A rapid HPLC method used to establish pediatric reference intervals for vitamins A and E

BackgroundSerum concentrations of the fat-soluble vitamins A (retinol) and E (tocopherol) are measured to assess deficiency and, in the case of vitamin A, toxicity. We modified our existing HPLC method for analyzing vitamins A and E by using a high throughput analytical column and small diameter tubing to reduce analysis time. The modified HPLC method was used to establish pediatric reference intervals for these vitamins.MethodsSerum or plasma proteins were precipitated with ethanol. A and E vitamins were extracted into hexane, evaporated under nitrogen, dissolved in absolute ethanol, and analyzed by HPLC with ultraviolet detection.ResultsThe modified HPLC method correlated well with the existing method. Data analysis from the reference interval study resulted in age-dependent intervals for retinol and non-age-dependent intervals for retinyl palmitate, α-tocopherol, and γ-tocopherol. Gender-based reference intervals were not necessary.ConclusionsWe validated a rapid HPLC method for analyzing vitamins A and E that decreased run-time by 60%, mobile phase consumption by 39%, and sample injection volume by 50%. The modified method was used to establish pediatric reference intervals for vitamins A and E in samples from 1136 healthy children aged 7 to 17 y.