Validation and application of a simple and rapid stability-indicating liquid chromatographic assay for the quantification of caffeine from human saliva

A clinical trial is currently underway to examine the efficacy of using caffeine citrate to prevent intermittent hypoxemia in late preterm neonates. Determining caffeine concentration using saliva in this population would be preferable as it is less invasive than plasma sampling, but a suitable method of analysis is required. This paper presents the development and validation of a rapid, efficient and reproducible stability-indicating high-performance liquid chromatography (HPLC) method and extraction protocol for the quantification of caffeine present in saliva. The stability of extemporaneously prepared caffeine citrate solutions (at 20-25 degrees C) was determined, along with the stability of caffeine spiked saliva samples (at 20-25 and 2-8 degrees C), to ensure the suitability of the developed method in the analysis of clinical trial samples. Protein precipitation using acetonitrile ensured the complete removal of salivary proteins and resulted in extraction recovery of >= 95% for all samples. The HPLC assay following extraction was linear (R-2>0.99) over the range 0.3-50 mu g/mL (lower limit of quantification 0.3 mu g/mL). The accuracy of the quality control samples was 94-100% and the relative standard deviation (RSD) was <7% for all samples. Caffeine-spiked saliva samples were stable for three freeze-and-thaw cycles pre-extraction and up to 72 hr post-extraction. The extraction and HPLC methods described were thus suitable for the analysis of clinical trial samples from the Latte Dosage Trial. All caffeine solutions were physically and chemically stable, with concentrations at the end of the three-month test period being within 4% of the baseline concentrations, indicating appropriateness for use as clinical trial medications.