高效液相色谱质谱联用检测血浆莎巴比星的基质效应影响因素

Factors influencing matrix effects of sabarubicin and its major metabolite in human plasma during liquid chromatography-tandem mass spectrometry analysis

目的:考察EDTA-K2和肝素锂抗凝血浆不同前处理方法和液相条件对于高效液相色谱质谱联用法检测莎巴比星及其代谢产物M3基质效应的影响。方法:以含不同抗凝剂的空白血浆配制低、中、高浓度样品,乙酸乙酯或氯仿液液萃取,流动相:A:0.1%甲酸水,B:乙腈,等度洗脱或梯度洗脱。莎巴比星、M3和内标多柔比星检测离子对分别为:m/z 644→130,m/z 646→333.2,m/z 544→360。采用提取后加入法,评价各浓度莎巴比星和M3在2种抗凝剂血浆中基质效应的差异以及方法优化对基质效应的影响。结果:低浓度时,EDTA-K2组莎巴比星和M3基质效应分别为123%和110%,肝素锂组分别为143%和160%。基质效应在不同抗凝剂组间差异有统计学意义(莎巴比星:P<0.05;M3:P<0.05)。氯仿萃取肝素锂抗凝血浆中莎巴比星低、中、高浓度基质效应分别为142%,94%和78%,M3分别为123%,102%和89%。梯度洗脱莎巴比星低、中、高浓度基质效应分别为108%,81%和73%,而M3分别为93%,83%和73%。最终低浓度莎巴比星和M3基质效应分别为85.4%和98.7%,高浓度分别为88.5%和96.0%。结论:肝素锂抗凝血浆中莎巴比星及其代谢物M3的基质增强效应更强,且在不同浓度存在较大差异,优化前处理方法以及液相方法后基质效应得到显著改善。

Objective： To investigate the influence of EDTA-K2 or lithium heparin anticoagulant plasma,different extraction agents and liquid chromatograph methods on matrix effects of sabarubicin and M3 during analysis by high performance liquid chromatography mass spectrometry. Methods： Prepared plasma samples of sabarubicin and M3 at 3 concentrations in plasmas containing different anticoagulants were extracted by ethyl-acetate or chloroform.Gradient elution or isocratic elution composed of A： 0. 1% formic acid water and B： acetonitrile were adopted,anda gradient elute were developed. Detection was performed by electrospray ionization（ ESI） in the positive ionization mode by multiple reaction monitoring（ MRM） of the transitions at m / z 644→130 for sabarubicin,m / z 646→333. 2for M3 and m / z 544→360 for internal standard doxorubicin. Post-extraction spiking method was adopted to investigate the impacts of these conditions on matrix effects of sabarubicin and M3. Results： At lower concentration,the matrix effects of sabarubicin and M3 in EDTA-K2 anticoagulant plasma were respectively 123% and 110%,while it is 143% and 160% in in lithium heparin anticoagulant plasma. There was a statistically significant difference between anticoagulant plasmas（ sabarubixin： P 0. 05,M3： P 0. 05）. Matrix effects for chloroform extracted sabarubicin and M3 at 3 concentrations in lithium heparin anticoagulant plasma were 142%,94% and 78% for sabarubicin,and 123%,102% and 89% for M3,respectively. After adoption of gradient elute,the matrix effects were 108%,81% and 73% for sabarubicin,and 93%,83% and 73% for M3,at 3 concentrations. The matrix effects in ultimate method were 85. 4% and 98. 7% for sabarubicin and M3 at low concentration,and 88. 5% and96. 0% at high concentration. Conclusion： The matrix effects are more serious for sabarubicin and M3 in lithium heparin anticoagulant plasma,which have large difference in multi-concentration. Improvement of pretreatment method and liquid chromatograph method could decrease matrix effects.