Nucleotides are present in cells in different forms:
- Apolar free heterocyclic bases adenine, guanine, cytosine, and uracil (RNA) or thymine (DNA);
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Apolar free ribo- and deoxyribonucleosides
purines: adenosine/deoxyadenosine, guanosine/deoxyguanosine, inosine;
pyrimidines: cytidine/deoxycystidine, uridine, thymidine; - Negatively charged free ribo- and deoxyribonucleotides mono-, di- and triphosphates (AMP/dAMP, GMP/dGMP, IMP, CMP/dCMP, UMP, dTMP, ADP/dADP, GDP/dGDP, CDP/dCDP, UDP, ATP/dATP, GTP/dGTP, CTP/dCTP, UTP)
- Polymeric negatively charged nucleic acids RNA and DNA composed of ribo- and deoxynucleotides monophosphates, respectively.
There are several analytical methods for separating and analyzing nucleotides:
-
Anion Exchange Chromatography allows the separation and
quantification of nucleotides mono-, di- and tr-phosphates.
😒 However, this method does not allow the analysis of corresponding bases and nucleosides that can be present along with negatively charged nucleotides. -
Acidic hydrolysis of nucleotides to corresponding bases followed by
separation of heterocyclic bases.
😒 However, this approach does not allow to discriminate whether heterocyclic bases result from the degradation of ribo- or deoxynucleotides or even from nucleic acids degradation. -
Ion-paired chromatography is a technique that allows to separate
both apolar (bases and nucleosides) and negatively charged compounds
(nucleotides mono- di- and triphosphates) in one-run.
😀 This technique overcomes challenges faced by other ion chromatography methods;
😒 but requires careful selection of ion-pairing reagents and has limited column lifetime.
Using ion-paired chromatography with diode array detection (HPLC-UV), NOVOCIB provides analytical service for complete dietary nucleotides spectra characterization (bases, nucleosides, nucleotides mono-, di- and triphosphates).
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Dietary Nucleic acids DNA and RNA quantification:
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UV spectroscopy
Nucleic acids can be quantified by measuring the absorbance at 260
nm and 280 nm with 1A 260 corresponding to 50µg/ml of dsDNA, to
~40µg/ml RNA and to 33µg/ml ssDNA.
😒 This method can be applied only to purified DNA or RNA. -
Acid hydrolysis/ chromatography
Acidic hydrolysis of nucleic acids in perchloric or formic acid
leads to the formation of purine and pyrimidine bases that are
analyzed by LC-MS/MS or HPLC-UV chromatography.
😒 Using environmental pollutant perchlorate or toxic formic acid together with high cost are major limitations of this powerful analytical method. -
Enzymatic/HPLC-UV
Nuclease digestion of DNA and RNA into constituent nucleotides NMP
and dNMP prior to HPLC-UV analysis.
😀 This technique overcomes environment and cost challenges of acidic hydrolysis;
😒 requires careful selection of conditions to achieve complete nucleic acid digestion.
To quantify dietary nucleic acids NOVOCIB uses enzymatic/HPLC-UV approach where nucleic acids are converted enzymatically to 5'NMP and analyzed before and after nuclease treatment. Nucleic acid concentration is calculated as a difference in 5'NMP concentration before and after nuclease.
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Our analytical system
Agilent 1120 series HPLC liquid chromatograph fitted with binary pump, vacuum degasser, well-plate autosampler, thermostatic column compartment and multiple wavelength and diode array detector. Run and data acquisision are controlled by Agilent Chem Station software. Calibrations are performed with standards prepared in mobile phase and with standards mixed with cell extracts, which are run immediately before and after every series of samples. Peak assignment of different bases, ribonucleosides and ribonucleoside monophosphatesis is done by comparing both retention times and characteristics of UV absorption spectra (254/280 ratio) with those of standards. The area of individual peaks was measured using ChemStation software (Agilent).