Solvent Mixture and Mobile Phase

Among different solvents, acetonitrile provides better recovery for a wide range of mycotoxins and matrices. However, there are reports of using methanol as a suitable co-extraction solvent in this field [27]. Different proportions of acetonitrile, methanol and water have been studied to discover an efficient extraction procedure with high recoveries for all analytes. In addition, to obtain the best chromatogram with the lowest noise signal, different mobile phases were used.

To optimize the extraction step, different proportions of acetonitrile and water were applied. Three volume ratios, acetonitrile:water (80:20, v:v), (60:40, v;v) and (50:50, v:v) were employed and acetonitrile:water (50:50, v:v) was confirmed as the most appropriate volume ratio. Various proportions of acidic water (0.1%, 0.2%, 0.3%, 0.5% and 1%) were tested and the extraction with equal proportion of acetonitrile and water containing 0.3% formic acid was suitable for all acidic and neutral mycotoxins.

Different various mobile phases and additives were evaluated, including acetonitrile and methanol (mobile phase) and formic acid, ammonium formate, ammonium fluoride, acetic acid and ammonium acetate (additive). Methanol containing formic acid, ammonium formate, and ammonium fluoride was found to give superior peak shape compared to acetonitrile. The reconstitution mixture was added at the end of the sample preparation step because the compatibility of mobile phases with final extraction solvent leads to improved signal response and prevents peak fronting and unstable retention time compared with no reconstitution. Since the nature of polar compounds makes them prone to dissolve in more polar solutions, reconstitution of samples with polar mobile phases is strongly recommended.

In this research, the combination of acetonitrile and acidic water as an extraction solvent at the beginning of the sample preparation and reconstitution with methanol:water (50:50, v:v) provides higher recoveries and better resolution for all 14 mycotoxins.