Estimation of uncertainty from method validation data: Application to a reverse-phase high-performance liquid chromatography method for the determination of amino acids in gelatin using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent

• Published in: Journal of pharmaceutical and biomedical analysis 2016-09, Vol.129, p.389-397
• Main Authors: Azilawati, M.I, Dzulkifly, M.H., Jamilah, B., Shuhaimi, M., Amin, I.
• Format: Article
• Language: English
• Subjects: 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent; Amino acids; Amino Acids - chemistry; Aminoquinolines - chemistry; Carbamates - chemistry; Chromatography, Reverse-Phase - methods; Estimation of uncertainty; Gelatin; Gelatin - chemistry; Hydroxyproline - chemistry; Indicators and Reagents - chemistry; Reference Standards; Reproducibility of Results; Uncertainty
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2016.07.012

[Display omitted] •No study had reported the uncertainty evaluation for amino acid analysis in gelatin matrix.•Estimation of uncertainty was done according to LGC/VAM protocol.•Using mainly the method validation data (precision and trueness).•Other sources uncertainties were measurements in standard preparation.•The findings data can be used as reference for collaborative study. A detailed procedure for estimating uncertainty according to the Laboratory of Government Chemists/Valid Analytical Measurement (LGC/VAM) protocol for determination of 18 amino acids in gelatin is proposed. The expanded uncertainty was estimated using mainly the method validation data (precision and trueness). Other sources of uncertainties were contributed by components in standard preparation measurements. The method scope covered a single matrix (gelatin) under a wide range of analyte concentrations. The uncertainty of method precision, μ(P) was 0.0237–0.1128pmolμl−1 in which hydroxyproline and histidine represented the lowest and highest values of uncertainties, respectively. Proline and phenylalanine represented the lowest and highest uncertainties value for method recovery, μ(R) that was estimated within 0.0064–0.0995pmolμl−1. The uncertainties from other sources, μ(Std) were 0.0325, 0.0428 and 0.0413pmolμl−1 that were contributed by hydroxyproline, other amino acids and cystine, respectively. Hydroxyproline and phenylalanine represented the lowest and highest values of expanded uncertainty, U(y) that were determined at 0.0949 and 0.2473pmolμl−1, respectively. The data were accurately defined and fulfill the technical requirements of ISO 17025:2005.