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

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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.

HPLC Method for Asparagine, L-Cysteine, Cysteine, Proline, Valine, D-Valine, Methionine, L-Methionine, Isoleucine, D-Isoleucine, DL-Isoleucine, D-Leucine, Phenylalanine, Tryptophan, Histidine, Arginine, Amino Acids, Leucine, L-Threonine on Primesep 100 by SIELC Technologies

High Performance Liquid Chromatography (HPLC) Method for Analysis of Asparagine, L-Cysteine, Cysteine, Proline, Valine, D-Valine, Methionine, L-Methionine, Isoleucine, D-Isoleucine, DL-Isoleucine, D-Leucine, Phenylalanine, Tryptophan, Histidine, Arginine, Amino Acids, Leucine, L-Threonine.

Amino acids are the building blocks of proteins. Based on their dietary requirement, they are classified into essential and non-essential amino acids. Essential amino acids cannot be synthesized by the human body in sufficient quantities and must be obtained from the diet. Non-essential amino acids, on the other hand, can be synthesized by the body and are not dependent on dietary intake.

It’s worth noting that while these amino acids are considered “non-essential” for adults under normal circumstances because the body can synthesize them, there are situations where some may become “conditionally essential.” This means that under certain conditions like illness, stress, or trauma, the body might not produce them in sufficient quantities, and dietary intake becomes necessary. Arginine, for instance, is considered conditionally essential, especially during periods of rapid growth, illness, or trauma.

Amino acids can be retained, separeted and analyzed on a Primesep 100 mixed-mode stationary phase column using an isocratic analytical method with a simple mobile phase of water, Acetonitrile (MeCN), and a sulfuric acid (H2SO4) as a buffer. This analysis method can be detected in the UV regime at 200 nm.

HPLC Method for Valine, D-Valine, Methionine, L-Methionine, Isoleucine, D-Isoleucine, DL-Isoleucine, L-Isoleucine, D-Leucine, Phenylalanine, Histidine, L-Histidine hydrochloride monohydrate, Tryptophan, Amino Acids, L-Threonine on Primesep 100 by SIELC Technologies

High Performance Liquid Chromatography (HPLC) Method for Analysis of Valine, D-Valine, Methionine, L-Methionine, Isoleucine, D-Isoleucine, DL-Isoleucine, L-Isoleucine, D-Leucine, Phenylalanine, Histidine, L-Histidine hydrochloride monohydrate, Tryptophan, Amino Acids, L-Threonine.

L-Threonine is an essential amino acid with the chemical formula C₄H₉NO₃.  It cannot be produced within the body and must be obtained through consuming it. It’s found in many protein-rich foods, including but not limited to eggs, meat, dairy, legumes, and seeds. It is necessary in the body as a building block of protein like collagen and elastin. The two proteins are crucial for skin, hair, and connective issue.

L-Valine is an essential amino acid with the chemical formula C₅H₁₁NO₂. It cannot be produced within the body and must be obtained through consuming it. It’s found in foods including but not limited to nuts, legumes, whole grains, and seeds. It is especially beneficial for athletes.  It is important for muscle repair,  growth, and energy regulation.

DL-Methionine is an essential amino acid with the chemical formula C₅H₁₁NO₂S.  It cannot be produced within the body and must be obtained through consuming it. It is required for protein synthesis. It also helps build and repair tissue including, but not limited to, skin, hair, muscles, and nails. In a veterinary context, DL-Methionine is used to address bladder issues in dogs.

L-Isoleucine is an essential amino acid with the chemical formula C₆H₁₃NO₂.  It cannot be produced within the body and must be obtained through consuming it. It is a building block of protein that are essential for muscle growth, repair, and other bodily functions.  It also helps regulate blood sugar levels and supports the immune system. It is found in foods like meat, fish, eggs, dairy, beans, lentils, nuts, and seeds.

L-Leucine is an essential amino acid with the chemical formula C₆H₁₃NO₂. It cannot be produced within the body and must be obtained through consuming it. It stimulates production of protein that are essential for muscle building and repair. Meats are the easiest way to get L-Leucine in significant amounts.

L-Phenylalanine is an essential amino acid with the chemical formula C₆H₉NO₂. It cannot be produced within the body and must be obtained through consuming it. It is typically found in high protein foods such as meat, eggs, and fish. Outside of being important for creation of protein, it is also used in treatment for skin disorders and depression.

L-Histidine is an essential amino acid with the chemical formula C₉H₁₁N₃O₂. It cannot be produced within the body and must be obtained through consuming it. It s fundamental for repair of damaged tissue, growth of muscles, and making of blood cells. Outside of protein, it also has the unique property of being able to act as a buffer to help maintain stable pH levels in the body. Sources of it include meat, fish, dairy products, beans, and nuts.

L-Tryptophan is an essential amino acid with the chemical formula C₁₁H₁₂N₂O₂. It cannot be produced within the body and must be obtained through consuming it. Like the other essential proteins, it is a building block for protein and muscle tissue, but it is also converted in the body into serotonin, which affects mood. L-Tryptophan is also used in treatments for severe PMS symptoms, depression, and insomnia. It is naturally found in red meat, poultry eggs, and dairy.

Valine, D-Valine, Methionine, L-Methionine, Isoleucine, D-Isoleucine, DL-Isoleucine, L-Isoleucine, D-Leucine, Phenylalanine, Histidine, L-Histidine hydrochloride monohydrate, Tryptophan, Amino Acids, L-Threonine can be retained and analyzed using the Primesep 100 stationary phase column. The analysis utilizes an isocratic method with a simple mobile phase consisting of water and acetonitrile (MeCN) with a sulfuric acid buffer. Detection is performed using UV.

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.