination of bivalirudin in human plasma:①Plasma sample preparation: to0.2mL plasma in the1.5mL centrifuge tube,50μL of methanol,50μL of IS (2.166μg/mL), and0.6mL of methanol were added. The mixture was vortex-mixed thoroughly for1min, and then centrifuged at13,000rpm for10min. After centrifugation, the supernatant fluid was then transferred into another set of clean glass tubes and evaporated to dryness at40℃under a gentle stream of nitrogen. The residue was reconstituted in100μL of acetonitrile:water (20:80, v/v), and transferred to200μL glass vials. An aliquot of5μL was injected into the HPLC-MS/MS system for analysis.②Mass spectrometry: Triple-quadruple tandem mass spectrometric detection was carried out on a Sciex API4000Qtrap mass spectrometer (Applied Biosystems, CA, USA). The ESI source was set in positive ionization mode. Quantification was performed by multiple reaction monitoring (MRM) of the transitions of m/z1091.0→m/z650.3for bivalirudin and m/z662.2→m/z328.7for nafarelin (IS). The MS settings were:ion source voltage:5500V; ion source temperature:600℃; collision gas (N2):medium; curtain gas:15psi; nebulizer gas:60psi; auxiliary gas:55psi. Data acquisition and processing were performed with Analyst1.5software (Applied Biosystems, USA).③Liquid chromatography:The chromatography was performed on a SHIMADZU LC-20A DXR UFLC system with an autosampler and column oven enabling temperature control of the analytical column. Zorbax Eclipse C18column (50mm x2.1mm I.D,3.5μm, Agilent) column was employed. The column temperature was maintained at40℃. The isocratic mobile phase was acetonitrile-0.2%formic acid20:80(v/v) at a flow rate of0.35mL/min. The autosampler was conditioned at room temperature and the injection volume was5μL.④Subjects selected:The pharmacokinetic study was approved by the local Ethics Committee and all volunteers gave their signed informed consent to participate in the study according to the principles of the Declaration of Helsinki.The standards of subjects selected:30healthy volunteers, gender parity, aged and weight between20～27and42～70respectively. Subjects are restrictly controlled from having the past medical history and the addiction to alcohol. They shouldn’t use any drugs14days before the test. Tobacco, alcohol and caffeine are forbidden during the clinical trials in the hospital for drawing the blood. Strenuous exercise is also controlled.⑤administration sample collection:The pharmacokinetic study was a single-dose, open-label, randomized, complete three-way crossover study. Each subject was intravenous administrated0.25,0.5,1.0mg/kg bivalirudin (a formulation made in China) in the first, second and third period, respectively. Blood samples were collected before and5,10,15,30,45,60,90,120,180,240, and300min post-dosing. Samples were centrifuged and plasma was separated and stored at-70℃until analysis.⑥Samples analysis:Unknown plasma samples were processed according to plasma sample preparations. A standard curve was established every day with low, middle, and high quality control (QC) samples. The same batch of the QC samples and the unknown plasma samples were calculated by the standard curve. The accuracy of QC samples should be in the range of±15%.⑦Data analysis:The maximum plasma concentration (Cmax) and their time were noted directly. The elimination rate constant (ke) was calculated by linear regression of the terminal points of the semi-log plot of plasma concentration against time. Elimination half-life (t1/2) was calculated using the formula t1/2=0.693/ke. The area under the plasma concentration-time curve (AUC0-t) to the last measurable plasma concentration (Ct) was calculated by the linear trapezoidal rule. The area under the plasma concentration-time curve to time infinity (A UC0-∞) was calculated as:AUC0-∞=A UCo-t+Ct/ke.(2) Determination of metformin and rosiglitazone in human urine:①Urine sample preparation:To a0.20mL aliquot of urine sample in1.5mL centrifuge tube,50μL of internal standard (IS)(1121ng/mL),50μL of methanol,50μL of water, and400μL acetonitrile were added. The mixture was vortex-mixed thoroughly for1min and then centrifuged at13000rpm. for10min. An aliquot of the supernatant was directly injected into the HPLC-MS/MS system.②Mass spectrometry:Detection was performed on a Sciex API4000Qtrap MS system (Applied Biosystems Sciex, Ontario, Canada) equipped with a Turbo Ionspray interface. Mass spectrometer settings in positive-ion mode (ESI+) were:voltage at5000V, temperature at400℃, collision gas (N2) at medium, curtain gas at20, ion source gases at40and60. Quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z130.1→m/z60.1for metformin, m/z358.2→m/z134.9for rosiglitazone and m/z206.3→m/z59.9for phenformin. Data acquisition and processing were performed with the Analyst software.③Liquid chromatography: Chromatographic separation was performed on an SHIMADZU LC-20A DXR UPLC with an autosampler and a column oven that enabled temperature control of the analytical column. A ZorBax Eclipse XDB-C18column (50mm×2.1mm,3.5μm) was employed and maitained at room temperature. The mobile phase consisted of acetonitrile-10mM ammonium acetate+0.1%Formic acid (20:80, v/v) at an isocratic flow rate of0.25mL/min. The injection volume was5μL.④Subjects selected:The pharmacokinetic study was approved by the local Ethics Committee and all volunteers gave their signed informed consent to participate in the study according to the principles of the Declaration of Helsinki.The standards of paticipants selected:10healthy volunteers, gender parity, aged and weight between20～27and42～70respectively. Subjects are restrictly controlled from having the past medical history and the addiction to alcohol. They shouldn’t use any drugs14days before the test. Tobacco, alcohol and caffeine are forbidden during the clinical trials in the hospital for drawing the blood. Strenuous exercise is also controlled.⑤administration sample collection:The pharmacokinetic study was a single-dose, open-label, randomized, complete three-way crossover study. Each subject was orally administrated the following doses:500mg of metformin and2mg of rosiglitazone. Urine samples were collected0-2、2-4、4-6、6-8、8-12、12-24h post-dosing. Samples were centrifuged and stored at20℃until analysis.⑥Samples analysis:Unknown urine samples were processed according to plasma sample preparations. A standard curve was established every day with low, middle, and high quality control (QC) samples. The same batch of the QC samples and the unknown urine samples were calculated by the standard curve. The accuracy of QC samples should be in the range of±15%.⑦Data analysis: The elimination rate constant (ke) was calculated by linear regression of the terminal points of the semi-log plot of urine concentration against time. Elimination half-life (ti/2) was calculated using the formula ti/2=0.693/ke.ResultsIn plasma:The linear calibration curves were obtained over the concentration range of1.006-2011ng/mL for bivalirudin. The intra-and inter-day precision values (RSD) were3.2%～5.8%and6.0%～9.5%and the accuracy (RE) was2.7%～7.8%at all three QC levels. The method was applied to study the pharmacokinetics of30healthy volunteers after intravenous administrated of0.25,0.5,1.0mg of bivalirudin. Mean peak plasma levels (Cmax) of2016.5±535.97ng/mL,3788.8±797.7ng/mL,6759±1959.4ng/mL and7max of0.091±0.021h,0.1±0.035h,0.1±0.053h were observed. The mean t1/2value of0.66±0.14h,0.55±0.30h,0.75±0.41was obtained, AUC0-t and AUC0-∞were calculated to be909.0∞152.3μg·h/mL,1755.8±235.5μg·h/mL,3135.4±350.9μg·h/mL and955.7±146.9μg·h/mL,1798.1±248.7μg·h/mL, 3199.3±378.4μg·h/mL respectively.In urine:The standard calibration curves for metformin and rosiglitazone were linear in the concentration range of202.0-20200ng/ml and1.054-263.5ng/mL in human urine. The intra-and inter-day precision values (RSD) were3.7%～5.8%and4.7%～5.2%. The accuracy (RE) was-5.7%～0%and-4.8%-3.1%at all three QC levels. The main parameters obtained after single dose of500mg metformin and2mg rosiglitazone to10healthy volunteers were as follows:the value t1/2of3.14±0.5h, Urinary excretion rate of31.846±18.517%respectively.It has been reported that it was feasible to the determination of the concentrations of bivalirudin in the plasma and metformin and rosiglitazone in the urine by HPLC-MS/MS in human. Firstly, the time of each sample was only4.5min for simultaneous determination of bivalirudin in plasma or3.5min for simultaneous determination of rosiglitazone and metformin in urine. Sample throughput was greatly improved about250～300biological samples per day. Secondly, MRM can simultaneously detect the parent ion and the product ion. The Parent ion was collided and the product ion was selected to monitor. The background noise was significantly reduced. Meantime, the specificity and sensitivity was high. The lower limit of quantification for bivalirudin, metformin and rosiglitazone were1.006ng/mL,202.0ng/mL and1.054ng/mL. In addition, the sample preparation was simple that only involving one-step protein precipitation with acetonitrile or methanol. In one word, this method was simple, fast, economic, sensitivity and can be successfully applied to the pharmacokinetic study of bivalirudin, metforminand and rosiglitazone in healthy volunteers.ConclusionThe methods were proved to be of high specificity, fast, sensitivity and have been successfully utilized for the evaluation of the pharmacokinetics of bivalirudin, metformin and rosiglitazone.