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Lutein HPLC Method Comparison and Validation
Author(s) -
Craft Neal E,
Springs Donna G,
Chavan Jayanthi
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.689.5
Subject(s) - lutein , saponification , zeaxanthin , chromatography , chemistry , carotenoid , xanthophyll , food science , high performance liquid chromatography
Lutein is one of two pigments that accumulate in the macula of the human eye. It is yellow in color and a member of the carotenoid family. Lutein (and zeaxanthin) filter blue‐light and thereby protect the eye from damage. Lutein is very ubiquitous in plants but often masked by chlorophylls. Major dietary sources include spinach, kale, broccoli, eggs, peppers, corn and peas; however people seldom consume adequate amounts. As such, lutein has been incorporated into many dietary supplements generating a need for accurate measurement of this antioxidant pigment. Objectives To compare and validate two HPLC methods for lutein analysis which use different columns and mobile phases. To compare cold and heated alkaline hydrolysis (saponification). Methods Acceptance criteria were established as Accuracy: 90–110%; Reproducibility: within‐day <5% CV, day‐to‐day <6% CV. Both Silica and Diol HPLC columns were tested. Mobile phases were 75 hexanes/25 ethyl acetate and 96 hexanes/4 IPA, respectively. Both systems were monitored at 448 or 450nm. Saponification was necessary for samples containing lutein esters or high oil and low lutein content. Both cold and heated (56°C and 70°C) saponification methods were compared. Results For both methods, calibration curves from 0.15–35 ppm were linear with r 2 > 0.998. Recovery of low, mid and high lutein concentrations from spiked blank oil samples was 96–103%. Within day precision was 0.57–2.74% for esterified lutein and 0.74–3.84% for free lutein. Day‐to‐day precision was ≤3.1% for esterified lutein and ≤2.8% for free lutein. Heated saponification led to 9% less trans‐lutein and 4% less total lutein concentration compared to cold saponification. Conclusions Both methods met all of the criteria set forth for validation. Within day precision was less than 5% and day‐to‐day precision was less than 6%. Accuracy, as measured by spike recovery, was within the specification of 90–110%. Results were more accurate using refrigerated or 56°C saponification compared to 70°C. Both methods are aligned and produce similar results for lutein in oil‐based products. Additionally, zeaxanthin can be measured in both methods. Support or Funding Information Funding supplied, in part, by OmniActive Health Technologies.