色谱与质谱参数的优化对免疫抑制剂定量灵敏度的影响

doi:10.3969/j.issn.1674-8115.2021.11.009

摘要/Abstract

摘要：

目的·考察色谱条件中的流动相及质谱离子源参数对环孢菌素A（cyclosporine A，CsA）、他克莫司（tacrolimus，TaC）、西罗莫司（sirolimus，SiR）及依维莫司（everolimus，EvE）4种常见免疫抑制剂药物的液相色谱-质谱分析定量结果的影响。方法·基于超高效液相色谱-串联质谱（ultra-high performance liquid chromatography-tandem mass spectrometry，UPLC-MS/MS）技术，通过改变流动相及离子源参数，评价不同参数设置对免疫抑制剂峰形及色谱峰响应的影响；通过考察标准曲线线性及最低定量限（limit of quantitation，LOQ）检验不同离子源温度下方法的灵敏度。结果·甲醇作为有机相能提供更窄的峰宽；水相中添加甲酸铵（ammonium formate，AF）能够明显提高色谱峰响应，高浓度的AF反而能一定程度地抑制色谱峰响应，因此选择5 mmol/L的AF作为流动相改性剂。离子源温度的优化能够明显改善色谱峰峰形和定量灵敏度，250 ℃下方法具良好的线性（r²>0.99）和灵敏度（LOQ=0.05 ng/mL）。结论·建立了基于UPLC-MS/MS技术的4种免疫抑制剂同步定量分析方法，并通过优化流动相及离子源参数便捷且显著地提高了定量灵敏度。该方法中AF的添加和离子源温度是影响定量分析的关键参数，这为基于液相色谱-质谱法的其他化合物的高灵敏度定量方法开发和优化提供新的思路。

关键词: 免疫抑制剂, 超高效液相色谱-串联质谱, 离子源温度, 定量分析

Abstract:

Objective·To investigate the influence of mobile phase in chromatographic conditions and mass spectrometry ion source parameters on the quantitative results of four common immunosuppressant drugs including cyclosporine A (CsA), tacrolimus (TaC), sirolimus (SiR) and everolimus (EvE) by liquid chromatography-mass spectrometry.

Methods·Based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology, the effect on peak shape and response under different parameter settings were evaluated by changing the mobile phase and ion source parameters; the sensitivity of the method under different ion source temperatures was detected by linearity of the standard curve and limit of quantitation (LOQ).

Results·The narrower peak width was obtained when methanol was used as organic phase; the addition of ammonium formate (AF) to the aqueous phase observably improved the peak response but inhibited partly when the concentration of AF excessed; therefore, 5 mmol/L AF was chosen as the mobile phase modifier. The optimization of the ion source temperature significantly improved the chromatographic peak shape and quantitative sensitivity. The quantitative method had nice linearity (r²>0.99) and sensitivity (LOQ=0.05 ng/mL) under 250 ℃ ion source temperature.

Conclusion·A simultaneous quantitative analytical method for four immunosuppressant drugs is established based on UPLC-MS/MS technology and the quantitative sensitivity has been conveniently and significantly improved by optimizing the mobile phase and ion source parameters. The addition of AF in the mobile phase and the ion source temperature are key parameters that affect the quantitative analysis and provide new ideas for the development and optimization of high-sensitivity quantitative methods for other compounds based on liquid chromatography-mass spectrometry.

Key words: immunosuppressant drug, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), ion source temperature, quantitative analysis

中图分类号:

R331.9

孟爽, 周立, 付勤, 夏立, 孟丽媛. 色谱与质谱参数的优化对免疫抑制剂定量灵敏度的影响[J]. 上海交通大学学报(医学版), 2021, 41(11): 1461-1469.

Shuang MENG, Li ZHOU, Qin FU, Li XIA, Li-yuan MENG. Influence of optimization of chromatographic and mass spectrometric parameters on the quantitative sensitivity of immunosuppressive drugs[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(11): 1461-1469.

图/表 6

表1 4种免疫抑制剂MRM方法参数

Tab 1 Parameters of MRM method of four immunosuppressant drugs

Immunosuppressant drug	Q1 （m/z）	Q3 （m/z）	CE/V	DP/V	CXP/V
CsA	1 219.9	1 203.0	32	80	13
TaC	821.6	768.6	30	80	15
SiR	931.7	864.6	25	80	17
EvE	975.8	908.7	25	80	20

图1 4种免疫抑制剂在不同流动相体系下色谱峰峰形及色谱响应的比较Note： A. CsA. B. SiR. C. TaC. D. EvE. TW—total width; Area—chromatographic peak area; ACN—acetonitrile; MeOH—methanol.

Fig 1 Comparison of chromatographic peak shapes and chromatographic responses of four immunosuppressant drugs under different mobile phase systems

表2 不同缓冲盐体系对4种免疫抑制剂色谱峰的影响

Tab 2 Influence of different buffer salt systems on the chromatographic peaks of four immunosuppressant drugs

Compound
	5 mmol/L			10 mmol/L			20 mmol/L			5 mmol/L			10 mmol/L			20 mmol/L
	Area	TW	RT	Area	TW	RT	Area	TW	RT	Area	TW	RT	Area	TW	RT	Area	TW	RT
CsA	3.03×10⁶	0.40	5.82	1.68×10⁶	0.43	5.83	1.68×10⁶	0.53	5.84	2.23×10⁶	0.34	5.81	1.74×10⁶	0.42	5.84	9.19×10⁵	0.42	5.85
SiR	2.75×10⁶	0.19	5.30	1.94×10⁶	0.18	5.30	2.35×10⁶	0.18	5.31	2.60×10⁶	0.18	5.30	1.90×10⁶	0.20	5.31	1.31×10⁶	0.19	5.32
TaC	5.92×10⁷	0.71	5.12	5.29×10⁷	0.60	5.12	5.30×10⁷	0.68	5.14	6.00×10⁷	0.61	5.13	4.56×10⁷	0.65	5.15	3.99×10⁷	0.64	5.16
EvE	3.05×10⁶	0.19	5.33	2.08×10⁶	0.20	5.34	2.48×10⁶	0.19	5.35	2.15×10⁶	0.19	5.35	1.95×10⁶	0.20	5.36	1.63×10⁶	0.19	5.35

图2 质谱离子源参数对4种免疫抑制剂色谱峰响应的影响Note： A. IS. B. TEM. C. CUR. D. Gas 1. E. Gas 2. F. CAD.

Fig 2 Influence of mass spectrometry ion source parameters on the chromatographic peak response of four immunosuppressant drugs

图3 质谱TEM对4种免疫抑制剂色谱峰峰形的影响

Fig 3 Influence of mass spectrometry TEM on the chromatographic peak shape of four immunosuppressant drugs

图4 TEM对4种免疫抑制剂标准曲线线性的影响Note： A. 250℃. B. 500 ℃. From top to bottom are the standard curves of CsA, SiR, TaC and EvE.

Fig 4 Influence of TEM on the standard curve linearity of four immunosuppressant drugs

参考文献 27

1	Di Maira T, Little EC, Berenguer M. Immunosuppression in liver transplant[J]. Best Pract Res Clin Gastroenterol, 2020, 46-47: 101681.
2	Ivulich S, Westall G, Dooley M, et al. The evolution of lung transplant immunosuppression[J]. Drugs, 2018, 78(10): 965-982.
3	Lim MA, Kohli J, Bloom RD. Immunosuppression for kidney transplantation: where are we now and where are we going?[J]. Transplant Rev (Orlando), 2017, 31(1): 10-17.
4	Ericson JE, Zimmerman KO, Gonzalez D, et al. A systematic literature review approach to estimate the therapeutic index of selected immunosuppressant drugs after renal transplantation[J]. Ther Drug Monit, 2017, 39(1): 13-20.
5	Johnston A. Equivalence and interchangeability of narrow therapeutic index drugs in organ transplantation[J]. Eur J Hosp Pharm, 2013, 20(5): 302-307.
6	Adams DH, Sanchez-Fueyo A, Samuel D. From immunosuppression to tolerance[J]. J Hepatol, 2015, 62(1): S170-S185.
7	Posfay-Barbe KM, Baudet H, McLin VA, et al. Immunosuppressant therapeutic drug monitoring and trough level stabilisation after paediatric liver or kidney transplantation[J]. Swiss Med Wkly, 2019, 149: w20156.
8	Strobbe G, Pannier D, Sakji I, et al. Advantages of everolimus therapeutic drug monitoring in oncology when drug-drug interaction is suspected: a case report[J]. J Oncol Pharm Pract, 2020, 26(7): 1743-1749.
9	Sommerer C, Suwelack B, Dragun D, et al. An open-label, randomized trial indicates that everolimus with tacrolimus or cyclosporine is comparable to standard immunosuppression in de novo kidney transplant patients[J]. Kidney Int, 2019, 96(1): 231-244.
10	Chen L, Song Q, Chen Y, et al. Tailored reconstituted lipoprotein for site-specific and mitochondria-targeted cyclosporine A delivery to treat traumatic brain injury[J]. ACS Nano, 2020, 14(6): 6636-6648.
11	Sottani C, Grignani E, Mazzucchelli S, et al. Development and validation of a simple and versatile method for the quantification of everolimus loaded in H-ferritin nanocages using UHPLC-MS/MS[J]. J Pharm Biomed Anal, 2020, 191: 113644.
12	Freudenberger K, Hilbig U, Gauglitz G. Recent advances in therapeutic drug monitoring of immunosuppressive drugs[J]. Trac Trends Anal Chem, 2016, 79: 257-268.
13	Zhang Y, Zhang R. Recent advances in analytical methods for the therapeutic drug monitoring of immunosuppressive drugs[J]. Drug Test Anal, 2018, 10(1): 81-94.
14	Li W, Li R, Liu H, et al. A comparison of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-multiplied immunoassay technique (EMIT) for the determination of the cyclosporin A concentration in whole blood from Chinese patients[J]. Biosci Trends, 2017, 11(4): 475-482.
15	Becker A, Backman JT, Itkonen O. Comparison of LC-MS/MS and chemiluminescent immunoassays for immunosuppressive drugs reveals organ dependent variation in blood cyclosporine a concentrations[J]. Clin Chim Acta, 2020, 508: 22-27.
16	Mei S, Wang J, Chen D, et al. Simultaneous determination of cyclosporine and tacrolimus in human whole blood by ultra-high performance liquid chromatography tandem mass spectrometry and comparison with a chemiluminescence microparticle immunoassay[J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2018, 1087-1088: 36-42.
17	Krnáč D, Reiffová K, Rolinski B. A new HPLC-MS/MS method for simultaneous determination of cyclosporine A, tacrolimus, sirolimus and everolimus for routine therapeutic drug monitoring[J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2019, 1128: 121772.
18	Gong ZS, Wu ZH, Xu SX, et al. A high-throughput LC-MS/MS method for the quantification of four immunosu-ppressants drugs in whole blood[J]. Clin Chim Acta, 2019, 498: 21-26.
19	Pablo AH, Breaud AR, Clarke W. Analysis of immunosuppressant drugs in whole blood by liquid chromatography-tandem mass spectrometry (LC-MS/MS)[J]. Curr Protoc Toxicol, 2020, 84(1): e92.
20	Brase RA, Spink DC. Enhanced sensitivity for the analysis of perfluoroethercarboxylic acids using LC-ESI-MS/MS: effects of probe position, mobile phase additive, and capillary voltage[J]. J Am Soc Mass Spectrom, 2020, 31(10): 2124-2132.
21	Kruve A, Kaupmees K. Adduct formation in ESI/MS by mobile phase additives[J]. J Am Soc Mass Spectrom, 2017, 28(5): 887-894.
22	Liang Y, Guan T, Zhou Y, et al. Effect of mobile phase additives on qualitative and quantitative analysis of ginsenosides by liquid chromatography hybrid quadrupole-time of flight mass spectrometry[J]. J Chromatogr A, 2013, 1297: 29-36.
23	Soleilhac A, Dagany X, Dugourd P, et al. Correlating droplet size with temperature changes in electrospray source by optical methods[J]. Anal Chem, 2015, 87(16): 8210-8217.
24	Kruve A. Influence of mobile phase, source parameters and source type on electrospray ionization efficiency in negative ion mode[J]. J Mass Spectrom, 2016, 51(8): 596-601.
25	Xu JD, Xu MZ, Zhou SS, et al. Effects of chromatographic conditions and mass spectrometric parameters on the ionization and fragmentation of triterpene saponins of Ilex asprella in liquid chromatography-mass spectrometry analysis[J]. J Chromatogr A, 2019, 1608: 460418.
26	Bittersohl H, Herbinger J, Wen M, et al. Simultaneous determination of protein-unbound cyclosporine A and mycophenolic acid in kidney transplant patients using liquid chromatography-tandem mass spectrometry[J]. Ther Drug Monit, 2017, 39(3): 211-219.
27	Silvester S. Mobile phase pH and organic modifier in reversed-phase LC-ESI-MS bioanalytical methods: assessment of sensitivity, chromatography and correlation of retention time with in silico logD predictions[J]. Bioanalysis, 2013, 5(22): 2753-2770.

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[5]	贾晓媛, 王伟铭. IgA肾病的治疗进展[J]. , 2012, 32(3): 361-.
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[7]	姚洁洁, 詹维伟, 陈曼, 等. 超声造影定量评价兔肾缺血再灌注损伤[J]. , 2010, 30(4): 428-.