Simultaneous determination of caffeine and its metabolites in rat plasma by UHPLC‐MS/MS

Abstract

Caffeine is a widely consumed psychostimulant with several mechanisms of action and various positive and negative effects on organisms. Caffeine undergoes extensive hepatic metabolism to form main metabolites such as theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid. However, interspecies diversities have been observed in caffeine metabolism. In the present study, we developed a sensitive and straightforward ultra-high-performance liquid chromatography-tandem mass spectrometry method to quantify caffeine and its primary metabolites, namely theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid in rat plasma. After extraction of analytes using micro solid-phase extraction plate, analytes were separated by elution gradient on the Acquity UPLC HSS T3 (50 × 2.1 mm, 1.8 μm) column over 4 min. The detection was done on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring modes using a positive electrospray ionization interface.The method was successfully validated according to the European Medicine Agency guideline over a concentration range of 5-1500 ng/ml for caffeine, 5-1200 ng/mL for theobromine, and 2.5-1200 ng/mL for theophylline, paraxanthine, and 1,3,7-trimethyluric acid. The developed method was applied to analyze samples from animal experiments focusing on the metabolism and effects of caffeine and caffeine-containing beverages.

Simultaneous quantification of caffeine and its main metabolites by gas chromatography mass spectrometry in horse urine

“…The simultaneous analysis of caffeine and its metabolites has been recommended (Mendes et al, 2019). It has been reported that caffeine and its metabolites can successfully be analyzed together in some studies, but the close chromatographic retention times for theophylline and paraxanthine that generate similar product and precursor ions can be challenging (Chen et al, 2017; Kertys et al, 2021). In this study, a derivatization method was used to separate paraxanthine and theophylline to accurately analyze these two compounds in samples.…”

“…By using an automated solid‐phase extraction device, this analysis can be done easily. The common features of the cartridges used in the extraction were that they are effective over wide pH ranges, they allow the simultaneous extraction of substances with different properties and have been used caffeine analyses before (Kertys et al, 2021). It is possible to use Oasis MCX cartridges, but the absorbance value is lower than that for the other cartridges.…”

“…Previously published methods showed that theophylline and paraxanthine formed the same fragmentation using LC-MS or LC-MS/MS instruments (m/z 181.1/124.1; Kertys et al, 2021;Mendes et al, 2019, Ptolemy et al, 2010. These two substance could not be distinguished from each other owing to chromatographic interference and in most of the studies, only one of them was selected and analyzed.…”

“…Otherwise, interference between paraxanthine and theophylline can cause erroneous quantitative analysis of theophylline. In previous studies where these two metabolites were separated chromatographically, specific columns (Choi et al, 2013;Kertys et al, 2021), mobile phases (Aresta et al, 2005), long separation time, e.g. ; 45-25 minutes (Caubet et al, 2002(Caubet et al, , 2004Huang et al, 2012) and changes in instrument conditions (Chen et al, 2017) were applied to quantitatively analyze them accurately or analysis was performed by loading paraxanthine and theophylline separately (Mendes et al, 2019).…”

A high‐throughput liquid chromatography‐tandem mass spectrometry method for simultaneous determination of direct oral anticoagulants in human plasma

“…However, several disadvantages should be recognized, such as calibration to the particular drug, the imprecise results at low plasma levels, and none of the methods being specific enough for quantifying all DOACs in a single run [10]. LC-MS/MS presents a golden standard and powerful technique for multiple drug analysis [11][12][13], including DOACs monitoring in preclinical drug development [14,15]. Due to the high degree of specificity, sensitivity, and selectivity over the coagulation-based assays, the use of LC-MS/MS in clinical laboratories has significantly increased in past years for DOAC monitoring [16].…”