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Sep 1, 2010 (Vol. 30, No. 15)

Less Is More for Mass Spec Sample Prep

Goals Set to Increase the Throughput, Cost-Effectiveness, and Overall Analytical Efficiency

  • Analyzing Dried Blood Spots

    Dried blood spots (DBS) preserved on filter paper represent a complex matrix used in drug development and newborn blood screening. DBS are an easy way to collect, transport, and store blood samples for testing without the need for refrigeration.

    Analyzing compounds in the blood sample can be challenging due to matrix effects caused by the complex makeup of blood and by the components of the paper. Joseph Stankovich, an intern in Gary Van Berkel’s lab at Oak Ridge National Laboratory, described an automated, high-throughput technique called liquid extraction surface analysis (LESA) using Advion BioSystems’ TriVersa Nanomate® nano-electrospray ionization (nano-ESI) MS system for analyzing drugs in DBS.

    The success of MS analysis following direct liquid sample extraction from the DBS will depend on the solubility of the analyte and the ionization efficiency, as well as the characteristics of the surface, including the size, shape, topography, and hydrophobicity of the blood spot.

    Several techniques have been used to facilitate liquid extraction of DBS, including coating the surface of the filter paper with silicone, creating wells on the surface (for example, by printing a wax pattern on the paper, followed by heating, causing the wax to form shallow wells), or punching out intact spots and putting the filter paper disks directly in the wells of a microtiter plate where the extraction then takes place.

    Stankovich showed that all three of these methods yielded comparable results in terms of analyte quantification (in this instance of the drug propranolol), down to 10 ng/mL with an internal standard spiked directly into the liquid blood before spotting. Propranolol was quantified down to a level of 20 ng/mL with addition of an internal standard in methanol on top of the spots after the blood had dried—simulating a practical laboratory situation.

    The third technique, punching out the DBS, was the only one that allowed the researchers to apply an internal standard after the initial application of dosed blood and to achieve acceptable accuracy and reproducibility.

    Stankovich and colleagues concluded that LESA is a viable method for automated analysis of DBS, and that several techniques can be used to facilitate sample extraction. LESA enabled quantitative sample analysis in less than two minutes per sample.

  • Emerging from MIST

    Joanna Pols, associate principal scientist at Merck Research Laboratories, presented a study on the development and optimization of an LC-MS method to separate the major metabolites of a drug candidate, as drug developers face the challenges presented by the MIST guidelines.

    In her talk, Pols described the goal of a method-development study as generating a metabolite-rich matrix of 14C-labeled compounds that can be used for LC-MS method development and optimization.

    Pols outlined several objectives of sample processing: removal of co-eluting/interfering endogenous components; quantitative extraction and recovery of the parent drug and its metabolites; spiking of 14C-labeled parent drug into blank matrix to assess the extraction recovery of the parent drug; and concentration of the sample to prepare it for LC-MS.

    A variety of extraction methods can be used, the most common being SPE, solvent extraction with protein precipitation, and liquid-liquid extraction. Direct injection may also be possible depending on the body fluid being analyzed.

    “Sample reconstitution after extraction is an often overlooked, critical step,” said Pols. Following extraction, the supernatants are combined and evaporated to near dryness under vacuum to achieve a final volume of 100–500 µL. Based on the results of stability experiments, Pols and colleagues determined that both the pH and concentration of biological matrices are important factors affecting degradation and that drying extracts under vacuum conditions in some cases is preferable to drying under nitrogen.

    Once the samples are ready for metabolite profiling they can be analyzed using standard MS instruments such as triple quadrupole or Orbitrap (Thermo Fisher Scientific) systems, optimizing the processing conditions for the parent compound. Pols emphasized the value of starting with a robust, well-understood method that can then support all future metabolite-profiling studies throughout the development life cycle of a particular compound.


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