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I am a beginner chemistry student studying anticoagulant rodenticides. The literature shows that high-performance liquid chromatography (HPLC) with fluorescence or UV light is the most commonly used method for the detection of residues in tissue samples. I want to understand on a basic, conceptual level why this particular analytical method is the preferred method.

I understand that HPLC can separate complex organic compounds and enable detection at extremely low concentrations, but I do not know whether there are other analytical methods that can also do this.

Is this particular analytical method more effective in detecting the presence of residues from anticoagulant rodenticide in tissue samples? If so, why? Is the answer related to the chemical properties of the compounds in rodenticides (hydroxycoumarins and indandiones)?

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  • $\begingroup$ Without a doubt there are other techniques that could be used. But chemistry employs the KISS principle. A HPLC setup is much cheaper to buy and easier to maintain than say a GC/MS. // If the compound is fluorescence you get a better S/N ratio than measuring absorption, and also relatively few compounds are fluorescent so there is an additional specificity factor. $\endgroup$ – MaxW Apr 23 at 4:08
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UV(/VIS) spectroscopy is the most common HPLC detection method, it's quite cheap and powerful but you can only detect substances which absorb light in the range you are using for detection. You also don't get any real structural information, most of the time identification is only possible using reference material. But most HPLC systems will be equipped with such a detector.

Fluorescence detectors can be used for fluorescent molecules and usually you got a much lower limit of detection. Like for UV/VIS you don't get structural information, but identification works quite well, since you can selectively only detect molecules with the used excitation and emission wavelengths.

Other detectors would be for example refractive index detector, which only really work well if HPLC is run isokratic or MS, which gives you structural information.

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  • $\begingroup$ In this case the technique is also very adequate since the aromatic structures of hydroxycoumarins and indandiones will all absorb in the UV spectrum. I guess at least some of them might be fluorescent too. With a good collection of standards and a column with high resolution power there's no need for costly detectors such as MS. $\endgroup$ – Variax Nov 14 '16 at 17:06
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Is this particular analytical method more effective in detecting the presence of residues from anticoagulant rodenticide in tissue samples?

Yes, it is :)

  1. You might want more than just detection: quantitative determination.
  2. You might want to determine a one or more compounds that are only available in traces.

Extraction of the relevant compounds from tissue samples will remove the biological background and allows for aggregation/enrichment prior to HPLC.

HPLC will allow to separate structurally similar rodenticides.

Fluorescence is extremely sensitive and will help to quantify trace amounts.


Update

In Multicomponent Determination of 4-Hydroxycoumarin Anticoagulant Rodenticides in Blood Serum by Liquid Chromatography with Fluorescence Detection, published in J. Anal. Toxicol., 1991, 15, 126-129 (DOI, free PDF), this has been demonstrated for the separation and fluorescence detection ($\lambda_\mathrm{ex} = 318~\mathrm{nm}, \lambda_\mathrm{em} = 390~\mathrm{nm}$) for a mixture of

  • warfarin
  • coumatetralyl
  • difenacoum
  • brodifacoum and
  • bromadiolone
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Is this particular analytical method more effective in detecting the presence of residues from anticoagulant rodenticide in tissue samples?

It is not by any means more effective then other methods. The reason for popularity of HPLC is it's fairly universal nature, being adaptable to a variety of anayltical problems. This makes it very popular and widely available in analytical labs. Other separation techniques like capillary electrophoresis would also work for those analytes (also fluorescence and absorbance detector).

Is the answer related to the chemical properties of the compounds in rodenticides (hydroxycoumarins and indandiones)?

Some of those compunds show fluorescnce, which can be detected with high sensitivity but that doesnt make HPLC the obvious choice. Other separation techniques are possible too.

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