EN
Determination of Caffeine in Human Plasma by Using Liquid Chromatography-Tandem Mass Spectrometry
Abstract
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine caffeine (CAF) in human plasma. The plasma samples were extracted by protein precipitation using CAF-D3 as an internal standard (IS). The chromatographic separation was performed on GL Sciences InertSustain C18 Column (4.6 x 50 mm, 5 µm) maintained at 40 °C with a mobile phase consisting of formic acid, water, and methanol at a 1 mL/min of flow rate using two separate lines. CAF was detected and identified by mass spectrometry with electrospray ionization (ESI) in positive ions and multiple-reaction monitoring (MRM) mode. The MRM transitions of m/z 195.10 > 138.00 for CAF and 198.10 > 141.10 for IS were used for quantification. The standard curve was linear in the range of 10 - 10000 ng/mL for CAF. The within-batch precision and accuracy were evaluated by analyzing QC samples at five different concentration levels with six replicates in a batch. The between-batch precision and accuracy were determined by analyzing three different batches. The within-batch accuracy and precision was -8.76% - 9.61% and 0.95% - 7.22%, respectively. The between-batch accuracy and precision was -7.47% -1.42% and 1.83% - 8.66%, respectively. The results of the intra- and inter-day precision and accuracy studies were within the limits. The validated method applied to a pharmacokinetic study and the test product containing 60 mg CAF administered to total of 12 subjects. The mean ± SD of maximum plasma concentration (Cmax) was found to be 147.94 ± 139.39 ng/mL and the mean ± SD of area under the plasma concentration-time curve from zero to last measurable concentration (AUC0-tlast) was found to be 1119.59 ± 1468.30 h.ng/mL for the fasting conditions. The median time to reach peak plasma concentration (Tmax) was found to be 12.00 (6.50 - 12.00). The developed and validated method can be used for bioavailability and bioequivalence studies in human plasma samples.
Keywords
References
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Details
Primary Language
English
Subjects
Pharmaceutical Analytical Chemistry
Journal Section
Research Article
Publication Date
September 29, 2023
Submission Date
June 24, 2023
Acceptance Date
August 28, 2023
Published in Issue
Year 2023 Volume: 44 Number: 3