A rapid, highly sensitive and roubst spectrofluorimetric method was developed for trace analysis of silymarin
(SLM) in active pharmaceutical ingredient (API), pharmaceutical preparations and human plasma. The proposed
method is based on reaction of SLM with a novel reagent; 3-amino-5-pyridin-3-yl-1,2,4-triazole (3-APT); in the
presence of 0.04 M sodium hydroxide. The formed fluorescent product was formed within 5 min and was measured
at 504 nm after excitation at 390 nm. All reaction parameters were optimized and the proposed method
was validated according to ICH guidelines. The developed method was linearly correlated at the concentration
range of 0.05–8 μg mL−1 with good correlation coefficient 0.9993, limit of detection 10.79 ng mL−1 and limit
of quantitation 32.71 ng mL−1. The relative standard deviations %RSD values were 1.59–2.69% and 1.47–2.62%
in case of intra- and inter-day precision, respectively. Computational molecular modeling and NMRspectroscopy
were used to identify the reaction mechanismbetween SLMand 3-APT. The proposed methodwas employed for
determination of SLMin API or bulk material, pharmaceutical capsules and sachets. Further, the method was sensitive
enough to be applied for analysis of the free (unconjugated) SLMflavonolignans in human plasma samples.