兴奋剂检测中吗啡和可待因的LC-MS/MS定量方法研究

Quantitation of Morphine and Codeine in Human Urine by HPLC-MS/MS for Doping Control

目的：建立WADA禁用清单中阈值物质吗啡及相关物质可待因的HPLC-MS/MS定量方法并进行方法验证。方法：尿样加内标后,加入磷酸盐缓冲液（0.1 mol/L Na2HPO4和0.1 mol/L Na H2PO4水溶液p H=6.8）及β-Glucuranidase水解过夜,加入固体缓冲剂（Na HCO3/K2CO3=3/2,w/w,p H=9.5）和提取液（乙醚/异丙醇=9/1,v/v）萃取,有机相于氮气流下吹干,用初始流动相定容后进样于LC/MS/MS。使用耐受高p H值的Waters XBridgeTMC18色谱柱,以乙腈/氨水（p H≈10）作为流动相体系,使用乙腈的起始浓度为2%,最终浓度为98%,运行时间为10min的梯度洗脱条件,采用多反应离子监测正离子模式,以286→201（morphine）,300→171（codeine）,289→165（morphine-D3）为定量离子。结果 ：吗啡和可待因在尿中的定量限分别为0.05μg/m L和0.005μg/m L,线性范围分别为（r^2〉0.99）0.10-10μg/m L和0.05-10μg/m L,吗啡定量的日内精密度和日间精密度（CV）分别为2.64%和4.01%,可待因为2.22%和2.01%。吗啡和可待因在1.0μg/m L浓度时的方法回收率分别为98.47±2.73%和99.41±4.12%。定量检测误差均低于5%。结论：本方法使用一个步骤同时测定尿中禁用物质morphine的总量,并可同时定量检测尿样中的codeine,数据处理简单;简化了前处理操作步骤,定量结果的精密度得到了提高;采用Bridge ^TM C18色谱柱和高p H值流动相,改善了morphine在普通C18柱上不保留、仪器响应和保留时间重现性较差、峰形拖尾较为严重的问题。方法重现性好,测量结果准确可靠,满足WADA技术文件的要求,应用于本实验室常规检测和WADA的外部质量评估计划,效果良好。

Objective This study aimed at development of a simple and accurate method for quantification of morphine and codeine by HPLC-MS/MS technique. Method The human urine samples with internal standard were hydrolyzed overnight with β-Glucuranidase at p H 6.8（0.1 mol/L Na2HPO4 and 0.1 mol/L Na H2PO4）,and then pretreated with a single-step liquid-liquid（Diethyl ether/Isopropanol=9/1,v/v）extraction at p H 9.5（Na HCO3/K2CO3=3/2, w/w）. The supernatants were blown to dry with nitrogen gas and dissolved with the initial mobile phase,and finally detected by HPLC-MS/MS with a Xbridge^TMC18 column and high p H value mobile phase system（NH4OH/ACN,p H ≈10）. Using positive ions and multiple reaction monitoring（MRM）mode,the 286 →201（morphine）, 300 →171（codeine）and 289 →165（morphine-D3）were selected as quantitative ions. Results After determination of a series of spiked urine samples, the limit of quantitation of morphine and codeine in urine was 0.05μg/m L and 0.005μg/m L. The linear ranges（r^2〉0.99）were 0.10-10μg/m L for morphine and 0.05 -10μg/m L for codeine. The intra- and inter-day precisions of morphine and codeine were 2.64% and 4.01%,and 2.22% and 2.01%,respectively. The average recoveries of morphine and codeine at the concentration of 1.0μg/m L were 98.47 ±2.73% and 99.41 ±4.12%,respectively. The analytical biases for morphine and codeine were both less than 5%. Conclusion A simple,reliable and accurate method for quantitation of morphine and codeine by liquid chromatography-tandem mass spectrometry was established,and met the requirement of WADA.