Validated HPLC Method for Determination of Caffeine Level in Human Plasma using Synthetic Plasma: Application to Bioavailability Studies

Abstract

Several high-performance liquid chromatography (HPLC) methods have been described for the determination of caffeine in human plasma. However, none have been cross validated using synthetic plasma. The present study describes a simple and reliable HPLC method for the determination of the caffeine level in human plasma. Synthetic plasma was used to construct calibration curves and quality control samples to avoid interference by caffeine commonly present in donor's human plasma. After deproteination of plasma samples with perchloric acid, caffeine and antipyrine (internal standard, IS) were separated on a Waters Atlantis C18 column using a mobile phase of 15 mM potassium phosphate (pH 3.5) and acetonitrile (83:17, v/v), and monitored by photodiode array detector, with the wavelength set at 274 nm. The relationship between caffeine concentrations and peak area ratio (caffeine-IS) was linear over the range of 0.05-20 μg/mL. Inter-run coefficient of variation was ≤ 5.4% and ≤ 6.0% and bias was ≤ 3% and ≤ 7% using human and synthetic plasma, respectively. Mean extraction recovery from human plasma of caffeine and the IS was 91% and 86%, respectively. Caffeine in human plasma was stable for at least 24 h at room temperature or 12 weeks at -20 °C, and after three freeze-thaw cycles. The method was successfully applied to monitor caffeine levels in healthy volunteers with correction of caffeine levels using the mean ratio of the slopes of the calibration's curves constructed using human and synthetic plasma.

Determination of Caffeine in Surface Water Using Solid Phase Extraction and High Performance Liquid Chromatography

“…The drawback of using buffer solution is to clog the column with the salt of buffer if there is no enough washing. Our study also compared with other previous reported work in terms of solvent consumption [20]. Chowdhury et al, reported that caffeine was separated after 20 min run at 1.0 mL which means each run could consume 20 mL [21], compared to our study, only 1.0 mL was consumed at 0.3 mL/min for each run.…”

Validation and Vitamin C Testing in Crystal Guava (Psidium guajava L.) With Variations of Origin With the HPLC Method (High Performance Liquid Chromatography)

“…The obtained R value is at 0.9911, whereas according to AOAC (2013), the suggested correlation coefficient is ≥ 0.99. That result fulfills linearity requirements and demonstrates that the test with the HPLC method has good linearity, therefore the linear regression equation can be used to determine levels of vitamin C in the samples (Alvi and Hammami 2011).…”

“…Validation Methods include: System suitability test, Linearity, and Precision (Alvi and Hammami, 2011).…”

An impedimetric molecularly-imprinted biosensor for Interleukin-1β determination, prepared by in-situ electropolymerization on carbon screen-printed electrodes

“…The impedance response was recorded for each concentration by covering in a drop-like fashion the biosensors with an electron mediator solution of 5.0 × 10 −3 M of potassium hexacyanoferrate III, ( Interfering solutions of Myoglobin (Myo) and Immunoglobulin G (IgG) were prepared in PBS, with A concentration 6.0 × 10 −9 M, as IL-1β. Synthetic serum was also prepared according to [19] and consisted of NaCl (145 × 10 −3 M), KCl (4.5 × 10 −3 M), CaCl 2 (32.5 × 10 −3 M), MgCl 2 (0.8 × 10 −3 M), Urea (2.5 × 10 −3 M), and Glucose (4.7 × 10 −3 M) in aqueous solution.…”