Validation of an Aqueous Normal Phase Chromatography Method for the Analysis of Erqothioneine in Commercial Mushrooms

Several types of commercially available mushrooms were analyzed for quantification of ergothioneine using a Cogent Diamond Hydride column (100 x 4.6 mm) at a flow rate of 1 mL/min, with a deionized water:acetonitrile:0.1% formic acid mobile phase using an aqueous normal phase (ANP) gradient. The ANP mode retains polar compounds in similar mobile phase conditions as HILIC, but differs greatly in the retention mechanism while providing more favorable conditions; that is, faster column equilibration time, a reduced concentration of buffers or complex additives, and no requirement of derivatization steps to retain polar compounds. Different solvents (methanol, acetone, acetonitrile) in deionized water, as well as different additives (acetic acid, formic acid, ammonium acetate, ammonium formate), were chosen for the mobile phase optimization process. Figure 2c shows the result of the water extraction, and Figure 2d is the chromatogram obtained for the ethanol extraction. Because of the greater signal provided by the ethanol procedure, it was selected for use in the optimized protocol.