Method establishment and clinical practice for concentration determination of caffeine and its metabolites in urine
- VernacularTitle:咖啡因及代谢产物的尿药浓度测定方法建立及临床应用
- Author:
Xianglong CHEN
1
,
2
;
Yang ZHAO
2
;
Qiongye HUANG
2
;
Mingqing XU
2
;
Yue LI
3
;
Chao LU
3
;
Luning SUN
2
;
Yongqing WANG
1
Author Information
1. School of Pharmacy/Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy，Xuzhou Medical University，Jiangsu Xuzhou 210004，China
2. Research Division of Clinical Pharmacology，the First Affiliated Hospital of Nanjing Medical University，Nanjing 210029，China
3. Dept. of Pediatrics，Jiangsu Women and Children Health Hospital，Nanjing 210036，China
- Publication Type:Journal Article
- Keywords:
caffeine;
metabolites;
apnea of prematurity;
urinary drug concentration;
HPLC-MS/MS
- From:
China Pharmacy
2023;34(18):2233-2237
- CountryChina
- Language:Chinese
-
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-13C3-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 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， 0.050-50.0，0.050 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 （r＞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 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.
