Bedford VA

Bedford VA has established protocols for profiling redox-active compounds at low ng/mL plasma quantities, below limits-of-detection usually achieved by LC-QQQ analyses. The laboratory employs methods to screen for amines and polyamine, aromatic amines including the tyrosine, tryptophan and purine pathways(Shurubor et al., 2005), sulfur amino acids such as SAM and SAH, DNA and RNA markers of oxidative damage(Bogdanov et al., 2003), small peptides and fat soluble vitamins (Acworth et al., 2008). Briefly, after thawing at 0°C, 125 ul blood plasma aliquots are extracted with 500 ul degassed acetonitrile (w/ 0.4% acetic acid) at -20°C, centrifuged at 21,000x g for 20 min at 4°C. The supernatant is freeze dried and reconstituted in the running buffer for HPLC separations via counter ion-reverse phase columns and quantified using an ESA multiarray electrochemistry detector. Platform performance is monitored with tissue specific pools that have been tracked over the past six years. Study specific quality controls are used by pooling an aliquot of each sample (‘reference design’ study) to assess data precision and for time normalization (stretching) across the entire data sets. QC measurements are randomized and followed by authentic standard mixtures of 80 metabolites for establishing quantification reference points. Chromatograms are background corrected to eliminate base line drifts. Retention time precisions are ±15 s across all chromatograms of 110 min duration. Data files are aligned against a single reference sample using a two-step stretching protocol under control of the ESA software vs CEAS 512. For all identified metabolites, reference calibration curves are used for quantification reports in ng/ml and estimated for unknowns by the total coulombs per peak assuming a molecular weight of 200 and a two-electron charge transfer. Results are reported as digital maps and exported as csv. Results reflect the self-similarity of personal plasma profiles (Figure 4) in drug response phenotyping over weeks of treatment, indicating the usefulness of n=1 clinical trials (using the patient as self-control before and after treatments).

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