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AOAC Final Action Method 2016.02 (Biotin)
Page 13 of 16
SUMMARY AND CONCLUSION
The SLV and the MLT data provide systematic scientific evidence for a simple, selective, accurate and
precise method as a potential candidate reference method for dispute resolution for the determination of
total biotin in all forms of infant, adult, and/or pediatric formula. The method fully meets the intended
purpose and applicability statement by complying with standard method performance requirement outlined
in AOAC SPIFAN SMPR 2014.005.
The method was applied to a cross section of matrix types and established acceptable precision and
accuracy. The analytical platform is inexpensive and the method can be used in almost any labs worldwide
with basic facilities. The immunoaffinity column (IAC) clean-up extraction is the key step to successful
analysis. Although R-Biopharm IAC was used for the MLT, alternative IAC was compared during SLV with
comparable results. The performance parameters of the method are compared with AOAC SPIFAN SMPR
2014.005 and the key points are summarised in the Table 2016.02I.
Table 2016.02I: Comparison of method performance with SMPR 2014.005
Parameters
SMPR 2014.005
AOAC 2016.02
Comments
Analytical Range
0.1 - 150 µg/100g 0.1 - 300 µg/100g
Analytical range of the proposed method is wider based on
the sample weight and loading volume for IAC extraction.
Limit of Quantitation
≤0.1 µg/100g
≤0.1 µg/100g
The proposed method meets the LOQ of 0.1 µg/100g as
required by SMPR 2014.005.
Repeatability (RSD
r
)
>1 µg/100g: ≤6% >1 µg/100g: ≤5%
Repeatability of the proposed method is better than the
SMPR 2014.005.
Reproducibility (RSD
R
)
>1 µg/100g:
≤12%
>1 µg/100g:
≤10%
Reproducibility of the proposed method is better than the
SMPR 2014.005.
Horwitz Ratio (HorRat)
0.5 to 2.0
0.3 to 0.5
Excellent precision performance rating.
Recovery (>1µg/100g)
90 to 110%
95 to 105%
The spiked recoveries by the proposed method are better
than the recovery range specified by SMPR 2014.005.
NIST SRM 1849a
199 ± 13
197 ± 10
NIST 1949a results are within the certified limits.
RECOMMENDATIONS
A complete AOAC International MLT study workbook along with the statistical report and draft copy of the
study report summarising the outcomes of this collaborative study were submitted with the recommendation
that the AOAC First Action Official Method 2016.02 be accepted as a SPIFAN endorsed AOAC Official
Final Action Method
.
NOTES FROM THE STUDY DIRECTOR
I would like to place on record my learnings and experience with the collaborators during the course of the
study. I believe that ideas for improvement, feedback and challenges are valuable information for future
enhancement to the method in some way or other.
Filtrate for IAC Clean-up
: One of the participants commented that the filtrate for IAC clean-up is not clear,
suggested that if the same extraction would be also efficient if performed at pH 4, which would precipitate
many of the proteins, yielding a clear filtrate. My laboratory tested few SPIFAN matrices using sodium
acetate solution 0.4M, pH 4.0 (In to a 2000mL volumetric flask, weigh 108.8 g sodium acetate trihydrate.
Add about 1800mL water. Dissolve. Add 50mL acetic acid, and adjust pH to 4.0 with acetic acid, dilute to
volume with water). The filtrate was clear, IAC clean-up was faster and the biotin results were comparable
to the current method. However this change was not advised to any of the collaborators and the participant
was advised to proceed with current method as published as First Action Official AOAC 2016.02.
IAC loading speed
: Two laboratories obtained lower biotin results for practice samples initially were
advised to follow IAC loading by gravity as documented in the method and the results came back higher
and comparable to other labs.
2016.02 (BIOTIN) FINAL ACTION REVIEW
FOR ERP USE ONLY
DO NOT DISTRIBUTE