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

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.