OBJECTIVE

2

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

2

Using caffeine-

13

C

3

-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

2

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

2

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.