Study On Pharmacokinetics Of Compound Metoprolol In Healthy Volunteers

AIM: To develop and validate a rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the method based on simultaneous quantitation of hydrochlorothiazide and metoprolol in human plasma and to apply the method in a pharmacokinetic study of a combination dosage form of hydrochlorothiazide and metoprolol in 30 healthy volunteers.STUDY METHOD: Hydrochlorothiazide (HCT), metoprolol (MET) and two internal standards (I.S.) 5-bromouracil (BRU) and tramadol (TRA) were extracted from human plasma by liquid- liquid extraction using diethyl ether-dichloromethane (60:40, v/v), and separated on a Venusil MP-C18 column using a mobile phase of methanol-ammonium acetate (10 mM)-formic acid (50:50:0.05,v/v/v) at a flow rate of 0.8 mL/min. The column eluant was split 1:1 (v/v) so that approximately 0.4 mL/min entered the mass spectrometer. Under these conditions, HCT and BRU co-eluted at 1.74-1.82 min and MET and TRA co-eluted at 2.44-2.46 min. The cycle time of the assay was 3.0 min. It included a switch from the negative to the positive ion mode at 2.1 min. Detection was carried out by liquid chromatography–tandem mass spectrometry in the MRM mode with an ESI interface. MRM was performed using the folllowing mass transition ion-pairs, HCT: m/z 296.1→269.0; MET: m/z 268.3→116.2; BRU: m/z 188.8→78.8; TRA m/z 264.3→58.2. Assay validation showed the assay performed within the guidelines of the FDA.Thirty healthy volunteers were randomly divided into three groups (n=10; 5 men, 5 women), and administered either one tablet (HCT 25 mg, MET 50 mg) or two tablets twice a day for a total of 8 days. The concentrations of hydrochlorothiazide and metoprolol in plasma samples at different times were determined by LC/MS/MS to generate plasma concentration-time profiles. Pharmacokinetics parameters based on these plasma concentration-time data were statistically analyzed to determine differences in pharmacokinetic parameters between the low and high dose, the single and multiple doses, males and females.RESULTS: A rapid,sensitive and robust LC/MS/MS method has been developed and validated for the quantitation of HCT and MET in human plasma. This method was linear in the concentration range 3.00–1000 ng/mL for both analytes with a lower limit of quantitation (LLOQ) for both of 3.00 ng/mL. Accuracy was in the range±3.79% and the intra- and inter-day precision was <6.17%. The sample preparation method was simple and cost-effective. Stability studies showed that both drugs were stable. This method satisfies the relevant national standards and is applicable to pharmacokinetic studies of a combination of HCT and MET.After administration of 1 or 2 tablets of the combined dosage form, pharmacokinetic parameters of HCT were as follows(Mean±SD): T_max: 2.3±0.8h and 2.0±0.4 h; C_max: 215.5±54.09 ng/mL and 544.80±98.27 ng/mL; t_1/2: 7.93±1.21 h and 8.29±1.05h; CL: 262.5±63.7 mL/min and 244.2±59.2 mL/min; Vd: 180.4±50.6L and 176.1±61.6 L; AUC0-t: 1638.65±398.98 ng·h/mL and 3533.97±863.18 ng·h/mL; AUC0-∞: 1690.44±402.22 ng·h/mL and 3593.95±869.38 ng·h/mL.Pharmacokinetic parameters of MET were as follows: T_max: 1.6±0.6 h and 1.5±0.5 h;C_max: 116.68±42.98 ng/mL and 179.36±101.04 ng/mL; t_1/2: 4.42±1.25 h and 4.42±1.25 h; CL: 416.4±545.6 mL/min and 2580.7±1903.1 mL/min; Vd: 211.4±124.0 L and 686.1±373.8 L; AUC0-t: 912.62±602.69 ng·h/mL and 1032.30±819.40 ng·h/mL; AUC0-∞: 957.26±616.69 ng·h/mL and 1073.58±826.06 ng·h/mL.After administration of a multiple dose of the combination, pharmacokinetic parameters for HCT and MET were as follows: T_max: 1.6±0.6 h and 1.47±0.50 h; C_max: 397.70±158.57 ng/mL and 157.11±65.38 ng/mL; t_1/2: 10.15±1.70 h and 5.28±1.16 h; CL: 127.5±26.8 mL/min and 755.8±394.9 mL/min; Vd: 114.6±41.8L and 314±113.9L; AUC0-τ: 2211.27±495.86 ng·h/mL and 1036.84±435.65 ng·h/mL。Statistical analysis of pharmacokinetic parameters showed there was no effect on gender (P>0.05).There was no difference in pharmacokinetic parameters of HCT (P>0.05) for doses of one and two tables, and HCT showed linear pharmacokinetics. There was no difference in t_1/2 and T_max for MET. But, multiple doses showed C_max and AUC_0-∞ increased less then expected, but the CL and Vd were significantly increased with the dose.After single and multiple doses,statistical analysis showed no difference in the pharmacokinetics of MET (P>0.05). DF decreased and R was1.41, indicating there was slight accumulation of MET. As for HCT, C_max and AUC0-τincreased significantly,whereas CL and Vd decreased significantly,and t_1/2 was prolonged. Although DF decreased, the accumulation of HCT was not negligible, suggesting that the time interval-τshould be adjusted to reduce drug accumulation.