液相色谱-电喷雾串联质谱分析人全血中环孢素的基质效应评估及其机理探究

目的:评估液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)分析人全血中环孢素的基质效应,并探究其发生机理。方法:全血裂解物的蛋白沉淀上清液用作全血基质。分别用乙腈水溶液和全血基质配制等浓度环孢素A(CyA)、环孢素C(CyC)的质控样品。分别在添加H^+、NH4^+和Na^+的流动相中,用LC-ESI-MS/MS的MRM模式检测质控样品中CyA,CyC的M+H]^+、M+NH4]^+和M+Na]^+(M=CyA,CyC)峰面积,以其计算基质效应因子;用MS Scan模式检查质控样品中CyA、CyC色谱流出峰的质谱离子组成情况,用于判断基质效应原因。结果:在添加H^+、NH4^+和Na^+的流动相中,全血基质中CyA、CyC基质效应因子分别为-76.6%^-54.0%、-86.0%^-55.3%和-42.8%^-34.1%。在加H+、加NH+4流动相中,标准液加全血基质的CyA、CyC色谱峰的质谱中出现丰度很高的M+Na]^+和M+K]^+,在加Na^+流动相中它们出现丰度很高的M+K]^+,而对应的定量离子M+H]^+、M+NH4]^+或M+Na]^+的丰度显著降低。结论:全血基质对环孢素的LC-ESI-MS/MS分析产生明显的基质效应,其原因与基质中Na^+、K^+同添加在流动相中用于辅助电离的H^+、NH4^+或Na^+竞争与环孢素加合,导致定量目标加合离子丰度显著降低有关。

Exploration of the matrix effect of human whole blood on analysis of cyclosporins by liquid chromatography-electrospray ionization-tandem mass spectrometry

OBJECTIVE To evaluate the matrix effect of human whole blood on analysis of cyclosporins by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), and explore its mechanism.METHODS The supernatant of protein precipitation of the lysate was used as the substrate.The quality control samples of cyclosporine A (CyA) and cyclosporine C (CyC) at equal concentrations were prepared with acetonitrile aqueous solution and matrix, respectively.In the mobile phase added with H⁺, NH4⁺ and Na⁺, respectively, the M+H]⁺, M+NH4]⁺ and M+Na]⁺ (M=CyA, CyC) peak areas of CyA and CyC in the quality control samples were detected using the MRM mode of LC-ESI-MS/MS, and the matrix effect factor was calculated; the mass spectral ion composition of CyA and CyC chromatographic effluent peaks in the quality control samples was examined using the MS Scan mode to determine the cause of matrix effect.RESULTS In the mobile phase with H⁺, NH₄⁺ and Na⁺, matrix factors of CyA and CyC in blood matrix were -76.6% to -54.0%, -86.0% to -55.3%, and -42.8% to -34.1%, respectively. Abundant M+Na]⁺ and M+K]⁺ appeared on the MS scan spectra of CyA and CyC in blood matrix eluted by the mobile phases with H⁺ and NH₄⁺, and abundant M+K]⁺ appeared on the MS scan spectra of CyA and CyC in blood matrix eluted by the mobile phases with Na⁺. The abundance of corresponding quantitative ions M+H]⁺, M+NH₄]⁺ or M+Na]⁺ decreased significantly.CONCLUSION Blood matrix has severe matrix effect on the analysis of cyclosporins by LC-ESI-MS/MS. The main reason is the competition between Na⁺, K⁺ in matrix and H⁺, NH₄⁺ or Na⁺ in the mobile phase in the formation of adducts with cyclosporins, which results in significant decrease in the abundance of quantitative adduct ions.