Screening method and content measurement method for bordetella pertussis tracheal cytotoxin
A technology of tracheal cytotoxin and determination method, which is applied in the field of vaccine quality evaluation, and can solve problems such as difficulty in implementing the detection method of tracheal cytotoxin of B. pertussis
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Embodiment 1
[0092] 1. Experimental instruments and equipment:
[0093] High pressure binary pump, degasser, autosampler, column oven and triple quadrupole mass spectrometer.
[0094] 2. Experimental reagents:
[0095] The reference substance Bacillus pertussis tracheal cytotoxin was purified in the laboratory, and its structure was confirmed by multi-stage characterization of high-resolution mass spectrometry.
[0096] 3. Detection conditions:
[0097] Chromatographic column: stationary phase Ⅰ, R1 is silica gel;
[0098] Mobile phase: A-contain 10mmol / L ammonium formate aqueous acetonitrile (adjust pH to 4.3 with formic acid), the volume concentration of acetonitrile in the acetonitrile aqueous solution is 95%; B-10mmol / L ammonium formate aqueous solution (adjust pH to 4.3 with formic acid) ;
[0099] Specifically, the final concentration of ammonium formate in mobile phase A in aqueous acetonitrile is 10 mmol / L.
[0100] Gradient: 0-5min 80%A-20%A; Column temperature: 30°C; Flow ra...
Embodiment 2
[0125] The content of Bacillus pertussis tracheal cytotoxin in the pertussis toxin product of manufacturer 1 is detected, and the detection conditions are as follows:
[0126] Stationary phase: with stationary phase described in embodiment 1;
[0127] Mobile phase: A-contain 10mmol / L ammonium formate aqueous acetonitrile (adjust pH to 4.3 with formic acid), the volume concentration of acetonitrile in the acetonitrile aqueous solution is 95%; B-10mmol / L ammonium formate aqueous solution (adjust pH to 4.3 with formic acid) ;
[0128] Specifically, the final concentration of ammonium formate in mobile phase A in aqueous acetonitrile is 10 mmol / L.
[0129] Gradient: 0-5min 80%A-20%A; Column temperature: 30°C; Flow rate: 0.3mL / min; Injection volume: 10μL. LC-MS conditions: Ion source: ESI positive and negative ion scanning simultaneously, nebulizer flow rate 3L / min, heater flow rate 10L / min, interface temperature 200°C, DL temperature 230°C, heating module temperature 400°C, dryi...
Embodiment 3
[0132] The content of Bacillus pertussis tracheal cytotoxin in the intermediate product of pertussis toxin from manufacturer 1 is tested under the following conditions:
[0133] Stationary phase: stationary phase II;
[0134] Mobile phase: A-contain 10mmol / L ammonium formate aqueous acetonitrile (adjust pH to 4.3 with formic acid), the volume concentration of acetonitrile in the acetonitrile aqueous solution is 95%; B-10mmol / L ammonium formate aqueous solution (adjust pH to 4.3 with formic acid) ;
[0135] Specifically, the final concentration of ammonium formate in mobile phase A in aqueous acetonitrile is 10 mmol / L.
[0136]Gradient: 0-5min 5%A-30%A; Column temperature: 50°C; Flow rate: 0.3mL / min; Injection volume: 10μL. LC-MS conditions: Ion source: ESI positive and negative ion scanning simultaneously, nebulizer flow rate 3L / min, heater flow rate 10L / min, interface temperature 200°C, DL temperature 230°C, heating module temperature 400°C, drying gas flow rate 10L / min mi...
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