Dear researchers
I want to analysis a microbial derived crude extract and find the dominant bioactive metabolite within. As I don’t know anything about the bio-compound composition and likely provide many fractions. Hence, it would be crucial for me to get an obvious and clean chromatogram which shows dominant peaks individually. If I carry out LC-Mass analysis is it satisfactory? How can I enhance the detection? The crude extract is a polar compound and soluble in organic solvents.
Dear Sir. Concerning your issue about the best chromatography method for detection of a dominant metabolite in unknown crude extract. Unknown compounds in polar fractions of Arabidopsis thaliana crude leaf extracts were identified on the basis of calculations of elemental compositions obtained from gas chromatography/low-resolution quadrupole mass spectrometric data. Plant metabolites were methoximated and silylated prior to analysis. All known peaks were used as internal references to construct polynomial recalibration curves of from raw mass spectrometric data. Mass accuracies of 0.005 ( 0.003 amu and isotope ratio errors of 0.5 ( 0.3% (A + 1/A), respectively, 0.3 ( 0.2% (A + 2/A), could be achieved. Both masses and isotope ratios were combined when the elemental compositions of unknown peaks were calculated. After calculation, compound identities were elucidated by searching metabolic databases, interpreting spectra, and, finally, by comparison with reference compounds. Sum formulas of more than 70 peaks were determined throughout single GC/ MS chromatograms. Exact masses were confirmed by high-resolution mass spectrometric data. More than 15 uncommon plant metabolites were identified, some of which are novel in Arabidopsis, such as tartronate semialdehyde, citramalic acid, allothreonine, or glycolic amide. I think the following below links may help you in your analysis:
https://pdfs.semanticscholar.org/5089/f552caaa5f5d53974ba66f8691617be88b09.pdf
https://www.omicsonline.org/open-access/methods-of-analysis-extraction-separation-identification-and-quantification-of-carotenoids-from-natural-products-2157-7625-1000193.php?aid=77160
https://pubs.acs.org/doi/abs/10.1021/ac991142i
Thanks
LC-MS is a very efficient method to determine the major components of a crude, make sure your sample is 0.5 mg/ml or less when injecting into LC-MS. avoiding concentrated sample is crucial. What i would do is an LC-MS using a slow gradient over 30 minutes or so. I believe running an HPLC-DAD using the same gradient and the same column as a complementary to the LC-MS is a good idea too.
I will advise to run an LC-MS, using HPLC-PDA detector will be great. You may prepare 1mg/ml of your crude extract and inject not more than 5 ul, slow gradient condition, water-acetonitrile, over 45 minutes. Start with 10% acetonitrile for 5 min, ramp to 40% acetonitrile in 5 min, keep for 5 min, ramp to 60% acetonitrile in 5 min, keep it for 5 min, ramp to 80 acetonitrile for 5 min, keep for 5 min, ramp to 100% acetonitrile for 5 min, keep for 5 min. From this first trial, should the separation not be good, you will know how to adjust your separation method for a better separation. Thereafter, with the peak areas or intensities obtained, you will be able to calculate the relative quantitation of the different compounds detected.
Hello Sheida Heidarian
I think GC-MS with library data is the best option for identified un-knowns any crude plant sample. TLC only show the number of compounds in highly percentage but GC-Ms will give you the all compounds library with structure.
hope understand the answer.
I think you have more than one choice you can do GC-MS, HPLC or LCMS to take an idea about the compounds that you have in the crude extract
GC-MS coupled with HPLC can give an idea of major components in the crude extract.
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