2.3. LC/MS/MS separation and analyses for all-trans-retinoic acid and 4-hydroxy retinoic acid.

All solvents employed for sample extractions and liquid chromatography were LC/MS or LC grade and were purchased from Thermo Fisher Scientific (Pittsburgh, PA). All measurements were carried out on a Waters Xevo TQ MS ACQUITY UPLC system (Waters, Milford, MA). The system was controlled by MassLynx software version 4. 1 (Waters, Milford, MA).

All-trans-retinoic acid (atRA) was extracted using a two-step, acid-base extraction [74], with minor modifications. Briefly, 0.5 ml of 0.025 M KOH in ethanol was added to 250 μl of tissue homogenate, which contained 50 mg of wet tissue. Five nanograms of pentadeuterated all-trans-retinoic acid (atRA-d5) dissolved in absolute ethanol was added to each extract as an internal standard. The aqueous phase was extracted with 5 ml of hexane. The organic phase containing nonpolar retinoids (retinol and retinyl esters) was removed. Thirty microliters of 4 M HCl was then added to the aqueous phase, and polar retinoids, including retinoic acid, were removed by extraction into 5 ml hexane. The hexane was removed under N₂. Extracts were resuspended in 70 μl of acetonitrile and transferred to amber LC/MS vials (Waters, Milford, MA). Only glass containers, pipettes, and calibrated syringes were used to handle and process retinoic acid. Samples were maintained at 4°C in the autosampler, and 5 μl was loaded onto a Waters ACQUITY UPLC HSS C18 column (2.1 mm inner diameter × 100 mm with 1.8 μm particles), preceded by a 2.1 × 5 mm guard column with the same packing material (Waters). The column was maintained at 40°C by a column heater. The flow rate was 300 μl/min in binary gradient mode with the following mobile phase gradient: initiated with 32% phase A [H₂O, containing 0.1% formic acid] and 68% mobile phase B [acetonitrile, containing 0.1% formic acid]; the gradient was maintained for 6.3 min. The acetonitrile content of the solvent was increased linearly to 85% over 6.4 min and maintained until 9.5 min, increased to 100% to wash the column for 2 min, and then decreased acetonitrile to 68%. All-trans-retinoic acid (atRA) eluted between 8.2 and 8.4 min. Positive ESI-MS/MS mass spectrometry was performed using the following parameters: capillary voltage 3.8 kV, source temperature 150°C, desolvation temperature 500°C, desolvation gas flow 800 l/h, collision gas flow 0.15 ml/min. Optimized cone voltage was 16 V, collision energy for multiple reaction monitoring mode (MRM) was 18 eV, and the following transitions were used: atRA for quantification, m/z 301.16 → 123.00; atRA for verification, m/z 301.16 → 205.03; and atRA-d5, m/z 306.15 → 127.03.

4-Hydroxy all-trans-retinoic acid (4-HO atRA) was extracted using chloroform/methanol. Briefly, 500 μL of tissue homogenate containing 100 mg of liver tissue were mixed with 2 ml chloroform and 1 ml methanol containing 5 ng of atRA-d5 as an internal standard. The mixture was vortexed well and centrifuged at 3,000 g for 10 minutes. The lower organic phase was transferred to another clean glass tube using a Pasteur pipette. Two ml of chloroform was added to the residual aqueous phase, followed by vortex mixing and centrifugation again at 3,000 g for 10 min, to extract any remaining lipids. The lower organic phases were pooled and evaporated under nitrogen. The extracted lipids were reconstituted in 30 μL of acetonitrile/methanol (vol:vol = 1:1) and transferred to LC/MS autosampler vials (Waters, Milford, MA) for injection. The sample was maintained at 4°C in the autosampler and a volume of 5 μl was loaded onto a Waters ACQUITY UPLC HHS C18 column (2.1 mm inner diameter × 100 mm with 1.8 μm particles, Waters, P/N 186003533), and a 2.1 × 5 mm guard column with the same packing material (Waters, P/N 186003981). The column was maintained at 40°C. The flow rate was 300 μl/min in binary gradient mode with the following mobile phase gradient: initiated with 50% phase A (H₂O containing 0.1% formic acid) and 50% mobile phase B (acetonitrile, containing 0.1% formic acid). Acetonitrile content was linearly increased to 100% over 5 min and maintained till 10 min. Then the content of acetonitrile was reduced to 50% and maintained for 2 min before the next injection. Species of interest were eluted between 8.2 and 8.4 min. Positive ESI-MS/MS with multiple reaction monitoring (MRM) mode was performed using the following parameters: capillary voltage, 4 kV; source temperature, 150 °C; desolvation temperature, 500 °C; desolvation gas flow, 1000 L/hr; and collision gas flow, 0.18 mL/min. Optimized cone voltage was 20 V, collision energy for multiple reaction monitoring mode (MRM) was 20 eV. The following transitions were used: 4-HO atRA, m/z 299.3 → 95.0; and atRA-d5, m/z 306.15 → 127.03.