Analysis of Vitamin A Stable Isotope Tracers using LC-MS/MS

The retinol isotope dilution (RID) method estimates the total body stores (TBS) of vitamin A in humans through stable isotope dilution with the body's vitamin A pool. For this, it is critical to accurately and efficiently determine the plasma isotopic ratio of labeled to non-labeled retinol through mass spectrometry. To avoid extensive and time-consuming extraction and/or purification procedures, such as preparative HPLC and derivatization, LC-MS/MS is employed to conduct fast, sensitive, and simultaneous analysis of labeled and non-labeled retinoids. The method utilizes two levels of detection: (i) an initial mass/charge (m/z) separation of parent (precursor) ions, followed by (ii) detection of fragmented daughter (product) ions. This results in high sensitivity, with retinol detection limits as low as 6 fmol on-column. Despite the advantages of tandem mass spectrometry, a liquid chromatographic separation is required to separate retinol from retinyl esters since the terminal functional groups are lost during ionization, resulting in similar parent (m/z of 269) and daughter ion fragmentation patterns. The article describes the isolation of endogenous and labeled (¹³C or ²H) retinol from plasma by solvent extraction, followed by quantification by LC-MS/MS under atmospheric pressure chemical ionization (APCI) in positive ion mode.