A scheme for obtaining standards and HPLC quantification of leafy vegetable carotenoids

Reliability of data obtained by HPLC directly depends on the accuracy of the calibration. A major difficulty is obtaining and maintaining pure standards. This work presents a strategy for isolating standards by open column chromatography and quantification by HPLC, using leafy vegetables as examples. The purity of standards evaluated by HPLC was 91–97% for neoxanthin, 95–98% for violaxanthin, 97–100% for lactucaxanthin, 92–96% for lutein and 90–97% for β-carotene. Calculation procedures were also evaluated, demonstrating that results obtained with one-point recalibration, straight-line equation (until 30 days after construction of the full calibration curves) and response factors relative to β-carotene were similar (CVs of 1.6–4.0%), being well below between-sample lot natural variation (CVs of 6.1–42.5%). The scheme proposed is relatively low-cost, provides a constant supply of carotenoid standards, including those unavailable commercially, and high sample throughput.