Application of Rhodamine 800 for Reversed Phase Liquid Chromatographic Detection Using Visible Diode Laser-Induced Fluorescence

This study reports the application of rhodamine 800, a far-red dye, suitable for excitation using visible diode laser-induced fluorescence (VDLIF) detection. A reagent synthesized from rhodamine 800 was evaluated as a precolumn reagent for derivatization with amino-containing analytes. The derivative of this reagent with primary amine analytes showed a loss of fluorescence. Rhodamine 800 was then applied as a mobile phase additive in the indirect mode for quantitation of valproic acid in plasma using reversed phase HPLC in combination with VDLIF detection. A visible diode laser (output power 8.50 mW) temperature-tuned to oscillate at 674.70 nm was used as a light source for a laboratory constructed HPLC fluorescence detector. A liquid/liquid extraction procedure was applied to human blank plasma. The selectivity of this method was validated by demonstration of a lack of interfering peaks in extracts of plasma (n = 3 sources). A calibration curve for valproic acid between 40 and 200 μg/mL was shown to be linear (r = 0.9932). The recoveries of valproic acid at concentrations of 50 and 100 μg/mL were evaluated and determined to be 73 and 72%, respectively. The precision and accuracy (n = 5) of the assay was within 7.0% RSD and 8.0% difference from the spiked concentration, respectively. The limits of detection (S/N = 3) for extracted and unextracted valproic acid were 15.0 and 11.54 μg/mL, respectively. The theoretical (C lim) and practical (C det) limits of detection in the detector flow cell for unextracted valproic acid at a S/N = 1 were found to be within 15%.