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
Journal of Chromatography B(2009)
Abstract
An isocratic RP-HPLC method for the determination of retinol and α-tocopherol in serum, with fluorescence and UV/VIS detection, respectively, was developed and validated according to international guidelines. Detection (retinol 0.015mg/L, α-tocopherol 0.29mg/L) and quantification (retinol 0.05mg/L, α-tocopherol 0.95mg/L) limits were determined. Repeatability was <3.3% and <2.9% and intermediate precision was <4.6% and <3.2%, for retinol and α-tocopherol, respectively. Certified reference materials were utilised to assess bias and guarantee traceability to SI units. Expanded uncertainties (retinol 8.9%; α-tocopherol 7.9%), estimated according to the EURACHEM/CITAC guide from method validation data, satisfied fit-for-purpose requirements based on biological variability.
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Key words
Serum,Retinol,α-Tocopherol,HPLC,Validation,Uncertainty of measurement,Biological variability,Quality specifications
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