Optimization of an extraction procedure for the simultaneous quantification of riboflavin, nicotinamide and nicotinic acid in anchovies (Engraulis enrasicolus) by high-performance liquid chromatography–tandem mass spectrometry

A new analytical method that uses high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed for the first time for the analysis of vitamins B2 (riboflavin) and B3 (nicotinamide and nicotinic acid) in anchovies. Samples were extracted using four different extraction procedures: acidic hydrolysis (method 1), acidic hydrolysis plus peptide precipitation (method 2), acidic plus enzymatic hydrolysis (method 3) and enzymatic hydrolysis (method 4), and method 1 was found to be very good in terms of recoveries and quantitative results. From an analytical point of view, the separation of three analytes was achieved using a Kinetex Hilic analytical column (100 mm × 4.6 mm i.d.) with isocratic elution in a very short time (2.5 min or chromatography run). The recovery percentages (for method 1) obtained by spiking the matrices at 2.5 and 20 mg kg−1 with a standard mixture of the three analytes were in the range 96.2-105.3%; the correlation coefficients (R2) were always higher than 0.9996, and limits of quantification (LOQs) for all compounds were in the range 1–5 μg kg−1. Once validated, method 1 was applied to the analysis of 28 anchovy samples, both fresh and canned ones.