咖啡因及代谢产物的尿药浓度测定方法建立及临床应用  

Method establishment and clinical practice for concentration determination of caffeine and its metabolites in urine

作　　者：陈相龙 赵杨 黄琼叶 徐铭卿 李悦 陆超 孙鲁宁 王永庆 CHEN Xianglong;ZHAO Yang;HUANG Qiongye;XU Mingqing;LI Yue;LU Chao;SUN Luning;WANG Yongqing(School of Pharmacy/Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy,Xuzhou Medical University,Jiangsu Xuzhou 210004,China;Research Division of Clinical Pharmacology,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China;Dept.of Pediatrics,Jiangsu Women and Children Health Hospital,Nanjing 210036,China)

机构地区：[1]徐州医科大学药学院/江苏省新药研究与临床药学重点实验室,江苏徐州210004 [2]南京医科大学第一附属医院临床药理研究室,南京210029 [3]江苏省妇幼保健院儿科,南京210036

出　　处：《中国药房》2023年第18期2233-2237,共5页China Pharmacy

基　　金：国家卫生健康委计划生育药具不良反应监测中心/江苏省卫生健康发展研究中心开放课题(No.JSHD2021004,No.JSHD2021011)。

摘　　要：目的建立同时测定尿液中咖啡因及3种代谢产物茶碱、副黄嘌呤和可可碱浓度的方法并应用于临床。方法以咖啡因-13C3-d3为内标,尿液样本经乙腈沉淀蛋白后,采用高效液相色谱-串联质谱(HPLC-MS/MS)技术测定咖啡因及3种代谢产物的浓度。以Waters ACQUITY UPLC?BEH HILIC为色谱柱,60 mmol/L乙酸铵溶液(A)-乙腈(B)为流动相进行梯度洗脱,流速为0.5mL/min,柱温为38℃,进样量为2μL。采用电喷雾离子源,以多反应监测模式进行正离子扫描,用于定量分析的离子对分别为m/z 195.1→110.0(咖啡因)、m/z 181.1→124.0(茶碱)、m/z 181.1→124.0(副黄嘌呤)、m/z 181.1→138.0(可可碱)、m/z 198.1→140.1(内标)。采用上述方法测定19例早产儿呼吸暂停(AOP)患儿尿液中咖啡因及3种代谢产物的浓度。结果咖啡因、茶碱、副黄嘌呤、可可碱检测质量浓度的线性范围分别为0.200~200、0.050~50.0、0.050~50.0、0.100~100μg/mL(r均大于0.990),定量下限分别为0.200、0.050、0.050、0.100μg/mL;日内、日间精密度的RSD均不高于10.37%,基质因子为85.68%~109.90%,提取回收率为93.53%~109.40%(RSD均小于15%),稳定性试验的RSD均小于15%。19例AOP患儿尿液中咖啡因及3种代谢产物的质量浓度分别为(27.346±7.951)、(0.351±0.223)、(0.428±0.395)、(0.472±0.374)μg/mL。结论所建HPLC-MS/MS法操作简单、灵敏度高,可用于AOP患儿尿液中咖啡因及3种代谢产物浓度的测定。OBJECTIVE To establish a method for concentration determination of caffeine and its three metabolites,theophylline,paraxanthine and theobromine in urine,and apply it in clinical practice.METHODS Using caffeine13C3-d3 as internal standard(IS),and the urine samples were protein precipitated with acetonitrile;HPLC-MS/MS method was adopted to determine the concentrations of caffeine and its three metabolites.The determination was performed on Waters ACQUITY UPLC®BEH HILIC column with mobile phase consisting of 60 mmol/L ammonium acetate(A)-acetonitrile(B)(gradient elution)at the flow rate of 0.5 mL/min.The column temperature was set at 38℃,and the sample size was 2μL.The electrospray ionization detection was operated in a positive mode by multiple reaction monitoring.The detection ions for quantitative analysis were m/z 195.1→110.0 for caffeine,m/z 181.1→124.0 for theophylline,m/z 181.1→124.0 for paraxanthine,m/z 181.1→138.0 for theobromine,and m/z 198.1→140.1 for IS.The above method was used to determine the concentrations of caffeine and its three metabolites in the urine of 19 infants with apnea of prematurity(AOP).RESULTS The linear ranges of mass concentration of caffeine,theophylline,paraxanthin and theobromine were 0.200-200,0.050-50.0,0.0500-50.0,and 0.100-100μg/mL,respectively.The lower limits of quantification were 0.200,0.050,0.050 and 0.100μg/mL(r>0.990),respectively.RSDs of intra-day and intraday precision were not above 10.37%,and matrix factors were 85.68%-109.90%;extraction recoveries were 93.53%-109.40%(RSD≤15%),and RSDs of stability tests were all lower than 15%.The concentrations of caffeine and its three metabolites in the urine of 19 cases were(27.346±7.951),(0.351±0.223),(0.428±0.395)and(0.472±0.374)μg/mL,respectively.CONCLUSIONS The established HPLC-MS/MS method is simple,sensitive and can be used for the determination of caffeine and its three metabolites in urine samples of AOP.

关 键 词：咖啡因 代谢产物 早产儿呼吸暂停 尿药浓度 高效液相色谱-串联质谱法 

分 类 号：R969.1[医药卫生—药理学]