Automated Sample Prep
Lisa Calton, Ph.D., technical manager at Waters, presented a poster illustrating the automation of sample preparation for the analysis of plasma metanephrines by UPLC/MS/MS. She found that automated sample preparation improves repeatability, minimizes human error, and can enable traceability of samples with an onboard barcode reader.
Dr. Calton used Oasis µElution SPE technology in 96-well plate format with the Tecan Freedom EVO 100 liquid-handling system to quantify plasma metanephrines. These assays are typically performed by HPLC using electrochemical detection—a method that involves labor-intensive pretreatment with long analytical run times.
Plasma metanephrine is a marker for pheochromocytoma, a disease whose primary symptom is high blood pressure, with reported diagnostic specificity of 92% and sensitivity of 98%. Screening every patient who presents with high blood pressure using the labor-intensive HPLC metanephrine assay is not feasible. However, Dr. Calton and her colleagues demonstrated the ability to process a large number of samples with minimal operator involvement.
There are a number of polar basic compounds that are important in clinical, pharmaceutical, and forensic applications that may be quantified with some method optimization. “The use of LC/MS/MS in clinical laboratories has increased rapidly over the last five years. The technology offers improved selectivity and sensitivity compared to traditional immunoassay methods widely used in clinical laboratories.”
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.”
AB Sciex has been investigating mass spec quantitation for some time. Christie Hunter, Ph.D., presented her research results using the TripleTOF 5600 System to perform MRM-like analysis. The goal of the experiment was to improve specificity by eliminating noise and interferences in an analysis of proteins in a complex matrix such as human plasma.
“What’s unique about this instrument relative to other mass spec instruments is that you can collect data at high resolution in full scan MS/MS mode at very high acquisition rates. This opens the door to a number of new workflows such as this high-resolution MRM-like workflow.”
The TripleTOF can mimic MRM/SRM data acquisition by performing looped high-resolution MS/MS experiments, then fragment ion XICs are generated by post acquisition processing to produce MRM-like data. The data was first extracted with a wide fragment ion-extraction window, simulating MRM specificity. Then, the data extraction was performed on the same data with a very tight window, generating very high resolution data.
“We were able to completely remove some very bad matrix interferences by leveraging the higher resolution,” Dr. Hunter said. “We improved the LLOQ by a factor of 40 in the peptides cases where we could remove significant interference using resolution.”