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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

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