Rapid Isocratic HPLC Method and Sample Extraction Procedures for Measuring Carotenoid,Retinoid, and Tocopherol Concentrations in Human Blood and Breast Milk for Intervention Studies

Vitamin A deficiency continues to be a major public health concern in the developing world effecting over 200 million people. Interventions including carotenoid-rich fruits and vegetables, containing α- and β-carotene, and β-cryptoxanthin, are being tested for their potential to alleviate vitamin A deficiency in several countries. Thus, there is a need for a faster isocratic reverse-phase high pressure liquid chromatography (HPLC) method than the methods currently available. Efficient extraction procedures for biological samples are also valuable to save time, materials, and cost. We developed an HPLC method and compared several extraction methods for carotenoids, retinoids, and vitamin E in plasma and human breast milk. The method uses an Agilent 1100 system equipped with a Waters Spherisorb ODS2 column (3 × 125 mm 3 μm) and similar guard column (ThermoScientific ODS2 3 × 20 mm 3 μm). Mobile phase of 7:2:1 acetonitrile:dichloromethane:methanol was run at 0.5 mL min^?1, and run times were complete in approximately 8 min. This rapid method uses little solvent and provides excellent results in the analysis of these fat-soluble nutrients. Analyte measurements were repeatable using standards and plasma and were accurate compared to NIST certified and reference serum values. The validated HPLC method was applied to the analysis of plasma samples from a vitamin A intervention study.