Evaluation of detection methods for the reversed‐phase HPLC determination of 3′,4′,5′‐trimethoxyflavone in different phytopharmaceutical products and in human serum

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Abstract
Quantitative determination of the major compound, 3′,4′,5′‐trimethoxyflavone (1), in plant extracts, in tablets of Flos and of Radix Primulae veris and in human serum has been accomplished using reversed‐phase HPLC with UV, fluorescence and mass spectrometric (MS) detection. Compared to UV detection, fluorescence detection showed greater selectivity, was 10‐fold more sensitive and allowed the determination of 1 in human serum after sample pre‐treatment by solid‐phase extraction. MS detection of 1 using electrospray ionisation (ESI) interface could be improved by substituting trifluoroacetic acid with the more polar and less conductive additive acetic acid, giving rise to a 230‐fold improvement in analyte detectability at the cost of an increase of only 45% in the peak width of the eluting peak at its half height. Further optimisation of the acetic acid concentration showed the highest signal intensity at 1.25% for HPLC‐atmospheric pressure chemical ionisation (APCI)‐MS and at 0.75% for HPLC‐ESI‐MS. The optimised MS method proved to be extremely selective, 50 times more sensitive than UV detection and 5 times more sensitive than fluorescence detection. Furthermore, fragment‐ion spectra produced by collision induced dissociation‐MS have been used as “fingerprints” for identifying compounds in the highly complex mixtures examine. Copyright © 2001 John Wiley & Sons, Ltd.