ATRA was quantified in serum as previously described (43 (link)). Briefly, 100 μl of serum and 1 μl of ATRA-d5 [50 μg/ml; internal standard, Toronto Research Chemicals (TRC)] were transferred to a 15-ml glass culture tube and extracted in 200 μl of acetonitrile (Sigma-Aldrich) and 2-μl formic acid (Sigma-Aldrich) and 10 ml of hexanes (Sigma-Aldrich). The hexane extracts was separated by centrifugation, dried under nitrogen flow, and reconstituted in 100 μl of acetonitrile for LC-MS/MS analysis. The whole process was performed on ice and under yellow light to avoid the degradation.
The concentration of ATRA from serum was measured using the AB Sciex 4000 QTRAP LC-MS/MS System equipped with atmospheric-pressure chemical ionization (APCI) in positive ion mode. Briefly, ATRA was separated with the 2.1 mm by 100 mm Supelcosil ABZ + PLUS column (3 μm; Sigma-Aldrich) with the following running solvents: A, H2O with 0.1% formic acid; B, acetonitrile with 0.1% formic acid. Quantification is based on peak area ratio of ATRA to the ATRA-d5 and the ATRA-d5 concentration.
The concentration of ATRA from serum was measured using the AB Sciex 4000 QTRAP LC-MS/MS System equipped with atmospheric-pressure chemical ionization (APCI) in positive ion mode. Briefly, ATRA was separated with the 2.1 mm by 100 mm Supelcosil ABZ + PLUS column (3 μm; Sigma-Aldrich) with the following running solvents: A, H2O with 0.1% formic acid; B, acetonitrile with 0.1% formic acid. Quantification is based on peak area ratio of ATRA to the ATRA-d5 and the ATRA-d5 concentration.
