Universal Sample Preparation Unlocking Multimodal Molecular Tissue Imaging.

A new tissue sample embedding and processing method is presented that provides downstream compatibility with numerous different histological, molecular biology, and analytical techniques. The methodology is based on the low temperature embedding of fresh frozen specimens into a hydrogel matrix composed of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) and sectioning using a cryomicrotome. The hydrogel was expected not to interfere with standard tissue characterization methods, histologically or analytically. We assessed the compatibility of this protocol with various mass spectrometric imaging methods including matrix-assisted laser desorption ionization (MALDI), desorption electrospray ionization (DESI) and secondary ion mass spectrometry (SIMS). We also demonstrated the suitability of the universal protocol for extraction based molecular biology techniques such as rt-PCR. The integration of multiple analytical modalities through this universal sample preparation protocol offers the ability to study tissues at a systems biology level and directly linking results to tissue morphology and cellular phenotype.

Application of novel solid phase extraction-NMR protocols for metabolic profiling of human urine.

Metabolite identification and annotation procedures are necessary for the discovery of biomarkers indicative of phenotypes or disease states, but these processes can be bottlenecked by the sheer complexity of biofluids containing thousands of different compounds. Here we describe low-cost novel SPE-NMR protocols utilising different cartridges and conditions, on both natural and artificial urine mixtures, which produce unique retention profiles useful for metabolic profiling. We find that different SPE methods applied to biofluids such as urine can be used to selectively retain metabolites based on compound taxonomy or other key functional groups, reducing peak overlap through concentration and fractionation of unknowns and hence promising greater control over the metabolite annotation/identification process.

Analysis of wastewater samples by direct combination of thin-film microextraction and desorption electrospray ionization mass spectrometry.

An analysis method for aqueous samples by the direct combination of C18/SCX mixed mode thin-film microextraction (TFME) and desorption electrospray ionization mass spectrometry (DESI-MS) was developed. Both techniques make analytical workflow simpler and faster, hence the combination of the two techniques enables considerably shorter analysis time compared to the traditional liquid chromatography mass spectrometry (LC-MS) approach. The method was characterized using carbamazepine and triclosan as typical examples for pharmaceuticals and personal care product (PPCP) components which draw increasing attention as wastewater-derived environmental contaminants. Both model compounds were successfully detected in real wastewater samples and their concentrations determined using external calibration with isotope labeled standards. Effects of temperature, agitation, sample volume, and exposure time were investigated in the case of spiked aqueous samples. Results were compared to those of parallel HPLC-MS determinations and good agreement was found through a three orders of magnitude wide concentration range. Serious matrix effects were observed in treated wastewater, but lower limits of detection were still found to be in the low ng L(-1) range. Using an Orbitrap mass spectrometer, the technique was found to be ideal for screening purposes and led to the detection of various different PPCP components in wastewater treatment plant effluents, including beta-blockers, nonsteroidal anti-inflammatory drugs, and UV filters.

Analysis of biological fluids by direct combination of solid phase extraction and desorption electrospray ionization mass spectrometry.

A novel, solid phase extraction (SPE)-based sample preparation method was developed for desorption electrospray ionization (DESI) mass spectrometry. Conventional SPE sample preparation was followed by a custom elution procedure. The eluate was evaporated from the closing frit of the cartridge using a gas jet. Thus the analyte was concentrated on the surface of the frit, which is ideal for DESI analysis. Application of the above SPE protocol allowed the concentration of the analyte content of up to 1 L liquid sample into a 1 mm diameter circular spot. The sample preparation procedure can improve the overall sensitivity of the method by up to 6 orders of magnitude if the sample volume is sufficient. The device has been tested using aqueous solutions of Rhodamine 116; the limit of detection was comparable to the LOD of electrospray analysis. Methodology was tested for drug monitoring applications in human serum. Levels of Cyclosporine A were determined using a 0.1 mL serum sample. Dynamic range of the method exceeded 3 orders of magnitude; the detection limit was below the therapeutic serum concentration of the drug.