High Performance Liquid Chromatography (HPLC) Analysis

ILM and RET tissue samples obtained as described above were homogenized in 250–500 μl of methanol using a micro tissue grinder. Samples were subjected to sonication, carried out with a Branson Sonifier. After that, the samples were centrifuged at 1,000 g for 10 min and filtered on 0.45 μm syringe filters (0.45-μm Spartan filters, Schleicher and Schuell, Dassel, FRG). The filtrates were transferred into clean test tubes and subjected to a gentle stream of nitrogen till dryness. The dried residue was reconstituted in 100 μl of mobile phase by vortexing for 120 s, filtered again on 0.45 μm filters, and, finally, 10–20 μl aliquots were injected into the chromatographic apparatus (each sample was analyzed in triplicate).

HPLC analyses were performed on an Agilent 1260 Infinity II chromatographic system (Agilent Technologies) equipped with a ChemStation OpenLab software (M8307AA), a quaternary pump (G7111B), a diode array detector (DAD, G7111B), and a thermostated column compartment (G1316A). Chromatographic separations were performed using a Kinetex XB-C18 column (100 Å, 100 mm × 4.60 mm, 2.6 μm, Phenomenex). BBG, Me-BBG, and PBB^® were analyzed using three different isocratic ternary mobile phases consisting of (A) acetonitrile, (B) methanol, and (C) triethylamine 0.3% (v/v) in water (pH adjusted to 3.0 with trifluoroacetic acid) with different proportions (A:B:C) as follows: BBG 49:5:46 (v/v/v); Me-BBG 54:5:41 (v/v/v); PBB^® 62:5:33 (v/v/v). The flow rate was set at 0.5 ml/min and the column was thermostated at 24°C during the analysis. UV spectra were recorded in the range 200–800 nm and chromatograms were acquired at 613 and 316 nm. The analytical method was validated according to ICH guidelines (ICH Guideline, 2005). Identification of the dye molecules (BBG, Me-BBG, and PBB^®) was determined by HPLC–DAD analysis by comparing the retention times and the UV spectra of the analyzed samples with those of reference standards. Potential interferences from endogenous matrix constituents of ILM and RET tissues were excluded by analyzing the chromatograms of control tissues (untreated eyes), used for HPLC calibration. Furthermore, peak-purity tests (Gilliard and Ritter, 1997; Stahl, 2003) were used to demonstrate the absence of coeluting peaks. Peak-purity tests were done with OpenLab software (Agilent Technologies) using photodiode-array detector spectra. Eleven-point calibration curves were set up for all standards to test the linearity of the UV-DAD response. Linear regression was performed, using OpenLab software (Agilent Technologies), on the calibration data points of each dye tested to determine slope, intercept, and correlation coefficient (R²). Calibration standards were prepared by spiking 10 μl of appropriate working solutions of the respective dye standard into control (untreated eyes) ILM and retinal samples. Calibration standards were processed as reported in the sample preparation procedure and analyzed by HPLC. The limits of detection (LOD) and quantification (LOQ) were determined in biological samples spiked with progressively lower concentrations of the analytes. The LOD and LOQ were established with an S/N ratio of 3:1 and 10:1, respectively (n=6). Precision and accuracy were determined at three concentration levels in ILM and RET within the calibration range by assaying replicates (n = 5) of samples spiked with the three tested dyes (25,800 and 12,800 ng/ml), and processed as the unknowns. To determine intra- and inter-day precision and accuracy, matrix samples were analyzed in replicates on the same day and on three different days, respectively. Intra- and inter-day precision was reported as relative standard deviation (RSD%), with an acceptability limit set at ≤15%. Accuracy was calculated by comparison of mean assay results with the nominal concentrations, with acceptability limits set at ±15%.