Targeted Approach to Toxicology
Dr. Stone from Agilent addressed the need for pain-management assays to assess and adjust individual medication doses. The assay can also be used for monitoring illicit drug use. Dr. Stone used LC/MS/MS assays to analyze more than 80 compounds, achieving sensitivity of one-tenth the cut-off level for most analytes. The method incorporated dynamic MRM capabilities to increase the number of analytes and decrease the analysis time while maintaining high-quality data.
The method paired a Zorbax Poro Shell 120 column with the Agilent 6430 QqQ Mass Spectrometer. Analytes included benzodiazepines, opiates, amphetamines, analgesics, and other pain-management drugs with their metabolites.
Dr. Stone said that the innovation in the assay is not that it’s ultrafast (although at 6 minutes, it is very fast), but that it is so comprehensive. It can process up to 85 analytes at a time. Testing for that many analytes would tax the resources of a conventional analytic laboratory.
Dr. Stone credited the dynamic MRM technique for the ability to look at so many analytes. With a predetermined window of retention time, the instrument can predict which analytes will come up when, and monitors those analytes specifically.
“It allows the instrument to spend more time looking at each of the MRM transitions, which improves the statistics of the response of each one and gives better quality, more reliable data.”
Bryan Krastins, Ph.D., from the Thermo Fisher Scientific Biomarker Research Initiatives in Mass Spectrometry (BRIMS) Center, presented a poster on the development of a synthetic quality control standard for sample proteolysis. Many protein samples are prepared for mass spectrometry using proteolytic digestion. However, the results of such a digestion can be highly variable, and those results will affect the outcome of the mass spec experiment. Dr. Krastins developed a standard protein that could be used to monitor the quality of proteolytic digestion for mass spectrometry applications.
The synthetic protein contained 42 amino acids containing eight isotopically labeled arginine or lysine residues. The protein produces four peptides upon digestion, which can be used to create calibration curves for SRM limit of quantitation (LOQ) and LLOQ measurement. Additionally, spiking the synthetic protein into a protein sample allows its use as an internal standard for the efficiency, reproducibility, and quality of the digested sample.
According to Dr. Krastins, the standard offers a way of gauging the success of the digestion step. “Even if we get 80 percent digestion, it provides a better understanding to show how far to completion the experiment went. It’s another number you can build into your assay to more accurately quantify peptides and proteins you want to target in your assay, it’s basically a quality control of your enzymatic digestion step.”