Figure 3: The stability of retinol, atRA and 13-cisRA in blood and plasma.

Panel (A) shows the effect of time from blood draw to separation of plasma from blood on retinoid stability. The blood was drawn to heparin vacutainer tubes (green top), immediately light protected with foil, and inverted. Blood samples were kept on ice for 0, 15, 30, and 60 minutes post-blood draw, followed by centrifugation for 10 min at 1,000 × g to separate plasma. Panels (B-D) show the analysis of storage conditions on retinol (B), atRA (C) and 13-cisRA (D) recovery from human plasma samples. For these analyses aliquots of plasma samples collected above were stored at −20°C or −80°C for up to 90 days. At designated time points, aliquots were thawed and analyzed by LC-MS/MS. For analysis, samples were spiked with an IS mixture (atRA-d₅, 13-cisRA-d₅, and ROL-d₈), protein precipitated with acetonitrile (1:1 v/v), centrifuged and analyzed as described in the protocols section. Analyte peak areas were normalized to their respective IS. Data is shown as means ± SD for three replicates per time point and peak area ratios are presented as % of t = 0 days. Shaded regions denote ±15% around t=0 starting measurement to indicate range of acceptable analytical variability.