Tryptophan

High Performance Liquid Chromatography (HPLC) Method for Analysis of Mixture of Tryptophan and its Catabolites on Primesep 100 by SIELC Technologies

Separation type: Liquid Chromatography Mixed-mode

Tryptophan and its catabolites participate in several biological pathways, having roles in protein synthesis, serving as precursors to bioactive molecules, and influencing several physiological processes. Here’s an overview considering a mixture of tryptophan and its catabolites:

Tryptophan:

• Essential Amino Acid: Tryptophan is a precursor to several important compounds, including serotonin and melatonin.
• In Protein Synthesis: Incorporated into proteins during protein synthesis.

Catabolites:

1. Serotonin:

• Neurotransmitter: Regulates mood, appetite, and sleep, among other functions.
• Derivative: Melatonin, which regulates the sleep-wake cycle.

2. Kynurenine Pathway (Major catabolic pathway of tryptophan):

• Kynurenine: An intermediate and precursor to several bioactive compounds.
• Kynurenic Acid: An NMDA receptor antagonist, believed to have neuroprotective effects.
• Xanthurenic Acid: Its physiological roles are still being explored, but it’s often studied for its relation to diabetes and neurological conditions.
• 3-Hydroxykynurenine: Can generate reactive oxygen species, potentially contributing to cellular stress.
• Quinolinic Acid: A neuroactive metabolite that can act as an NMDA receptor agonist.

3. Indoleamine 2,3-dioxygenase (IDO) Pathway:

• Tryptophan can be degraded into several catabolites via the IDO pathway, influencing immune response and cell proliferation.

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Tryptophan and its Catabolites can be retained, separated and analyzed on a Primesep 100 mixed-mode stationary phase column using an gradient analytical method with a simple mobile phase of water, Acetonitrile (MeCN), and a sulfuric acid as a buffer. This analysis method can be detected using UV at 220 nm.

High Performance Liquid Chromatography (HPLC) Method for Analyses of Mixture of Tryptophan and its Catabolites

Condition

Description

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Essential amino acids cannot be made by the body. As a result, they must come from food.
The 9 essential amino acids are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.

Tryptophan is an essential amino acid used in the synthesis of proteins. Tryptamine is structurally similar to tryptophan. Serotonin is a neurotransmitter derived from tryptophan. Dimethyltryptamine and 5-MeO-DMT are psychedelic substances derived from tryptamine. All the compounds have similar structures and can present difficulties to separation in reverse-phase HPLC. They can be separated using the Primesep 100 mixed-mode column in gradient analysis with acetonitrile (ACN) and water mobile phase with ammonium formate (AmFm) buffer, making the method MS-compatible. The amines can also be UV detected at 280nm.

Closely related compounds like amino acids can be separated on an Obelisc R column by various buffers depending on the amount of baseline separation required. By choosing different buffers, the separation between compounds can be adjusted based on application needs, especially those that require low organic concentration in the mobile phase. UV detection at 250nm.

The Separation of Phenylalanine, Glucosamine and Tryptophan demonstrates how the small changes to the organic and/or buffer concentrations in the mobile phase affect the retention of compounds on a mixed-mode column by hydrophobic, ion-exchange and ion-exclusion mechanisms in reverse-phase HPLC chromatography. Evaporative Light Scattering Detector (ELSD) used

Separating basic, acidic and zwitterionic compounds in one run in reverse-phase HPLC can be very challenging. The methods might require the use of ion-pairing reagents and complex gradients that can make MS-compatibility difficult. Obelisc R column which has both positive and negative ion-pairs embedded in the stationary phase allows for fine tuning and separation of a wide range of compounds with different ionic properties. Acids, bases, amino acids and neutral compounds were separated isocratically in one run using a simple MS-compatible mobile phase of acetonitrile (ACN) and water with Ammonium Acetate (AmAc) buffer. Can also be UV detected at 250nm.