Background Clearance of caffeine depends upon specific hepatic metabolic pathways and its measurement, which quantitates liver metabolic function, requires multiple samples for up to 3 days. Herein we describe a method for measuring deuterated isotopes of caffeine to allow determination of clearance from single samples of serum.
Methods Caffeine concentrations range from 100 to 6,000 ng/mL 24 h after a single oral dose of 300 mg. Deuterated caffeine (D3 and D9), unlabeled caffeine, and phenacetin (500 ng/mL) were added to five separate samples of calf serum and extracted with methylene chloride after alkalinization. The methylene chloride layer was dried and reconstituted in 50 μL of acetone. Compounds were analyzed by gas chromatography-mass spectrometry with an initial oven temperature of 40°C for 0.55 min, increasing at 50∫/min to 280°C, held isothermally at 280°C for 4 min, and quantified by selected ion monitoring (m/z 179, 194, 197, and 203) using calibration curves with phenacetin as internal standard.
Results The correlation coefficients for the calibration curves were 0.995, 0.996, and 0.995 for unlabeled, D3 and D9 caffeines, respectively. X ± SD and coefficients of variance (CV) for unlabeled caffeine (2,800 ng/mL) and D3 and D9 (400 ng/mL each) were 2,800 ± 109, 3.9%; 411 ± 18, 4.4%; and 385 ± 16 ng/mL, 4.2%, respectively. Instrument precision was 99.50%, 99.38%, and 99.51%, respectively. These concentrations reflect expected concentrations in human serum 4 h after an oral dose of 300 mg of total caffeine at a molar ratio D3 (or D9): unlabeled caffeine of 1:7. X ± SD, CV of unlabeled caffeine (600 ng/mL), and D3 and D9 (150 ng/mL each) were 539 ± 61, 11%; 143 ± 12, 8.4%; and 135 ± 16 ng/mL, 12% with precision of 98.73%, 99.43%, and 99.22%, respectively. These concentrations reflect expected concentrations in serum 24 h after an oral dose of 300 mg total caffeine with a molar ratio of D3 (or D9): unlabeled caffeine of 1:4.
Conclusions Our method precisely and accurately quantifies caffeine and deuterated isotopes over concentration ranges achieved after oral dosing with 300 mg caffeine.
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