A rapid GC-FID method was developed to simultaneously determine residual levels of triethylamine(TEA),1,1,3,3-tetramethylguanidine(TMG),and diisopropylamine(DIPA)in the synthetic route of an active pharmaceutical ingredient(API).Due to the severe absorption of amines on GC stationary phases,GC columns with various stationary phases were evaluated for optimal peak shape and reproducibility.The final conditions used the Agilent CP-Volamine column to resolve the three amines in 12 min.Various inlet liners were also screened to further improve the sensitivity of the analysis.The Restek Siltek? liner was selected to achieve the desired detectability for the method.The quantitation limits were 4,3,and 4 μg/mL for TEA,DIPA,and TMG in the presence of API,respectively.All three amines showed good linearity(r＞0.999)and recoveries(＞90％)over the concentration range of 3 to 16 μg/mL.The testing of residual amines was initially performed at the penultimate stage of the synthesis.However,this work demonstrates that TMG can act as a proton sponge to react with salicylic acid,the counter ion of the penultimate,to form a volatile component that elutes at a different retention time.Consequently,in the final method,these three amines were monitored in the final API to circumvent the matrix interference.Key parameters of the method were qualified per method validation requirements in ICH guidelines.The method was successfully applied for batch testing during development and implemented as an in-process control procedure at manufacturing sites.

Objective To investigate the role of miR-10a in CD4+CD25+Treg-mediated immunosuppression during sepsis and its potential role in immunotherapy for sepsis.Methods Sepsis mouse model was established by cecal ligation and puncture(CLP).Balb/c mice of clean grade were sacrificed 1,3,5,and 7 days after operation.Blood as well as spleen samples were harvested at given intervals.The splenic CD4+CD25+Treg cells and CD4+T cells were isolated by MACS microbeads.Cells were cultured,and phenotypes were analyzed by flow cytometry.The miR-10a expressed in Treg cells were detected by Real-time PCR.After administration of LV-mmu-miR-10a-5p-inhibition,the immunosuppressive function have been detected.Statistical analyses were performed using one-way analysis of variance (SPSS 19.0,Chicago,USA) test followed by Dunnett-t test to compare among three or more groups or by Student's t-test to compare between two groups.Results The percentages of splenic Tregs (CD4+CD25+/CD4+T) was (7.34±1.2)％ in normal group,and the increase in percentage of Tregs in spleen has been observed in septic mice (P＜0.05).The mean fluorescence intensity (MFI) of Foxp3+Treg was increased in septic mice compared with sham group (P＜0.05).The expression of miR-10a was significantly elevated on CLP 1-7 day (P＜0.05).After down-regulation of miR-10a in septic mice,the percentages of Tregs (CD4+CD25+/CD4+T) was significantly increased in septic mice (P＜0.05),the MFI of Foxp3+Treg was increased in septic mice compared with control group (P＜0.05).The CD4+T cell proliferative activity in CLP-induced mice was significantly suppressed on CLP 3 day compared with sham group (P＜0.05).After down-regulation of miR-10a in septic mice,the CD4+T cell proliferative activity was significantly suppressed compared with control group (P＜0.05).Conclusions Treg plays a critical role in immunosuppression in septic mice.Inhibition of miR-10a in vivo could enhence immunesuppression of CD4+CD25+Treg.Therefore miR-10a may participate in the regulation of CD4+CD25+Treg immunosuppression in sepsis and become the target for immunotherapy.