Synthesis of <sup>13</sup>C<sub>2</sub>-Retinyl Acetate and Dose Preparation for Retinol Isotope Dilution Tests

The retinol isotope dilution (RID) test is the most sensitive method to assess vitamin A status by estimating total liver reserves, considered the reference standard. For gas chromatography-combustion-isotope ratio mass spectrometry detection, ¹³C is added to the retinol moiety. The synthetic procedure for ¹³C-retinyl acetate begins with the naturally occurring β-ionone. Briefly, C18-tetraene ketone is synthesized from β-ionone and purified. If a ¹³C4-retinyl acetate is desired, the carbanion of ¹³C2-labeled triethylphosphonoacetate can be used to add two ¹³C's to the β-ionone moiety before adding acetone's three carbons to synthesize the C18-tetraene ketone. If a ¹³C3-retinyl acetate is desired, the three carbons of the acetone could be labeled with ¹³C. For the most commonly used ¹³C2-retinyl acetate for future analysis by gas-chromatography-combustion isotope ratio mass spectrometry, a modified Wittig-Horner procedure is used, and unlabeled C18 tetraene ketone is reacted with the carbanion of ¹³C2-labeled triethylphosphonoacetate to add two-¹³C's to the 14 and 15 positions of the retinol molecule. The resulting ethyl ester is reduced to the alcohol and esterified to ¹³C2-retinyl acetate with acetic anhydride. The all-trans isomer is purified on 8%-water-deactivated alumina using relatively innocuous, highly volatile, solvents-hexanes and diethyl ether-which are removed by rotary evaporation and sonification of oil doses. The product is characterized by ultraviolet-visible (UV-Vis) spectroscopy, thin layer chromatography (TLC), and high-pressure liquid chromatography (HPLC) against analytical standards. The concentrated dose is stored dissolved in soybean oil at -80 °C. Final doses are diluted in soybean oil (~200 µL/dose) and quantified by UV-Vis spectroscopy against the vehicle soybean oil. Doses are measured for delivery to humans with a positive displacement pipette or tuberculin syringes for the RID test. Using appropriate assumptions, total body stores are calculated, and total liver reserves are estimated.