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AOAC Final Action Method 2016.02 (Biotin)
Page 3 of 16
Table 2016.02B: AOAC SPIFAN Matrices
Samples
Product Description
Batch / Lot #
Blind Duplicate Codes
Practice 1
Infant Formula Powder Partially Hydrolysed Soy Based
410457651Z
SWUO667
SWUO667
Practice 2
Infant Formula Powder FOS/GOS Based
50350017W1
URTF231
URTF231
MLT - 1
Infant Formula Powder Partially Hydrolysed Milk Based
410057652Z
KDOX966
ATAN351
MLT - 2
Infant Elemental Powder
00795RF
ECHL425
UOPM297
MLT - 3
Infant Formula RTF Milk Based
EV4H2R
XKIP216
HYJU890
MLT - 4
Adult Nutritional RTF High Fat
00729RF00
DYLB360
ZMQM883
MLT - 5
Infant Formula Powder Milk Based
4044755861
NSRB999
JSDT587
MLT - 6
Infant Formula Powder Soy Based
E10NWZC
TJHR217
OACN211
MLT - 7
NIST SRM 1849a
CLC10-b
KGSZ273
LTCT316
MLT - 8
Adult Nutritional Powder Low Fat
00859RF00
LYNY751
PZGP859
MLT - 9
Child Formula Powder
00866RF00
RQXQ518
GVPE615
MLT - 10
Toddler Formula Powder Milk-Based
4052755861
EFXN778
BFA0941
MLT - 11
Infant Formula Powder Milk Based
K16NTAV
CULF358
GBZC169
MLT - 12
Adult Nutritional RTF High Protein
00730RF00
FPTE312
DOMY545
Homogeneity assessment of AOAC SPIFAN product matrices were performed by analysing several active
nutritional ingredients. The testing was performed by Covance Laboratories Inc. in one of their facilities.
The homogeneity report was provided to the study director which is carefully evaluated before commencing
the MLT programme.
The participating laboratories were requested to analyse two practice samples in duplicate using the first
action method provided and report the results to the study director. It was communicated to the laboratories
that any deviation, such as necessity to substitute reagents, columns, apparatus or instruments, must be
duly recorded and reported. Electronic templates were provided to the participants for data reporting.
Moreover raw data was requested wherever necessary. After review, the study director identified the
laboratories which have the capability to run the analysis successfully. The study director also had
discussion with those laboratories produced unacceptable data to see any technical reasons which can be
resolved within reasonable time frame to include them in the second part of the study.
The qualified laboratories were then asked to analyse the MLT samples on two different days following a
carefully designed protocol provided. The results were submitted to the study director for evaluation.
Unless otherwise specified in the protocol, all powdered samples were analysed on a reconstituted basis,
using 25 grams of sample into 225 grams with water, as stated in the method. An electronic template was
provided for data reporting including system suitability, linearity, peak areas of the standard curve as well
as of the sample extracts. Furthermore, detailed information on the different weights and volumes used
during sample preparation as indicated in the method, as well as raw data (chromatograms of standards
and samples) were requested. Laboratories are asked to report final biotin and biocytin results in
µ
g/100 g
to two decimal places.
After data collection, outliers were detected using Cochran and Grubbs tests. The number and coded
identity of statistical outlier laboratories is included in the final report. Average biotin concentrations,
standard deviations of repeatability (Sr) and relative standard deviations of repeatability (RSDr) were
estimated from blind duplicates in MLT samples. The blind coded duplicates were analysed on the same
day. Standard deviations of reproducibility (SR), relative standard deviations of reproducibility (RSDR), and
HorRat values (RSDR/predicted RSDR) were also determined.
The analytical method provided to the laboratories for the MLT is the same as it is published in Journal of
AOAC Volume 99, Number 4, 2016, pages 1110 to 1112 which is codified as First Action AOAC 2016.02.
Extensive details of the sample preparation, chromatography, calculation, reporting criteria were specified
in the method and the protocol provided to the laboratories. None of the laboratories that participated in the
MLT recorded any modifications or deviations from the documented procedure. This information was
requested to assess the suitability of the method for further approval as Final Action AOAC method.
2016.02 (BIOTIN) FINAL ACTION REVIEW
FOR ERP USE ONLY
DO NOT DISTRIBUTE