| 1. The analytical method of domperidone and omeprazole in rat’s plasma and mice’s tissueA liquid chromatography coupled with tandem mass spectrometric(LC-MS/MS) method was developed and validated for the quantification of domperidone in rat’s plasma and mice’s tissue. Chromatography was performed with a mobile phase consisting of methanol-15 mmol·L-1 ammonium acetate(90:10, v/v/v).The plasma concentration of domperidone were determined by taking ambroxol as internal standard. A triple quadrupole tandem mass spectrometer equipped with Turbo Ionspray Source was used as detector and was operated in the positive ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 426.3→m/z 175.2 and m/z 379.1→m/z 264.0 was performed to quantify domperidone and the internal standard ambroxol, respectively. The calibration curve of domperidone in rat’s plasma and mice’s tissue were linear in the range of 1.0-500 ng·mL-1 and 0.01-5.0μg·g-1, respectively. The lower limit of quantification were 1.0 ng·mL-1 and 0.01μg·g-1, respectively. The intra-and inter-day precision(RSD) were below 13% and 10%, and the accuracy were between±6% and±4 %, respectively. The recovery of extraction were more than 63% and 69%, respectively.An HPLC-UV method was developed for the quantification of omeprazole in rat’s plasma and mice’s tissue. The chromatographic separateion was performed on the colume of Diamonsil C18 (200mm×4.6mm,5μm) with UV detection at 302 nm, and the mobile phase was methanol-0.05 mol·L-1 potassium dihydrogen phosphate buffer (65:35, v/v/v),flow rat at 800μL·min-1.The plasma concentration of omeprazole were determined by taking phenacetin as internal standard.The calibration curve of omeprazole in rat’s plasma and mice’s tissue were linear in the range of 0.05-10.0μg·mL-1 and 0.5-100μg·g-1, respectively. The lower limit of quantification were 0.05μg·mL-1 and 0.5μg·g-1, respectively. The intra-and inter-day precision(RSD) were below 10% and 8%, and the accuracy were between±10% and±4%, respectively. The recovery of extraction were more than 76% and 71%, respectively.2. Study on the pharmacokinetics of domperidone in rat’s plasmaThe blood concentration of domperidone reaches the maximum between 0.57 to 0.93 h after i.g. administration to healthy rat at the dosage of 0.9,1.8 and 3.6 mg·kg-1. There had no relationship with Tmax and dosage. Cmax were 57.8±6.75,103.0±26.6 and 214.1±28.8 μg·L-1, MRT0-t were 7.58±1.02,8.00±0.75 and 5.76±0.99 h, AUC0-t were 341.1±57.7, 452.0±90.8 and 873.5±142.5μg·h·L-1, Vd/F were 6.13±2.41,8.06±2.91 and 6.62±1.22 L, Cl/F were 0.47±0.08,0.77±0.13 and 0.82±0.15 L·h-1, t1/2 were 9.38±4.82,7.26±1.92 and 5.70±1.29 h, respectively. The variance of the ratio of Cmax, AUC0-t to the dose in 3 groups showed there have no significance difference with each other(P>0.05), which showed that the process of domperidone in rat conformed to the character of linear pharmacokinetics characteristic. It shows a two-compartment model characteristics in rats of domperidone. The half-life of absorption phase was low, and the half-life of elimination phase was more longer than distribution phase. It shows a higher affinity to tissue according to Vd.3. Study on the pharmacokinetics in rat’s plasma and distribution in mice’s tissue of domperidone combined with omeprazoleThe pharmacokinetics parameters of domperidone in healthy rats after i.g. administration to domperidone alone at the dosage of 1.8 mg·kg-1 and combination of omeprazole (3.6 mg·kg-1)and domperidone (1.8 mg·kg-1) were as follow:Tmax were 0.80±0.11 and 1.20±0.45 h., Cmax were 103.0±26.6 and 72.6±9.23μg·L-1, MRT0-t were 8.00±0.75 and 9.36±0.82 h, AUC0-t were 452.0±90.8 and 465.0±30.3μg·h·L-1, Vd/F were 8.06±2.91 and 8.80±2.97 L, Cl/F were 0.77±0.13 and 0.71±0.06 L·h-1, t1/2 were 7.26±1.92 and 8.58±2.94 h. The statistic analysis showed that Cmax of combined group decreased by 29.5%(P<0.05), and the rank sum test showed that Tmax of combined group decreased significantly (P<0.05). There were no significant differences of the other pharmacokinetics parameters between the two groups (P>0.05).After i.g. administration to domperidone alone at the dosage of 2.6 mg·kg-1 and combination of omeprazole (5.2 mg·kg-1) and domperidone (2.6 mg·kg-1) in healthy mices, the tissue concentration of domperidone reached the maximum in stomach,intestine and liver at 0.25 h. And the highest concentration distributed in kidney and heart attained at 0.75 h after i.g. administration. The orders of the concentration in each tissue at 0.25,0.75 and 12 h were as follow:stomach> intestine>liver> kidney>heart, stomach> intestine> kidney> heart> liver, kidney, heart> intestine> liver> stomach, respectively. There were no significant differences of the concentration of domperidone between the two groups(P>0.05).The pharmacokinetics parameters of omeprazole in healthy rats after i.g. administration to omeprazole alone at the dosage of 3.6 mg·kg-1 and combination of omeprazole (3.6 mg·kg-1)and domperidone (1.8 mg·kg-1) were as follow:Tmax were 2.10±0.22 and 1.40±0.22 h., Cmax were 3.62±1.10 and 3.53±0.81μg·mL-1, MRT0-t were 3.73±0.23 and 3.24±0.21 h, AUC0-t were 14.0±4.19 and 11.7±7.36 mg·h·L-1, Vd/F were 149.9±51.3 and 120.1±47.2 mL, Cl/F were 54.2±17.6 and 48.2±12.6 mL·h-1, t1/2 were 1.98±0.56 and 1.73±0.30 h. The statistic analysis showed that MRT0-t of combined group decreased significantly, and the rank sum test showed that Tmax of combined group decreased significantly(P<0.05). There were no significant differences of the other pharmacokinetics parameters between the two groups(P>0.05).After i.g. administration to omeprazole alone at the dosage of 5.2 mg·kg-1 and combination of omeprazole (5.2 mg·kg-1) and domperidone (2.6 mg·kg-1) in healthy mices, the tissue concentration of omeprazole reached the maximum in stomach and intestine at 1 h. And the highest concentration distributed in heart, kidney and liver attained at 2 h after i.g. administration. The orders of the concentration in each tissue at 1,2 and 8 h were as follow:stomach> intestine>heart>liver> kidney, stomach> intestine> liver> kidney> heart, stomach, intestine> liver, kidney> heart, respectively. The statistic analysis showed that the concentration of omeprazole of combined group increased by 57%(P<0.05) in kidney. There were no significant differences of the concentration of omeprazole between the two groups(P>0.05).4. The pharmacokinetics and bioavailability of domperidone in humanAn HPLC-MS/MS method has been established for the determineation of domperidone in human plasma. Chromatography was performed with a mobile phase consisting of methanol-water-formic acid (95:5:0.05,v/v/v),delivered at 100μL·min-1.The plasma concentration of domperidone were determined by taking ambroxol as internal standard. A triple quadrupole tandem mass spectrometer equipped with Turbo Ionspray Source was used as detector and was operated in the positive ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 426.4→m/z 175.0 and m/z 379.0→m/z 264.0 was performed to quantify domperidone and the internal standard ambroxol, respectively. The calibration curve of domperidone in human plasma were linear in the range of 0.5-50 ng·mL-1. The lower limit of quantification were 0.5 ng·mL-1. The intra-and inter-day precision(RSD) were below 14%, and the accuracy were between±7%. The recovery of extraction were more than 61%.A single oral dose of 10mg domperidone for the test and reference preparations were administrated to 20 healthy volunteers in a randomized cross-over design to study its pharmacokinetics and relative bioavailability. Main pharmacokinetic parameters of domperidone for the test and reference drugs were as follows:Tmax were 0.75±0.24 and 0.83±0.28 h, Cmax were 17.0±4.83 and 15.9±4.18μg·L-1, MRT0-t were 8.01±1.64 and 7.95±1.40 h, AUC0-t were 104.5±28.6 and 104.1±31.4μg·h·L-1, Vd/F were 1186.1±523.6 and 1044.0±326.6 L, Cl/F were 90.6±26.0 and 96.2±26.8 L·h-1, t1/2 were 9.78±4.66 and 7.70±1.88 h. The relative bioavailability of the test drug is 94%-109%. The main pharmacokinetics parameters were analyzed by ANOVA after log transformation. Adopted two one-side test and (1-2α) confidential interval test, AUC0-t, AUC0-∞and Cmax of test preparation both rejection the assumption of inequivalence. Cmax of test preparation was 70 %-143% of reference preparation by 90% confidence interval test. AUC0-t and AUC0-∞of test preparation was 80%-125% of reference preparation by 90% confidence interval test. So the test and reference preparations were bioequivalence. It shows a similar characteristics in two-compartment model and Vd in healthy volunteers compared to rats. The larger standard deviation of bioavailability and pharmacokinetics parameters of domperidone shows that there have an obviously individual differentce.
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