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.
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