A highly sensitive and selective high pressure liquid chromatography with tandem mass spectrometry (HPLC/MS-MS) method for the direct peptide reactivity assay (DPRA).

While the HPLC/UV (high performance liquid chromatography coupled with ultra-violet spectrometry)-based DPRA (Direct Peptide Reactivity Assay) identifies dermal sensitizers with approximately 80% accuracy, the low selectivity and sensitivity of the HPLC/UV-based DPRA poses challenges to accurately identify the sensitization potential of certain chemicals. In this study, a high performance liquid chromatography coupled with tandem mass spectrometry (HPLC/MS-MS)-based DPRA was developed and validated according to the test guideline (OECD TG 442C). The final results were compared with the results from the traditional HPLC/UV-based guideline DPRA. This HPLC/MS-MS-based DPRA demonstrated similar performance compared to HPLC/UV-based DPRA using known dermal sensitizers and non-sensitizers according to the test guideline (OECD TG 442C). Following the validation, a challenge set of chemicals with either overlapping retention time with peptides, or higher hydrophobicity or chemicals potentially forming non-covalent interactions with peptides were assessed for dermal sensitization potential using both methods and the results were compared to existing in vivo data. The HPLC/MS-MS-based DPRA correctly predicted these chemicals as sensitizers or non-sensitizers; however, the HPLC/UV-based DPRA resulted in false-positive predictions for hydrophobic substances, chemicals with UV peaks overlapping with those of the peptide(s), and compounds that non-covalently interact with the peptides. These findings demonstrate the broader applicability and better sensitivity and selectivity of the LC/MS-MS-based DPRA over the traditional HPLC/UV-based guideline DPRA.

Analytical methods impact estimates of trichloroethylene's glutathione conjugation and risk assessment.

The glutathione (GSH) conjugates, S-(1,2-dichlorovinyl)-glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC), have been implicated in kidney toxicity and kidney cancer from trichloroethylene (TCE) exposure. Considerable differences in blood and tissue levels of DCVG and DCVC have been reported, depending on whether HPLC/UV (High Performance Liquid Chromatography-Ultraviolet) or HPLC/MS (HPLC-Mass Spectrometry) was used. A side-by-side comparison of analytical results with HPLC/UV and HPLC/MS/MS (High Performance Liquid Chromatography-Tandem Mass Spectrometry) detection was undertaken to quantitatively compare estimates for DCVG and DCVG using rat and human tissues. For the HPLC method, DCVG and DCVC were initially derivatized with fluorodinitrobenzene (DNP). The results from the HPLC/UV method showed that derivatized-DCVC eluted at the solvent front and could not be quantified. Derivatized-DCVG, however, was quantified but significant interference was observed in all four control tissues (rat blood, liver, kidney; and human blood), resulting in average spike recoveries of 222-22,990%. In contrast, direct analysis of spiked tissues by HPLC/MS/MS resulted in recoveries of 82-127% and 89-117% for DCVG and DCVC, respectively. These differences in analytical results were further confirmed in tissues from TCE-treated rats, e.g., DCVG levels in rat liver were 18,000 times higher by HPLC/UV as compared to HPLC/MS/MS. Fraction collection of the derivatized-DCVG peak (obtained with the HPLC-UV method), followed by peak identification via an HPLC/UV/Q-TOF/MS/MS method, identified DNP-derivatized endogenous glutamate as the primary interfering substance that contributed to and exaggerated recoveries of DCVG. Thus, estimates of DCVG based on the HPLC/UV methods are not reliable; they will over-estimate the formation of the GSH conjugates of TCE and will artifactually exaggerate the potential cancer risk in humans from TCE exposure. Therefore, it is recommended that any characterization of cancer risks from TCE exposure attributable to the GSH conjugates of TCE rely on results obtained with the more specific and reliable HPLC/MS/MS method.