3-Amino-5-pyridin-3-yl-1,2,4-triazole, a novel fluorescence probe for trace analysis of silymarin in bulk material, pharmaceutical dosage forms and human plasma: Further insights on reaction mechanism using computational molecular modeling and NMR spectroscopy.

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 NMR spectroscopy were used to identify the reaction mechanism between SLM and 3-APT. The proposed method was employed for determination of SLM in API or bulk material, pharmaceutical capsules and sachets. Further, the method was sensitive enough to be applied for analysis of the free (unconjugated) SLM flavonolignans in human plasma samples.