Table 1.

Overview of the key characteristics of evaluated HILIC-MS methods.

Specification	HILIC-MS	HILIC-MS with D2O Co-Infusion	Partial HILIC-HDX-MS	Full HILIC-HDX-MS
Mobile Phase A	H2O 10 mM Ammonium Formate 0.125% Formic Acid	H2O 10 mM Ammonium Formate 0.125% Formic Acid	D2O 10 mM Ammonium Formate 0.125% Formic Acid	D2O 10 mM D5-Ammonium Formate 0.125% D2-Formic Acid
Mobile Phase B	95% Acetonitrile/5% H2O 10 mM Ammonium Formate 0.125% Formic Acid	95% Acetonitrile/5% H2O 10 mM Ammonium Formate 0.125% Formic Acid	95% Acetonitrile/5% D2O 7.5 mM Ammonium Formate 0.125% Formic Acid	95% Acetonitrile/5% D2O 7.5 mM D5-Ammonium Formate 0.125% D2-Formic Acid
Resuspension Solvent	Acetonitrile/H2O (4:1, v/v) with CUDA, Val-Tyr-Val (IS)	Acetonitrile/H2O (4:1, v/v) with CUDA, Val-Tyr-Val (IS)	Acetonitrile/D2O (4:1, v/v) with CUDA, Val-Tyr-Val (IS)	Acetonitrile/D2O (4:1, v/v) with CUDA, Val-Tyr-Val (IS)
Column Flow Rate	0.4 mL/min	0.4 mL/min	0.4 mL/min	0.4 mL/min
Co-Infusion Solvent	—	D2O	—	—
Co-Infusion Flow Rate	—	0.05, 0.1 mL/min	—	—