Analysis of <sup>13</sup>C Enrichment of Vitamin A in Biological Samples by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry

Vitamin A is critical for health, but assessment of vitamin A status is challenging. Vitamin A circulating in blood is homeostatically controlled and often does not reflect vitamin A deficiency or excess. Use of ¹³C-vitamin A allows tracing and quantifying the total body vitamin A stores and additional information on pharmacokinetics and metabolism. This protocol describes the extraction, purification, and determination of ¹³C-isotopic enrichment of vitamin A in serum, breast milk, and tissue samples using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Analysis using GC-C-IRMS provides sensitive and precise enrichment values, particularly for samples with ¹³C enrichments near natural abundance levels. This enables flexibility in study designs to use smaller tracer doses and longer-term pharmacokinetic sampling. Briefly, to 0.4 to 1 mL serum, ethanol is added to precipitate proteins, C-23 β-apo-carotenol is added as an internal standard, and hexane is used to extract retinoids. The extract is subsequently resuspended in methanol and purified using HPLC. The retinol fraction is then resuspended in hexane and injected into the GC-C-IRMS system. Atom % (At%) is directly calculated in reference to carbon dioxide, which is calibrated against a sucrose standard using an elemental analyzer. Other tissue types that have been analyzed by GC-C-IRMS include breast milk and liver after saponification. Solid phase extraction techniques can be used to assist in the purification of these or other complex matrices. Furthermore, an elemental analyzer can be attached to the IRMS to analyze food samples for total ¹³C content.