| AIM: To establish feasible dissoluble method and standard procedure of sGLP-1; to investigate the stability of standard solution under three stored conditions of 20℃, 4℃and -20℃; to establish detected method of HPLC-UV and feasible SPE pre-processing method of sGLP-1 with high concentration in biologic matrix; to research for detected method of HPLC-MS/MS and pre-processing method of sGLP-1 with low concentration in biologic matrical samples; to make a exploratory study of quantitative detected method of sGLP-1 in biologic base matrix.METHOD: According to primary structure of sGLP-1, a bottle of sGLP-1 powder is dissolved by water, acetonitrile, acetonitrile-water(50:50,v/v), 1 M sodium acetate, 0.01 M sodium hydroxide, 0.1 M sodium hydroxide, PBS buffer solution for pyro-steam sterilization. To inspect different chromatographic peaks of different dissolved methods and to determinate the final dissolved method of the drug and the standard operated procedure.The standard solution of sGLP-1 is stored at 20℃,4℃,-20℃respectively, and is measured at the moment of preparation, 24 h, 48 h, 72 h, one week, two weeks and one month. Three samples of every condition are derected by HPLC-UV. Every area of peek of drug is compared to the area of peek of control article for stability research.The final detected conditions of HPLC-UV are determinated through optimizing the mobile phase gradient of HPLC-UV initial condition. To investigate ultrafiltration and solid phase extraction for biologic metrical sample, meanwhile to establish every elutriants and eluants in order to select the most effective elutriants and eluants.The HPLC-MS/MS method is explored initially. To optimize the first class mass spectrum parameter fragment, ion source parameter (gas temperature, gas flow, nebulizer, capillary), the second class mass spectrum parameter collision energy, EMV, sensitivity parameter MS1 Res & MS2 Res one by one. To optimize chromatographic condition including selection of chromatographic column, flow rate, column temperature, concentration of formic acid, mobile phase gradient. Seven serial standard solutions for three continuous days by adopting the optimal result of HPLC-MS/MS method are measured. They are1 ng/mL,5 ng/mL,10 ng/mL,50 ng/mL,100 ng/mL,300 ng/mL,500 ng/mL. 1/x weighting linear regression of peak area and concentration is used to calculate the standard curve and r value. The method of quantitating sGLP-1 in biologic matrix is researched for the first time using precipitation of protein and solid phase extraction.RESULT: The peak of the standard reserved solution dissolved by 0.1 M NaOH and 0.1 M HCl (pH=5.71) is best. This dissolving method is the standard dissolving schedule. The result of essay shows: the standard reserved solution can be stored for 72 h at 20℃, at lest two weeks at 4℃and one month at -20℃.The HPLC-UV method is established with the limit of detection of 2μg/mL, as while as feasible SPE pre-processing method of sGLP-1 with high concentration in biologic matrix.The mass spectrum parameter and chromatographic parameter of HPLC-MS/MS have been optimized. After determinated seven serial standard solutions, the results demonstrate that the linearity is good among 1~500 ng/mL. But during the research the column was changed for another one with the same tipe to do and repeat the same experiments, the results are different from the former. It show that the reproducibility of HPLC-MS/MS method is not good. And there are endogenous interferes in the sGLP-1 biologic metrical samples. After all, this investigation has provided references for further research.CONCLUSIONS: The research has established the dissolving method of sGLP-1 and the standard dissolving schedule. The result of essay shows: the standard reserved solution can be stored for 72 h at 20℃, at lest two weeks at 4℃and one month at -20℃. So the concentration of sGLP-1 can be kept stable while preparing standard solutions and measuring them. The HPLC-UV method and feasible SPE pre-processing method of sGLP-1 with high concentration in biologic matrix have been established. HPLC-MS/MS method and pre-processing method of sGLP-1 with low concentration in biologic matrical samples have been explored.
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