Post-column mobile phase adjustment: A strategy to eliminate the contradiction between liquid chromatography and mass spectrometry in the determination of flavonoids in rat plasma

• Compared with reported method, sensitivity was significantly enhanced.
• Rapid determination of six flavonoids in rat plasma within 5 min.
• Improved peak shape by the reduction of longitudinal diffusion.
• Post-column mobile phase adjustment was systematically discussed and validated.
• Eliminate the contradiction between LC and MS in negative ESI mode.

Flavonoids are a group of important naturally occurring polyphenolic compounds with a wide range of biological effects. In this study, a sensitive liquid chromatography tandem mass spectrometry method was developed to simultaneously determine multiple active flavonoids, including quercetin (Que), kaempferol (Kae), apigenin (Api), isorhamnetin (Iso), luteolin (Lut), and naringenin (Nar), in rat plasma. To achieve a satisfied peak shape and LC separation, formic acid with the concentration between 0.05 and 0.2%, or in some case 5%, was generally used to acidify the LC mobile phase in reported studies. Here we found that even 0.05% formic acid could lead to strong mass signal suppression, and the absence of formic acid could reverse the signal suppression but cause serious peak tailing. There is an irreconcilable contradiction between liquid chromatography (LC) and mass spectrometry (MS). In order to simultaneously satisfy LC and MS, LC mobile phase with 0.00075% formic acid and post column mobile phase adjustment with 0.0677% ammonium solution in isopropanol were applied. Compared with the conventional method with mobile phase containing 0.05% formic acid, the mass signal response of Que, Kae, Api, Iso, Lut, Nar, and Oka increased 26.2, 18.6, 13.6, 23.5, 17.5, 15.6 and 15.4 fold, respectively. In addition, the post column mobile phase addition exhibited the better peak shape for the reduction of analytes longitudinal diffusion. The method has been fully validated according to FDA guidelines within the linear range between 0.328 ng mL−1 and 168 ng mL−1, and successfully applied to a pilot pharmacokinetic study of rats after administering 5.43 g kg−1 Pollen of Brassica campestris.

A rapid method for the simultaneous determination of six flavonoids in rat plasma was developed. Compared with conventional method (orange TIC (total ion chromatography)), current strategy (green TIC) significantly enhanced the mass signal intensity.Figure optionsDownload as PowerPoint slide