The application of ion‐pair high performance countercurrent chromatography monitored by off‐line LC‐ESI‐MS/MS injections to study betalain metabolite from Vietnamese red dragon fruit (Hylocereus polyrhizus)

The pre‐purified betalain extract from Vietnamese red dragon fruits (Hylocereus polyrhizus) was fractionated by a high performance countercurrent chromatography (HPCCC) device using the ion‐pair forming biphasic solvent of tert‐butylmethylether/n‐butanol/acetonitrile/1% aqueous trifluoroacetic acid (2:2:1:5, v/v/v/v). The J‐type HPCCC centrifuge with a 240g centrifugal force field was able to fractionate seven pigments from a 350 mg enriched crude pigment fruit extract, resulting in purified fractions for qualitative identification by LC‐ESI‐MS/MS. This approach fractionated the main betacyanin epimers namely 15R/15S‐betanin (m/z 551), 15R/15S‐phyllocactin (m/z 637), and apiosyl‐15R/15S‐betanin (m/z 683) from the solely existing betaxanthin indicaxanthin. In addition, few minor betacyanins such as 2’‐apiosyl‐15R/15S‐phyllocactin (m/z 769) were concentrated in a few fractions. That method also allowed the better detection of cacti fruit pigment 15R/15S‐hylocerenin (m/z 695), and 15R/15S‐betanidin 5‐O‐β‐sophoroside (m/z 713). However, the separation of these highly polar compounds was incomplete due to insufficient force of the ion‐pairing effect of trifluoroacetic acid (TFA).