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

CHEN Xianglong; ZHAO Yang; HUANG Qiongye; XU Mingqing; LI Yue; LU Chao; SUN Luning; WANG Yongqing

doi:10.6039/j.issn.1001-0408.2023.18.11

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

更新时间：2025-09-26

- Method establishment and clinical practice for concentration determination of caffeine and its metabolites in urine
- ZHONGGUO YAOFANG Vol. 34, Issue 18, Pages: 2233-2237(2023)
- 作者机构：
  
  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
- 作者简介：
- 基金信息：
- DOI：10.6039/j.issn.1001-0408.2023.18.11
  CLC： R969.1
- Received：24 April 2023，
  
  Revised：2023-07-24，
  
  Accepted：10 August 2023，
  
  Published：30 September 2023
- 稿件说明：
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陈相龙,赵杨,黄琼叶,等.咖啡因及代谢产物的尿药浓度测定方法建立及临床应用[J].中国药房,2023,34(18):2233-2237.

CHEN Xianglong,ZHAO Yang,HUANG Qiongye,et al.Method establishment and clinical practice for concentration determination of caffeine and its metabolites in urine[J].ZHONGGUO YAOFANG,2023,34(18):2233-2237.
陈相龙,赵杨,黄琼叶,等.咖啡因及代谢产物的尿药浓度测定方法建立及临床应用[J].中国药房,2023,34(18):2233-2237. DOI： 10.6039/j.issn.1001-0408.2023.18.11.

CHEN Xianglong,ZHAO Yang,HUANG Qiongye,et al.Method establishment and clinical practice for concentration determination of caffeine and its metabolites in urine[J].ZHONGGUO YAOFANG,2023,34(18):2233-2237. DOI： 10.6039/j.issn.1001-0408.2023.18.11.

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-

-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0.5 mL/min，柱温为38 ℃，进样量为2 μL。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、0.100～100 μg/mL（

均大于0.990），定量下限分别为0.200、0.050、0.050、0.100 μg/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。

Abstract

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 caffeine-

-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 2 μL. The electrospray ionization detection was operated in a positive mode by multiple reaction monitoring. The detection ions for quantitative analysis were

195.1→110.0 for caffeine，

181.1→124.0 for theophylline，

181.1→124.0 for paraxanthine，

181.1→138.0 for theobromine， and

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， 0.050 0-50.0， and 0.100-100 μg/mL， respectively. The lower limits of quantification were 0.200， 0.050， 0.050 and 0.100 μg/mL （

＞0.990）， respectively. RSDs of intra-day and intra-day 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 concentration

s 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

Keywords

references

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