Leading the Way in Life Science Technologies

GEN Exclusives

More »

Feature Articles

More »
May 15, 2012 (Vol. 32, No. 10)

Simplifying Complex Biological Matrices

  • Emerging Trend for LC-SPE Sorbents

    Click Image To Enlarge +
    The combination of eVol and MEPS can be used to infuse extracted analytes directly into the MS-ESI source without any additional modifications. [Emily Hilder, ACROSS, University of Tasmania/ SGE Analytical Science]

    “We are starting to see the use of LC-SPE (solid-phase extraction) sorbents to perform separations in the protein and proteomics areas. It is an emerging trend,” comments Rob Freeman, business manager, chromatography, SGE Analytical Science.

    Conventional SPE started with disks and tubes for sample volumes from 50 mL to 1 L, using either gravity or a vacuum to move the solvent through the large sorbent bed. SGE Analytical Science has combined the advantages of SPE with automation by developing a digital syringe, eVol®, with an embedded miniaturized SPE cartridge.

    MEPS™, micro-extraction by packed sorbent, incorporates a cartridge, which holds a few mg of sorbent, into a removable needle syringe. This solid-phase-extraction technique can be used for protein sample preparation manually, just like a standard syringe, or interfaced with gas or liquid chromatography systems through an autosampler. In addition, recently, the combination of eVol and MEPS was used to infuse extracted analytes directly into the MS-ESI (electrospray ionization) source, without any additional modifications.

    The specific sorbents and particle pore sizes (pore size corresponds to surface area), impact the sizes of proteins and peptides that are retained. The typical MEPS pore size is 120 angstroms, which is larger than the conventional SPE pore sizes of 60–80 angstroms.

    The miniaturized format works with small-volume biological samples, 10–1,000 µL, improves efficiencies, and virtually eliminates solvent use and waste.

    The sorbent, a spherical silica particle with an attached polymer such as C4, C8, or C18, separates and extracts through hydrophobicity and retentive interaction. SGE Analytical Science is working on extending MEPS capabilities for protein sample preparation using polymer monoliths in collaboration with Emily Hilder, Ph.D., at the Australian Centre for Research on Separation Science.

  • Still a Long Way to Go

    There is still significant work to be done in the area of protein sample preparation and absolute protein quantification.

    According to Dr. King, “We have to understand what we are really measuring and what we are talking about. Results from a ligand-binding assay are not going to be the same as from a MS with a surrogate peptide workflow, which is not going to give you the same answer as a top-down measurement, looking at the whole intact protein in a MS.”

  • Small Molecules vs. Proteins

    If the focus is on small molecules and not protein-based biologics, the HybridSPE®-Phospholipid can be used to clean up both proteins and phospholipids in just one step.

    “The conventional procedures for small molecule sample preparation from biological samples include protein precipitation, liquid-liquid extraction, and solid-phase extraction. None of these techniques specifically target the removal of phospholipids, which are a well-known cause of matrix effects in LC-MS analysis of biological samples,” explains Dr. Xiaoning Lu, Ph.D., senior scientist, Supelco division of Sigma-Aldrich.

    In conventional SPE the compound of interest is absorbed onto the sorbent and then eluted off. In HybridSPE-Phospholipid the compounds of interest go through the phase.

    In a typical procedure, users load samples on the HybridSPE-Phospholipid device, add organic solvents and mix it with the samples, then pull the sample through the device by vacuum or positive displacement pressure. The precipitated proteins are filtered by a 5 µm PTFE frit and a 0.2 µm filter membrane. Phospholipids attach to the sorbent, a zirconia-coated silica bed. The protein- and phospholipid-free samples are then ready for injection and analysis in the LC-MS.

    HybridSPE-Phospholipid is available in a variety of formats, 96-well plates, cartridges, and microliter pipette tips, for implementation into automated processes. Although unique in phospholipid removal, the HybridSPE-Phospholipid technique is very similar to the standard protein precipitation method and easily incorporated into the laboratory workflow.

  • Sample-Prep Changes for Production

    Click Image To Enlarge +
    The MARS 6 with One Touch Technology has nearly 80 pre-programmed methods for batch processing of similar raw material and finished product samples, CEM reports.

    USP Method 233, Elemental Impurities Procedures, is the proposed laboratory procedure to replace the antiquated USP Method 231, Heavy Metals, for sample preparation and analysis of raw materials and finished products for pharmaceutical drug products, including those from natural sources and rDNA.

    USP Method 233 uses current analytical instrumentation, ICP-OES (inductively coupled plasma—optical emission spectroscopy), ICP-MS and microwave, instead of hot plates, muffle furnaces, and the technician’s eyes for visual determinations. Advances in ICP require cleaner sample to achieve lower detection limits.

    The cost to the pharmaceutical industry for the method changeover is significant: instrument purchases, microwave digestion and ICP method development, determination of which analytes to analyze, training and documentation.

    “Some samples are very simple to digest; others, like raw materials containing biological molecules, can be more challenging. CEM’s goal is to make it as easy as possible for a technician to walk up to the microwave, safely put their vessels together, and add the right amount of sample and acid. Our extensive built-in training aids and pre-programmed methods help achieve this,” explains Jason Keith, product manager, CEM.

    The Discover SP-D digests one sample at a time and can handle, in an automated process, varying samples where different acids and digestions temperatures are needed.

    The MARS 6 with One Touch™ Technology batch processes similar samples and has nearly 80 preprogrammed methods. Sensors count the number and type of vessels to determine the amount of microwave power needed to reach the digestion temperature for the selected method.

Related content

Be sure to take the GEN Poll

Cancer vs. Zika: What Worries You Most?

While Zika continues to garner a lot of news coverage, a Mayo Clinic survey reveals that Americans believe the country’s most significant healthcare challenge is cancer. Compared to other diseases, does the possibility of developing cancer worry you the most?

More »