A method for determining activity-inducing substances in polypeptides
A technology for inducing substances and activities, applied in the field of determination of active inducing substances in polypeptides, can solve problems such as no quality standards to check, and achieve the effects of good accuracy, strong specificity and improved efficiency
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Embodiment 1
[0024] A method for measuring activity-inducing substances in polypeptides, comprising the steps of:
[0025] S1. Take an appropriate amount of IPTG reference substance, add 50% acetonitrile to dissolve it, and make a solution containing 0.1 μg of IPTG per 1 mL. Solution, as need testing solution;
[0026] S2, prepare two parts of IPTG reference substance solutions in parallel, detect through UPLC-MS method, obtain the peak area of IPTG in the reference substance solution respectively, calculate the ratio of the concentration of peak area and reference substance solution, obtain the response factor of IPTG;
[0027] S3. Get the test solution and detect it by UPLC-MS method to obtain the peak area of the test solution, and use the response factor to calculate the IPTG content in the test solution.
[0028] Specifically, the chromatographic conditions of the UPLC-MS method in steps S2 and S3 are as follows: a HILIC chromatographic column is used, the column temperature is 3...
Embodiment 2
[0034] The linear range investigation of the assay method that embodiment 1 provides
[0035] Take 10mg of IPTG in a 10mL volumetric flask, dissolve and dilute to the mark with 50% acetonitrile, shake well, and use it as reference substance stock solution 1; accurately measure 100 μL of reference substance stock solution 1, place it in a 10mL volumetric flask, and dilute with 15% acetonitrile to the scale line, shake well, as the reference substance stock solution 2; accurately measure 1000 μL of the reference substance stock solution 2 and place it in a 10mL volumetric flask, dilute with 15% acetonitrile to the mark line, shake well, and use it as the reference substance stock solution 3; Pipette 0.1mL, 0.2mL, 0.5mL, 0.8mL, 1mL, 1.5mL, and 2mL of reference substance stock solution 3 into 10mL volumetric flasks, dilute to the mark with 15% acetonitrile, shake well, and mark them as L-10 , L-20, L-50, L-80, L-100, L-150 and L-200.
[0036] The assay method that embodiment 1 pr...
Embodiment 3
[0040] The sample addition recovery test of the assay method that embodiment 1 provides
[0041] Through the method of sample addition and recovery, analyze the accuracy of the assay method provided in Implementation 1, take 2 mg of the polypeptide preparation, accurately weigh, and parallel 11 copies, take 2 of them, and measure the IPTG content in the sample; take 3 of them, Dilute with L-50, constant volume and mix uniformly, as 50% standard addition recovery rate solution; take 3 parts of them, respectively dilute with L-100, constant volume and mix uniformly, as 100% standard addition recovery rate solution; Among them, 3 parts were diluted with L-150, constant volume and mixed uniformly, as 150% standard addition recovery rate solution. Measured by the UPLC-MS method of Example 1, the results are shown in Table 3, the results show that the IPTG recovery rate is 109~122%, and the recovery rate RSD% of 9 needles is 4, showing that the assay method provided by Example 1 has...
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