AOAC SPIFAN Review Team (August 2016)

Single Lab Validation of a Method for the Determination of β‐

Galactooligosaccharides in Infant Formula & Adult Nutritionals / Sean Austin (NRC,

Lausanne)

25 Jul 2016

CONFIDENTIAL

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Page 12 / 35

2.3. Validation Design

2.3.1

Linearity

Calibration solutions of maltotriose (0.005 nmol/mL – 0.9 nmol/mL) at 11 different levels, were

prepared in triplicate all containing the laminaritriose internal standard (at 0.304 nmol/mL). The

ratio of the peak areas (maltotriose / laminaritriose) was plotted against the concentration of

maltotriose (since the laminaritriose concentration remained constant throughout there was no

need to plot the ratio of the concentrations on the x‐axis). A linear model was used to fit the data

for calibration purposes.

The relative residuals were calculated and plotted against the analyte concentration.

2.3.2

Precision

All samples from the SPIFAN kit were analysed in duplicate on a single day, and five of the samples

were found to contain GOS; The Placebo Infant Formula RTF, Milk Based and the following fortified

products: Infant Formula Powder Partially Hydrolysed, Milk Based, Infant Formula Powder Milk Based,

Infant Formula Powder FOS/GOS Based, Infant Formula RTF, Milk Based. In addition, infant formula

products from other companies and a Nestlé reference sample were also analysed giving a total of 8

matrices for the precision study.

Each of the GOS‐containing samples from the SPIFAN kit, and the two samples purchased from the

supermarket, were reconstituted as instructed in the SPIFAN guideline and analysed in duplicate, on

at least six different days, using 2 different instruments (same model) and 2 different columns (same

stationary phase and manufacturer, but different lot) by 2 different analysts. At the same time the

Nestlé reference sample was also analysed (without reconstitution). SD(r) and SD(iR) were calculated

from the data obtained using the equations below:‐

SD(r)

 





x x

r SD

 







SD(iR)

 

1 b SD iR SD





Where:

n is the number of (single or duplicate) determinations

is the individual result within the set of single determinations with i going from 1 to n ;

and x

are the two results within the set of duplicate determination with i going from 1 to n ;

SD(b) is the standard deviation between the means of duplicates

VALIDATION REPORT

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