HPLC Salicylic Acid

Basic questions from students; resources for projects and reports.

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Hi,
I am trying to quantify SA in infected leaf tissue. However, as I am new to this type of work I am a little confused as to the use of an internal standard. I am spiking all my samples with a known amount of pure SA so that I can eliminate variations between injected samples. So, I have two questions:
1) How do I calculate the percentage recovery of my SA from my samples to test how efficient my extraction procedure is?
2) If all my samples (plant extracts) are spiked with pure SA, how is this showing how much SA is produced by the plant following infection - Am I not just measuring the SA that was spiked in?
If anyone can help that would be great as I am very confused!
Thanks in advance.
1) Usually run a sham extraction using only your standard without actual leaf tissue. Run the standard before sham then review the difference.

2) You cannot use SA as an IST for SA. You must use another compund that can be distinguished from SA.
superstarjetjaguar wrote:
1) Usually run a sham extraction using only your standard without actual leaf tissue. Run the standard before sham then review the difference.


I assume this means a blank extraction, haven't heard that term before.


superstarjetjaguar wrote:
2) You cannot use SA as an IST for SA. You must use another compound that can be distinguished from SA.


Correct. But why is there need to use an internal standard (don't you have an autosampler?) ?

Salicylic acid analysis is pretty straightforward using external standard (we rarely use internal standards, with modern HPLC equipment).

Do NOT use acetylsalicylic acid as internal standard, that will contain some salicylic acid already.
Thank you for your reply but what do you mean by an external standard?
An external standard is one that is prepared outside of your sample. For instance, you make up your SA standards at known concentrations in water and run them with your method. You generate your concentration-response curve with all of the associated statistics. Now, you prepare a sample and run that. You take the response for SA in your sample and use the calibration data to calculate the concentration in the sample.

When you're extracting an analyte from a sample like plant tissue, it's good to have an internal standard that is chemically similar to the analyte but easily separable from the analyte. You spike the internal standard into the plant matter (not always a straightforward process) and then extract it as you would. This type of procedure assumes that even if you don't get 100% extraction, you get a proportional amount of each. If you know what that concentration of your internal standard gives as far as response goes in an external standard, if it's comparable in your extract you're probably doing pretty well.

To evaluate your extraction for SA you might do serial extractions on the same sample. You'll get the most out in the first extraction but there will likely be some left behind. More will come in the second extraction but the instrument response will be less. Keep extracting until you are getting very little SA in the extract compared to the first extract. You can get an idea of your recovery that way. If you get 90% of the material in the first shot, then you can probably quit. You might need to do 2 to get 95%. Now you're starting to reach the error in the analysis. To me, when you get that close, there's no point in doing more extractions.

The other thing you can do is use what is called the "method of standard addition". You can extract your tissue sample with a solvent that contains a known amount of SA. It needs to be more than what's expected from your sample. You extract the sample with just water and also the same mass of sample with the water+SA and run them both. The response factor is:

RF = (Ri - Ro)/Ci

where Ri is the response for SA in the SA+water+sample extract, Ro is the response from the sample+water extract, and Ci is the amount of SA you added in the SA+sample+water extract. Then:

Co = Ro/RF

where Co is the concentration of SA in your sample. The drawback here is you have to do it twice (spiked and unspiked).

More than one way to skin the cat! Good luck.
rb6banjo wrote:
An external standard .......Good luck.


rb6banjo - thanks for helping him out. I was going to respond "go back to college, learn something this time"
Now that the issue about IS is solved, a little practical comment: Free salicylic acid is usually present in very low amounts in plants (in the ng/g fresh weight range). This implies that few detectors are able to detect and quantify it. LC-MS/MS or possibly GC-MS after derivatization are the methods of choice and in both cases, the use of an internal standard, preferably labelled, would be highly recommended.
In line with Gaetan Glauser's comment, indeed, vast majority of plants have rather LOW salicylic acid content.

When I was a massspec facility manager with an educational institution, I had couple biologists (sorry) coming up asking for a quick measurement of SA at levels at what turned out to be low ng/g. They were not happy to hear it would not be a two hour or two day for that matter, project...

Plain vanilla HPLC (i.e., LC-UV), given the presence of a multitude UV-absorbing compounds, might not be the tool of choice here. Possible avenues to pursue? Attach some fluorescent label or MS (or MS/MS) based detection perhaps?

I'll be happy to share any wisdom I might have, let me know.
Alexei Gapeev
Millis Scientific, Inc.
gapeev@millisscientific.com
Tel. 877-844-2635
You can buy isotopically labelled salicylate (deuterated) which works very well as an internal standard.
I've used an Agilent 1946 single quad for salicylate measurements in plant samples; you don't need anything particularly sophisticated.

Even with an internal standard, personally I think the original poster is right to check the success of their extraction; it's embarrassing realising that you're trying to assay only 1% of the starting material, not to mention the huge errors you'll meet if you do so. You can do it either by checking the internal standard peak in samples versus a neat injection of internal standard, or if you wish to check recovery without internal standard, you can do a proper recovery experiment. Although a blank extraction with known spike of analyte isn't a bad idea, it's not a complete demonstration of a good extraction: it doesn't test whether your analyte binds to some gunk in the sample and gets centrifuged out, for example. I always recommend taking some material, dividing it into several batches, and spiking some batches with a known amount of analyte. Then extract them all, and look to see whether the difference between spiked and unspiked batches matches the expected size of the spike. The earlier in the extraction process that you can add the spike, the more sources of error you will be detecting.
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