| Objective:To establish a method for the simultaneous determination of dexmedetomidine,dezocine,midazolam and its metabolite 1-hydroxymidazolam in Beagle dog plasma by ultra-high performance liquid chromatography tandem mass spectrometry（UPLC-MS/MS）,and to study the pharmacokinetics of dexmedetomidine,dezocine,midazolam and 1-hydroxymidazolam in beagle dogs.To establish an UPLC-MS/MS method for the simultaneous determination of parecoxib and its metabolite valdecoxib in beagles and study the effects of dexmedetomidine on the pharmacokinetics of parecoxib and its metabolite valdecoxib in beagles.Methods:（1）The plasma of beagle dogs was precipitated with acetonitrile.The mobile phase was acetonitrile-0.1%formic acid（gradient elution）,and the flow rate was 0.4ml/min.In the positive ion mode（ES+）,dexmedetomidine,dezocine,midazolam and their metabolites 1-hydroxymidazolam and internal standard were scanned by multiple reaction monitoring（MRM）.The parent and daughter ions used for quantification were dexmedetomidine m/z 201.10→94.90,dezocine m/z 246.20→147.00,midazolam m/z 326.10→291.13,1-hydroxymidazolam m/z 341.96→324.00,the internal standard m/z 285.00→154.00,respectively.Six beagles were given dexmedetomidine,dezocine and midazolam（dexmedetomidine 0.2μg/kg,intravenous injection slowly,dezocine 0.33 mg/kg,intramuscular injection,midazolam 0.15 mg/kg,intramuscular injection）.At 0.08,0.17,0.33,0.67,1,1.5,2,3,4,6 h after injection,1.0 ml of blood was collected and put into the heparinized EP tube.The established UPLC-MS/MS method was used to simultaneously detect dexmedetomidine,dezocine,midazolam and 1-hydroxymidazolam in Beagle plasma.The main pharmacokinetic parameters of the four analytes were calculated by DAS2.0.（2）Six beagle dogs were used in this study.In the first cycle,6 beagle dogs were intramuscularly injected with 0.33 mg/kg dezocine and 0.15 mg/kg midazolam.At0.08,0.17,0.33,0.67,1,1.5,2,3,4,6,9 and 12 h after injection,1.0 ml of blood was collected into heparinized EP tube.Blood samples were centrifuged immediately and plasma was collected and stored in refrigerator at-20℃（group A）.After a week of cleaning,the same 6 beagle dogs were given 2μg/kg dexmedetomidine intravenously for 7 consecutive days.On day 7,6 beagles were intramuscularly injected with 0.33mg/kg dezocine and 0.15 mg/kg midazolam.At 0.08,0.17,0.33,0.67,1,1.5,2,3,4,6,9 and 12 h after injection,1.0 ml of blood was collected into heparinized EP tube.Blood samples were centrifuged immediately and plasma was collected and stored in refrigerator at-20℃（group B）.The plasma concentrations of dezocine,midazolam and 1-hydroxymidazolam in beagle plasma were determined by UPLC-MS/MS.The main pharmacokinetic parameters of the 3 analytes were calculated by E DAS 2.0.The pharmacokinetic parameters between group A and group B were compared.（3）The plasma of beagle dogs was precipitated with acetonitrile.The mobile phase was acetonitrile-0.1%formic acid（gradient elution）,and the flow rate was 0.4ml/min.In the negative ion mode（ES-）,the two analytes and internal standard were scanned by multiple reaction monitoring（MRM）.The parent and daughter ions for quantification were parecoxib m/z 369.1→119.1,valdecoxib m/z 313.0→118.0,and internal standard m/z 380.0→316.0,respectively.Six beagles were used in this study.In the first cycle,6 beagle dogs were intramuscularly injected with 1.33 mg/kg parecoxib.Venous blood was drawn at different time points,plasma was separated and stored for testing（Group C）.After a week of cleaning,the same 6 beagles were given 2μg/kg dexmedetomidine intravenously for 7 consecutive days.On day 7,dexmedetomidine was injected 0.5 hours after dexmedetomidine injection,and 1.33mg/kg parecoxib was injected intramuscularly.Then venous blood was drawn at different time points,plasma was separated and stored for testing（Group D）.The established UPLC-MS/MS method was used to simultaneously detect parecoxib and valdecoxib in Beagle plasma.The main pharmacokinetic parameters of the two analytes were calculated by DAS 2.0.The pharmacokinetic parameters between group C and group D were compared.Results:（1）The UPLC-MS/MS method for the simultaneous determination of dexmedetomidine,dezocine,midazolam and 1-hydroxymidazolam showed good linearity.The RSD of intra day and inter day precision was less than 10.09%,and the intra day and inter day accuracy re were between-4.66%and 7.78%.The method has high recovery and good stability,and the matrix effect does not affect the detection.（2）After intramuscular injection of dezocine,dezocine was absorbed rapidly.The Cmax of dezocine in group B was 32.48%higher than that in group A,AUC（0-t）and AUC（0-∞）were 64.44%and 65.86%higher than those in group A,t1/2was prolonged from 0.74 h to 2.27 h,and the apparent distribution volume（Vz/F）and clearance rate（CLz/F）were also decreased.After intramuscular injection of midazolam,midazolam was absorbed rapidly and metabolized into 1-hydroxymidazolam.The Cmax of midazolam in group B was 43.81%higher than that in group A,AUC（0-t）and AUC（0-∞）were 75.81%and 65.46%higher than those in group A,t1/2was prolonged from 1.18h to 2.64 h,and the apparent distribution volume（Vz/F）and clearance rate（CLz/F）were also decreased.At the same time,Cmaxof 1-hydroxymidazolam in group B was29.81%lower than that in group A,but t1/2was prolonged from 1.27 h to 2.34 h,AUC of 1-hydroxymidazolam in two groups did not change significantly.（3）The method for simultaneous determination of parecoxib and its metabolite valdecoxib in Beagle plasma by UPLC-MS/MS has good linearity.The intra day and inter day precision is less than 8.07%,and the standard error RE range is-1.20%to2.76%.The method has high recovery rate,good stability,and the matrix effect does not affect the detection.（4）After intramuscular injection of parecoxib,parecoxib was absorbed rapidly and converted into active metabolite valdecoxib.There was no significant difference in the pharmacokinetic parameters of parecoxib between group C and Group D.The Cmax and AUC（0-∞）of valdecoxib in group D were 37.79%and 36.19%higher than those in group A,t1/2increased from 2.44 h to 2.91 h,and the apparent distribution volume（Vz/F）and clearance rate（CLz/F）were also decreased.Conclusion:（1）This study established an UPLC-MS/MS method for the simultaneous determination of dexmedetomidine,dezocine,midazolam and 1-hydroxymidazolam in Beagle plasma.The method is accurate,reproducible and specific,and meets the requirements of biological sample detection.It is suitable for the pharmacokinetics and drug drug interactions of dexmedetomidine,dezocine,midazolam and1-hydroxymidazolam.（2）Dexmedetomidine can inhibit the metabolism of dezocine and midazolam in beagle dogs,and increase the exposure of dezocine and midazolam in beagle dogs.（3）The established UPLC-MS/MS method for the simultaneous determination of parecoxib and its metabolite vardicib in Beagle plasma was accurate,reproducible and specific.It was suitable for the pharmacokinetics and drug-drug interaction of parecoxib and valdecoxib.（4）Dexmedetomidine can inhibit the metabolism of valdecoxib in beagle dogs and increase the exposure of valdecoxib in beagles,but it does not affect the pharmacokinetics of parecoxib. |
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