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Baby Milk Nucleotide Analysis
Nucleotides are naturally present in milk (i.e. 2-5% of the non-protein nitrogen in Human milk). However, after studies had suggested that nucleotides have positive effects in infant nutrition, manufacturers have got used to adding nucleotides in baby food, for several years now and particularly in milk powder. Unsurprisingly, in a context where food additives are more and more subjected to regulation, nucleotide additions in baby milk powders do or are about to enter under the control of strict rules.
Nucleotides are naturally present in milk (i.e. 2-5% of the non-protein nitrogen in Human milk). However, after studies had suggested that nucleotides have positive effects in infant nutrition, manufacturers have got used to adding nucleotides in baby food, for several years now and particularly in milk powder. Unsurprisingly, in a context where food additives are more and more subjected to regulation, nucleotide additions in baby milk powders do or are about to enter under the control of strict rules.
Other services for nucleotide analyses are available. Please, see also our Customized Nucleotide Profiling Analysis.
Aim: Determination of the content of nucleotides monophosphate in baby milk powder, particularly CMP, UMP, GMP, IMP and AMP
IMPORTANT: Client-specified alterations can be accommodated
Nucleotides additives in baby food are generally monophosphate nucleotides. NOVOCIB usually analyzes CMP, UMP, GMP, IMP and AMP contents in baby milk, but other nucleosides or nucleotides can be quantified upon request.
Nucleosides & nucleotides separation and analysis:
The extraction procedure of nucleotides has been optimized for milk powder analysis. It involves a specific extraction protocol from the powder matrix, a fixation-washing-elution step on a strong anion exchange (SAX, Supelco) cartridge and a filtration through a 0.45µm filter membrane (Roth).
Separation method is based on what is routinely performed for cell extract analysis, with slight adjustments of buffers pH and concentrations. Nucleotides are separated by ion-pairing HPLC (Agilent 1100) system with a Zorbax EclipsePlus C18 column and quantified using an Agilent ChemStation software
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IMPORTANT: Client-specified alterations can be accommodated
Nucleotides additives in baby food are generally monophosphate nucleotides. NOVOCIB usually analyzes CMP, UMP, GMP, IMP and AMP contents in baby milk, but other nucleosides or nucleotides can be quantified upon request.
Nucleosides & nucleotides separation and analysis:
The extraction procedure of nucleotides has been optimized for milk powder analysis. It involves a specific extraction protocol from the powder matrix, a fixation-washing-elution step on a strong anion exchange (SAX, Supelco) cartridge and a filtration through a 0.45µm filter membrane (Roth).
Separation method is based on what is routinely performed for cell extract analysis, with slight adjustments of buffers pH and concentrations. Nucleotides are separated by ion-pairing HPLC (Agilent 1100) system with a Zorbax EclipsePlus C18 column and quantified using an Agilent ChemStation software
| Linearity - Limit of Detection:
Nucleotides monophosphate (NMP) standards are from Sigma-Aldrich, MPBiochemicals or Roche. 1mg/ml NMP stock solution is prepared in water and kept in the freezer (-20°C). Working standards are prepared daily at concentrations of 0.25, 0.5, 1.25, 2.5, 5, 10, 25 and 37.5 mg/100 g by dilution of stock solution with mobile phase A. The limit of detection is defined as the concentration where RSD (relative standard deviation) is <20%. A typical chromatogram of 5 nucleotide standards is shown in Figure 1. The related linearity and LOD results are shown in Table 1. |
Table 1: Linearity and LOD of NMP
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| Figure 1: Superposed spectra of 5 concentrations of standards (0.25, 0.5, 1.25, 2.5 and 5mg/100g).
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| Recovery and Reproducibility:
The extraction of milk powder samples is done with and without spiked nucleotide monophosphate (NMP) (2.5mg of each NMP per 100g powder) for every provided sample. |
Table 2: Recovery of 5 spiked NMP's in a baby milk powder
(4 measurements)
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| Figure 2: Superposed chromatograms of milk powder without exogenous nucleotide (blue) and of a milk powder spiked with NMP at 2.5mg/100g (red).
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