This protocol can be used to perform an extraction from brain tumors in or next to a server on using solid phase microextraction fibers. This technique enables the extraction of small molecules directly from resected tumors and provides an opportunity for rapid intraoperative diagnostics by coupling extraction devices directly to analytical instrumentation. To prepare the solid phase microextraction device, trim the coding of each solid phase microextraction device probe with mixed mode or C18 coatings to an appropriate length according to the size of the tumor.
And soak the probes and a 50 50 methanol water solution for at least one hour before the sample collection. This protocol focuses on brain tumors but the strategy can be implemented for the diagnosis of many different cancers. The technology is fully portable, so there is no need for any additional devices.
As soon as possible after tumor removal immerse the probe fibers with LCMS grade water for five seconds before inserting the fibers as far as possible into the brain tumor tissue sample, to ensure that the entire extraction phase is located inside the tumor. Leave the probes within the tissue for precisely 30 minutes to eliminate sources of error due to the presence of artifacts from sources other than the sample tumor, place conditioned probe fibers on the table for the same period of time. During the extraction, label HPLC vials to be used for probe storage after the extraction.
At the end of the extraction, immerse the fibers in LCMS grade water for three seconds to remove any blood or cell debris and insert each probe through the bottom of the septa of a single HPLC vial cap to immobilize the fibers. Then cap the vials with the immobilized fibers and place the vials into an appropriate transportation container. Upon laboratory arrival, immediately placed the vials into a minus 30 or minus 80 degrees Celsius freezer for no more than three or five years respectively.
Once all of the samples from a single experiment have been collected, place the vials containing the mixed mode fibers at room temperature. and label two milliliter vials for desorption place a glass insert into each vial and add 300 microliters of freshly prepared 80 20 acetonitrile water solution to each insert. Fully immersed the coding of each probe in individual vials of the desorption solvent and agitate the vials for 120 minutes at 1, 200 revolutions per minute on a vortex.
Once the desorption is complete, remove the caps from the vials and set aside 10 microliter aliquots from each file containing extract from the tumor to prepare a quality control sample. Then close the vials with new caps and place the vials into the four degrees Celsius auto sampler of a liquid chromatography high resolution mass spectrometer. To prepare the samples for Lipidomic Analysis place the vials containing the C 18 fibers at room temperature and label two milliliter HPLC vials for the disruption place silanized glass inserts into the vials and add 200 microliters of freshly prepared 50 50 isopropanol methanol solution to each insert.
Fully immerse each probe coating into the solvent and agitate the samples for 50 minutes at 1, 200 revolutions per minute on a vortex. At the end of the time, stop the desorption by removing the caps and then set aside a 10 microliter aliquots from each file containing extract from the tumor to prepare the quality control sample and close the vials with new caps. Then, place the vials into the four degrees Celsius auto sampler of the liquid chromatography high resolution mass spectrometer.
The reproducibility of the instrumental analysis was determined to be very good based on the tight clustering of the quality control samples on the principal component analysis plot. These lipidomics data for samples collected from patients with gliomas and meningiomas highlight the ability of the tumor samples to be distinguished according to their histological origin and malignancy. As illustrated with these metabolomics data, gliomas can be further divided based on their degree of malignancy and or according to the presence or absence of mutations of interest.
Statistical analysis also permits the selection of specific compounds within the studied groups. It is essential to carefully control the extraction time and to maintain reproducible conditions for all of the samples. Alternatively, samples can be dissolved from the fiber directly to Ms without chromatographic separation allowing the targeted analysis of specific markers and shortening the entire procedure to several minutes.
