Development and validation of a simple isotope dilution-liquid chromatography-tandem mass spectrometry method for detecting vitamins A and E in serum and amniotic fluid

摘要

Accurate and simple measurements of vitamins A(all-trans retinol) and E(alpha.-tocopherol) are essential for ensuring the appropriate supplementation and diagnosis of diseases related to vitamin A or E deficiency. High-performance liquid chromatography is a widely used method; however, few studies have reported an isotope dilution-liquid chromatography tandem mass spectrometry method(ID-LC-MS/MS) for detecting vitamins A and E in serum, let alone amniotic fluid. Here, we developed and validated an accurate and precise measurement procedure based on ID-LC-MS/MS for detecting vitamins A and E in serum and amniotic fluid. In this method, only 50 mu L of serum/amniotic fluid was used. After simple protein precipitation and extraction, all-trans retinol and alpha-tocopherol were analyzed by ID-LC-MS/MS using positive electrospray ionization in multiple reaction monitoring mode. Vitamins A and E status was evaluated using the validated method in 65 apparently healthy volunteers and 58 pregnant women in the second trimester. The within-run and total coefficients of variation(CVs) of all-trans retinol were 2.8%-3.9% and 4.7%-6.0%, respectively, in serum; and 5.0%-7.3% and 5.4%-7.4%, respectively, in amniotic fluid ; and the values for alpha-tocopherol were 3.2%similar to 4.3% and 3.3% similar to 4.9% in serum and 6.2%-7.1% and 8.0%-9.1% in amniotic fluid. The accuracy bias of the ID-LC-MS/MS method was less than 5.0% for both all-trans retinol and alpha-tocopherol. Mean levels of all-trans retinol and alpha-tocopherol in the 65 apparently healthy volunteers were 0.504 +/- 0.126 mg/L and 12.038 +/- 2.501 mg/L, respectively. A significant positive correlation was observed between serum and amniotic fluid all-trans retinol (r=0.277, P=0.035) in the pregnant women in the second trimester, while no correlation with alpha-tocopherol (P=0.290). None of the volunteers developed vitamin A or E deficiency. In conclusion, an ID-LC-MS/MS method for measuring both serum and amniotic flu