Review Team (February 2016).pdf

Fonterra Co-operative Group

Page 14

Bias Against Reference Method

As part of initial method validation the LC-MS/MS was evaluated for bias against an HPLC-UV method

based on AOAC 2002.05 [3]. A range of infant formula samples was tested by AOAC 2002.05 (modified)

and by the LC-MS/MS method. The

-value of 0.09 indicates that at the 0.05% level of confidence, no bias

was found (Table 13). Bias against a reference method is not defined as a parameter within the SMPR.

Table 13: Results for bias experiment against AOAC 2002.05

Parameter

Reference Method

LC-MS/MS Method

Mean Reference Method

10.5 μg hg

-1

10.8 μg hg

-1

Standard Deviation Reference Method

3.18 μg hg

-1

3.66 μg hg

-1

Number of Replicates (n)

95% Confidence Interval Reference Method

10.0

–

11.0 μg hg

-1

10.2

–

11.4 μg hg

-1

Mean of Paired Differences

-0.3

Standard Deviation of Paired Differences

1.27

stat

1.73

Degrees of Freedom

-Value

0.09

Chromatography

A typical MRM chromatogram for a sample is given in Figures 7

–

8. The advantages of using the PTAD

derivatisation strategy are that many compounds (such as plant sterols) isobaric with vitamin D

and

vitamin D

and are excluded from detection since they lack the conjugated diene structure and do not form

adducts. Two epimers, 6S and 6R, are produced by derivatization of vitamin D, since the PTAD reagent

reacts with the

s-cis

diene moiety from both the α

and β

-sides; the ratio of 6S/6R is approximately 4:1 [5].

The 6S/6R epimers co-elute using the described chromatographic conditions.

VitD-18 (February 2016)

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