Collaborative Study Report: Method 2011.20 Nucleotides by HPLC-UV
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Lab 5: The method performs to stated applicability and “works exactly how it is written”. There are no
recommendations to amend the AOAC method and no safety concerns. However, the “only major
observation we had was around the importance of the specified pH values. We did a miniature experiment
around the pH of Mobile Phase A and found if the pH was higher, 0.3 pH units, than the specified value then
the elution sequence changed for AMP and TMP. Usually TMP will elute just before AMP however this was
the other way around when the pH was higher and the elution times were extended by approximately 0.5
minutes.”
Lab 7: Some concerns expressed about the value of the extinction coefficient for CMP. Follow-up work may
be needed.
Lab 9: There are no specific safety concerns. The method performs to stated applicability and there are no
concerns about method’s reproducibility. Suggest adding “centrifuge of 8000 rpm/min 5 min at 4 °C before
anion-exchange procedure.” Problems were found with starch-based samples.
Lab 10: The method performs to stated applicability, “however, UMP accuracy in some product matrices is
around the 90% lower SMPR limit”. No safety concerns with method. “We recommend a decrease in the
amount of sample loaded onto the SPE column to the extent that the SRM gives accurate recoveries and
the method is then more universally applicable to varied product matrices”.
Conclusions
A multi-laboratory collaborative study of AOAC First Action 2011.20, an HPLC-UV method for the analysis of
nucleotides in infant formula, was undertaken. The method was applied to a number of different infant
formula matrices and demonstrated acceptable precision for nucleotide supplemented infant formulas.
Recommendations
On the basis of the results of this study, it is recommended that AOAC First Action 2011.20 be endorsed as
Final Action for the determination of adenosine 5′-monophosphate (AMP), cytidine 5′-monophosphate
(CMP), guanosine 5′-monophosphate (GMP), inosine 5′-monophosphate (IMP), and uridine
5′-monophosphate (UMP) in infant formula and be entitled: AOAC Official Method 2011.20 Nucleotides in
Infant Formula.
Acknowledgements
I am grateful to the following collaborators and their colleagues for their participation in this study:
Greg Jaudzems, Nestlé, OH, USA
George Joseph, AsureQuality, Auckland, New Zealand
Sabine Meng Jensen, Eurofins; Vejen, Denmark
Sam Murray and Paul McNabb, Cawthron Institute, Nelson, New Zealand
Sheila Saldo and Holly Huo, Fonterra, Waitoa, New Zealand
Steve Tennyson and Scott Christiansen; Perrigo Nutritionals, Georgia, VT, USA
