A Buffered LC-MS Method for Resolving and Quantifying Albiflorin and Paeoniflorin

Albiflorin and paeoniflorin are bioactive isomers found in Paeoniae Radix Alba, a key component of Traditional Chinese Medicine. Their accurate and simultaneous quantification is essential for pharmacological research. Standard separation methods often rely on LC-MS using a mobile phase containing 0.1% formic acid. This study demonstrates that the use of 0.1% formic acid generates a secondary peak for albiflorin that exhibits an identical mass-to-charge ratio and similar fragmentation pattern as the main peak. To address this, an LC-MS method was developed using a Kinetex phenyl-hexyl column with a gradient elution using a buffered mobile phase of 10 mM ammonium acetate and acetic acid (pH 4.4) in water and methanol. The method was validated for linearity, precision, accuracy, and sensitivity. It successfully separated paeoniflorin and albiflorin preventing the formation of the albiflorin artifact. The method demonstrated good linearity over a concentration range of 1-50 μg/mL. Applicability was tested through the analysis of a Paeoniae Radix Alba extract. The developed LC-MS method enables accurate and simultaneous quantification of albiflorin and paeoniflorin by eliminating the formation of a second albiflorin peak. This makes the method potentially suitable for pharmacological studies of Paeoniae Radix Alba and other plants containing albiflorin and paeoniflorin.