ArticleAnalytical and bioanalytical chemistry2026
Analysis of human chorionic gonadotropin glycoforms by nano-liquid chromatography-mass spectrometry: novel strategies based on mobile phase additive and adduct dissociation.
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Abstract
The nano-LC-HRMS method previously developed for the analysis of human chorionic gonadotropin (hCG) glycoforms at the intact level involved trifluoroacetic acid (TFA) as ion-pairing agent in the LC mobile phase to favor separation, but resulted in both a significant decrease in the mass spectrometry (MS) signal and in complex mass spectra due to the presence of numerous adduct forms leading to a time-consuming manual data treatment and contributing to the MS signal decrease. To find the best balance between LC resolution, ESI ionization, and absence of adduct detection, two main strategies were here explored for the first time for hCG characterization. To enhance MS signal and reduce adduct formation, a decrease from 0.05 to 0.025% of TFA concentration was first assayed. It led to an efficient increase in MS signal intensities, but high glycoform/adduct ratios were still detected. The switch from TFA to difluoroacetic acid (DFA) 0.05% significantly improved the intensity of the signal in MS and significantly decreased adduct ratios (from 30 to 10%), despite some co-elution of hCG alpha (hCGα) and beta (hCGβ) glycoforms during LC separation. Keeping DFA as an ion-pairing agent, the All Ion Fragmentation mode of the Orbitrap mass spectrometer efficiently dissociated the remaining adducts leading to an increase in MS signal intensity by a factor 3 to 11 for hCGα glycoforms and 6 to 23 for hCGβ glycoforms. With these conditions, 76 hCGα and 200 hCGβ glycoforms were detected, resulting in a significant increase in the number of glycoforms detected compared with the initial method: a twofold increase for hCGα and a sixfold increase for hCGβ, thereby enabling a more detailed characterization of hCG glycosylation.
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