To date, sample preparation of solid materials requiring ultrasonic extraction has mostly been performed manually. This is in part due to the fact that automation of the sonication step has not previously been available; however, another reason is that the solid particulate can create a suspension in the extraction solvent, which can easily block a syringe, making automated collection of the extract and subsequent injection to the GC or LC unreliable.
In this application note, an automated sample preparation protocol was developed for the ultrasonic extraction of glycosides and phenolic compounds from plant material for a metabolomics study. The implementation of screen filters to prevent blockage of the MPS syringe along with filtration of the extract using a 0.45 μm filter enabled direct injection of the sonicated and filtered samples to an LC-MS system.
For a metabolomics study of glycosides and phenolic compounds in a plant material, 86 samples were prepared using the automated Prep Sequence described above. After preparation, the samples were introduced to the LC-MS system, and data acquisition was performed. Of the 86 samples, 18 were quality control (QC) samples that were used to assess the reproducibility of the sample preparation protocol. Both targeted and untargeted analyses were performed. For targeted analysis, the internal standard and a number of known compounds were selected, and the area reproducibility was calculated; the results were excellent.
It should be noted that for metabolomics studies, the cutoff for area RSD values is 30%. The targeted analysis results obtained from the QC samples gave an area RSD of less than 14% for the low intensity peaks, well within the limit for metabolomics data, and less than 6% for Rutin and Chlorogenic acid. Untargeted analysis was performed on the QC samples by performing molecular feature extraction and plotting the area RSD values against RSD limits. The results from the untargeted analysis were also excellent.
This application note describes the development of an automated ultrasonic extraction protocol for the extraction of glycosides from plant material. In vial screen filters were successfully utilized to prevent blockage of the MPS syringe. Extracts were aspirated from inside the screen filter inserts, and taken through a further automated filtration step based on 4 mm 0.45 μm syringe filters before being injected into an LC/MS system. Following analysis of the quality control samples used in a metabolomics study, it was determined that the results obtained were highly reproducible.
Please review the full study for further details.