论文部分内容阅读
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(r2>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: To establish a HPLC-MS / MS method for quantitative determination of codeine morphine and its related substances in WADA banned list. Methods: The urine sample was added with phosphate buffer solution (0.1 mol / L Na2HPO4 and 0.1 mol / L Na H2PO4 solution p H = 6.8) and β-Glucuranidase was hydrolyzed overnight. Solid buffer (Na HCO3 / K2CO3 = (Ether / isopropanol = 9/1, v / v). The organic phase was air-dried under a stream of nitrogen and sample-in with the original mobile phase On LC / MS / MS. A Waters XBridge TM C18 column with high p H values was used with a mobile phase system of acetonitrile / ammonia (p H ≈ 10) using a gradient of 2% acetonitrile, 98% final, and 10 min running time Elution conditions, using multiple reactive ion monitoring positive mode, with 286 → 201 (morphine), 300 → 171 (codeine), 289 → 165 (morphine-D3) as a quantitative ion. Results: The limit of quantitation of morphine and codeine in urine were 0.05 μg / mL and 0.005 μg / mL, respectively. The linear range was 0.10 ~ 10μg / mL and 0.05 ~ 10μg / mL for r2> 0.99, Intra-day and intra-day precision (CV) for morphine quantitation were 2.64% and 4.01%, and codeine was 2.22% and 2.01%, respectively. The recoveries of morphine and codeine at the concentration of 1.0 μg / mL were 98.47 ± 2.73% and 99.41 ± 4.12%, respectively. Quantitative detection of errors are less than 5%. Conclusion: This method uses a single step to simultaneously determine the total amount of morphine in the urine and quantitatively detect codeine in urine simultaneously. The data processing is simple, the pretreatment steps are simplified, the precision of quantitative results is improved, Bridge TM C18 column and high p H mobile phase, improved morphine retention on a normal C18 column, poor reproducibility of instrument response and retention times, and severe peak tailing. The method has good reproducibility, the measurement results are accurate and reliable, meets the requirements of WADA technical documents, and is applied to routine laboratory tests and WADA external quality assessment plan with good results.