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目的建立高效液相色谱串联三重四级杆质谱法检测重组铜绿假单胞菌外毒素A(recombinant Pseudomonas aeruginosa exotoxin A,r EPA)蛋白原液中氨苄西林残余量。方法用乙腈沉淀蛋白质,离心后收集上清,采用高效液相色谱串联三重四级杆质谱法上样分析,色谱条件:色谱柱Zorbax SB-C18 Rapid Resolution HT,1.8 Micron2.1 mm×100 mm,流动相30%乙腈,流速0.1 ml/min;质谱检测条件:多重反应监测(multiple reaction monitoring,MRM),正离子模式,ESI电压3 500 V,碎裂电压110 V,碰撞能10,气体流速8 L/min,母离子的质荷比(mass to charge ratio,m/z)=350.1,定量子离子m/z=105.8,定性子离子m/z=159.9。对建立的方法进行系统适用性、专属性、精密度、稳定性验证,确定该方法的线性范围及最低检出限、定量限,并进行基质效应试验。结果该方法试验参数符合检测要求,对氨苄西林的检测专属性强,当氨苄西林浓度在10~100 ng/ml范围内,r2>0.999 00,最低检出限为5 ng/ml,最低定量限为10 ng/ml。氨苄西林加标试验的回收率在98%~110%之间,重复性及日间精密度RSD均小于5.5%;冻融稳定性、样品前处理后的稳定性、短期稳定性试验中,样品回收率均在95%~120%之间;以超纯水5倍稀释的r EPA蛋白原液作为溶剂配制的3个不同浓度的氨苄西林对照品溶液的回收率均接近120%,RSD值在3%~5%之间,准确度和精密度良好。结论该方法系统适用性好,灵敏度高,专属性强,准确度和精密度好,检测时间短,是一种易于实现仪器自动化分析的方法。
OBJECTIVE To establish a high performance liquid chromatography tandem triple quadrupole mass spectrometry for the determination of residual ampicillin in recombinant Pseudomonas aeruginosa exotoxin A (r EPA) protein stock solution. Methods The protein was precipitated with acetonitrile and the supernatant was collected after centrifugation. The samples were analyzed by high performance liquid chromatography tandem triple quadrupole mass spectrometry. The chromatographic conditions were: column Zorbax SB-C18 Rapid Resolution HT, 1.8 Micron 2.1 mm × 100 mm, Mobile phase of 30% acetonitrile at a flow rate of 0.1 ml / min; mass spectrometry detection conditions: multiple reaction monitoring (MRM), positive ion mode, ESI voltage 3500V, fragmentation voltage 110 V, collision energy 10, gas flow 8 Mass to charge ratio (m / z) = 350.1, m / z = 105.8 and m / z = 159.9 for the parent ion. The established method for system suitability, specificity, precision, stability verification, to determine the linear range of the method and the minimum detection limit, limit of quantification, and matrix effect test. Results The test parameters of the method met the test requirements and the ampicillin test was highly specific. When the ampicillin concentration was in the range of 10-100 ng / ml, r2> 0.99900, the lowest detection limit was 5 ng / ml, and the lowest limit of quantification 10 ng / ml. The recoveries of ampicillin spiked test ranged from 98% to 110% with repeatability and daytime RSD of less than 5.5%. The freeze-thaw stability, stability after sample pretreatment, and short-term stability test The recoveries were between 95% and 120%. The recoveries of three different concentrations of ampicillin reference solution with r-EPA protein stock solution diluted 5-fold with ultrapure water as solvent were both close to 120% with RSD values of 3 % ~ 5%, accuracy and precision are good. Conclusion The method has the advantages of good system suitability, high sensitivity, strong specificity, good accuracy and precision, and short detection time. It is an easy way to automate the analysis of instruments.