Collaborative Study Report: Method 2011.20 Nucleotides by HPLC-UV
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Method Applicability
The Nucleotides SMPR applicability statement:
“
Determination of nucleotides in all forms of infant, adult, and/ or pediatric formula (powders, ready-to-feed
liquids, and liquid concentrates). For the purpose of this SMPR, nucleotides are defined as adenosine
5′-monophosphate (CAS 61-19-8), cytidine 5′-monophosphate (CAS 63-37-6), guanosine 5′-monophosphate
(CAS 85-32-5), inosine 5′-monophosphate (CAS 131-99-7), and uridine 5′-monophosphate (CAS 58-97-9). It
would also be desirable to measure the corresponding nucleosides: adenosine (CAS 58-61-7), cytidine
(CAS 65-46-3), guanosine (CAS 118-00-3), inosine (CAS 58-63-9), and uridine (CAS 58-96-8).
” (4)
The method has been demonstrated to be suitable for the analysis of nucleotides in a wide range of
supplemented infant formulas, as illustrated with the range of different matrices used in this collaborative
study. The method was demonstrated to be unsuitable for samples containing endogenous nucleotide
levels only. Nucleosides are an optional nutrient defined by the SMPR and are not determined with this
method. The method may be applicable to adult nutritional products; however, such are generally not
fortified with nucleotides since they are not considered an essential dietary nutrient for adults.
The method applicability in Final Action Method should read: Applicable to the determination of nucleotides
adenosine 5′-monophosphate (AMP), cytidine 5′-monophosphate (CMP), guanosine 5′-monophosphate
(GMP), inosine 5′-monophosphate (IMP), and uridine 5′-monophosphate (UMP) in nucleotide supplemented
infant formulas.
Safety Considerations
There are no major hazards beyond those typical found in chemistry laboratories. Use appropriate safety
equipment when handling acids, bases, and solvents. Refer to MSD sheets for detailed safety instructions
for each chemical used. Beware of high vacuum when using the SPE manifold; do NOT exceed the
manufacturer’s recommended maximum vacuum.
Comments from Collaborators
A summary of each laboratory’s performance was sent to participants along with an invitation to make
comments on the performance of the method in their laboratory. The following comments were received:
Lab 2: “The method performs very well. It is very linear, accurate, precise, and sensitive”. No safety
concerns “as long as appropriate personal protective equipment (PPE) is used and precautions are taken
when handling the potassium hydroxide, phosphoric acid, and methanol”. No changes to method
recommended as “the method performs very well”. “Nice job on the method development for both sample
preparation and chromatographic separation”.
Lab 3: The method performs to stated applicability. There are no specific safety concerns. Modify
published First Action method to use EDTA as salt and not free acid as is described in method protocol.
