Pharmacokinetics Profile Of Morphine-6-Glucuronic Acid Injection

Objective:In this paper,a method of high performance liquid chromatography-mass spectrum（HPLC-MS/MS）is established to determine the drug concentration of biological sample in vivo of morphine-6-glucuronic acid（M6G）,which is aimed at examining the in-vivo absorption,distribution,metabolism and excretion of M6G injection,so as to provide a scientific basis for a further research and development.Methods:（1）The analyte（M6G）and internal standard（YN-7507）in the sample of rat plasma were firstly extracted by a solid phase extraction plate and then separated by the Atlantis dC18（2.1×150 mm,5μm）chromatographic column,followed by a positive ion detection through electrospray ionization in the form of multiple reaction monitoring.The ion pair used for quantitative analysis was respectively m/z:462.2→286.1（M6G）and478.11→268.2（YN-7507）.（2）24 rats with half males and half females were selected and divided into 3 dose groups,4 animals/gender for each group,the administration dose of each group was respectively 2、4 and 6 mg/kg（in M6G dihydrate）,their administration volumes were all 4 mL/kg,the administration was done within 10-20 s.The animals were sampled in blood before the administration and on minute 2,5,15,30 and on hour 1,2,4,6 and 8 after administration,this experiment used HPLC-MS/MS method to analyze the M6G concentration in the rat plasma,the lower limit of quantitation of the method was 5ng/mL.the non-compartment model（NCA）method of WinNonlin 6.2 pharmacokinetic data analysis software was used to analyze the plasma concentration and calculate the pharmacokinetic parameters and the metabolic feature of M6G in the animal body after administration was examined.（3）30 Sprague-Dawley rats were selected with a 50-50percentage of female and male and randomly divided into 5 groups according to the gender sector,and 5 rats/gender were set up as a blank group.The animals were intravenously injected with M6G by the dosage of 4 mg/kg,the animals in distribution group were killed by euthanasia on hour 0.5,2 and 4 after administration,3 animals were killed at each time point and their plasma,brain,heart,liver,spleen,lung,kidney,stomach,small intestine,fat,muscle and gonad（male:testicle;female:ovary）were collected.The animals in bile group and part of animals in blank group replaced were collected in bile on hour 0.5,2,4 and 8after administration;the animals in the urine-faces group were collected in urine and faces on hour 4,8,24,48 and 72 after administration,3 animals/gender for each time piont.HPLC-MS/MS method was used to detect the M6G concentration in plasma sample,at the same the method of detecting M6G in each tissue,bile,urine and faces was partly verified with reference of this method,and the M6G concentration in the biological matrix above was measured.Results:（1）This method’s M6G linear range was 5-1000 ng/mL,the accuracy at each point of standard curve sample was between 92.36%and 104.18%,their linear parameters were all more than 0.99.the mean accuracy of quality-control sample（15、120、800 ng/mL）was between 98.76%and 103.40%,except for the off-limit sample,the rest samples had an accuracy between 87.32%and 111.60%.The intra-batch and inter-batch precision（%CV）ranged from 3.90%to 9.63%and 4.04%to 8.79%,respectively.the lower limit of quantitation of M6G analytic method was 5 ng/mL.the mean precision of lower limit quantitative sample was between 101.50%and 109.60%,except for the off-limit sample,the rest lower limit quantitative samples had an accuracy between 87.00%and 119.40%.The intra-batch precision for LLOQ ranged from 8.33%to 11.15%,and the inter-batch precision was 10.09%.The extraction recovery rate of low,moderate and high-concentration M6G samples was 87.51-118.27%,the precision（%CV）of extraction recoveries ranged from 6.98%-8.35%,matrix effect was 88.96%-115.82%,the precision（%CV）of matrix effect ranged from 4.91%-9.54%.The recovery of 100-fold dilution samples ranged from 101.08%-108.09%,the precision（%CV）of dilution integrity was2.35%.（2）Based on 2、4、6 mg/kg（in M6G dehydrate）administration dosage,after a single intravenous injection of M6G to rats with a concentration of 0.5、1、1.5 mg/mL,the M6G t_1/2was respectively 0.41、0.40、0.39 h,C_max was respectively 7417.16、15496.22、21920.37ng/mL,AUC(_0-8h)was 2020.0、3832.8、5513.9 h·ng/mL,AUC_inf was 2022.7、3839.1、5523.4 h·ng/mL,Vd was 573.87、612.39、608.56 mL/kg,Cl was 999.78、1069.31、1112.92mL/h/kg,MRT was 0.26、0.26、0.25 h.The animals were intravenously injected with M6G by 2、4、6 mg/kg（in M6G dehydrate）,the mean Cmax rate of male and female M6G was respectively 1.03、0.97、1.16,mean AUC_（0-8h）rate was 0.95、0.81、1.04.The animals were intravenously injected with M6G within a range of 2-6 mg/kg（in M6G dehydrate）,the peak concentration of M6G in the animal plasma and AUC increased as the dosage increased.The dosage rate between three groups was 1:2:3,the mean C_max rate of male animal M6G was 1:2.15:2.77,mean AUC_（0-8h）rate was 1:2.05:2.62;the mean C_max rate of female animal M6G was 1:2.03:3.13,mean AUC_（0-8h）rate was 1:1.74:2.85;the mean C_max rate of overall animal M6G was 1:2.09:2.96,mean AUC_（0-8h）rate was 1:1.90:2.73.（3）0.5hour after The animals were intravenously injected with M6G by the dosage of 4 mg/kg,M6G was distributed in all tissues,its relative concentration peaked in small intestine,followed by kidney,stomach and brain,it had a relatively lowest concentration in brain,the M6G concentration in each organ tissue in proper order was small intestine>kidney>stomach>liver>lung>gonad>muscle>fat>spleen>heart>brain.On hour 2after administration,the concentration of M6G rapidly went down in the plasma and all tissues,but in the brain tissue,M6G concentration went down slower,which went down by less than half relative to hour 0.5 after administration.On hour 4 after administration,except for stomach and small intestine,the M6G concentration in other tissues and plasma were all lower than or close to the quantitative lower limit.On hour 8 after administration,the mean accumulated excretion rate of M6G prototype in bile was 27.70%,the dead animals were culled,the mean accumulated excretion rate of M6G prototype was 42.77%.On hour 72 after administration,the mean accumulated excretion rate of M6G prototype in urine was 23.90%,0.04%in stool.Part of the confirmations of result of HPLC-MS/MS method of detecting the M6G in tissues,bile,urine and stool of rats revealed that,this method met the requirement of biological analysis,and was suitable for the determination of M6G in the biological matrix samples in this experiment.Conclusion:the HPLC-MS/MS method is sensitive,convenient and has a good reproducibility in measuring the M6G concentration in established biological samples,it is suitable for the detection of M6G concentration in biological sample and for pharmacokinetic study.After the rats were intravenously injected with M6G,they were widely distributed with M6G,which can be detected in all the tissues and had a relatively high concentration in small intestine,kidney,stomach and liver and a relatively low concentration in brain,M6G is removed fast in all the tissues,and it decreases as the blood concentration decreases,but it decreases relatively less in brain tissue,M6G is removed relatively slower in the brain tissue on hour 0.5 to 4 after administration.Very small amount of prototype M6G is excreted by stool,part of M6G prototypes are excreted by bile and urine.