On-line solid-phase extraction–HPLC–fluorescence detection for simultaneous determination of puerarin and daidzein in human serum

• Published in: Talanta (Oxford) 2010-09, Vol.82 (4), p.1212-1217
• Main Authors: Liu, Ying-Kun, Jia, Xiao-Yan, Liu, Xiao, Zhang, Zhi-Qi
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Calibration; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography, High Pressure Liquid - methods; Daidzein; Exact sciences and technology; HPLC–fluorescence detection; Human serum; Humans; Isoflavones - blood; On-line solid-phase extraction; Other chromatographic methods; Puerarin; Reference Standards; Reproducibility of Results; Response surface methodology; Spectrometry, Fluorescence - methods
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2010.06.038

Response surface methodology (RSM) was applied to the optimization of on-line solid-phase extraction (SPE) parameters, and an automated system of on-line SPE coupled with high-performance liquid chromatography (HPLC) with fluorescence detection was developed for the determination of puerarin and daidzein in human serum. The human serum sample of 50 μL was injected into a conditioned C18 SPE cartridge, and the matrix was washed out with acetonitrile–KH 2PO 4–triethylamine buffer (0.01 M, pH 7.4) (3:97, v/v) for 3 min at a flow rate of 0.25 mL/min. Then the target analytes were eluted and transferred to the analytical column. A chromatographic gradient elution was programmed with the mobile phase consisting of acetonitrile and KH 2PO 4–triethylamine buffer, and the analytes were determined with a fluorescence detector at excitation wavelength of 350 nm and emission wavelength of 472 nm, respectively. The proposed method presented good linear relations (0.85–170 μg/mL for puerarin and 0.2–40 μg/mL for daidzein), satisfactory precision (RSD < 8%), and accredited recovery (92.5–107.8%).