Single reference quantitative analysis of xanthomonasin A and B in Monascus yellow colorant using high-performance liquid chromatography with relative molar sensitivity based on high-speed countercurrent chromatography

Monascus yellow (MY) is a natural yellow food coloring. The main components from MY are xanthomonasin A (XA) and xanthomonasin B (XB) for natural yellow colorant of food additives. However, few chromatographic assays of XA and XB exist in food additive products because of unavailable standards for calibration curves. In this study, the single reference (SR) quantitative analysis of XA and XB in MY product is proposed by high-performance liquid chromatography with photodiode array detection (HPLC/PDA) using relative molar sensitivity (RMS). Moreover, high-speed countercurrent chromatography (HSCCC) purification with 1H quantitative NMR (qNMR) evaluation is necessary to separate the two analytes for the RMS to be demonstrated. For HSCCC separation, the biphasic solvent system (hexane/ethyl acetate/methanol/0.1% formic acid in water, 1/5/1/5) was used to obtain XA and XB fractions that were subjected to qNMR for the determination of their contents in each test solution. Using these solutions and SR solution of carbazochrome acid (CBZ), the RMS of XA and XB are calculated from slopes ratios of calibration curves (three ranges from 0 to 177 μM for XA and 0-126 μM for XB, r2 > 0.998). The averaged RMS of XA/CBZ and XB/CBZ were 8.75 ± 0.07 and 14.8 ± 0.26, respectively. The concentrations of XA and XB in MY can be determined from RMS, peak area and content of CBZ added in the samples; the concentrations were found to be 7.26 μmol/g and 2.53 μmol/g, respectively. The performance of HPLC/PDA using RMS was compared with an absolute calibration curve method. This developed HPLC/PDA using RMS is simple and reliable quantification that does not require native XA and XB standards based on HSCCC purification and qNMR evaluation.