NOVOCIB's Method Validation

Introduction

Nucleotides are considered semi-essential nutrients, as endogenous synthesis does not always meet physiological demand, particularly during periods of rapid growth, immune activation, or intestinal repair. In infants, dietary nucleotide supplementation has been shown to enhance humoral antibody responses to vaccination, as demonstrated by Pickering et al. (1998). In aquaculture, nucleotide-fortified feeds are widely recognized to improve intestinal health, growth performance, immune competence, gut integrity, and stress resistance in fish and shrimp.

On the other hand, nucleotides and nucleic acids are also a major source of purines, and excessive human consumption may lead to overproduction of uric acid. This is particularly relevant for single-cell proteins (SCP), a new sustainable alternative to animal or plant proteins. SCPs are produced from fast-growing microbial biomass and purified by microfiltration, a process that concentrates not only proteins but also polymeric DNA and RNA, resulting in high nucleotide content. FAO/WHO recommended that SCP intended for human consumption should be processed to reduce nucleic acids to ~2% of dry matter, because high RNA intake increases purine load and uric-acid production.

To ensure compliance with nutritional and regulatory requirements, a new analytical method was developed and validated to accurately quantify nucleotides and nucleic acids in complex matrices. The method is applicable to nucleotide ingredients, yeast and SCP protein as well as final feed products, enabling robust quality control and monitoring of purine load in formulations containing microbial proteins.

Analytical Method Overview

NOVOCIB's analytical method integrates two complementary modern technologies to achieve accurate quantification of free nucleotides, nucleosides, bases, and polymeric nucleic acids (RNA and DNA) in complex biological ingredients and feed matrices.

Analytical Standards

Since commercially available nucleobases, nucleosides, and nucleotide mono-, di-, and triphosphates may contain unspecified amounts of residual salts and water, NOVOCIB verifies the true molar concentration of each standard using published molar absorption coefficients (see tables below). This verification step ensures a reproducible concentration of analytes from different sources for the HPLC calibration. The integrity of every standard is controlled by HPLC analysis.

How to check the concentration of purified nucleotide by absorbance

1. Prepare a 100 mM stock solution in autoclaved deionized water^* using the molecular weight provided by the supplier.
2. Choose the appropriate wavelength on the UV-spectrophotometer.
3. Make a blank using a quartz 1-cm cuvette (plastic cuvettes must not be used for short-UV wavelengths) filled with a buffer of the desired pH.
4. Prepare a 0.05 mM (50 µM) solution of the nucleotide in the same buffer^**.
5. Measure the absorbance of the 50 µM solution.
6. Compare the obtained absorbance value with the molar extinction coefficient (ε) to calculate the actual analyte concentration.

^* Some nucleosides and bases are insoluble in water but can be dissolved in 0.1–0.5 M KOH.
^** Intermediate dilutions may be required to prepare a 50 µM solution from a 100 mM stock.

Quality Control Sample

To avoid any loss of bases, nucleosides, or nucleotides during the pretreatment step, sample preparation is limited to solubilization in sterile deionized water followed by deproteinization and injection into the ion-paired reverse-phase (IP-RP) HPLC system, allowing direct separation and quantification of the whole spectrum of bases, nucleosides and nucleotides present in the sample (Fig. 9).

A powder of concentrated nucleotides, a commercial product derived from Saccharomyces cerevisiae (26.2 g/100 g according to the supplier and 31.1 g/100 g according to NOVOCIB), is used as a Quality Control Sample run immediately after standards STD1 (NMP), STD2 (Nucleosides) and STD3 (Bases), both before and after the analyzed samples.

Table 7 presents the nucleotide quantification results obtained using NOVOCIB's IP-RP method compared with the manufacturer's data. For purine NMPs — AMP, GMP and IMP — the values are similar. In contrast, NOVOCIB's values for the pyrimidine nucleotides UMP and CMP are higher, most likely because pyrimidine nucleotides are not degraded under NOVOCIB's conditions, unlike during acidic hydrolysis. Table 8 shows the relative standard deviations of peak area for the Quality Control Sample analyzed six times, together with the acceptance criteria (Fig. 9).

Table 7. Nucleotide content per 100 g (S. cerevisiae concentrate)

Compound	NOVOCIB g/100g	Acid Hydrol. g/100g
Cytosine	0.00	—
Uracil	0.17	—
Guanine	0.00	—
Hypoxanthine	0.00	—
Adenine	0.18	—
Cytidine	0.34	—
Uridine	0.85	—
Guanosine	1.91	—
Inosine	0.00	—
Adenosine	1.62	—
CMP	6.10	5.00
UMP	7.83	5.00
GMP	8.86	8.00
IMP	0.00	0.20
AMP	8.27	8.00
Total 5'-NMP	31.07	26.2

CMP–AMP rows given without salt/water.

Table 8. QC repeatability (% RSD)

Compound	% RSD n=6	Criteria
CMP	1.3%	<5%
UMP	2.3%	<5%
GMP	2.5%	<5%
IMP	2.2%	<5%
AMP	2.2%	<5%

All compounds meet the <5% acceptance criterion.

All compounds meet the <5% acceptance criterion.

Chromatogram superposition of a Saccharomyces cerevisiae-derived nucleotide-concentrated commercial product at 254 nm and 280 nm

Method Performance

Selectivity

Selectivity of this method is ensured by the excellent separation of nucleotides, achieved through the high purity of the TBA ion-pairing reagent, its optimized concentration in buffers A and B, and the superior performance of the Zorbax Eclipse Plus C18 column (Agilent).

Specificity

Specificity of this method relies on accurate nucleotide identification by comparing retention times and UV absorption spectra (254/280 ratio), which are characteristic for each nucleotide. Spectra at 254 nm and 280 nm are recorded using a diode-array detector (Agilent). Run control and data acquisition are performed using Agilent ChemStation software.

Chromatogram of 15 mixed standard solutions of nucleobases, nucleosides and NMP at 250 nm

Superposition of UV spectra at 254 nm and 280 nm for 15 mixed standards of nucleobases, nucleosides and NMP with 254/280 area ratio

Linearity and Repeatability

Linearity and repeatability were evaluated using three independent calibration curves prepared from mixtures of standards: STDB1 (CMP, UMP, GMP, IMP, AMP), STDB2 (cytosine, uridine, guanosine, inosine, adenosine), and STDB3 (adenine, guanine, hypoxanthine, cytidine, uracil). Each mixture was analyzed at the indicated concentrations, with six repeated measurements per level. Tables 4–6 summarize the coefficients of determination for all calibration curves and the relative standard deviations of peak areas obtained at the lowest tested concentration (25 µM) for nucleotides, nucleosides, and bases. All compounds exhibited good linearity, with the highest correlations observed for 5'-NMP, which represents the primary focus of this study.

Table 4. Coefficients of determination obtained with calibrated standards solutions of nucleotides monophosphates

Composition	Linearity (r²)	% RSD
CMP	0.9998	10.5%
UMP	0.9994	0.9%
GMP	0.9999	3.1%
IMP	0.9999	6.8%
AMP	0.9999	1.2%
XMP	0.9999	2.5%

STD1 (NMP), 25–500 µM, peak area at 254 nm.

Superposition of HPLC chromatograms of six nucleotide monophosphate standards

Calibration curves for six nucleotide monophosphates showing linear peak-area responses

Table 5. Coefficients of determination obtained with calibrated standards solutions of nucleosides

Composition	Linearity (r²)	% RSD
Cytidine	0.9995	0.9%
Uridine	0.9997	2.8%
Guanosine	0.9999	11.7%
Inosine	0.9999	13.6%
Adenosine	0.9997	2.8%
Xanthosine	0.9999	2.8%

STD2 (Nucleosides), 25–500 µM, peak area at 254 nm.

Superposition of HPLC chromatograms of six nucleoside standards

Calibration curves for six nucleosides showing linear peak-area responses

Table 6. Coefficients of determination obtained with calibrated standards solutions of bases

Composition	Linearity (r²)	% RSD
Adenine	0.9998	2.8%
Guanine	0.9999	0.4%
Hypoxanthine	0.9995	3.5%
Cytosine	0.9994	5.9%
Uracil	0.9991	10.0%
Xanthine	0.9994	1.2%

STD3 (Bases), 25–500 µM, peak area at 254 nm.

Superposition of HPLC chromatograms of six nucleobase standards

Calibration curves for six nucleobases showing linear peak-area responses