Reversed-phase peptide separation in hydrogen deuterium exchange (HDX) mass spectrometry (MS) must be done with conditions where the back exchange is the slowest possible, the so-called quench conditions of low pH and low temperature. To retain maximum deuterium, separation must also be done as quickly as possible. The low temperature (0 &degC) of quench conditions complicates the separation and leads primarily to a reduction in separation quality and an increase in chromatographic backpressure. To improve the separation in HDX MS, one could use a longer gradient, smaller particles, a different separation mechanism (for example, capillary electrophoresis), or multi-dimensional separations such as combining ion mobility separation with reversed-phase separation. Another way to improve separations under HDX MS quench conditions is to use a higher flow rate where separation efficiency at 0 &degC is more ideal. Higher flow rates, however, require chromatographic systems (both pumps and fittings) with higher backpressure limits. We tested what improvements could be realized with a commercial UHPLC/UPLC system capable of ~20,000 psi backpressure. We found that a maximum flow rate of 225 uL/min was possible and that higher flow rate clearly led to higher peak capacity. HDX MS analysis of both simple and particularly complex samples improved, permitting both shorter separation time, if desired, and providing more deuterium recovery.

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