Suplex pkb 100 column

Tissue and cellular RA values were quantified by LC/MS/MS [46 (link)], with modified LC conditions. LC was done using a Suplex pkb-100 column (Supelco, 2.1 x 250 mm, 5 μm particles). LC was eluted with a gradient of 80% acetonitrile/20% water/0.1% formic acid for 3 min, followed by a linear gradient to 95% acetonitrile/5% water/0.1% formic acid over 9 min, held for 4 min, returned by linear gradient to the original mobile phase over 1 min and held 8 min, all at 0.4 mL/min. Tissue and primary cell retinal values were measured after O-ethyloxime derivatization, using an ultra-high performance liquid chromatography MS/MS assay with a 5 fmol lower limit of detection [47 (link)]. Retinol was measured with a LC/UV assay [48 (link)].

Fucoxanthin was quantified using the HPLC method described by Gille et al. [28 ] with slight modifications. Briefly, the purified fucoxanthin was resolved in pure ethanol with BHT (250 mg/L) and compared to a commercial analytical standard (16337, Sigma-Aldrich, St. Louis, MO, USA) using reverse-phase HPLC with a Suplex pKb 100 column (5 µm, 250 × 4.6mm, Supelco, Bellefonte, PA, USA). Samples (injection volume 5 µL) were analyzed using a HPLC (1200 Infinity, Agilent, Santa Clara, CA, USA) equipped with a multi-wavelength UV detector at 450 nm and a flow rate of 1 mL/min. The gradient used for the method is described in detail elsewhere [29 (link)].
Additionally, fucoxanthin from P. tricornutum was analyzed and compared to the commercial standard by UHPLC-DAD (1290 Infinity, Agilent Technologies) using a Zorbax Eclipse Plus C18 (2.1 × 50 mm) column with a particle size of 1.8 µm. Mobile phase A contained water with 0.1% formic acid and mobile phase B consisted of methanol with 0.1% formic acid. The gradient used is shown in Table 1. The fucoxanthin was detected at 450 nm and analyzed in a mass spectrometer (LTQ XL, Thermo Scientific) using ESI in full scan mode from 200 to 1000 m/z at a temperature of 275 °C and −9.0 V. The m/z values (see supplementary material, S1) were compared to the analytical standard and to literature [30 (link)].