New method for caffeine quantification by planar chromatography coupled with electropray ionization mass spectrometry using stable isotope dilution analysis

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Abstract
A new high‐performance thin‐layer chromatography/electrospray ionization mass spectrometry (HPTLC/ESI‐MS) method for the quantification of caffeine in pharmaceutical and energy drink samples was developed using stable isotope dilution analysis (SIDA). After sample preparation, samples and caffeine standard were applied on silica gel 60 F254 HPTLC plates and over‐spotted with caffeine‐d3 used for correction of the plunger positioning. After chromatography, densitometric detection was performed by UV absorption at 274 nm. The bands were then eluted by means of a plunger‐based extractor into the ESI interface of a single‐quadrupole mass spectrometer. For quantification by MS the [M+H]+ ions of caffeine and caffeine‐d3 were recorded in the positive ion single ion monitoring (SIM) mode at m/z 195 and 198, respectively. The calibration showed a linear regression with a determination coefficient (R2) of 0.9998. The repeatability (RSD, n = 6) in matrix was ≤ ± 3.75%. The intermediate precisions (RSD, n = 2) for two samples of different brand names were determined three times and ranged between RSD ±0.68% and ±2.64% (sample 1) and between ±3.44% and ±8.60% (sample 2). The method accuracy was evaluated by comparing the results obtained by HPTLC/SIDA‐ESI‐MS with those from the validated HPTLC/UV method. The results for pharmaceutical and energy drink samples were (ng/band) 99.82 ± 3.75 and 338.09 ± 4.87 by HPTLC/SIDA‐ESI‐MS and 104.74 ± 1.51 and 334.86 ± 5.63 by HPTLC/UV. According to the F‐test (homogeneity of variances) and the t‐test (comparison of means) the two methods show no significant difference. The detection and quantification limits were 75 and 250 µg L−1 (0.75 and 2.5 ng/band), respectively, which were a factor of 13 lower than those established for HPTLC/UV. The positioning error (RSD ±6%) was calculated by comparing HPTLC/SIDA‐ESI‐MS with HPTLC/ESI‐MS. However, using SIDA the positioning error was nullified. HPTLC/SIDA‐ESI‐MS was demonstrated to be a highly reliable method for the quantification of compounds by planar chromatography coupled online with mass spectrometry. Copyright © 2007 John Wiley & Sons, Ltd.

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