In the analysis of plant hormones in alcoholic extracts by UPLC-MSMS, SPE columns are usually employed as a cleaning step. However, this step is also presented as a source of loss of target metabolites and some publications even mention the use of these columns. I wonder whether working with filtrations of 0.22 microns and injections up to 5 microliters really will compromise the life of the UPLC C18 column. Is there a possibility to omit this step? Whereas the impairment occurs in the UPLC column, can it be concluded that the cost of the SPE to avoid this burden will actually be lower than the cost of replacing the column after injections?
Hi Henrique !. As I know.. SPE cartridges are used to "concentrate" the sample more than "cleaning". Of course, many people (including myself) use SPE to separate molecules with different polarity index, i.e using increasing gradient steps in order to elute different compounds in different fractions. But the main use of SPE must be to concentrate the sample. If you use filtrations and normal injections you do not have to worry about column integrity. You only must be careful with: Column pressure limits and the pH of the solvents.
I mean, you can avoid SPE step, but if you are using this step a a concentration step, then it will be important for further determinations.
Best wishes !!
Probably you can use a refillable column and reduce the cost if you destroy it and even washing the column is not helping?
To concentrate the extract you can sensitively use a vacuum evaporater before injecting.
Thank you for your answers. Leandro, I know that SPE cartridges are used primarily to "concentrate" the sample than "cleaning". But as the tissue samples are rich in pigments (chlorophyll, anthocyanins, carotenoids), the use of SPE was more with the emphasis on the separation of these substances to avoid that they can dirty the structures of MS (cone, hexapolo, quadrupole, etc. .). Considering that my concern with dirt column and MS are unnecessary, I can use nitrogen gas to concentrate the samples, reducing the costs of that stage. What is your opinion? Christian, I have tested evaporater vacuum, but occurs oxidation and reduction of target metabolites.
As I understand you right, during the evaporation of your solution the targeted metabolites got degraded?
Do you know the target molecules or are they completly new?
If you know them, you should estimate if they get reduced OR oxidiced and then adjust the whole procedure. For example working under nitrogen or with some protecting agents. If you do not know them, you have the laboriuos way to etablish a new protocol in front of you.
We have done exactly this with several different analytes and matrices. Assuming that you have sufficient sensitivity to avoid the preconcentration step and you have a guard column installed, then we have seen absolutely no degradation of the analytical column. You do have to change out your guard column and cap filter more frequently, but they're a lot cheaper than SPE cartridges and your time.
Did you have recovery data of your target compounds and it retained well on C-18? For UPLC, the cleanliness of the sample is very important so I still recommend any cleanup procedure. If your target compound size is much different from major matrices, sephadex cleanup is a good recovery method.
From my own experience a SPE cleanup step is not really necessary for phytohormone analysis by UPLC-MSMS.
As for system stability/column degradation: I frequently run crude methanolic extracts of plant tissue and only use 0.2u filters as the final sample preparation step. I did not have problems with column degradation or system contamination. I use a switch valve to transfer only the range of the chromatographic run that contains the hormones into the MS, this increases the ion source cleaning intervals substantially.
We achieve the required sensitivity for phytohormone concentration by evaporative sample concentration (i do not observe degradation during the concentration step) and MRM conditions in the MS
Thank you for the answers, which were extremely important to my decision. I just want to know what is the brand/model of the rotary evaporator that Rayko is using, considering that already employs protocol analysis of hormones routinely?
I'm currently using a Labconco CentriVap system, but have used other brands before. They all worked fine, just make sure to use a gentle conditions (moderate temperature) to prevent sample loss.
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