OMB Meeting Book_9-11-14

Method Performance

The initial phase of method evaluation within participating laboratories involved the analysis of a practice

sample. The NIST 1849a CRM was selected for this purpose for a number of reasons: (i) it was readily

available in most laboratories and hence the method set-up and evaluation could begin immediately without

awaiting samples to be shipped; (ii) since the expected values are known, participants could get instant

feedback on whether the method was working appropriately in their hands; (iii) it provides additional

confidence that there is no bias in the operation of the method amongst all participants. All participating

laboratories provided acceptable data for the practice sample and once the test sample set was delivered,

the participants could begin the analysis at their earliest convenience; Tables 13–15.

The results submitted by participants and the statistical analysis for each sample is given in Tables 16–27.

All 12 collaborators returned acceptable standard calibration parameters based on linear regression correlation coefficients (r 2 : 0.9971–1.0000). Laboratory 9 had problems with Sample 2 and the results were

excluded from dataset. All remaining data were statistically analysed using the AOAC protocol for overall

mean, intra-laboratory repeatability (S r

), repeatability relative standard deviation (RSD r

), inter-laboratory

reproducibility (S R

), reproducibility relative standard deviation (RSD R

), and HorRat (2). Cochran (p = 0.025,

1-tail) and Grubbs (single and double, p = 0.025, 2-tail) tests were utilized to determine outliers. In some

instances statistical outliers were identified but where deemed reasonable to do so, these were retained in

the data set for calculation of method precision.

Nucleotide concentrations in nucleotide supplemented infant formula powders ranged from at 4.4– 13.1 mg hg -1 for CMP, 2.7–4.6 mg hg -1 for UMP, 0.9–1.8 mg hg -1 for GMP, 1.5–3.9 mg hg -1 for IMP, and 3.1–5.2 mg hg -1 for AMP. RSD r values obtained ranged from 1.1–2.7% for CMP, 1.4–5.4% for UMP, 1.6– 3.9% for GMP, 1.1–1.8% for IMP, and 1.1–3.9% for AMP. RSD R values obtained ranged from 7.1–8.7% for CMP, 7.9–9.0% for UMP, 2.8–7.7% for GMP, 5.5–10.3% for IMP, and 3.0–7.8% for AMP. In all instances of

nucleotide supplemented infant formulas, the repeatability and reproducibility are within limits set in the

Nucleotides SMPR (3). Acceptable reproducibility was also demonstrated with HorRat values for the

method ranging from 0.3–1.0 (recommended range 0.5–2.0) (2). The soy-based infant formula was not

fortified with nucleotides and therefore contained endogenous levels only. The precision for this sample is

poor, but this is to be expected given nucleotide concentrations are near or below the method detection limit

(4).

Collaborative Study Report: Method 2011.20 Nucleotides by HPLC-UV

Page 16