Chromatography Forum

I want to Quantify plant hormones (ABA, JA and SA) by LCMS/MS.In most of the hormone exatraction and quantification protocol isotope labelled standard of the corresponding hormones are used as Internal standard in LCMS. However in my case I don't have isotope labelled Internal standards. Can anybody suggest me internal standards for ABA, JA and SA other than the labelled phytohormones?

In general terms, you should use as I.S. a compound that is as chemically similar to the analyte as possible.

Labelled standards are the most similar, and are followed by a compound from the same chemical class, e.g. -CH3CH2CH2 ester or -CH3 instead of -CH3CH2 ester; another example would be a steroid as I.S. for vitamin D.

Is it possible to carry out plant phytohormone (Jasmonic acid, Abscisic acid, Salicylic acid) Quantification in LCMS/MS without Internal standard?
And if yes how far the quantification is acceptable?

Since labelled internal standard are commercially available for these hormones, if you don't use them, you run the risk that when you publish your work, a reviewer will raise concerns about your method.

There are things you can do to mitigate the concerns.
(1) If you can use chemically similar compounds as internal standards, that may help.
(2) you can make multiple runs of each sample, including unlabelled hormone standard in some runs. If you imagine making two runs, one with added hormone, the other not, then the difference between the two runs is a measure of the signal produced by the amount of hormone you added, in situ in the sample. You are measuring the ionisation efficiency in the real sample. This is a reasonable way to compensate for varying ionisation efficiency in a batch of samples. It is not as good as an internal standard because it cannot compensate for losses during extraction.
(3) For which reason, if you cannot use an internal standard, you should also carry out recovery experiments on your extraction method. This means taking plant material and adding hormone to it at the earliest stage you can in your extraction procedure, and then comparing the measured amount you get back at the end with the amount you added (you do, of course, need to do a parallel extraction without added hormone, and subtract the natural abundance from the total in the other sample, to find only the amount that was added).

Thank you JMB and lmh for your reply.

Can anyone list me some compounds which can be used as Internal standard in LC MS/MS for (Jasmonic acid, Abscisic acid, Salicylic acid) . Although labelled standards are commercially available and they are too expensive so I cannot afford them.

Thank you Imh for the reply.....Can you list me some compounds which can be used as Internal standard in LC MS/MS for (Jasmonic acid, Abscisic acid, Salicylic acid) . Although labelled standards are commercially available and they are too expensive so I cannot afford them.

Since labelled internal standard are commercially available for these hormones, if you don't use them, you run the risk that when you publish your work, a reviewer will raise concerns about your method.

There are things you can do to mitigate the concerns.
(1) If you can use chemically similar compounds as internal standards, that may help.
(2) you can make multiple runs of each sample, including unlabelled hormone standard in some runs. If you imagine making two runs, one with added hormone, the other not, then the difference between the two runs is a measure of the signal produced by the amount of hormone you added, in situ in the sample. You are measuring the ionisation efficiency in the real sample. This is a reasonable way to compensate for varying ionisation efficiency in a batch of samples. It is not as good as an internal standard because it cannot compensate for losses during extraction.
(3) For which reason, if you cannot use an internal standard, you should also carry out recovery experiments on your extraction method. This means taking plant material and adding hormone to it at the earliest stage you can in your extraction procedure, and then comparing the measured amount you get back at the end with the amount you added (you do, of course, need to do a parallel extraction without added hormone, and subtract the natural abundance from the total in the other sample, to find only the amount that was added).

If you cannot use the labeled I.S., then you have to select a very chemically similar compound---consider Acetyl-salicylic acid (aspirin) or similar, but your I.S. has to be chromatographically resolved from the analytes of interest.

AND

Preferably, the I.S. should have a similar MS fragmentation to the I.S. (i.e. losses of similar neutral groups etc.)

Search the Aldrich catalogue and ChemSpider for suitable candidates
(www.chemspider.com; now owned by Royal Society of Chemistry).

JMB

Please suggest me If I choose the Internal standard given below , will they work or will they be compatible.


Analyte ------------------------- Internal standard
Abscisic acid-------------------------Naphthaleneacetic acid
Salicylic Acid------------------------ Naphthaleneacetic acid or 5-fluorosalicylic acid (5-FSA) or 3,4-DHBA
Jasmonic Acid-----------------------???????????????????????????????????????????????????????????????

Please suggest me , If I choose the above Internal standards will they be compatible with the corresponding analytes or not?

And also please suggest me some Internal standard for Jasmonic acid other than Dihydrojasmonic acid or any other labelled form.