This research validates RP-HPLC fluorimeter and OPA-IBLC derivatization parameters to lower limits of detection (LOD) and quantitation (LOQ). Three-dimensional fluorometric scans of 25 μM OPA-IBLC derivatized histidine and glutamic acid in 0.4 M sodium borate buffer pH 10.4 showed peak fluorescence occurs at 368- to 445-nm and 336- to 452-nm λex - λem, respectively. When diluted to 12.5 μM, the fluorescence maxima shifted to 230- to 450-nm and 234- to 451-nm λex - λem, respectively. These results are consistent with the reported literature. Additionally, the ~360 λex shows linearity at higher OPA-IBLC derivatized amino acid concentrations than the ~230 nm λex peak due to differential quenching—a feature that should be considered during calibration. Additional experiments should vary pH, ionic strength, and buffer composition to determine if fluorometric results change in different chemical environments.

Finding optimal OPA-IBLC derivatized amino acid analysis parameters with three-dimensional fluorometric scans for HPLC method validation

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