Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormones.

Aim: Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. Results: The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.4 kDa. Results show comparable performance between two platforms in sensitivity, accuracy and linearity. For some peptides, HRMS provided lower background interference. The benefit of increased sensitivity using microflow UPLC was also demonstrated. Conclusion: HRMS is a versatile platform capable of both basic characterization and reliable quantitation in complex matrices. Microflow UPLC provides lower LLOQs than conventional flow systems, even with less sample volume injected.

Impurity profiling quality control testing of synthetic peptides using liquid chromatography-photodiode array-fluorescence and liquid chromatography-electrospray ionization-mass spectrometry: the obestatin case.

Following several conflicting publications, the inability to reproduce the original findings on in vitro obestatin binding and activation of GPR39 receptors was recently reported by its discoverers, and several hypotheses to rationalize these findings were proposed. Based on one of these postulations (i.e., presence of impurities), peptide identity and impurity profiles were thoroughly evaluated on obestatin peptides obtained from five different manufacturers, as used by the different research groups. We found that one of the products examined was in reality a totally different peptide and that the quality of two-thirds of the other peptides was insufficient for in vitro and in vivo experiments (i.e., peptide purity less than 95% and/or individual impurities exceeding 1%). These observations question the divergent conclusions reported in the literature about the activity of obestatin. Therefore, we strongly recommend appropriate quality control testing before using any peptides for biomedical research purposes.