Chemical Diversity of Nucleotides

Total Potentially Available Nucleotides (TPAN) is the sum of all nucleotides that can be liberated from nucleic acids and nucleotide-containing compounds in a food or biological sample after complete enzymatic hydrolysis. It includes:

• Free nucleotides already present (e.g., AMP, GMP, CMP, UMP).
• Nucleotides bound in RNA and DNA, which are released upon hydrolysis of nucleic acids.
• Nucleotide derivatives (such as nucleosides or nucleoside monophosphates) that can be converted into nucleotides.

The TPAN concept was first introduced for milk nucleotides (Leach et al., 1995*), where a dedicated method measured total potentially available nucleosides including free nucleosides, free nucleotides, nucleotide-containing adducts (such as NAD and UDP-glucose), and nucleotide polymers, primarily RNA. With this method, Leach et al. reported averages of 72 and 68 mg/L TPAN in milk samples from American and European women, respectively. According to Tressler et al. (1998), free nucleotides in human milk represent less than half of TPAN, with RNA around 43% and free nucleosides around 39.9%**.

(1) J L Leach, J H Baxter, B E Molitor, M B Ramstack, M L Masor Total potentially available nucleosides of human milk by stage of lactation. Am J Clin Nutr 1995 . Jun;61(6):1224-30

(2) Tressler, R., Ramstack, M., White, N., Molitor, B., & Masor, M. (1998). Total potentially available (ribo)nucleosides (TPAN) in human milk from Asian women. Journal of Pediatric Gastroenterology and Nutrition, 26(5)

Traditional Acid Hydrolysis Method

The conventional approach uses perchloric acid hydrolysis followed by reverse-phase HPLC:

1. Strong acid hydrolyzes nucleic acids, nucleotides, and nucleosides into free bases.
2. Bases are separated by reverse-phase HPLC.

Advantages: Simple, fast, widely used.

Limitations:

• Non-specific: cannot distinguish free NMP from nucleosides or nucleic acids.
• Functional mismatch: RNA-rich samples appear identical to free NMP-rich samples.
• Harsh conditions: acids degrade/modify analytes, introducing artifacts.
• Environmental impact: corrosive waste requiring special disposal.

Nucleotide analysis Traditional Acid Hydrolysis Method

NOVOCIB’s Enzymatic Hydrolysis + Ion-Paired HPLC

NOVOCIB’s method couples enzymatic hydrolysis with ion-paired reverse-phase HPLC and diode array detection:

1. Ion-paired chromatography separates bases, nucleosides, and nucleotides (mono-, di-, triphosphates) in one run.
2. Nucleic acids (RNA/DNA) are enzymatically hydrolyzed to NMP/dNMP and quantified before and after nuclease treatment.

Advantages:

• Specific and accurate: distinguishes nucleotides, nucleosides, bases, and nucleic acids.
• No chemical modification of analytes.
• Relevant: reflects true composition (taste-active NMP vs inert RNA).
• Greener: reduced environmental impact compared to acid hydrolysis.

Why enzymatic-HPLC? Acid hydrolysis is fast but blind, collapsing all compounds into bases. Enzymatic-HPLC is slower but precise, preserving distinctions critical for food chemistry, nutrition, and pharmacology.