Optimization of conditions for the isolation and preparation of glucosinolates from Maca by high-speed counter-current chromatography with orthogonal experiments

Glucosinolate is an important bioactive substance in Maca. In our study, two glucosinolates named glucotropaelin (GTL) and glucolimnanthin (GLI) were successfully separated from Maca with semi-preparative high-speed counter. current chromatography (HSCCC). For a best separation and better stationary phase retention, orthogonal experiments were carried out with the flow-rate of the mobile phase, ammonium sulfate concentration of biphasic solvent system, injection volume of crude extracts and the centrifugal speed of the apparatus as the four main factors. The separation was performed with the biphasic solvent system composed of normal butanol-acetonitrile-200 g/L ammonium sulfate solution (1:0.5:2.4, v/v/v) and the upper phase was used as the stationary phase and the lower phase as the mobile phase. The detection wavelength was set at 229 nm. The obtained optimum preparation conditions were that the mobile phase flow-rate was 2 mL/min, 500.00 mg crude extract was dissolved in 15 mL mobile phase, the ammonium sulfate concentration was 200 g/L and the centrifugal speed of the apparatus was 900 r/min. Under these conditions, a high efficiency separation of HSCCC was achieved, and 157.72 mg/kg of GTL and 31.93 mg/kg of GLI were obtained from the crude sample with a high stationary phase retention of 57.6%. The high performance liquid chromatography (HPLC) analysis showed that the purity of GTL was 97.9%. The identification and quantification of glucosinolates were completed by HPLC and mass spectrometry (MS). The established separation method is simple, less adsorption loss, lower cost and suitable for large scale separation of glucosinolates from Maca.