Comparison of the quantification of a therapeutic protein using nominal and accurate mass MS/MS.

BACKGROUND: The quantification of proteins and peptides in in vivo samples is a critical part of supporting the drug development process for biotherapeutics. LC-MS/MS using tandem quadrupole mass spectrometers is well established as the technology of choice for the quantification of small-molecule drugs and their metabolites in biological fluid. The application of accurate mass MS for quantification in a DMPK environment has attracted considerable interest in recent years. MATERIALS & METHODS: In this article we describe and compare the application of LC-high-resolution MS and LC-selected reaction monitoring (SRM) for the quantification of a therapeutics proteins. RESULTS: The accurate mass instrumentation showed acceptable linearity and sensitivity to quantify the protein therapeutic to the level of 10 ng/ml. The accurate mass instrument was operated in accurate mass SRM using high resolution (SRM-HR), the assay was demonstrated to be linear over three orders of magnitude. By narrowing the mass window from 100 mDa to 40 mDa and then to 20 mDa the assay specificity was significantly improved, hence increasing the S/N and improving the assay sensitivity. CONCLUSION: The high-resolution instrument was demonstrated to be reproducible over the course of the assay. The accurate mass method sensitivity was determined to be within one order of magnitude of that obtained with a tandem quadrupole MS/MS assay.

Rapid analysis of dried blood spot samples with sub-2-µm LC-MS/MS.

The use of dried blood and dried plasma spots for storage and transportation of samples derived from clinical trials holds the promise to reduce cost, simplify storage and shipping as well as reducing animal usage. From the bioanalysts' point of view, these dried-paper samples add an extra layer of complexity to the analysis introducing extra matrix effects from the paper itself and sometimes from antiviral treatments applied to the card. In this article we demonstrate the use of the sub-2-µm particle LC-MS/MS for the bioanalysis of samples derived from a dried blood spot. The higher resolution provided by these small-particle separations allowed for greater resolution of the analyte from the endogenous components in blood samples and from the card-treatment chemicals. The method-development process was enhanced by the use of MS, which could simultaneously acquire full scan and multiple reaction monitoring data, allowing resolution from metabolites and endogenous matrix components. The use of this approach produced sensitivity levels in the 50-100 pg/ml range and analysis times in the 1-2 min range, which was five-times more sensitive and three-times faster than HPLC. This throughput and sensitivity makes this approach ideal for the analysis of preclinical and clinical studies derived from dried blood spots.

A novel LC-MS approach for the detection of metabolites in DMPK studies.

BACKGROUND: The profiling and quantification of drug metabolites in discovery and development bioanalysis studies is playing an increasingly important role in early candidate selection. Using a conventional tandem quadrupole mass spectrometer this activity normally requires several analytical runs to acquire the necessary analytical data. RESULTS: In this article we present the use of a new tandem quadrupole mass spectrometer equipped with a novel collision cell design, which allows the rapid switching between multiple reaction monitoring and full-scan MS mode. This approach allowed for the collection of multiple reaction monitoring data and full-scan data with no loss in sensitivity, with analysis times in the 1-2 min range. CONCLUSION: A modified approach of using the multiple reaction monitoring data to trigger the acquisition of full scan MS/MS data is described, where the data is collected on the trailing edge of the LC-MS peak, thus improving data quality and throughput.

Development of a high sensitivity bioanalytical method for alprazolam using ultra-performance liquid chromatography/tandem mass spectrometry.

A rapid, specific, assay was developed for the benzodiapine alprazolam in rat plasma using sub-2 µm particle liquid chromatography (LC) and tandem quadrupole mass spectrometry (MS/MS). The limit of quantification using protein precipitation was determined to 10 pg/mL, whereas the limit of quantification using solid-phase extraction (SPE) was determined to be 1.0 pg/mL. The assay was optimized for throughput and resolution of the analyte of interest from the hydroxy metabolite. During the method development process the plasma matrix signal was monitored, for lipids and other endogenous metabolites, to maximize signal response and minimize ion suppression. This was achieved by using a tandem quadrupole mass spectrometer equipped with a novel collision cell design which allowed for the simultaneous collection of full scan MS and multiple reaction monitoring (MRM) data. The lipid profile from the SPE process was significantly less than obtained with the protein precipitation approach.