Reverse-phase HPLC Purification for an Extremely Unstable Glucuronide Metabolite

ABSTRACT A pure β-​D-​Glucopyranosiduronic acid metabolite (≥ 98.0% purity and a single impurity ≤ 0.50%) was requested for biological studies. Due to its unusual instability, the purification of the glucuronide metabolite was extremely challenging. Initially, the crude sample (89% HPLC area purity) was purified on a Waters SunFire C8 OBD column with 40 mM ammonium acetate buffer and acetonitrile as the mobile phase under a gradient program. The purified glucuronide metabolite solid was obtained by evaporation and lyophilization. However, this procedure yielded the target compound with 97.6% HPLC area purity and did not meet the requirements. Through the investigation, lyophilization was identified as the key step for the purity of the metabolite, and further lyophilization resulted in an increased amount of the degraded impurities. To better understand the compound, stability studies of the purified metabolite were conducted under sample media, organic solvent, acid, base, and light exposure. The compound was observed to be extremely unstable in water, acid, base and methanol, and sensitive to light, but relatively stable in ammonium acetate buffer (pH 5.0). Taking into account compound stability and the initial purification method, the improved purification procedure was successfully developed and the purified glucuronide metabolite was obtained with 99.2% HPLC area purity and 0.39% of the largest single impurity.