48 / 123
AOAC Final Action Method 2016.02 (Biotin)
Page 8 of 16
The difference between the results of duplicate determinations tested on different days should not exceed
12% of the mean result
L. Uncertainty of Measurement
Uncertainty of the method was calculated as 7%, using appropriate statistical procedure (square root of the
sum of squares of the errors expressed as a percentage).
M. Limit of Quantitation
The LOQ was calculated based on the lowest working standard and dilution factor,
Limit of Quantitation (LOQ) was calculated based on the lowest working standard and the dilution factor.
LOQ = (1 x 100) / (20 x 50) = 0.1µg/100g (1ppb)
1 = 1µg/100mL lowest standard
100 = Volume (mL)
20 = 20g sample
50 = Volume (mL) loaded on immunoaffinity column
1= Final volume (mL)
RESULTS AND DISCUSSION
The laboratories were requested to analyse biotin and biocytin in the samples, however none of the
samples were found to have biocytin and only biotin results were reported and evaluated in the study. All
the biotin results presented in the report are expressed as
µg/100g
of sample as received basis from AOAC
International.
Three of the twelve laboratories (Labs 10, 11 and 12) could only report the results of practice samples prior
to the due date of the study, 27 Jan 2017. In general, the results from Lab 7 is showing low bias which was
investigated further. The possible reasons for the low bias could be the practice followed in sample
preparation especially the steps related to loading of immunoaffinity column and the elution technique
where triple backflush is specified. The investigation from Lab 7 reported that the analysts did not follow the
backflush technique effectively and that may have caused the incomplete elution of biotin from the
cartridges. The laboratory also reported that they had deviated from the protocol in reconstitution of powder
samples by taking 10g of powder in 80mL. The results from Labs 7, 10, 11 and 12 are included in the MLT
report and for statistical evaluation of the results to calculate precision of the analytical method.
Linearity and Range
The retention time of biocytin ranges from 3.3 minutes to 6.1 minutes and that of biotin from 13.8 min to
22.5 minutes. The retention times reported from Lab 9 stands out from other laboratories though the
chromatographic conditions were followed as documented. The variation is likely due to difference in the
hardware configuration of the liquid chromatographs used for the analysis. The information was not
available from Lab 10 at the time of the data processing.
All the participants used more or less the same calibration range 1 to 20 µg/100mL for biocytin and biotin
as specified in the method. The range showed excellent correlation coefficient among the participants of
not less than 0.999, confirming the linearity of the method over the calibration range (Table 2016.02C).
2016.02 (BIOTIN) FINAL ACTION REVIEW
FOR ERP USE ONLY
DO NOT DISTRIBUTE