Evaluation of uncertainty of measurement from method validation data: An application to the simultaneous determination of retinol and α-tocopherol in human serum by HPLC

An isocratic RP-HPLC method for the determination of retinol and a-tocopherol in serum, with fluorescence and UV/VIS detection, respectively, was developed and validated according to international guidelines. Detection (retinol 0.015 mg/L, alpha-tocopherol 0.29 mg/L) and quantification (retinol 0.05 mg/L, a-tocopherol 0.95 mg/L) limits were determined. Repeatability was < 3.3% and < 2.9% and intermediate precision was < 4.6% and < 3.2%, for retinol and a-tocopherol, respectively. Certified reference materials were utilised to assess bias and guarantee traceability to SI units. Expanded uncertainties (retinol 8.9%: a-tocopherol 7.9%), estimated according to the EURACHEM/CITAC guide from method validation data, satisfied fit-for-purpose requirements based on biological variability. (C) 2009 Elsevier B.V. All rights reserved.