Development of a Validated High-Performance Thin-Layer Chromatography (HPTLC) Analysis Protocol for Salivary Caffeine Used as a Probe Drug

CYP1A2 activity plays a critical role in the metabolism of drugs such as caffeine, clozapine, propranolol, and warfarin. In pharmacogenomic studies, caffeine is a probe drug of choice for CYP1A2 phenotyping. Due to the non-invasive nature of sampling, saliva is an alternative biofluid to plasma for monitoring caffeine levels. This study reports on a validated HPTLC method for quantifying salivary caffeine levels, which can support future studies on CYP1A2 phenotyping employing caffeine as a probe drug. The HPTLC method, using silica gel 60 F254 plates and acetone/toluene/chloroform (4:3:3, <i>v</i>/<i>v</i>/<i>v</i>) as the mobile phase, has detection and quantification limits of 2.42 and 7.34 ng/band, respectively. An optimised saliva processing protocol using a 1:1 dilution with methanol was also established. Five saliva sample sets collected 0-4 h after ingestion of 100 mg caffeine were analysed using the developed and validated HPTLC method, which demonstrated that salivary caffeine concentrations peak around 1 h post ingestion and then gradually decrease over the study period. Thus, the developed HPTLC method can be used to analyse caffeine levels in saliva and to support CYP1A2 phenotyping using caffeine as a probe drug.