α -tocopherol is the major vitamin E present in green plant tissues, more specifically in the membranes of chloroplasts and thylakoids.
I performed many protocols to detect and quantify α -tocopherol in Arabidopsis thaliana, tomato, tobacco and grape vine extracts using HPLC (C18 reverse-phase column, methanol as eluent, UV detection 292 nm). The problem is that only one protocol has worked and only on Arabidopsis extract !!! (De vasconcelos et al. 2010. Evaluating the potential of chestnut (Castanea sativa Mill.) fruit pericarp and integument as a source of tocopherols, pigments and polyphenols).
Any suggestion??
The answer depends! Usually, in validation of the method the LOQ is determined. As I recall it was about ~0.1 IU but it depends on the amount of mass your injecting on the column and the extraction procedure. We need more information in order to help you!
Hi Mr. Neagle,
I am using BHT (butylated hydroxy toluene) to extract tocopherol from 200 mg ground plant tissues (grinding with nitrogen liquid). 1 ml of extracted solution was injected. (see the reference above De vasconcelos et al. 2010).
Is there any method to determine the limit of quantification LOQ ??? How can I convert IU to mg ? Usually tocopherol is quantified as mass tocopherol per fresh weight of plant material.
What solvent are you using for your extraction medium? You may have to use fluorescence detector (FLD) but that means you have to eliminate Methanol and use Acetonitrile instead (since MeOH is known to quench fluorescence).
I am using BHT dissolved in heptane for extraction but yes, maybe I have to use FLD instead of UV detector. It is more reliable. No?
In which solvent is the sample diluted for injection? Ideally, it should be diluted in the mobile phase, because if it is in a stronger solvent, the signal tends to disappear.
The sample is diluted in BHT that is dissolved in heptane. BHT is used as an antioxidant to prevent the oxidation of tocopherol.
In addition to heptane you should be using a more polar solvent like IPA since you can have Vitamin E Acetate (while not natural but has a reduced solubility in Heptane). Fluorescence is more sensitive but has other precautions that must be taken (like always wearing Nitrile gloves, washing glassware with 0.1 N Nitric Acid...).
Bruce Neagle Thank you very much Mr. Neagle for your valuable answers.
Excuse me, but what about detective column? Do you use appropriate detecition columne. If yes, and if the level of α-tocopherol is low in extracts of these plants, maybe the HPLC method is not good enough, in your situtation I will try with GC-MS. But, firstly try to re-calibrate your HPLC device, with fresh new standard.
Thank you Mr. Srecec for your answer.
Yes, GC-MS is a good choice..
I am using an appropriate column for tocopherol detection: reverse-phase C18 and methanol as eluent, isocratic elution mode. What do you mean by HPLC re-calibration? Normally, I run diluted standard solutions of commercial tocopherol after checking pump efficiency.
Mohammad
What is your expected concentration (do an internet search for other plants)? Your detector (PDA/DAD) may not have enough sensitivity for the concentration in your sample matrix.
Yes, that's what I meant under the term re-calibration of HPLC. So, if doesn't work, you have no other choice than to use GC-MS.
The amount of α-tocopherol is variable depending on plant species . For example "lipstick tree" (plant from the Americas used to make paint and lipstick) contains nearly 1.8 mg/g FW tissue whereas in Oat we can found 5 µg/g FW.
In Arabidopsis thaliana whole plant (14 day-old from in vitro culture), I find nearly 70 µg/g FW. However in other plants as I said I cannot detect it and I do not know the reason!!!
Try a 'spike and recovery' experiment using your standard and extracted sample. This will demonstrate your HPLC is capable of the analysis. In the case were you have a 'high' %Recovery the tocopherol may be below your detection and quantitation limit (LOD and LOQ)(ng?). This may not be surprising since some plants only make tocopherol for their own use which may be very low in concentration. Remember, only a few plants make Vitamin C (Ascorbic Acid) at high levels like lemons that prevent the disease 'scurvy' in humans.
Yes, I agree with Bruce. That's it. The accumulation of tocopherol is definitely pretty questionable in some plant species, and you can find it only in traces. Sometimes the peaks are not vissible with HPLC devices which has the software for 2D chromatogram, such as, for instance HPLC UV-Vis, but only in 3D chromatogram such as chromatogram of HPLC-DAD. That was the reason why I suggested GC-MS.
Bruce Neagle
I will try it but is that means that I must add known amount of tocopherol in my sample tube that contains the extract from plant tissue and run it ?? Then I have claculate the difference between "sample+known amount of tocopherol" and the standard tube that contains only the known amount of tocopherol??
Use your standard to calculate the mass of tocopherol. Then, the calculation is;
% Recovery = 100% x [(sample+spike - sample)/spike]
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