超高效液相色谱法测定血浆华法林浓度及临床应用

doi:10.3877/cma.j.issn.2095-5820.2019.03.002

中华临床实验室管理电子杂志 ›› 2019, Vol. 07 ›› Issue (03) : 133 -136. doi: 10.3877/cma.j.issn.2095-5820.2019.03.002

所属专题：文献；

实验研究

超高效液相色谱法测定血浆华法林浓度及临床应用

唐江涛¹, 陈瑶², 白杨娟¹, 苗强¹, 邹远高¹^,^†(

)

^1. 610041　成都，四川大学华西医院实验医学科临床免疫室
^2. 610000　成都，四川省妇女儿童医院检验科

收稿日期:2018-08-02 出版日期:2019-08-28
通信作者: 邹远高

Determination of plasma warfarin concentration by ultra-high performance liquid chromatography and clinical application

Jiangtao Tang¹, Yao Chen², Yangjuan Bai¹, Qiang Miao¹, Yuangao Zou¹^,^†()

^1. Department of Laboratory Medicine, West China Hospital of Sichuan University, Chengdu 610041, China
^2. Department of Clinical Laboratory, Women′s and Children′s Hospital of Sichuan Province, Chengdu 610000, China

Received:2018-08-02 Published:2019-08-28
Corresponding author: Yuangao Zou
About author:
Corresponding author: Zou Yuangao, Email: zouyg69@126.com

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目的

建立超高效液相色谱法(ultra performance liquid chromatography，UPLC)测定血浆华法林浓度，探讨心脏换瓣术后患者华法林血药浓度与国际标准化比率(international normalized ratio，INR)测值的关系，以寻求更安全可靠的监测指标，指导临床抗凝治疗的合理用药。

方法

利用6-甲氧基萘乙酸作为内标，建立一种超高效液相色谱法，测定血浆中的华法林浓度。同时对79例样本的INR、华法林剂量和血浆华法林浓度进行相关性分析。

结果

华法林和内标的保留时间分别是2.2和1.1 min，血浆中华法林的平均提取回收率为96.0%，华法林浓度在15.6~4000 ng/ml具有良好的线性(R²=0.9996)，方法回收率为98.5%~99.9%，最低检测限为5.0 ng/ml。日内和日间精密度分别低于1.34%和2.69%。79例患者INR和华法林剂量相关性为r²=0.006(P=0.481)，INR和血浆华法林浓度为r²=0.006(P=0.497)，华法林剂量和血浆浓度相关性为r²=0.298(P=0.001)。

结论

该方法是一种简便、快速、准确、灵敏的定量测定人体血浆中华法林浓度的UPLC方法，华法林血药浓度测定对于心脏瓣膜置换手术后患者的抗凝治疗具有指导意义。

关键词: 华法林, 国际标准化比率, 超高效液相色谱法

Objective

To establish a method to determine warfarin concentration in plasma by ultra performance liquid chromatography (UPLC), and to explore the relationship between warfarin plasma concentration and international normalized ratio (INR) in patients after cardiac valve replacement, so as to seek more safe and reliable monitoring indicators and guide rational use of anticoagulant therapy.

Methods

An ultra performance liquid chromatography method has been developed for measuring warfarin concentration in plasma by using 6-methoxy naphthaleneacetic acid (6-MNA) as internal standard (IS). At the some time, the correlation of INR, warfarin dosage and plasma warfarin concentration in 79 samples was analyzed.

Results

The retention time of warfarin and the IS were 2.2 and 1.1 min, respectively. The average extraction recovery of plasma warfarin was 96.0%, The concentration of warfarin was 15.6~4000 ng/ml with good linearity (R²=0.9996). The overall accuracy of the method was 98.5%~99.9% and the lowest detection limit was 5.0 ng/ml. The intra-and inter-day variations were less than 1.34% and 2.69%, respectively. The correlation between INR and warfarin dose was r²=0.006 (P=0.481), INR and plasma warfarin concentration was r²=0.006 (P=0.497), and the correlation between warfarin dose and plasma concentration was r²=0.298 (P=0.001) in 79 cases of samples.

Conclusions

A simple, rapid, accurate and sensitive UPLC method for quantifying warfarin levels in human plasma was validated. The determination of warfarin blood concentration has guiding significance for anticoagulant therapy of patients after heart valve replacement.

Key words: Warfarin, International normalized ratio, Ultra performance liquid chromatography

图1 华法林血浆样品UPLC色谱图

表1 华法林UPLC检测方法精密度和准确度试验结果(n=20)

加入量(ng/ml)	日内精密度			日间精密度
加入量(ng/ml)	实测值(ng/ml)	RSD(%)	准确度(%)	实测值(ng/ml)	RSD(%)	准确度(%)
31.2	31.2±0.34	1.10	99.9	31.2±0.84	2.69	99.8
125	123.1±1.65	1.34	98.5	124.9±1.47	1.34	99.9
500	494.8±2.84	0.57	98.9	497.0±4.73	0.57	99.4
2000	1986.9±10.12	0.51	99.4	1995.7±7.61	0.38	99.8

表1 华法林UPLC检测方法精密度和准确度试验结果(n=20)

加入量(ng/ml)	日内精密度			日间精密度
加入量(ng/ml)	实测值(ng/ml)	RSD(%)	准确度(%)	实测值(ng/ml)	RSD(%)	准确度(%)
31.2	31.2±0.34	1.10	99.9	31.2±0.84	2.69	99.8
125	123.1±1.65	1.34	98.5	124.9±1.47	1.34	99.9
500	494.8±2.84	0.57	98.9	497.0±4.73	0.57	99.4
2000	1986.9±10.12	0.51	99.4	1995.7±7.61	0.38	99.8

图2 INR、华法林剂量和血浆华法林浓度之间的相关性分析结果

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