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AOAC First Action Method 2016.02 (Biotin) MLT Report
Page 12 of 16
4.5. Relative Standard Deviation of Reproducibility (RSD
R
)
Reproducibility expresses the precision among the laboratories. The relative standard deviation of
reproducibility (RSD
R
) exceeds the SMPR criteria of not more than 12% for all the 14 matrices analysed in
the study confirming the precision of the analytical method. The maximum RSD
R
noticed was 9.5% in the
MLT sample 1, which is partially hydrolysed milk based infant formula matrix. The reproducibility precision
confirms the suitability of the SPIFAN method AOAC 2016.02 as a strong candidate as a reference method
for global dispute resolution hence the inter-laboratory variation is minimal and exceeds the expectation of
the SMPR limits.
4.6. Horwitz Ratio (HorRat)
The Horwitz ratio (HorRat) is a normalized performance parameter indicating the acceptability of methods
of analysis with respect to among-laboratory precision (reproducibility). It is the ratio of the observed
relative standard deviation among laboratories calculated from the actual performance data, RSD
R
, to the
corresponding predicted relative standard deviation (PRSD
R
) calculated from the Horwitz equation. The
formula for the Horwitz ratio as presented in the
International Study Workbook
of AOAC International is
applicable only when the concentration is in the unit/unit form (e.g., µg/µg or g/g, etc.). When the analyte
concentration is a mass fraction amount as µg/100g, the appropriate factor for unit of measurement should
be selected to generate correct HorRat values. The factor selected in the study for the calculation is 1.00E-
08.
Under reproducibility conditions, the acceptable HorRat value range is 0.5 to 2 as per AOAC International.
The HorRat values obtained in this study were within 0.3 to 0.5 in all SPIFAN matrices except the NIST
SRM 1849a. The performance requirements of SPIFAN methods are generally tighter than routine
analytical methods and therefore PRSD
R
used in the formula may be slightly higher pushing the HorRat
values down. Nevertheless, the lower Horwitz ratios confirm the enhanced performance of the method and
homogeneity of the matrices used for the study. The NIST SRM 1849a recorded the lowest HorRat value of
0.21 and undoubtedly the most homogeneous certified reference material used in the MLT.
4.7. Accuracy (NIST SRM 1849a)
The accuracy of an analytical procedure expresses the closeness of agreement between the value which is
accepted either as a conventional true value or an accepted reference value and the value found. The
established procedure for accuracy assessment is by analysing samples with known concentrations such
as certified reference materials. The SPIFAN SMPR 2014.005 recommends NIST SRM 1849a for accuracy
evaluation and the SRM is included in the SPIFAN matrices as blind coded duplicates.
The NIST SRM 1849a results were all within the certified limits of biotin confirming the accuracy of the
method. It has been noticed that one of the duplicate results of NIST reference sample was slightly on the
low bias (185.41µg/100g) for Lab 7 but this was investigated as explained before. The NIST SRM 1849a
has comparatively higher biotin content and homogeneous therefore unlike other powder samples it was
not reconstituted with water and 1g powder was directly weighed for the analysis.
Matrix recoveries were carried out during SLV at different levels of calibration range and the recoveries
were within 95 to 105%.
4.8. Selectivity (Comparison with LCMS/MS)
Selectivity / specificity is the ability to assess unequivocally the analyte in the presence of components
which may be expected to be present. Typically these might include impurities, degradants, matrix, etc.
Selectivity of the method is confirmed by analysing the SPIFAN matrices by LCMS/MS method by one of
the laboratories participated in the MLT study (Lab 5). The method is a modified version of AOAC Official
Method 2012.16 for pantothenic acid and uses a UPLC platform with triple quadrupole mass spectrometer.
The biotin results by LCMS/MS method were closely comparable to the MLT results for the range of
SPIFAN matrices used in the study. Please refer to Table 9 for comparison data.
2016.02 (FEBRUARY 2017) BIO-02 MLT REPORT
FOR ERP USE ONLY
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