| Dirithromycin is a semi-synthetic derivative of erythromycin, and a 14-membered ring macrolide antibiotic,which is converted to an active metabolite 9-(S)-erythromycylamine. There are two main types of methods on the study of bioequivalence of dirithromycin at present:molecular concentration and biological effect.The former applies to a single chemical composition in vivo bioequivalence studies and the latter applies to multi-component studies,which has similar biological effects.As a result of different study,two research methods in the evaluation of bioequivalence of drugs may be different. LC-MS/MS(MS for short) and the microbiological method are the two types of the representative method.By mass spectrometry studies and microbiological assay methods of a single oral dose of 500mg of dirithromycin blood concentration in 20 healthy volunteers,respectively,we study bioequivalence of dirithromycin and study the two methods in the evaluation of bioequivalence dirithromycin of whether there are differences.Part one:The determination of erythromycylamine in plasma by LC-MS/MS Objective:To establish a method for the determination of erythromycylamine in plasma by LC-MS/MS.Methods:The concentration of erythromycylamine in plasma was determined by LC-MS/MS.Restek Allure C18(4.6×50mm, 5.0μm) column was used as analytical column.Mobile phase was composed methanol and water(0.5%formic acid)(50:50, v/v),at a flow rate of 0.5mL/min.10μL of solution was injected and analyzed at temperature 20℃.Mass spectrometry:ESI ion source,spray voltage at 5500V,spray temperature at 550℃, atomization gas:30L/min,curtain gas:20L/min,collision gas: 12L/min;MRM,m/z735.7→m/z577.5(erythromycylamine), m/z282.2→m/z212.2(phentolamine),CID voltage:36V (erythromycylamine) and 27V(phentolamine);dwell time: 200ms.SPE was used to treat blood samples.The validation included specificity,linearity,lower limit of quantitation, precision,accuracy,sample stability,extraction recovery rate and matrix effect.Results:The method had more specificity and the endogenous impurity in plasma did not interfere with the determination by MRM.With the selected condition of chromatography and mass spectrometry,erythromycylamine and internal standard had good separation,and the response of ions were stable and sensitive.The retention time was suitable and there were no obviously matrix effect.The linear range of method was 5.0-1000.0ng/mL,and the correlation coefficient was between 0.9977-0.9994,and the model regression equation was y=1.18×10-3x-1.49×10-3,r=0.9991(n=6).The lowest limit of quantitation was 5.0ng/mL.The inter-day RSD were not more than 4.8%,the intra-day RSD were not more than 4.6%and the RE were not more than 7.6%.The extraction recovery rate of low,middle and high standard samples were 91.7%,96.3%and 93.0%and internal standard was 92.1%(n=9).Samples laid without light at room temperature within 4h,laid after pretreatment at room temperature within 24h,three cycles of freeze-thaw and freezed within 60d(-40℃),had good stability and the RSD were not more than 4.2%.Conclusion:With blood samples treated by SPE,the determination method of erythromycylamine in human plasma by LC-MS/MS was specificity,sensitivity,stability and can be used for the study of PK of dirithromycin.Part two:Study on bioequivalence of dirithromycin enteric coated tablet by LC-MS/MSObjective:To study the pharmacokinetics characteristic of erythromycylamine after a single oral dose of 500mg dirithromycin in volunteers and test the bioequivalence on the basis of major pharmacokinetics parameters.Methods:20 volunteers random were divided into two group with two-period crossover design,and every group volunteers took orally PA twice random test and reference drug on an empty stomach,crossing over after interval.The twice trial cycle time was 14 days.To take blank blood sample before taking orally and take plasma samples at 1.5,2,3,4,5,6,8,10, 12,24,48,72,96h after taking orally and all were 14.Used DAS2.0 software to calculate PK parameters and data were processed with every volunteers curve fitting to get individual parameters,then calculate mean value.Used DAS2.0 software to statistics every formulation major PK parameters Cmax and AUC to variance analysis and bioequivalent statistics,Cmax and Tmax values were actuality measured,Cmax and AUC were double half T test after logarithm transformation and Tmax were applied with non parametric method.Results:In the process of determination samples,quality control samples including low,middle,high(10.0,100.0, 800.0ng/mL) were 45,more than 5%of the total,and the RE less than 15%.All batch samples accorded with technology requirement;With AIC method,weight as 1/C2,to get the minimum value,then simulated every volunteers blood drug concentration data.Most volunteers accorded with two-compartment model,with no obviously difference in two drug parameters,such as t1/2α,t1/2βand t1/2Ka,indicated that the two drug had no difference in metabolic process;t1/2=0.693/Zeta, AUC got by trapezoidal rule.Test and reference drug major PK parameters were:34.6 and 33.1h(elimination half life),4.4 and 4.7h(Tmax),249.2 and 265.9ng/mL(Cmax),3063.0 and 3166.5ng/mL·h(AUC0.96h),3480.6 and 3561.8ng/mL·h (AUC0-∞).The Cmax[Ln],AUC0-96h[Ln]and AUC0-∞[Ln]of erythromycylamine only had individual significant difference (P＜0.05),preparation and period of time without significant difference(P＞0.05) in analysis of variance.Cmax,AUC0-96h and AUC0-∞ two one-sided tests indicated that 90%of Cmax confidence interval of test drug was between 70%-143%.90% of AUC confidence interval of test drug was between 80%-125%.Test and reference drug had bioequivalence.Conclusion:The major pharmacokinetic parameters was basic concordance in this essay with reference reports.In 20 health volunteers random single oral administration dirithromycin enteric coated tablet(T) and reference dirithromycin enteric coated tablet(R) crossover trial,T and R have bioavailability by statistical analysis.The relative erythromycylamine bioavailability of T/R was 100.7±21.7%. LC-MS/MS method was stability,accurate and reliable.It can ensure to complete determination of samples in short time.Part three:Study on bioequivalence of dirithromycin by microbiological assay and the differences evaluation of bioequivalence of dirithromycin by LC-MS/MS methodObjective:To study pharmacokinetics of dirithromycin and compare the results got by LC-MS/MS to discuss the difference of absorption and transformation in human body.Methods:Micrococcus luteus as test organism with antibioticsⅡmedium(pH7.9±0.1) were used.The validation included specificity,linearity,lowest limit of quantitation,precision, accuracy,sample stability.The concentration of erythromycylamine was determined at different time after 20 volunteers oral administration single dose 500mg test and reference drug.DAS2.0 software was used to calculate PK parameters and data were processed with every volunteers curve fited to get individual parameters,and then calculated mean value.The two groups were judged first by the F test to determine whether the homogeneity of variance.Then the results of homogeneity of variance were judged by t test.Results:Blank blood plasma had no inhibition zone,other components in blood sample did not interfere with determination.The linear range was 15.0-500.0ng/mL and the coefficient correlation was between 0.9967-0.9985 and the model regression equation was y=13.86541gC-6.0765 r=0.9978(n=6).The lower limit of quantitation was 15.0ng/mL. The intra-day RSD were 8.0%,6.0%,4.8%and the inter-day RSD were 10.0%,11.2%and 8.2%and the recovery were 110.5%,103.7%and 106.6%.Samples freezed within 90d (-40℃) had good stability and the RSD were not more than 5.0%.Test and reference drug major PK parameters were 30.96 and 31.29h(elimination half life),4.4 and 4.7h(Tmax),256.5 and 268.3ng/mL(Cmax),3026.8 and 3080.0ng/mL·h(AUC0-96h), 3669.3 and 3728.8 ng/mL·h(AUC0-∞).The Cmax[Ln], AUC0-96h[Ln]and AUC0-∞[Ln]of erythromycylamine only had individual significant difference(P＜0.05),preparation and period of time without significant difference(P＞0.05) in analysis of variance.Cmax,AUC0-96h and AUC0-∞ two one-sided tests indicated that 90%of Cmax confidence interval of test drug was between 70%-143%.90%of AUC confidence interval of test drug was between 80%-125%.Test and reference drug had bioequivalence.Two methods in the evaluation of bioequivalence of dirithromycin had no significant difference.Conclusion:After 20 healthy volunteers oral administration 500mg of dirithromycin enteric coated tablet,the microbiological assay and mass spectrometry in the evaluation of bioequivalence dirithromycin had no significant difference. The relative bioavailability was 104.9±32.8%compared with the reference drug;to dirithromycin in the human body absorption process,the microbiological assay of measured plasma concentration was significantly higher than mass spectrometry,mainly due to determination of microbiological assay include dirithromycin and erythromycylamine after taking, and it was more in line with the actual situation of the human body,but dirythromycin in the body to eliminate the process of elimination half-life was no significant difference.
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