I am working with plant leaves water and ethanolic extract on the attachment and biofilm formation by oral bacteria. From many papers I read, almost all paper does not explain the methods of doing TMS derivatization of plant extract instead they only mention the protocol to prepare an extract from the raw sample and GCMS parameter settings. TMS derivatization is crucial in producing thermally stable and volatile compounds for better performance of GCMS. Can anyone experienced in the GCMS plant extract analysis explain the correct way to do TMS derivatization?
The following open access publication and the references therein will give you a reasonable oversight of the process. There are two approaches; methoxylation followed by TMS derivatisation or you can just do TMS derivatisation. The paper explains the differences. Make sure your solvents and sample are dry and be aware that some metabolites will yield more than one derivative. Good Luck!
Engel B, Suralik P,Marchetti-Deschmann M. Critical considerationsfor trimethylsilyl derivatives of 24 primarymetabolites measured by GC-MS/MS.Sep Sci plus.2020;3:407–418. https://doi.org/10.1002/sscp.202000025
Ong Thanasilan Firstly, try to dry your sample and re suspend if possible in a suitable solvent, like dicloromethane. Add a portion of your TMS reagent (the ratio can depend on manufacturer instructions) to a vial containing your sample. You can heat the mixture at around 75 degrees for about 30 minutes and then allow to cool. You can evaporate the solvent if necessary to remove excess derivatizatation reagent. You can then follow the standard GC protocol. Hope this helps, blessings.
Charles H Hocart I read the excellent paper by Engels you cited. While my relevant experience is very old and not documented, I could add some very likely advantages of the initial methoximation step, though sometimes the methoxamine may be optional:
1) Methoxamine (generally the HCl salt) is nearly always dissolved in pyridine, which is a good solvent for many compounds, including water. Pyridine invariably contains some water because it's terribly hygroscopic. That means the extract doesn't need to be completely dry, and the excess of silylating reagent, dissolved in the pyridine, will take care of the water.
2) If the extract happens to be a solid mass, the pyridine is likely to soften or dissolve it, facilitating the silylation reaction - and releasing any residual water. It can be difficult to get neat silylation reagents to react with lumpy extracts.
3) Depending on the extraction method, hydroxyacids such as 4-hydroxybutyrate and 3,4-dihydroxybutyrate (which we researched) may form the lactones. Some workers mistook these analytical artefacts for metabolites. We never encountered that, presumably because we always used methoximation followed by silylation; the slightly damp pyridine would be expected to ensure hydrolysis.
4) Pyridine, with or without methoxyamine HCl, should ensure well-defined reaction conditions for silylations in the presence of acids, bases or salts.
Finally, when using silylation cocktails, it may be advisable to have a suitable injection liner, and some means of diverting the solvent peak from the ion source. TMS derivatives of amino and carboxylate groups (for example) may be decomposed by traces of water if the silylating reagent is evaporated off and replaced by a solvent.
Oximation is a very efficient strategy for the derivatization of aldehydes and ketones just before the silylation. Oximes are classically prepared by the treatment of an aldehyde or ketone with hydroxylamine hydrochloride and this workflow is necessary to protect highly reactive aldehyde and ketone functional groups. For any omic based research, many compounds could be assumed as a prospective marker candidate and could be bearing an aldehyde or ketone group. Thus, they should be protected well and TMS addition should be versatile to gain high recovery...
İsmail Emir Akyildiz Methoximes tend to be preferred to oximes, partly of the relatively large molecular weight increment of TMS-oximes. I guess this was discussed in the early literature on steroid analysis.
Another disadvantage of oximes is the that the oximes of alpha-ketocarboxylic acids like alpha-ketoglutarate (Krebs cycle) are unstable with respect to decarboxylation.
On an unrelated point, tert-butyldimethylsilyl derivatives have been preferred to TMS for some purposes; it may be worth exploring the steroid literature on this subject. Hindered hydroxyls are not derivatised whereas TMS may give a mixture. Another advantage is that tBDMS derivatives are less likely to be hydrolysed if you want to isolate them before injection. It's a common protecting group in organic synthesis.
To complete the discussion I should have mentioned that, while tBDMS derivatives have longer GC retention times than TMS (which can be inconvenient), the electron ionisation spectra are usually dominated by the M-57 fragment. This was important before we had chemical ionisation, because it simplified the GC-MS profiles and provided better selectivity and detection limits with selected ion detection. The downside is less certainty (fingerprint information) on the identities of the analytes.
See the 1978 edition of Karl Blau & Graham S King, Handbook of Derivatives for Chromatography, Heyden, ISBN 0855012064
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