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

BACKGROUND: Serum 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.

METHODS: Serum 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.

RESULTS: The 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, alpha-tocopherol, and gamma-tocopherol. Gender-based reference intervals were not necessary.

CONCLUSIONS: We 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.