Protecting our food!

Quantitative analysis of Domoic Acid

Now that we know how dangerous this toxin is, how can we go about preventing another outbreak of Amnesic Shellfish Poisoning? 
Well, commercial seafood is currently monitored regularly for domoic acid, using High Performance Liquid Chromatography (HPLC) with UV detection to identify the toxin. 

What is HPLC? 
High Performance Liquid Chromatography (HPLC) is a method used to separate compounds dissolved in a solution. This is done by injecting the sample into the instrument, which will pass through a separating column that contains a mobile and stationary phase. According to the partitioning behaviour of each component, the components will stay at the stationary phase for different periods of times, eluting at different retention times, and hence separating the components.

1. Select Method
HPLC with UV detection was used to determine the concentration of Domoic acid using absorbance measurement at 242nm. Conditions of the HPLC are as follows:
-column: C18 reverse phase, 5 um, 250mm x 46mm
-temperature: 40 degrees
-flow: 1 ml/min

http://www.jascoinc.com/products/chromatography/hplc-systems/HPLC-Components/HPLC_Detectors/UV-Visible-Detectors


Why was measurement done at 242nm?
There is conjugation of double bonds in the Domoic Acid structure, acting as chromophores(Groups that absorb light in the UV range). As such, Domoic Acid can absorb light in the UV region.
By getting the absorption spectrum of Domoic Acid, the wavelength of maximum absorption was found to be 242nm. Wavelength of maximum absorption should be used in analysis in order to maximize sensitivity and minimize error.

Why Reverse Phase HPLC?
Domoic Acid contains 3 carboxyl groups, and 1 amine group, which makes it a very polar molecule, is only soluble in polar solvents. Reverse phase HPLC is made up of a polar mobile phase, that can dissolve domoic acid. It's non-polar stationary phase can be used to separate the compounds based on their polarity, with the more polar analytes eluted first, and the more non-polar analytes eluted late, as they interact with the non-polar stationary phase.
Acetonitrile was chosen as the solvent for this mobile phase because is it polar and has high UV quality, due to low absorbance typically at short wavelengths. Hence, acetonitrile is suitable for Reverse Phase HPLC using UV detector.



2. Obtain representative sample
Collect seafood and shellfish from the water body suspected of Domoic acid contamination.

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3. Prepare lab sample
For shellfish dissect it from its attached shell by using a clean scapel, and obtain about 100-150g of shellfish flesh. For fish, obtain 100-150g of fish meat and skin by removing all other parts including scales, tails, fins, guts, bones, including heads.

 

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4. Eliminate interferences

To remove dirty contaminants such as sand, salt on the seafood surface, rinse with fresh water and then drain them for 5 minutes to remove excess water. Homogenise the drained seafood in a high-speed blender until they are mixed well.

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5. Define replicate samples
Take three 4g samples of the homogenate and place them into 3 different graduate centrifuge tubes.

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6. Dissolve samples
Add 16ml extraction solvent of methanol:water 1:1 (v/v) to the 4 graduate centrifuge tubes and homogenize them at 10,000 rpm for 3 minutes.Centrifuge for at least 3,000 g for 10 min and filter the supernatant through dry methanol compatible 45um or 2um filter.

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7. Measure property of analyte
Firstly, we will need to obtain a calibration graph. Using standard pure DA solutions, we prepare a series of dilutions (at least 4), using the mobile phase to dilute. We then inject 20ul of each solution into the HPLC-UV instrument with acetonitrile:water, 1:9 (v/v) as the mobile phase, and record their respective areas of absorbance measurement according to their retention times. These areas are then used to plot a calibration graph of known concentrations against peak areas. However, it must also be noted that DA can isomerize to Epi-DA. As such, during calibration and quantification, the sum of both DA and Epi-DA must be taken into consideration.

Next, to measure the concentrations of DA in the 3 samples prepared, 20 ul of each sample is injected. The DA is separated from other components by the HPLC in the column and its absorbance measurement is taken at its wavelength of maximum absorbance, 242nm. The average of the 3 areas obtained can then be used to calculate the concentration of the sample.

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8. Calculate results
Now, we calibrate a graph of the DA and epi-DA, by plotting peak area against the known concentrations of the series of diluted DA standards. In order to be able to obtain accurate results, the coefficient of correlation of the calibration curve needs to be at least 99%.

The concentration of DA in the sample is measured by finding the concentration of DA corresponding to the area of the DA in the calibration graph.

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9. Estimating reliability of results
With regards to the usage of HPLC

• Since HPLC uses either an Autosampler or Rheodyne manual injector, a constant and reproducible volume is injected into the column, improving precision.
• A HPLC has good retention time reproducibility.
• Good sensitivity of domoic acid detection since absorbance measurement is made at wavelength at which domoic acid has maximum absorbance, which gives rise to a low limit of detection, making trace analysis possible.
• However, it is possible for other compounds to have an identical spectra with domoic acid.
  
  

With regards to calculations
We can ensure a high precision for the calibration graph by obtaining a coefficient of correlation of at least 99%. Higher accuracy and precision for the sample measurements can be further achieved by obtaining more sample results so errors can be minimized.

Citations:

1. Definition of high performance liquid chromatography. (n.d.). Retrieved from http://www.chemicool.com/definition/high_performance_liquid_chromatography_hplc.html

2. Eu harmonized standard operating procedure for determination of domoic acid in shellfish and finfish by rp-hplc using uv detection. (2008, 06). Retrieved from http://www.aesan.msps.es/CRLMB/docs/docs/procedimientos/EU-Harmonised-SOP-ASP-HPLC-UV_Version1.pdf

3. Acetonitrile. (n.d.). Retrieved from http://www.chromatography-online.org/topics/acetonitrile.html

More about the toxin, Domoic acid..

Domoic acid is a potent neurotoxin produced by a naturally occuring Harmful Algal Bloom. Specifically, it is made by a microscopic, red-brown, salt-water plant, or diatom calledNitzchia pungens. The toxin produced by these diatoms is concentrated in shellfish such as mussels and causes Amnesic Shellfish Poisoning when the contaminated shellfish are eaten. What makes this toxin more dangerous is the fact that it is not destroyed after cooking, which can cause many unsuspecting consumers to fall ill with Amnesic Shellfish Poisoning..

How dangerous is Domoic Acid?
Domoic acid is a toxin that affects the brain, specifically the  hippocampus, and affects both animals and humans. It may cause severe health implications.

Fig 1- Left: normal California sea lion brain section. Right: a not so normal one that has been affected by domoic acid exposure; notice the shrunken hippocampus in the center of the brain section

In humans..
Domoic Acid causes Amnesic Shellfish Poisoning(ASP).  ASP can be a life-threatening syndrome, and is characterized by both gastrointestinal and neurological disorders.
After consumption of the contaminated shellfish, Gastroenteritis usually develops.

Symptoms include:
-Nausea
-Vomiting
-Abdominal cramps
-Diarrhea.

In severe cases, neurological symptoms also appear, usually within 48 hours of toxic shellfish consumption.

Symptoms include:
-Dizziness
-Headache
-Seizures
-Disorientation
-Short-term memory loss
-Respiratory difficulty
-Coma


How does Domoic Acid work?

To understand this, we first have to introduce a new molecule- Glutamic acid.

Fig 2: Glutamic Acid structure

So what’s so important about this molecule?
Glutamate (the ionized form of glutamic acid) is a neurotransmitter, a molecule used to send a message from one nerve cell to another, and plays an important role in thought, learning, and memory. Binding of glutamate to a glutamate receptor causes the opening of channels allowing embedded on the membrane of the receiving nerve cell, the receptor opens channels in the membrane that allow calcium ions to flow into the cell. The influx of charge causes a voltage to build up across the cell membrane, and the nerve cell fires, passing the signal on to the next nerve cell.

However, too much glutamate acts as an excitotoxin- which kills cells by exciting them too much. This will eventually lead to an excitotoxic cascade, which plays an important role in brain injury and neurodegenerative diseases.

Now, back to Domoic Acid and how it causes damage to the brain.

Domoic acid’s structure is similar to glutamic acid.As a result, nerve cells mistakenly recognize domoic acid as glutamic acid- a fatal error.  Its five-sided ring makes it less flexible than glutamate, which causes it to bind very tightly to glutamate receptors. As a result, the excitatory effect of domoate is 30 to 100 times more powerful than that of glutamate, resulting in an excitotoxic cascade.

What is being done now to prevent another outbreak of Amnesic Shellfish Poisoning?
Luckily for us, commercial shellfish and seafood are now monitored regularly for domoic acid, using HPLC(High-Performance Liquid Chromatography) to identify the toxin. The screening and testing procedures have so far been successful- not a single instance of domoic acid poisoning in humans has been reported since the 1987 outbreak.

Read on to our next post to find out how we can carry out Quantitative Analysis on Domoic Acid!

Citations

1. Egglilng, S. (n.d.). Common names of amino acids. Retrieved from http://dl.clackamas.cc.or.us/ch106-05/common.htm

2. Foley, S. (2009, July 01). Domoic acid-toxipedia. Retrieved from http://toxipedia.org/display/toxipedia/Domoic Acid

3. Senese, F. (2010, February 15). General chemistry online: The poisoned needle. Retrieved from http://antoine.frostburg.edu/chem/senese/101/features/domoic.shtml

4. Human illness associated with hab. (n.d.). Retrieved from http://www.whoi.edu/science/B/redtide/illness/illness.html

5. Potera, C. (2009). Harmful algal blooms: An unexpected deep-sea diver. Environ Health Perspect, 117(6), Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2702427/

 6. The marine mammal centre: Domoic acid toxicity. (n.d.). Retrieved from http://www.marinemammalcenter.org/science/top-research-projects/domoic-acid-toxicity.html

Importance of Analytical Chemistry-What do you do when you’re dealing with an unknown, dangerous toxin?

Toxin strikes in Canada

In 1987, a mysterious syndrome struck in Canada. Over a hundred people became extremely ill within hours after dining on cultured blue mussels in restaurants around Prince Edward Island in Canada.

It quickly became apparent that this was no ordinary outbreak of food poisoning. Vomiting, cramps, diarrhea, and incapacitating headaches were followed by confusion, loss of memory, disorientation, and (in severe cases) seizures and coma. A few exhibited emotional volatility, with uncontrolled crying or aggressiveness. Three elderly victims died.

A tragic symptom of poisoning was the destruction of short term memory in about one quarter of the survivors. The most severely affected lost memories several years old. For twelve of the victims, the loss of short term memory was permanent.

This mysterious syndrome was called Amnesic Shellfish Poisoning. This sort of neurological damage due to food poisoning had never been encountered before. To prevent further injury and loss of life it was imperative that the toxic agent be isolated and identified as quickly as possible. A team of marine biologists and chemists was assembled by Canada’s Department of Fisheries and Oceans (DFO) to work on the problem.

Carrying out Qualitative Analysis on the unknown toxin

As the mussel samples were extremely complex, isolating the toxin was no easy task. Scientists first needed to separate the numerous compounds so the toxin could eventually be isolated. They did this through step by step physical separation methods, where mice were exposed to each fraction of the separation. Fractions found to be toxic were then retained for further analysis. Take a look at this diagram to see what methods they used! 

    

Fig 1: Identifying the toxin

Eventually, after a final separation of the sample by HPLC and a confirmation by high voltage paper electrophoresis, the isolated toxin underwent Mass Spectrometry for identification.

The toxin was found to be… Domoic Acid!

 

Fig. 2- Domoic Acid

So THIS compound was responsible for the mysterious food poisoning incident.

Learn more about this dangerous substance in our next post. Stay tuned!

Citations

1. Senese, F. (2010, February 15). General chemistry online: The poisoned needle. Retrieved from http://antoine.frostburg.edu/chem/senese/101/features/domoic.shtml

2. Habs and biotoxins: Marine biotoxins- domoic acid poisoning. (n.d.). Retrieved from http://www.nwfsc.noaa.gov/hab/habs_toxins/marine_biotoxins/da/index.html