HPLC Analysis of Betalains and Phenolics

Betalains and phenolic compounds were determined simultaneously by high-performance liquid chromatography, as reported previously by our research team [4 (link),10 (link),17 (link)]. A 1200 Series Agilent HPLC System (Agilent Technologies, Santa Clara, CA, USA) with a reverse-phase C18 column (Zorbax SB-C18, 250 × 4.6 mm i.d., S-5 µm; Agilent) at 25 °C was used. Mobile phase A was 1% formic acid (v/v) in ultrapure water, and mobile phase B was 1% formic acid (v/v) in methanol. Separation was achieved using an initial composition of 15% B during 15 min, increased to 25% within 10 min, subsequently ramped to 50% B within 10 min, increased to 75% B in 15 min, and finally followed by a decrease period of 15% B in 5 min prior to isocratic re-equilibration for 10 min. The flow rate was 0.8 mL/min, and the injection volume was 20 µL. The UV-visible photodiode array detector was set at four wavelengths to detect phenolic acids (280 nm), flavonoids (370 nm), betaxanthins (480 nm), and betacyanins (535 nm). UV/Vis spectra were additionally recorded between 200 and 700 nm. The HPLC-DAD was coupled to a mass spectrometry detector (LCMS SQ 6120, Agilent) with an electrospray ionization (ESI) source operating in positive ion mode. The drying gas was nitrogen at 3L/min at 137.9 KPa. The nebulizer temperature was 300 °C, and the capillary had 3500 V potential. The coliseum gas was helium, and the fragmentation amplitude was 70 V. Spectra were recorded m/z from 100 to 1000.
Further mass spectrometry analyses were performed in a maXis II LC-QTOF equipment (Bruker Daltonics, Bremen, Germany) with an ESI source and the same chromatographic conditions. The ESI-QTOF detector worked in positive ion mode and recorded spectra m/z from 50 to 3000. Operation conditions were 300 °C, 3500 V capillary voltage, 2000 V charging voltage, 2.0 bar nebulizer, and dry gas at 6 L/ min. MS/MS analysis used the bbCID (broad-band collision induces dissociation) method at 30 eV.
Compounds were identified according to their retention times, UV/Vis, and mass spectral data compared to those of commercial, semi-synthesized, or purified standards. Quantitation of most betalains, piscidic acid, rutin, isorhamnetin glycosides, and kaempherol glycoside was determined using the calibration curves of the corresponding isolated standards. Eucomic acid and derivatives were quantified using tyrosol standard [6 (link)]. Quercetin glycosides were quantified by using the rutin calibration curve. The identification of betalains and phenolic compounds in Opuntia stricta var. Dillenii samples are shown in Table 1.