Determination of Puberulic Acid in Monascus-Fermented Red Yeast Rice by LC-MS/MS Combined with Precolumn Derivatization

Puberulic acid (PA) is a mycotoxin produced by a species of <i>Penicillium</i>. It has received widespread attention as a significant contributor to the reported fatalities associated with red yeast rice dietary supplements. However, the detection of PA, especially at low concentration levels, poses a considerable challenge, with no detection methods reported thus far. Here, we present a simple and sensitive derivatization-based LC-MS/MS method, requiring no purification processes, for determination of PA in the red yeast rice. The methylating derivatization with trimethylsilyldiazomethane (TMSCHN₂) was performed to enhance its analytical performance. To achieve optimal detection sensitivity, the amount of solvent and TMSCHN₂ for the derivatization reaction, along with the reaction time, were individually optimized. Moreover, sample extraction solvent was carefully chosen to improve recoveries in real sample analyses. Comparatively, the proposed LC-MS/MS method achieved a superior detection sensitivity, over 100-fold higher than that of the liquid chromatography method. A good linear relationship within the concentration range of 5 ng/mL to 200 ng/mL (with a linear correlation coefficient of 0.99952) was demonstrated by the method validation. The average recovery rate was between 82.2% and 84.2%, and the repeatability (RSD of 2.1% to 10.4%, n = 6) was satisfactory. The derivatized PA remained stable within 48 h. The limit of detection and the limit of quantification could reach 2 μg/kg and 50 μg/kg, respectively. As a result, the method was successfully applied to detect PA in over 42 batches of the red yeast rice samples. It indicated a low risk of PA contaminations in the red yeast rice products made in China. Furthermore, its application to the other health food products containing red yeast rice demonstrated the applicability of the established method.