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Stress degradation studies using high-performance liquid chromatography (HPLC) was performed and validated for Droxidopa (L-DOPS). Droxidopa was susceptible to acid hydrolysis (0.1 N HCl), alkaline hydrolysis (0.15 N NaOH) and thermal degradation (105°C). It was found to be resistant to white light, oxidation and UV light exposure (72 h). The thermal, acid and alkali degradation impurities were detected with the retention time (RT) of 12.7, 19.25 and 22.95 min. Our HPLC method detected process impurities (2 R ,3 R )-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropionic acid (Impurity H), N-Hydroxypthalimide (Impurity N), (2 R ,3 S )-2-amino-3-(benzo[d][1,3]dioxol-5-yl)-3-hydroxypropionic acid (Impurity L) and L-threo n-phthaloyl-3-(3, 4-dihydroxyphenyl)-serine (Intermediate) with RTs of 3.48, 15.5, 25.76 and 28.0 min. The related substances were further characterized and confirmed by liquid chromatography–mass spectroscopy (LC–MS), and nuclear magnetic resonance spectroscopy analysis. Our HPLC method detected up to 0.05 µg/mL of Droxidopa with S/N &gt; 3.0 and quantified up to 0.10 µg /mL of Droxidopa with S/N ratio &gt; 10.0. Droxidopa was highly stable for 12 h after its preparation for HPLC analysis. Our newly developed HPLC method was highly precise, specific, reliable and accurate for the analysis of Droxidopa and its related substances.

Stability-Indicating Related Substances HPLC Method for Droxidopa and Characterization of Related Substances Using LC–MS and NMR