OMB Meeting Book (June 22, 2017)

AOAC Final Action Method 2016.02 (Biotin)

Page 10 of 16

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

, to the

corresponding predicted relative standard deviation (PRSD

) 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

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 standard reference material used in the MLT.

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%.

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 2016.02H for comparison data.

2016.02 (BIOTIN) FINAL ACTION REVIEW

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