For the analysis of the samples, 1 mL of urine was used. The samples were thawed at 4 °C and centrifuged at 14,000× g for 10 min at 4 °C. The supernatant was collected and 5 µL of internal standard solution (20 µM) was added. Then an SPE and elution in ethyl acetate were performed in the same way as for the retina samples in order to achieve a higher concentration of the analytes. Once evaporated, the dry extracts were reconstituted in 100 µL of H2O (pH 3, 0.1% v/v HCOOH):CH3OH (85:15 v/v). Finally, they were injected into the chromatographic system (UPLC-MS/MS).
Results were standardized by creatinine levels measured using a creatinine assay (MicroVue creatinine EIA, Quidel Corporation, San Diego, CA, USA) according to the manufacturer’s instructions.
Validation of the bioanalytical method in plasma and urine has been shown in previous studies [16 (link)] and the guidelines of the United States Food and Drug Administration were followed [17 ].
Results were standardized by creatinine levels measured using a creatinine assay (MicroVue creatinine EIA, Quidel Corporation, San Diego, CA, USA) according to the manufacturer’s instructions.
Validation of the bioanalytical method in plasma and urine has been shown in previous studies [16 (link)] and the guidelines of the United States Food and Drug Administration were followed [17 ].