Adenosine Monophosphate

Separation type: Liquid Chromatography Mixed-mode









High Performance Liquid Chromatography (HPLC) Method for Analysis of Adenosine monophosphate (AMP), Adenosine diphosphate (ADP), Adenosine triphosphate (ATP)




Adenosine mono-, di- and triphosphates are key components of the cell’s energy ecosystem. Adenosine triphosphate (ATP) is the main unit of energy used in the cell. When it is ‘spent,’ it is converted to adenosine diphosphate (ADP); it is often converted back to ATP to be reused. Adenosine monophosphate can be converted to ADP (and subsequently ATP), but it is also a key factor in RNA synthesis.
Using a Newcrom B mixed-mode column and a mobile phase consisting of water and acetonitrile with a sulfuric acid (H2SO4) buffer, adenosine mono-, di- and triphosphate can be separated, measured, and analyzed. This method can UV detect this family of compounds at 262 nm with very high resolution.

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Nucleotides are the monomers of DNA and RNA, composed of a five-carbon sugar, a nitrogenous base, and at least one phosphate group. Primesep SB is a good column for separating these highly polar compounds. Primesep SB is a reverse-phase column with strong embedded basic ion-pairing groups. Retention can me manipulated by adjusting acetonitrile, and the baseline separation can be achieved in under 6 minutes using a gradient. 

Nucleotides are important biological molecules which serve as subunits of nucleic acids. They are composed of a five-carbon sugar, a nitrogenous base, and at least one phosphate group. Nucleotides cannot be retained by reverse-phase chromatography without an ion-pairing reagent due to their highly polar nature. Primesep SB is capable of retaining and separating nine nucleotides. Primesep SB is a reverse-phase column with strong embedded basic ion-pairing groups.

A complex mixture of nicotinamide and related impurities was separated on Obelisc R mixed-mode column. Nicotinamide, methylnicotinamide, nicotinamide adenine mononucleotide, nicotinamide adenine dinucleotide, and adenosine monophosphate were baseline resolved in a 15 minute long method. This mixed-mode approach can be used for analysis of other nucleotides. Obelisc R trimodal column separates this complex mixture based on reversed-phase, cation-exchange and anion-exchange mechanisms. Retention is controlled by amount of ACN, buffer concentration and buffer pH. Additional selectivity can be gained by exploring various buffers within the same pH

Adenosine mono-, di and triphosphate are hydrophilic nucleotides which serve as building blocks of DNA and RNA. Each molecule consists of phosphate or phosphate groups, adenine and sugar ribose. Molecules are hydrophilic and lack a retention mechanism on traditional reversed-phase column. Three nucleotides were retained and separated on Primesep B2 reversed-phase anion-exchange column. retention time is controlled by buffer concentration and buffer pH. ADP and ATP require higher concentration of buffer to facilitate elution. Method can be used for LC/MS analysis of different nucleotides in various sample matrices (biofluids, plasma, blood, urine). Other detection techniques can be used for analysis. Method is reliable and robust and can tolerate interference from sample matrix. Additional sample preparation might be required.