Chromatography

Liquid chromatography tandem mass spectrometry (LC-MS) was carried out with a Shimadzu Prominence HPLC system and SCIEX 4000 mass spectrometer. An aliquot of 20 µl of each sample was injected onto an Luna 3 µm Phenyl-hexyl 200 x 2.0 mm column at 50^oC for gradient separation. Mobile phases were: A, ddH₂O with 0.1% Formic Acid; and B, Acetonitrile with 0.1% Formic Acid. Gradient schedule starts at 10% B and is linearly increased to 100% B at 3 minutes, then held at 100% B until 3.25 minutes, and then returned to 10% B at 3.4 at minutes and re-equilibration until 5 minutes. Time per sample injection was 5 minutes with a flow rate is 400µL/min.

Extraction Protocols:

Extraction protocols for sample processing are dependent on the matrices involved. Please see extraction methods section for more specific instructions.

Derivatization Protocol:

1. Mix 400µL or Samples + 44.4µL OPD (o-phenylenediamine) Prep (100µM OPD, 100µM DPTA, 71 ng/ml 5MQ, 4.5% TCA)
2. Vortex and Centrifuge Mixture briefly
3. Incubate in water bath at 65°C in dark for 1 hour
4. Centrifuge at end and add 200µL to autosampler vial

Method was adapted from the following reference:

References

[1] Assay of methylglyoxal and glyoxal and control of peroxidase interference
Paul J. Thornalley, Naila Rabbani
Biochemical Society TransactionsMar 20, 2014,42(2)504-510; DOI: 10.1042/BST20140009

Resources

This tab might be populated with additional information like PowerPoint slides from presentations at conferences, class presentations, grant associated links, and other components that would be directly tied to the method.