Full dietary nucleotides spectra by HPLC

nucleosides structure
Nucleotides are present in cells in different forms:
  • Apolar free heterocyclic bases adenine, guanine, cytosine, and uracil (RNA) or thymine (DNA);
  • 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:
  1. 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.
  2. 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.
  3. 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 high-performance liquid chromatography with diode array detection NOVOCIB provides HPLC analytical services of full spectra of nucleotides in cell extracts and in feed/ food products and ingredients.

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. Zorbax Extend-C18 4.6x150mm, 3.5μm particle size and guard column (Agilent). 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).

HPLC spectra
Fig. 1: Representative chromatogram of a mixture of nucleotides mono-, di- and triphosphates, nucleosides and heterocyclic bases separated using ion-paired reverse-phase HPLC coupled to a UV detector set at 254nm.
Photo HPLC Analysis and spectra
Dietary Nucleotides Analysis:

HPLC-UV analysis for full spectra of dietary nucleotides (bases, nucleosides and NMP, unhydrolyzed DNA and RNA nucleic acids) present in feed or ingredients

€ 420.00 / sample Inquiry
Celullar Nucleotides Analysis:

HPLC-UV analysis for full spectra of cellular nucleotides (bases, nucleosides, NMP, NDP and NTP) in cell extracts.

€ 350.00 / sample Inquiry
#S1200-05 Nucleic acid RNA and DNA quantification by HPLC-UV analysis
€ 300.00 / sample Inquiry
#S1200-06 HPLC-UV analysis for purines
€ 300.00 / sample Inquiry