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Validation of a method to determine lutein and lycopene levels in frozen human plasma by reversed‐phase HPLC
Author(s) -
Abadiano Judith,
Craft Neal,
Yachetti Stephen,
Burgher Anita
Publication year - 2013
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.27.1_supplement.850.7
Subject(s) - lutein , lycopene , chromatography , repeatability , high performance liquid chromatography , chemistry , detection limit , human plasma , carotenoid , food science
Objective This reversed‐phase HPLC method to measure lutein and lycopene in frozen human plasma was validated to support a study of infant plasma from three countries. Method Human plasma was maintained at −70 ± 10°C. Unspiked pooled infant plasma provided the Low Quality Control (QC) sample, Mid QC and High QC samples were prepared from adult plasma. Lutein and lycopene in plasma were separated by hexane/tetrahydrofuran extraction then quantified with isocratic reverse‐phase HPLC with visible detection. To validate the method, accuracy, repeatability (n=6) and intermediate precision (n=3) of QC samples were determined. ResultsLutein LycopeneNIST n=6 Low QC Mid QC High QC NIST n=6 Low QC Mid QC High QCMean (mcg/L) 46.4 50 142 292 352 62.3 305 458 RSD * (%) 3.4 5.3 7.2 7.5 2.8 6.4 6.0 8.3 CoA ** (mcg/L) 40 – 54300 – 380* RSD – Relative Standard Deviation** CoA – Certificate of AnalysisPlasma stability at −70° C for 38 weeks, mean lutein= 95.4% and lycopene=89.1% for the three QC sample pools. The Limit of Quantification (LOQ) for lutein = 5.46 mcg/L and for lycopene = 5.27 mcg/L and defined for this method as the sample concentration that corresponds to the lowest calibration standard. Conclusion The method has the sensitivity to detect lutein levels from 5.46–292 mcg/L and lycopene levels from 5.27–458 mcg/L in plasma for clinical trials. This is funded by Pfizer, Inc.