LC/DAD Analytical Method for Fingerprint Analysis of Erythrina velutina Alkaloids

Erythrina L. genus species are included in the Fabaceae family and comprises about 115 species. Phytochemical screenings have shown several compound groups for Erythrina species, such as alkaloids, catechins, steroids, flavonoids, phenols, saponins, tannins, triterpenoids, and xanthones. Among these compounds, the genus has a wide range of tetracyclic alkaloids isolated by different species, which are often used as chemical markers. The pharmacological activities described for Erythrina species highlight the action of these alkaloids on the central nervous system.[1] Despite the large number of publications on Erythrina‐type alkaloids, only a few studies about the development of analytical methods for these compounds have been reported, specially using high‐performance liquid chromatography (HPLC).[2] In addition, there are no studies that provide phytochemistry analysis of Erythrina species collected in Bahia. In this context, the present work aimed to optimize a method by high‐performance liquid chromatography coupled with photodiode‐array detection (HPLC/DAD) for fingerprint analysis of fraction of total alkaloids (FTA) of Erythrina velutina collected in Bahia. Leaves of E. velutina were collected at different trees in Boninal-BA and also different months of 2018 and 2019. The dried and milled leaves of E. velutina were extracted with hydrochloric acid 1% (1:40 w/v) under dynamic maceration. Then, extracts were washed with hexane:ethyl acetate (1:1 v/v) to remove the nonpolar constituents and the acid extract was alkalinized with NH4OH until pH 9–10 and partitioned with chloroform. The organic fractions were combined and dried in a rotavapor at 40 °C to produce the FTA. HPLC/DAD method was developed using Luna® C18 (5μm, 250 x 4.6 mm) column with a flow rate of 0.7 mL/min. A gradient system was optimized employing a mobile phase A of water:triethylamine:TFA (99.9:0.1 v/v; pH 3) and mobile phase B of acetonitrile:triethylamine (99.9:0.1 v/v). Different columns and mobile phases were used to optimize the separation of E. velutina alkaloids. The separation only occurred when TFA was associated with triethylamine; TFA was able to prevents hydroxyl groups ionization and triethylamine mask other ionized groups. The peak purity of the major peak was determined by Shimadzu software. The fingerprint analysis of E. velutina FTA showed a major peak at 30 min with an UV spectrum at 230 and 280nm, characteristic of erythrin alkaloids. In summary, this method can be useful for quality control analysis.