| Objective: To establish the LC-MS/MS determine method of landiolol in the plasma and application the pharmacokinetics research in Chinese healthy adults. To investigation the process of the distribution and elimination of landiolol in human body, determine the pharmacokinetics characteristics, observe the effect on landiolol to the heart rate of healthy people, analysis the correlation between PK and PD, providing the basis for drug registration and rational use of drug in clinical.Methods: An open, randomized, single-cycle test was designed. A total of 40 subjects were selected. Thirty healthy volunteers(15 males, 15 females) were divided into three groups(A, B, C) randomly using DAS software according the height and weight. Each volunteer intravenous push 1 min at first, and then intravenously guttae 10 min. Intravenous push doses of the three groups were 0.03 mg/kg/min, 0.06 mg/kg/min and 0.12 mg/kg/min, intravenously guttae doses were 0.01 mg/kg/min, 0.02 mg/kg/min and 0.04 mg/kg/min respectively. Steady-state dosing group of 10 people, intravenous push 1min at first, and intravenously guttae 40 min, according to the middle dose. Blood samples were collected from elbow vein before and after landiolol administration.Put the human blood samples in a tube with pyridostigmine as dispersion stabilizer. Then preserve the tube in ice water bath, centrifugate the sample quickly within one hour. In addition, pulse values were recorded at different time points in the test.To a 300 mL aliquot of plasma samples(including pyridostigmine bromide 40 μg) and IS(Bisoprolol) solution was added and the protein was precipitated with methanol. The supernatant was diluted with 1 m L water, and then obtained was transferred to solid phase extraction column which was activated and washed by 2 m L water, eluted by 1m L of methanol(containing0.1% formic acid). The elution was injected for LC-MS/MS. Separation was achieves on a MG S-5 C18 analytical column(4.6 mm×50 mm, 5 μm). The mobile phase was composed of 10mmol/m L ammonium formate solution(containing 0.1% formic acid)-methanol(35: 65, v/v). The flow rate was 0.3m L·min-1 and the column temperature was 25℃. Quadrupole mass detector with ESI source was used as the MS/MS detection in the positive ion mode. The monitored ions were m/z 510.1â†'m/z142.8, m/z510.1â†'m/z156.8 for landiolol,m/z326.1â†'m/z116.0 for bisoprolol(internal standard). Confirm the determine method under the above conditions, the methodological study contents include specificity, linear range, limit of quantification, precision, accuracy, extraction recovery, matrix effect and sample stability test.When the plasma samples of volunteers were measured, each analysis batch setup a standard curve. At the same time, determination some of QC samples, according to the results choice the data. Plasma concentrations of volunteers were recorded into Win Nonlin 6.2(Phoenix) program, pharmacokinetics parameters were calculated by non-compartmental model(NCA). Tmax of three dose groups was statistics by the Kruskal-Wallis H nonparametric rank sum test. Cmax, AUC and dose were carried out the correlation analysis. MRT, t1/2 and CL were statistics by variance analysis. Tmax of male and female volunteers in each dose groups were statistics by Wilcoxon non-parametric rank sum test. Cmax, AUC0-t, AUC0-∞ and t1/2 were statistics by independent sample t-test. The correlation analysis using Origin Pro 8 software, which was PK parameters(Ct) and PD index(heart rate) of landiolol compared with diversity of baseline.Results: The retention time of landiolol and internal standard were 2.28 min and 2.39 min respectively. Blank plasma did not interfere with the determination of the analyses. The linear concentration ranges of the calibration curves for landiolol was 0.4-500.0 ng·m L-1. The low limit of quantitication was 0.4 ng·m L-1. The standard curve equation is: Y=0.00467274+0.0319764X(R2=0.9984). The extration recovery rates of landiolol in low, middle and high concentrations were 103.5%, 91.4% and90.1% respectively. The recovery rate of the internal standard was 98%. The matrix effect of landiolol in the three concentrations was 0.7%,-1.4% and-7.0%. The matrix effect of the internal standard rate was 2.2%. The within-day RSD was less than 12.69%, and inter-day RSD was less than 9.43%.The main pharmacokinetic parameters of the three groups(A, B, C) were as follows: Tmax were(6.3±3.6),(7.7±3.8) and(9.6±3.1) min; Cmax were(183.1±45.1),(292.1±75.4) and(581.3±152.5) ng·m L-1; AUC0-t were(2340.4±471.9),(3936.8±779.0) and(7823.5±1545.9) ng·m L-1·min, respectively. The main pharmacokinetic parameters of D group were as follows: Tmax, Cmax and AUC0-t were(8.8±9.4) min,(362.4±61.2) ng·m L-1 and(11974.9±2447.6) ng·m L-1·min, respectively. Css was about 250 ng·m L-1.Tmax of the three groups(A, B, C) using Kruskal Wallis H nonparametric test. The results showed that there was no significant difference in statistically between the three dose groups(P>0.05). MRT were(4.6±1.2),(4.6±0.9) and(5.2±1.0) min, t1/2 were(6.3±1.1),(6.2±1.2) and(6.7±1.3) min, CL were(56.9±11.2),(68.0±15.2) and(67.7±11.5) m L·min-1·kg-1, there was no significant difference in statistically between the t1/2, MRT, and CL of the three dose groups by analysis of variance. It means that landiolol hydrochloride was first-order elimination kinetics in vivo. Cmax, AUC0-t and AUC0-∞ were related with the drug dose(P<0.001), and linear regression equation were Y=10340.4X+38.47(r=0.866), Y=141863.9X+397.02(r=0.919) and Y=143513.7X+398.72(r=0.919). Linear regression equation of males were Y=11659.3X+39.74(r=0.885)(P<0.001), Y=160951.4X+352.53(r=0.941) and Y= 163120.6X+348.53(r=0.942)(P<0.001). Linear regression equation of females were Y=9021.8X+37.22(r=0.907)(P<0.001), Y=122776.6X+441.50(r=0.963)(P<0.001) and Y=123906.8X+448.92(r=0.964)(P<0.001).Pharmacokinetic parameters of the three groups volunteers(A, B, C) statistical processed by SPSS13.0 according to gender. Tmax was detected by Wilcoxon nonparametric test, results showed no statistically significantdifferences between the three dose groups(P>0.05); Cmax, AUC0-t, AUC0-∞, t1/2 of each dose group of both male and female volunteers were used for statistical analysis using independent sample t-test. The results showed that: The rest of the parameters have no statistical significance(P>0.05).After administration, with the increase of the drug in the body, the heart rate dropped significantly, showing a good dose-effect relationship. There was a negative correlation trend between the heart rate and Ct, the regression equation was: Y=-0.012X-5.057(r=-0.314)(P<0.001).Conclusion: A sensitive, accurate and reproduce LC-MS/MS method had been established for determination of landiolol in human plasma. It has been applied to the pharmacokinetics research of landiolol.There was a significant positive correlation between Cmax, AUC and the dose, and showing a first-order to eliminate in the body; There was no significant difference in the pharmacokinetic parameters between female and male volunteers. Steady-state administration group about 20 min reached steady state after administration, steady state plasma concentration was about 250 ng·m L-1.After administration of A, B, C the three groups, with the increase of drug concentration the heart rate decrease, Analyze the correlation between changes values in heart rate compared to the baseline and Ct. There was a negative correlation between PK and Ct, showing a good dose-effect relationship. |
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