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蛋白质原液中氨苄西林残余量

Determination of residual ampicillin content in bulk of recombinant Pseudomonas aeruginosa exotoxin A by high performance liquid chromatography-mass spectrometry/mass spectrometry

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 develop a high performance liquid chromatography-mass spectrometry / mass spectrometry（LCMS / MS）method for determination of residual ampicillin content in bulk of recombinant Pseudomonas aeruginosa exotoxin（r EPA）. Methods The r EPA was precipitated with acetonitrile then centrifuged, and the supernatant was collected and analyzed by LC-MS / MS using Zorbax SB-C18 Rapid Resolution HT column（1. 8 Micron 2. 1 × 100 mm）. The 30 %acetonitrle was used as mobile phase at a flow rate of 0. 1 ml / min. The TQMS was run in multiple reaction monitoring（MRM）and positive ion mode. The parameters used were as follows： ESI voltage： 3 500 V; fragmentor： 110 V; collige energe： 10; gas flow rate： 8 L / min; the mass to charge ratio（m / z） of precursor ion： 350. 1; m / z of quantitative product ions： 105. 8; m / z of qualitative product ions： 159. 9. The developed method was validated for systemic suitability, specificity, precision and stability, and determined for minimum detection limit and quantitative detection limit, based on which matrix effect test was performed. Results The parameters of the developed method met the requirements for test. The method showed high specificity to ampicillin. The r2 value at an ampicillin concentration range of 10 ~ 100 ng / ml was more than 0. 999 00, while the minimum and quantitative detection limits were 5 and 10 ng / ml respectively. The spike recovery rate of ampicillin was 98 % ~ 110 %, while the RSDs in reproducibility and inter-precision tests were less than5. 5%. However, the recovery rates of samples in stability tests after freezing and thawing and after pre-treatment as well as short-term stability test were 95% ~ 120%. All the recovery rates of ampicillin controls at three concentrations, formulated using bulk of r EPA 5-fold diluted with ultra-pure water, were nearly 120%, with RSDs of 3% ~ 5%, indicating high accuracy and precision. Conclusion The method showed high systemic suitability, sensitivity, specificity, accuracy and precision, which was time-saving and suitable for automatic analysis.