HPLC-MS/MS法测定人血浆中地拉罗司
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摘要
目的:建立高效液相色谱-串联质谱(HPLC-MS/MS)法测定人血浆中地拉罗司的浓度,并在方法开发中对样品和HPLC-MS/MS系统中存在的Fe~(3+)对地拉罗司浓度测定的干扰进行了排除。方法:本方法在流动相和样品中加入适量的乙二胺四乙酸(EDTA),竞争性抑制地拉罗司与Fe~(3+)的络合,从而解决了地拉罗司检测过程中不稳定的难题。以米非司酮为内标,血浆样品经乙腈沉淀蛋白后进行HPLC-MS/MS分析。色谱条件:采用Hanbon Hedera ODS-2 (2.1 mm×150 mm, 5μm)色谱柱,流动相为甲醇-0.1%甲酸溶液(含0.04 mmol·L~(-1) EDTA)梯度洗脱,流速0.5 mL·min~(-1);质谱条件:电喷雾(ESI)正离子模式,多反应监测(MRM)方式检测,m/z 430.1→m/z 372.2(地拉罗司)、m/z 374.2→m/z 108.1(内标米非司酮)。结果:地拉罗司标准曲线线性范围为0.04～40μg·mL~(-1),线性关系良好;无内源性杂质干扰;地拉罗司3个浓度水平质控样品的批内和批间精密度RSD在2.9%～11.8%之间,准确度的偏差RE在-3.1%～4.0%之间;提取回收率较高;地拉罗司和内标在人血浆样品中无基质效应。结论:在流动相及样品中加入EDTA保证了地拉罗司在检测过程中的稳定性。该方法操作简单,具有良好的特异性、可重复性和可靠性,并且成功应用于10名中国健康受试者口服地拉罗司后的药动学研究。
OBJECTIVE To establish an HPLC-MS/MS method for the determination of the concentration of deferasirox in human plasma, and to eliminate the interference of Fe~(3+) which exist in prepared plasma samples and HPLC-MS/MS system in the determination of the deferasirox concentration during the period of the development process. METHODS Appropriate amount of ethylenediamine tetraacetate dihydrate(EDTA) was added to the mobile phase and samples to competitively inhibit the complexation of deferasirox with Fe~(3+), thereby solving the problem of instability in the detection process of deferasirox. Mifepristone was used as an internal standard(IS), plasma samples were precipitated with acetonitrile, and then were analyzed by HPLC-MS/MS. Chromatographic conditions: A Hanbon Hedera ODS-2(2.1 mm×150 mm, 5 μm) column was used with a mobile phase of methanol-0.1% formic acid solution(containing 0.04 mmol·L~(-1) EDTA) at a flow rate of 0.5 mL·min~(-1); MS conditions: electrospray(ESI) positive ion mode, multiple reaction monitoring(MRM) mode detection, m/z 430.1 →m/z 372.2(deferasirox), m/z 374.2→m/z 108.1(internal standard mifepristone). RESULTS The method showed good linearity over the concentration range of 0.04 to 40 μg·mL~(-1) for deferasirox with no endogenous impurity interference; the RSDs of intra-and inter-batch precision of the quality control samples at deferasirox's three concentration levels ranged from 2.9% to 11.8% and the deviations of accuracy were between-3.1% and 4.0%; the recovery was higher; deferasirox and the IS had no matrix effect in human plasma samples. CONCLUSION The addition of EDTA to the mobile phase and sample tended to stabilize the content of deferasirox,which ensured the stability of deferasirox in the detection process. The method is simple to perform with good specificity, reproducibility and reliability, and has been successfully applied to the pharmacokinetics of oral administration of deferasirox in 10 Chinese healthy subjects.
OBJECTIVE To establish an HPLC-MS/MS method for the determination of the concentration of deferasirox in human plasma, and to eliminate the interference of Fe~(3+) which exist in prepared plasma samples and HPLC-MS/MS system in the determination of the deferasirox concentration during the period of the development process. METHODS Appropriate amount of ethylenediamine tetraacetate dihydrate(EDTA) was added to the mobile phase and samples to competitively inhibit the complexation of deferasirox with Fe~(3+), thereby solving the problem of instability in the detection process of deferasirox. Mifepristone was used as an internal standard(IS), plasma samples were precipitated with acetonitrile, and then were analyzed by HPLC-MS/MS. Chromatographic conditions: A Hanbon Hedera ODS-2(2.1 mm×150 mm, 5 μm) column was used with a mobile phase of methanol-0.1% formic acid solution(containing 0.04 mmol·L~(-1) EDTA) at a flow rate of 0.5 mL·min~(-1); MS conditions: electrospray(ESI) positive ion mode, multiple reaction monitoring(MRM) mode detection, m/z 430.1 →m/z 372.2(deferasirox), m/z 374.2→m/z 108.1(internal standard mifepristone). RESULTS The method showed good linearity over the concentration range of 0.04 to 40 μg·mL~(-1) for deferasirox with no endogenous impurity interference; the RSDs of intra-and inter-batch precision of the quality control samples at deferasirox's three concentration levels ranged from 2.9% to 11.8% and the deviations of accuracy were between-3.1% and 4.0%; the recovery was higher; deferasirox and the IS had no matrix effect in human plasma samples. CONCLUSION The addition of EDTA to the mobile phase and sample tended to stabilize the content of deferasirox,which ensured the stability of deferasirox in the detection process. The method is simple to perform with good specificity, reproducibility and reliability, and has been successfully applied to the pharmacokinetics of oral administration of deferasirox in 10 Chinese healthy subjects.
引文
[1] Yang Y, Zhang J. Research progress on thalassemia in southern China [J]. Chin J Exp Hematol (中国实验血液学杂志), 2017, 25 (1): 276-280.
[2] Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators [J]. B World Health Organ, 2008, 86(6): 480-487.
[3] Yang Y, Dai BT. The Iron Overload and Iron-Cheleting Theraping of β-thalassanemia [J]. J Pediatr Pharm (儿科药学杂志), 2011, 17(3): 58-61.
[4] Abdel-Gadir A, Vorasettakarnkij Y, Ngamkasem H, et al. Ultrafast magnetic resonance imaging for iron quantification in thalassemia participants in the developing world [J]. Circulation, 2016, 134(5): 432-434.
[5] Li X, Zhang Q, Gao CJ, et al. Study on the effect of deferasirox and deferoxamine in treatment of iron overload in children with β-thalassemia major [J]. J Clin Exp Med (临床和实验医学杂志), 2018(1): 67-70.
[6] Rund D, Rachmilewitz E. Beta-thalassemia [J]. N Engl J Med, 2005, 353(11):1135-1146.
[7] Liu D, Chen NF, Chen ND, et al. Formulation optimization of deferasirox dispersible tablets [J]. China Pharm (中国药房), 2013(45): 4284-4286.
[8] Yang WJ, Huang KM, Shen YJ, et al. Therapy iron overload with deferasirox in 2～6 years old patients with β - thalassemia major: observe the safety in a year [J]. J Clin Exp Med (临床和实验医学杂志), 2014(9): 708-710.
[9] Ding WY, Tang KH, Wang YC, et al. An HPLC analytical method for determining related substances in deferasirox [J]. Chin J New Drugs(中国新药杂志), 2016(20): 2376-2381.
[10] Feng Y, Wu J, Gu JH, et al. Determination of the content and related substances of the deferasirox raw material by RP-HPLC [J]. J Chin Antibiotics (中国抗生素杂志), 2017, 42(8): 692-697.
[11] Lu MY, Wang N, Wu WH, et al. Simultaneous Determination of Plasma Deferasirox and Deferasirox-Iron Complex Using an HPLC-UV System and Pharmacokinetics of Deferasirox in Patients With β-Thalassemia Major:Once-daily Versus Twice-daily Administration [J]. Clin Ther, 2015, 37(8): 1751-1760.
[12] Galanello R, Piga A, Alberti D, et al. Safety, tolerability, and pharmacokinetics of ICL670, a new orally active iron-chelating agent in patients with transfusion-dependent iron overload due to beta-thalassemia [J]. J Clin Pharmacol, 2003, 43(6): 565-572.
[13] Pligoropoulou H, Vonaparti A, Panderi I. Hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometry for the quantification of deferasirox, an oral iron chelator, in human plasma [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2012, 893-894: 114-120.
[14] Song TS, Hsieh YW, Peng CT, et al. Development of a fast LC-MS/MS assay for the determination of deferiprone in human plasma and application to pharmacokinetics [J]. Biomed Chromatogr, 2012, 26(12): 1575-1581.
[15] Galanello R, Piga A, Cappellini MD, et al. Effect of food, type of food, and time of food intake on deferasirox bioavailability: recommendations for an optimal deferasirox administration regimen [J]. J Clin Pharmacol, 2008, 48(4): 428-435.
[2] Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators [J]. B World Health Organ, 2008, 86(6): 480-487.
[3] Yang Y, Dai BT. The Iron Overload and Iron-Cheleting Theraping of β-thalassanemia [J]. J Pediatr Pharm (儿科药学杂志), 2011, 17(3): 58-61.
[4] Abdel-Gadir A, Vorasettakarnkij Y, Ngamkasem H, et al. Ultrafast magnetic resonance imaging for iron quantification in thalassemia participants in the developing world [J]. Circulation, 2016, 134(5): 432-434.
[5] Li X, Zhang Q, Gao CJ, et al. Study on the effect of deferasirox and deferoxamine in treatment of iron overload in children with β-thalassemia major [J]. J Clin Exp Med (临床和实验医学杂志), 2018(1): 67-70.
[6] Rund D, Rachmilewitz E. Beta-thalassemia [J]. N Engl J Med, 2005, 353(11):1135-1146.
[7] Liu D, Chen NF, Chen ND, et al. Formulation optimization of deferasirox dispersible tablets [J]. China Pharm (中国药房), 2013(45): 4284-4286.
[8] Yang WJ, Huang KM, Shen YJ, et al. Therapy iron overload with deferasirox in 2～6 years old patients with β - thalassemia major: observe the safety in a year [J]. J Clin Exp Med (临床和实验医学杂志), 2014(9): 708-710.
[9] Ding WY, Tang KH, Wang YC, et al. An HPLC analytical method for determining related substances in deferasirox [J]. Chin J New Drugs(中国新药杂志), 2016(20): 2376-2381.
[10] Feng Y, Wu J, Gu JH, et al. Determination of the content and related substances of the deferasirox raw material by RP-HPLC [J]. J Chin Antibiotics (中国抗生素杂志), 2017, 42(8): 692-697.
[11] Lu MY, Wang N, Wu WH, et al. Simultaneous Determination of Plasma Deferasirox and Deferasirox-Iron Complex Using an HPLC-UV System and Pharmacokinetics of Deferasirox in Patients With β-Thalassemia Major:Once-daily Versus Twice-daily Administration [J]. Clin Ther, 2015, 37(8): 1751-1760.
[12] Galanello R, Piga A, Alberti D, et al. Safety, tolerability, and pharmacokinetics of ICL670, a new orally active iron-chelating agent in patients with transfusion-dependent iron overload due to beta-thalassemia [J]. J Clin Pharmacol, 2003, 43(6): 565-572.
[13] Pligoropoulou H, Vonaparti A, Panderi I. Hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometry for the quantification of deferasirox, an oral iron chelator, in human plasma [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2012, 893-894: 114-120.
[14] Song TS, Hsieh YW, Peng CT, et al. Development of a fast LC-MS/MS assay for the determination of deferiprone in human plasma and application to pharmacokinetics [J]. Biomed Chromatogr, 2012, 26(12): 1575-1581.
[15] Galanello R, Piga A, Cappellini MD, et al. Effect of food, type of food, and time of food intake on deferasirox bioavailability: recommendations for an optimal deferasirox administration regimen [J]. J Clin Pharmacol, 2008, 48(4): 428-435.