We report a fast, simple high-performance liquid-chromatographic assay for serum alpha-tocopherol, with use of a reversed-phase column and tocol as internal standard. Only 200 microliter of serum is required. Isocratic elution of the n-hexane extract is done within 6 min; total analysis time is less than 1 h. Within-day precision (CV) was 2.3% for 24 samples of a normal plasma pool (mean concn, 10.1 mg/liter). Day-to-day precision (CV) was 3.2%, measured for 20 days for a specimen with a concentration of 10.8 mg/liter. Analytical recovery of alpha-tocopherol from fortified serum was 89 to 100%. The lower detection limit of alpha-tocopherol is estimated to be 0.6 mg/liter. The specificity of the procedure was verified by spectrophotometry, gas-liquid chromatography, and combined gas chromatography/mass spectrometry of an eluate collected from the column.
A liquid-chromatographic assay for alpha, beta, and gamma-isomeric tocopherols in human serum is reported. The tocopherols and the internal standard (tocol) are absorbed into a silica gel column and are eluted in less than 10 min with n-hexane/isopropanol (99.4/0.6, by vol) at a flow rate of 1 mL/min. The complete analysis requires no longer than 30 min. Within-day precision (CV) was 1.4% (mean = 13.18 mg/L, n = 24), 7.4% (mean = 0.214 mg/L, n = 14), and 1.3% (mean = 1.01 mg/L, n = 14) for alpha-, beta-, and gamma-tocopherol, respectively. Day-to-day precision (CV) was 4.4% (mean = 9.85 mg/L, n = 10) for alpha-tocopherol, 9.1% (mean = 0.222 mg/L, n = 20) for beta-tocopherol, and 3.8% (mean = 1.00 mg/L, n = 20) for gamma-tocopherol. Extraction recoveries for alpha-, beta-, and gamma-tocopherol averaged 92.4 +/- 2.9% (n = 5), 91.4 4/- 7.6% (n = 5), an 92.0 +/- 4.1% (n = 4), respectively. The smallest injected amount detectable is estimated to be 0.03 micrograms for alpha-, and 0.04 micrograms for beta- and gamma-tocopherol.
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