Biogenic Amine Analysis Service

What is Biogenic Amine?

Biogenic amines are organic nitrogen compounds that are formed through the decarboxylation of amino acids by enzymatic action in living organisms. They are naturally occurring and play crucial roles in various physiological functions. The term "biogenic" refers to their biological origin, and "amines" denote their structure which contains one or more amine groups. These compounds are prominently found in foods like fish, cheese, and fermented products, where microbial action during storage and processing can lead to their formation.

There are several notable types of biogenic amines, including histamine, tyramine, dopamine, serotonin, and phenylethylamine. Each of these has distinct biological activities. For instance, histamine is involved in local immune responses and acts as a neurotransmitter. Tyramine can influence blood pressure, whereas serotonin is well-known for its role in mood regulation and gastrointestinal function. Dopamine, another key neurotransmitter, is essential for motor control and reward-motivated behavior.

The presence of excessive levels of biogenic amines in food can pose health risks, leading to symptoms such as headaches, hypertension, and allergic reactions when consumed. This is particularly a concern in food safety and quality control, making it important to monitor and regulate their concentrations. For example, histamine poisoning, also known as scombroid poisoning, is a common foodborne illness caused by the consumption of fish with high histamine levels.

Furthermore, biogenic amines are not just a concern in food but are also of great interest in medical and psychological research due to their significant role in the human nervous system. Disruptions in the balance of these amines can be linked to various disorders, such as depression, schizophrenia, and Parkinson's disease. Thus, understanding biogenic amines and their metabolic pathways is crucial for developing targeted treatments for these conditions.

Biogenic Amine Analysis by Creative Proteomics

Creative Proteomics offers comprehensive biogenic amine analysis services to meet the needs of diverse research and industry applications. These analyses are essential for understanding various physiological and pathological processes, as well as for ensuring food safety and quality. The specific project contents provided by Creative Proteomics in their biogenic amine analysis service include the quantification of common biogenic amines such as histamine, tyramine, putrescine, and cadaverine. These compounds are critical markers for monitoring microbial activity, spoilage, and fermentation processes in food products. Additionally, we provide analyses to measure levels of other biogenic amines including serotonin, dopamine, and norepinephrine, which are vital in neurochemistry and behavioral studies.

• Quantitative Analysis: Accurate measurement and quantification of biogenic amines in various sample matrices.
• Qualitative Analysis: Identification and profiling of biogenic amines present in samples using advanced analytical techniques.
• Method Development: Customized method development to optimize analysis for specific sample types and matrices.
• Consultation and Interpretation: Expert consultation and interpretation of results, providing actionable insights for research and quality assurance purposes.

List of Detectable Biogenic Amine

Biogenic Amine Assay Platforms

Creative Proteomics implements advanced Analytical Techniques to ensure the accurate and precise measurement of biogenic amines. Some of the primary analytical methods utilized include High-Performance Liquid Chromatography (HPLC), often coupled with fluorescence detection or mass spectrometry, which offers high sensitivity and specificity. Gas Chromatography-Mass Spectrometry (GC-MS) is employed for separating and identifying volatile biogenic amines due to its excellent resolving power and detection capabilities. For more complex biological matrices, techniques such as Liquid Chromatography-Mass Spectrometry (LC-MS) and Ultra-High-Performance Liquid Chromatography (UHPLC) are utilized to offer enhanced resolution and shorter analysis times.

Sample Requirements of Biogenic Amine Analysis

Deliverables for Biogenic Amine Analysis

Comprehensive Analytical Report: This report includes detailed quantitative results of all detected biogenic amines in the samples. It outlines concentrations measured in appropriate units (e.g., ppm, µg/g) and provides statistical analyses where applicable.

Qualitative Identification: Confirmation of the presence of specific biogenic amines through qualitative analysis methods. This includes chromatographic retention times, mass spectra (where applicable), and comparison against standard references.

Method Validation Package: Detailed documentation validating the analytical methods used, ensuring accuracy, precision, linearity, and detection limits. This package may include calibration curves, method blanks, and spike recovery experiments.

Consultation and Interpretation: Expert interpretation of the results with insights into the significance of detected biogenic amines. Our team provides contextual analysis related to regulatory compliance, product safety, or research implications.

Raw Data Availability: Access to raw data files upon request, facilitating independent verification or additional analyses by clients or regulatory authorities.

Confidentiality and Compliance: Assurance of confidentiality and adherence to regulatory requirements throughout the analysis and reporting process.

Title

Determination of Biogenic Amines in Fish and Fish Products Using Py-Tag Derivatization and LC–MS/MS

Background

Biogenic amines such as histamine, cadaverine, tyramine, and 2-phenylethylamine are important indicators of food quality and safety, particularly in fish and fish products. High levels of these amines can cause food poisoning and allergic reactions. Accurate and reliable methods to quantify these amines are crucial for food safety. This study focuses on developing and optimizing an LC–MS/MS method using Py-Tag derivatization to detect these biogenic amines in various fish and fish products.

Samples

The samples analyzed in this study included various fish and fish products purchased from supermarkets in Kanagawa Prefecture, Japan. These included tuna, dried horse mackerel, dried whole sardines, minced fish, and fish sauce. Additionally, reference and quality control samples with known concentrations of histamine were used to validate the method.

Technical Methods

Optimization of LC–MS/MS Analysis:

• Ionization Mode: Positive ionization electrospray MS/MS was chosen.
• Optimization: Cone voltage and collision energy were optimized using 1000 ng/mL of each biogenic amine derivatized with Py-Tag.
• Column and Mobile Phases: An ACQUITY UPLC CSH C18 column was used, with mobile phases consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B).
• Elution Gradient: The mobile phase gradient ensured good chromatographic separation of the derivatized biogenic amines.

Derivatization Optimization:

• Conditions: The derivatization was conducted with Py-Tag at 50°C.
• Time: The reaction was optimized by testing 15, 30, and 60 minutes. The optimal derivatization time was found to be 15 minutes.

Matrix Effects:

• LC–MS/MS Analysis: Matrix effects were assessed by comparing peak areas of derivatized amines in diluted sample extracts to those in the solvent.
• Derivatization Efficiency: The efficiency was evaluated by comparing peak areas of derivatized amines in sample extracts to those in a solvent, showing negligible impact from sample matrices.

Method Performance:

• Calibration Curve: Excellent linearity with correlation coefficients >0.999.
• Limit of Quantification (LOQ): 2 mg/kg for Him, Tym, and Pea; 10 mg/kg for Cad.
• Recovery Test: Trueness and repeatability were assessed through spiking experiments, with recovery rates between 87% and 104%.

Results

• Biogenic Amines Detection: Him and Tym were found in 35% of samples, Cad in 13%, and Pea in 10%.
• High Concentrations: The highest concentrations of Him (316 mg/kg) and other amines were found in dried whole sardines.
• Method Comparison: The developed method was compared with other LC–MS/MS methods and found to have advantages such as shorter derivatization time and ease of use without needing internal standards.
• Validation: The method's reliability was confirmed through the analysis of reference and quality control samples, showing consistency with known values.

Representative selected reaction monitoring (SRM) chromatograms for each derivatized biogenic amine of blank minced fish sample (top), minced fish sample spiked at LOQ levels (middle), and standard solutions at LOQ levels (bottom). Arrows show analyte of interest.

Reference

1. Shiono, Kouji, et al. "Simple and rapid determination of biogenic amines in fish and fish products by liquid chromatography–tandem mass spectrometry using 2, 4, 6-triethyl-3, 5-dimethyl pyrylium trifluoromethanesulfonate as a derivatization reagent." Journal of Chromatography A 1643 (2021): 462046.