HILIC and the Shift Beyond TFA in GLP-1 Analysis

This segment turns to hydrophilic interaction liquid chromatography (HILIC) as an alternative separation mode, and to a broader industry reckoning with the mobile-phase additives long considered the gold standard for peptide work. Piotr Alvarez positions HILIC as a complementary technique for drug substance and drug product analysis rather than a primary method, valuable specifically because it offers a genuinely orthogonal separation to reverse-phase chromatography; for GLP-1 agonists like semaglutide and tirzepatide, elution order under HILIC is essentially reversed compared with reversed-phase liquid chromatography (RPLC), and the same holds true for their impurities. That makes HILIC especially useful for resolving challenging impurities, such as stereoisomers or epimers, that reverse-phase methods fail to separate. Alvarez also notes that HILIC conditions tend to be considerably more mass spectrometry (MS)-friendly than traditional trifluoroacetic acid (TFA)-based reverse-phase separations, improving detection of low-level impurities.

That MS-compatibility point leads into a candid discussion of TFA’s drawbacks: despite delivering excellent peak shape and resolution, it causes ion suppression that can mask low-level impurities. Alvarez highlights difluoroacetic acid (DFA) as a valuable substitute that preserves separation performance while reducing suppression, though he notes that TFA and DFA are both PFAS compounds, or "forever chemicals," facing likely future regulatory restriction even if the analytical field hasn't felt that pressure directly yet. Sam Whitmarsh and Alvarez discuss whether HILIC represents a genuine path toward reducing reliance on PFAS-based ion-pairing agents, and Alvarez notes additional MS-compatible reverse-phase options, including ammonium acetate or formate mobile phases and newer core–shell, sub-2-micrometer column chemistries that reduce dependence on TFA altogether.

Nitish Sharma closes with his own additive guidance—formic acid, acetic acid, and ammonium formate or acetate among his team's preferred choices—while cautioning that TFA concentration must still be carefully limited to avoid compromising mass detector sensitivity.