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
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.
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.
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.
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.
https://encrypted-tbn3.google.com/images?q=tbn:ANd9GcTYIPwT2ENYLGuGIdpye-_oNdwBijdXA9W84Z4HAnyHShdGRN3F
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.
http://ars.els-cdn.com/content/image/1-s2.0-S0166445X05001505-gr2.gif
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.
http://ars.els-cdn.com/content/image/1-s2.0-S1568988307001473-gr1.jpg
9. Estimating reliability of results
With regards to the usage of HPLC
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.
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.
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.
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.
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.
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.html2. 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
