| Moxidectin and Imidacloprid are used for the prevention and treatment of parasitic diseases on the surface and in vivo of dogs and cats.In order to study the stability and pharmacokinetics of the transdermal preparation of Moxidectin and Imidacloprid,the following test contents were carried out:1.Establish a high performance liquid chromatography method for the detection of Imidacloprid in animal plasma.In order to facilitate the detection of Imidacloprid in animal plasma in laboratories without mass spectrometry conditions,a high performance liquid chromatography method for detecting Imidacloprid in animal plasma has been established.Plasma samples of rabbits,beagle dogs and rats were extracted with acetonitrile and centrifuged at 12000 r min-1 for 10 min.Transfer the supernatant to another centrifuge tube and blow dry with nitrogen at 50°C.After reconstituted with 1 m L of mobile phase,pass a 0.45μm water-based filter membrane to the sample bottle to be tested.The chromatographic conditions are:Agilent Exctend-C18 column（4.6 mm×150 mm,5μm）,column temperature was 25℃;mobile phase was methanol-acetonitrile-water（10:25:65,v/v/v）,flow rate was0.8 m L min-1;detection wavelength was 270 nm,injection volume was 10μL.The results showed that the limit of detection（LOD）and the limit of quantitation（LOQ）of Imidacloprid in the plasma of the three animals were both 2 ng m L-1 and 10 ng m L-1.In the concentration range of 0.01-100μg m L-1,the linear relationship was good（R2=1）.At 0.05,0.5,5,and 50μg m L-1 addition levels,the plasma recoveries of rabbits,beagle dogs and rats were 82%-115%,the intra-day coefficient of variation ranges from 0.93%to 7.56%,and the inter-day coefficient of variation ranges from 1.21%to 2.28%.Plasma samples were stable under the room temperature,repeated freeze-thaw and room temperature after treatment,with coefficients of variation below 10%.Studies have shown that the established high performance liquid chromatography method for the detection of Imidacloprid in animal plasma meets the methodological requirements,and can be used for simple,sensitive and rapid detection of Imidacloprid content in animal plasma and for the pharmacokinetic analysis of Imidacloprid.2.Establish a method for simultaneous detection of Moxidectin and Imidacloprid in rat plasma by liquid chromatography-tandem mass spectrometry.In order to study the pharmacokinetics of Moxidectin and Imidacloprid in rats,a method for the simultaneous detection of Moxidectin and Imidacloprid in rat plasma was established by liquid chromatography-tandem mass spectrometry.After the rat plasma sample was extracted with acetonitrile,it was centrifuged at 12000 r min-1 for 10 min.Transfer the supernatant to another centrifuge tube and blow dry with nitrogen at 50°C. After reconstituted with 1 m L of acetonitrile,pass a 0.45μm organic filter membrane to the sample bottle to be tested.The chromatographic conditions were:Agilent RRHD Eclipse-C18 column（2.1 mm×50 mm,1.8 um）,column temperature is 30℃;formic acid-acetonitrile（50:50,v/v）as mobile phase,flow rate is 20μL min-1,the injection volume was 5μL.Mass spectrometry conditions:positive ion mode,collision energy voltage of Moxidectin and Imidacloprid were 12 V and 10 V,capillary voltage were 2.5 V and 5 V,multi-reactive ion monitoring mode.The results showed that the LOD and LOQ of Moxidectin and Imidacloprid in rat plasma were both 0.1 ng m L-1 and 0.5 ng m L-1. Within the concentration range of 1-200 ng m L-1,the linear relationship between Moxidectin and Imidacloprid were good（R2>0.999）.The recoveries of Moxidectin and Imidacloprid at the addition levels of 1,5,10 and 50 ng m L-1 were 95.10%-103.45%and96.12%-103.44%,and the intra-day coefficient of variation were 0.45%-3.06%and 1.03%-4.16,the inter-day coefficient of variation were 0.62%-3.64%and.93%-4.04. Plasma samples were stable under the room temperature,repeated freeze-thaw and room temperature after treatment,with coefficients of variation below 10%.Studies have shown that the established liquid chromatography-tandem method for the simultaneous determination of Moxidectin and Imidacloprid in rat plasma met the requirements of methodology,and could be used for the determination of Moxidectin and Imidacloprid in rat plasma and the pharmacokinetic analysis of Moxidectin and Imidacloprid.3.Study on the stability of transdermal preparations of Moxidectin and ImidaclopridThree batches of samples were prepared and placed in the drug stability instrument for 10 days under the conditions of 60±2℃,4500±500 Lx,4500±500 Lx and 60±2℃ respectively,to determine the influencing factors test;the prepared three batches of samples were placed in the drug stability instrument for 6 months under the conditions of a temperature of 40±2°C and a relative humidity of 75±5%,and an accelerated stability test was carried out;the three batches of samples prepared were placed in a drug stability tester for 12 months at a temperature of 25±2°C and a relative humidity of 60±10%for long-term stability tests.Through character observation,HPLC detected the changes in the content of Moxidectin and Imidacloprid in the samples,and the stability of the self-made Moxidectin and Imidacloprid transdermal agents was tested and investigated.The results showed that there was no significant change in the properties of the samples,and the changes in the content of Moxidectin and Imidacloprid were both less than 5%,which met the requirements for the stability of pharmaceutical preparations.4.Study on the pharmacokinetics of transdermal preparations of Moxidectin and Imidacloprid in ratsEight rats weighing 231.50±17.90 g were selected,half male and half female. According to a single administration of 0.1 m L kg-1 bw percutaneously,the established liquid chromatography-tandem mass spectrometry was used to determine the drug concentration of Moxidectin and Imidacloprid in plasma,and Winnonlin 5.2 was used to calculate the pharmacokinetic parameters.After the administration,the rats were in good mental state and had no adverse reactions.Pharmacokinetic data analysis showed that the Tmax of Moxidectin and Imidacloprid in rats were 1.16±1.99 day and 0.90±2.06 day;Cmax were 453.99±168.44 ng m L-1 and 792.54±205.33 ng m L-1;T1/2βwere 25.76±7.81day and 27.15±17.41 day;CL were 4245.77±1701.65 m L day kg-1 and 10459.24±3864.14 m L day kg-1;Vd were 159770.43±87653.16 m L kg-1 and 390350.62±289867.83m L kg-1;Auc0-t were 1964.47±2631.76 day ng m L-1 and 2143.46±1016.95 day ng m L-1;MRT0-t were 28.50±11.91 day and 28.23±23.07 day.Some pharmacokinetic parameters of Moxidectin and Imidacloprid were different between male and female rats.The Cmax of Moxidectin in the plasma of male rats were extremely significantly lower than that of female rats（P<0.01）;the T1/2βand Vd of Imidacloprid in male rats were extremely significantly higher than that of female rats（P<0.01）. |
