干燥综合征患者全血中羟氯喹及其代谢物浓度与临床指标相关性研究

Determination of Hydroxychloroquine and its Metabolites in the Blood of Patients with Sjogren's Syndrome and the Correlation with Clinical Indexes

目的:建立一种高效液相色谱法检测全血中羟氯喹及其代谢物去乙基羟氯喹、去乙基氯喹的新方法,并分析干燥综合征患者血药浓度与免疫相关临床指标的相关性。方法:取100μL全血样本,加入内标,以两倍量的乙腈沉淀蛋白,离心取上清进样。用Zorbax SB-C_(18)色谱柱分离,流动相为0.2 mol·L^(-1)磷酸二氢钾-乙腈=15∶85(磷酸调至pH3.0),流速1 mL·min^(-1),检测波长254 nm,柱温35℃。考察该方法的专属性、标准曲线和定量限、精密度和准确度、提取回收率、稳定性;测定53例患者羟氯喹及其代谢物血药浓度,并收集患者白细胞水平、血小板计数、谷丙转氨酶、谷草转氨酶、血细胞沉降率等临床检验指标,用SPSS软件分析血药浓度与临床指标的关系。结果:羟氯喹、去乙基羟氯喹、去乙基氯喹、内标氯喹的保留时间分别在8.6,4.1,5.2 min和12.8 min左右,羟氯喹及其代谢物在3～3 000 ng·mL^(-1)范围内线性良好,最低定量限为3 ng·mL^(-1),批内、批间精密度、方法回收率符合要求;患者用药后白细胞水平有明显降低,其他临床指标无明显变化,血药浓度高组患者白细胞水平较高,其他指标在高低浓度组间没有明显差异。结论:该方法灵敏度高、结果准确、样品处理简单、分析快速,可用于临床检测。干燥综合征患者服用羟氯喹后白细胞计数有统计学差异,服药后高浓度组患者白细胞计数显著高于低浓度组患者。

OBJECTIVE To develop a high performance liquid chromatographic method for determination of hydroxychloroquine and its metabolites in the blood of patients with Sjogren’s syndrome and analyze their relationship with laboratory examination indexes.METHODS 100μL whole blood samples were spiked with internal standard,protein was precipitated with double amount of acetonitrile,and the supernatant was injected by centrifugation.The separation was performed on a Zorbax SB-C18 column with a mobile phase of 0.2 mol·L-1 potassium dihydrogen phosphate-acetonitrile=15∶85(pH adjusted to 3.0 with phosphoric acid)at a flow rate of 1 mL·min-1,a detection wavelength of 254 nm,and a column temperature of 35℃.and the detection wavelength was 254 nm.The specificity,limit of quantitation and standard curve,precision and accuracy,stability and recovery rate were also investigated.the plasma concentrations of hydroxychloroquine and its metabolites were determined in 53 patients,and clinical test indicators such as white blood cell level,platelet count,alanine aminotransferase,aspartate aminotransferase,and blood cell sedimentation rate were collected,and the relationship between plasma concentration and clinical parameters was analyzed using SPSS software.RESULTS The retention time of hydroxychloroquine,desethyl hydroxychloroquine,desethyl chloroquine and internal standard chloroquine were about 8.6 min,4.1 min,5.2 min and 12.8 min,respectively.The linearity of hydroxychloroquine and its metabolites was good in the range of 3~3000 ng·mL-1,with the lowest limit of quantification of 3 ng·mL-1.The intra-batch and inter-batch precision and method recovery rate met the requirements.After administration,the white blood cell level was significantly reduced,and no significant change was observed in other clinical indicators.The white blood cell level was higher in the high plasma concentration group,and there was no significant difference in other indicators between high and low concentration groups.CONCLUSION The method has exhibited high sensitivity with accurate results,simple sample processing and rapid analysis,and can be used for clinical detection.Patients with Sjogren’s syndrome had a statistically significant lower white blood cell count after taking hydroxychloroquine,and patients in the high concentration group had a significantly higher white blood cell count than patients in the low concentration group after taking the drug.