Determination of Luteolin and Apigenin in Herbal Teas by Online In-Tube Solid-Phase Microextraction Coupled with LC-MS/MS

Abstract

Herbal teas have attracted attention as functional beverages containing luteolin and apigenin, which exhibit antioxidant and anti-inflammatory effects. The objective of this study was to develop a sensitive online automated method to determine these flavones' contents in herbal teas using in-tube solid-phase microextraction (IT-SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). These compounds were extracted and concentrated by IT-SPME using a Supel Q PLOT capillary column and then separated and detected within 6 min using a CAPCELL PAK C18 MG III analytical column and a negative electrospray ionization-mode multiple-reaction monitoring system by LC-MS/MS. The detection limits (S/N = 3) for luteolin and apigenin were 0.4 and 0.8 pg mL^-1, respectively, and the calibration curves were linear in the range of 2-2000 pg mL^-1 with correlation coefficients above 0.9995, and intra-day and inter-day precisions with relative standard deviations below 2.9 and 3.6% (n = 6), respectively. The luteolin and apigenin in herbal tea were quantified using IT-SPME/LC-MS/MS following the acid hydrolysis of their glycosides. Among the 10 herbal teas tested, luteolin was detected in peppermint and sage at concentrations of 375 and 99 µg mL^-1, respectively, while apigenin was detected in German chamomile at 110 µg mL^-1, which were higher than in the other herbal teas. The method is expected to be a useful method for evaluating the efficacy of luteolin and apigenin in herbal teas as functional beverages.

Keywords: LC–MS/MS; apigenin; herbal teas; in-tube solid-phase microextraction; luteolin; online automated analysis.

Figure 1

Structures of luteolin, apigenin, and apigenin-d₅.

Figure 2

Schematic diagram of the improved IT-SPME LC-MS/MS system used in this study. (A) Adsorption of compound on stationary phase in capillary column, (B) desorption of compound by mobile phase solvent.

Figure 3

Effects of capillary coatings on IT-SPME of luteolin and apigenin. For each capillary column, 40 µL of a 100 pg mL⁻¹ standard solution was repeatedly injected 5 times.

Figure 4

Effects of pH of sample solution on IT-SPME of luteolin and apigenin. For each pH, 40 µL of a 100 pg mL⁻¹ standard solution was repeatedly injected 5 times.

Figure 5

Effects of number of sample injections on IT-SPME of luteolin and apigenin. For each injection number, 40 µL of a 100 pg mL⁻¹ standard solution was repeatedly injected.

Figure 6

MRM chromatograms obtained from standard solution by IT-SPME LC-MS/MS in the negative ion mode. IT-SPME LC-MS/MS conditions are described in the Experimental Section.

Figure 7

MRM chromatograms obtained from herbal tea by IT- SPME LC-MS/MS in the negative ion mode. IT-SPME LC-MS/MS conditions are described in the Experimental Section. MRM transitions are the same as in Figure 6.