HPLC Method for Separation of Biopterin, Rhamnopterin, Neopterin, Folic Acid and Aminopterin on Zodiac HST OG Column

Pterins are a class of heterocyclic compounds based on the pteridine ring system, which consists of a fused pyrimidine and pyrazine ring. Several biologically significant compounds, including Biopterin, Rhamnopterin, Neopterin, Folic Acid, and Aminopterin, fall under this structural class. Despite sharing a common backbone, these compounds exhibit diverse biological roles:

• Biopterin (Tetrahydrobiopterin): A crucial cofactor in the biosynthesis of neurotransmitters like dopamine, norepinephrine, and serotonin, as well as nitric oxide. Deficiencies in its recycling enzymes can result in phenylketonuria (PKU), a metabolic disorder.

• Neopterin: Produced by immune cells in response to interferon-gamma, neopterin serves as a biomarker for immune system activation. Elevated levels may indicate viral infections, autoimmune conditions, or malignancies.

• Folic Acid (Vitamin B9): Essential for DNA synthesis, repair, cell division, and growth. It must be acquired through diet or supplementation, as it is not synthesized by the human body.

• Aminopterin: An early antifolate chemotherapy agent that inhibits dihydrofolate reductase, thereby preventing folic acid activation and disrupting cell proliferation. It has been largely replaced by safer alternatives like methotrexate.

• Rhamnopterin: Though less well-documented, it is structurally related to other pterins and may share similar physicochemical properties.

These five pterin derivatives can be efficiently retained, separated, and analyzed using the Zodiac HST OG mixed-mode reversed-phase column (4.6 × 150 mm, 5 µm, 100 Å). The method employs a mobile phase comprising water, acetonitrile (MeCN), and ammonium formate as a buffer, making it fully compatible with UV detection at 275 nm, ELSD, Charged Aerosol Detection (CAD), or Electrospray Ionization Mass Spectrometry (ESI-MS).

The limit of detection (LOD) for each compound depends on the specific instrument configuration and laboratory conditions, and may vary even when similar methods are employed.