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'-trimethoxyflavon e (1), in plant extracts, in tablets of Flos and of Radix Primulae veris an d in human serum has been accomplished using reversed-phase HPLC with UV, f luorescence and mass spectrometric (RIS) detection. Compared to UV detectio n, fluorescence detection showed greater selectivity, was 10-fold more sens itive and allowed the determination of 1 in human serum after sample pre-tr eatment by solid-phase extraction. MS detection of 1 using electrospray ion isation (ESI) interface could be improved by substituting trifluoroacetic a cid with the more polar and less conductive additive acetic acid, giving ri se to a 230-fold improvement in analyte detectability at the cost of an inc rease 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 s ignal intensity at 1.25% far HPLC-atmospheric pressure chemical ionisation (APCI)-MS and at 0.75% for HPLC-ESI-MS. The optimised MS method proved to b e extremely selective, 50 times more sensitive than UV detection and 5 time s more sensitive than fluorescence detection. Furthermore, fragment-ion spe ctra produced by collision induced dissociation-MS have been used as "finge rprints" for identifying compounds in the highly complex mixtures examine. Copyright 2001 John Wiley & Sons, Ltd.