血浆羟氯喹浓度监测及在系统性红斑狼疮患者中的应用

Hydroxychloroquine Blood Concentration Monitoring and Application in Systemic Lupus Erythematosus Patients

目的建立超高效液相色谱-串联质谱(UPLC-MS/MS)法测定血浆中羟氯喹(HCQ)浓度,并分析系统性红斑狼疮(SLE)患者稳态血药浓度与给药剂量、疾病活动度(SLEDAI)、眼部等不良反应及临床指标的相关性。方法血浆样本加入内标物(奎宁,QN)后乙腈沉淀蛋白,离心取上清液于氮气流下吹干,再用0.1%甲酸-甲醇(75:25,V/V)复溶。以Poroshell 120 EC-C18(2.1 mm×100 mm,2.7μm)为固定相,0.1%甲酸溶液(A)、0.1%甲酸甲醇溶液(B)为流动相梯度洗脱,检测波长254 nm,流速0.25 mL·min^(-1),柱温35℃。采用电喷雾离子化,正离子-多反应监测模式扫描检测HCQ(m/z 336.4→247.2)和QN(m/z 320.2→247.2)浓度。运用该法检测53例SLE患者血浆HCQ浓度,并收集患者基本信息、给药剂量、SLEDAI、炎症、血常规、肝肾功能、凝血功能、血糖血脂等临床指标及不良反应发生情况,分析血浆HCQ浓度与上述指标的相关性。结果血浆HCQ浓度在5~4000 ng·mL^(-1)范围内线性关系良好,定量下限为5 ng·mL^(-1)。各浓度水平的批内、批间精密度相对标准偏差均小于10%,准确度为90%~110%,提取回收率为(89.42±2.27)%,稳定性良好。53例SLE患者HCQ平均血浆谷浓度为(591.10±288.05)ng·mL^(-1);高血药浓度患者SLEDAI较低,眼部不良反应出现较多。疾病缓解组(SLEDAI≤4)患者HCQ平均血药浓度高于疾病活动组(SLEDAI>4)(P<0.05)。治疗组(HCQ>500 ng·mL^(-1))患者,天冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)水平较亚治疗组(HCQ:10~500 ng·mL^(-1))低(P<0.05),血小板计数(PLT)高(P<0.01),其他临床指标没有显著差异。结论该方法灵敏、快速、简便、可靠、重现性好,适用于血浆HCQ浓度监测,为其个体化用药提供参考。维持高水平血浆HCQ浓度,可有效降低SLE患者疾病活动度及AST、ALP,升高PLT;可能增加眼部不良反应发生风险。

Objective Establish an ultra-performance liquid chromatography-mass spectrometry(UPLC-MS/MS)method to determine the concentration of hydroxychloroquine(HCQ)in the plasma of patients.Moreover,to analyze the correlation between the steady-state blood concentration and adverse reactions and clinical indicators,such as,dose,systemic lupus erythematosus activity index(SLEDAI),and ophthalmology.Methods The plasma sample was added to an internal standard(Quinine,QN)and precipitated by acetonitrile.The supernatant obtained via centrifugation was dried under nitrogen.Then,the acquired solid was redissolved with 0.1%formic acid aqueous solution and methanol(75:25,V/V).A Poroshell 120 EC-C18(2.1 mm×100 mm,2.7μm)chromatographic column was used as the stationary phases,and 0.1%formic acid acidified aqueous solution(phase A),and 0.1%formic acid acidified methanol(phase B)was used as the mobile phases for gradient elution.The detection wavelength was 254 nm,the flow rate was at 0.25 mL·min^(-1),and the column temperature was 35℃.A triple-quadrupole mass spectrometer conducted a quantitative analysis of HCQ with a positive-electrospray ionization source,monitored under a multiple reaction monitoring(MRM)mode.The extracted ions monitored following MRM transitions were m/z 336.4→247.2 for HCQ and m/z 320.2→247.2 for QN.The method was used to determine the HCQ concentration of 53 patients with systemic lupus erythematosus(SLE).In addition,the basic information,dosage of HCQ,SLEDAI,and the clinical indicators of the patients have been collected.Results The plasma concentration of HCQ was linear within the range of 5—4000 ng·mL^(-1).The lower quantitative limit for HCQ was 5 ng·mL^(-1).The relative standard deviations(RSDs)within and between batches at each concentration level were less than 10%.The accuracy was 90%—110%,and the extraction recovery rate was 89.42%±2.27%.The average plasma trough concentration was(591.10±288.05)ng·mL^(-1).Patients with a high plasma concentration of HCQ showed low SLEDAI and more ocular adverse reactions.The level of HCQ in patients with SLEDAI≤4 was higher than that with SLEDAI>4(P<0.05).The level of aspartate aminotransferase(AST)and alkaline phosphatase(ALP)were lower in patients with higher plasma HCQ levels.However,blood platelet(PLT)was higher in patients with higher plasma HCQ levels(P<0.05),while other indicators showed no significant difference among patients with different HCQ levels.Conclusion The method is sensitive,rapid,simple,reliable,and reproducible.Thus,it is suitable for monitoring HCQ blood concentration and provides a reference for its personalized medication.Maintaining high HCQ plasma concentration could effectively decrease SLEDAI,AST and ALP levels,but it also increases PLT level and the risk of ocular adverse reaction.