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-beta -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).