Gary A. Schultz

Surrogate matrix and surrogate analyte approaches for definitive quantitation of endogenous biomolecules

BACKGROUND Quantitation of biomarkers by LC-MS/MS is complicated by the presence of endogenous analytes. This challenge is most commonly overcome by calibration using an authentic standard spiked into a surrogate matrix devoid of the target analyte. A second approach involves use of a stable-isotope-labeled standard as a surrogate analyte to allow calibration in the actual biological matrix. For both methods, parallelism between calibration standards and the target analyte in biological matrix must be demonstrated in order to ensure accurate quantitation. RESULTS In this communication, the surrogate matrix and surrogate analyte approaches are compared for the analysis of five amino acids in human plasma: alanine, valine, methionine, leucine and isoleucine. In addition, methodology based on standard addition is introduced, which enables a robust examination of parallelism in both surrogate analyte and surrogate matrix methods prior to formal validation. Results from additional assays are presented to introduce the standard-addition methodology and to highlight the strengths and weaknesses of each approach. CONCLUSION For the analysis of amino acids in human plasma, comparable precision and accuracy were obtained by the surrogate matrix and surrogate analyte methods. Both assays were well within tolerances prescribed by regulatory guidance for validation of xenobiotic assays. When stable-isotope-labeled standards are readily available, the surrogate analyte approach allows for facile method development. By comparison, the surrogate matrix method requires greater up-front method development; however, this deficit is offset by the long-term advantage of simplified sample analysis.

Development of a nano-electrospray mass spectrometry source for nanoscale liquid chromatography and sheathless capillary electrophoresis

A Fisons Quattro I electrospray ionization (ESI) source has been modified to produce stable electrospray ion currents at flow rates as low as 50 nL/min. The original counter electrode and skimmer cone lens of the Fisons ESI source have been replaced with a spherical cone lens. This improved source provides a greater range of x,y,z positioning of a stainless steel tip resulting in a stable ion signal for flow rates of 50 nL/min to 2 μL/min. A tapered stainless steel electrospray tip (50 μm i.d.) was evaluated for mass spectrometry using nano-liquid chromatography (50 μm i.d., flow rate = 120 nL/min) and sheathless capillary electrophoresis. The analysis of a nonionic surfactant, octylphenol ethoxylate, was accomplished with both nanoscale separation techniques.

A novel chromatographic method allows on-line reanalysis of the proteome.

Liquid chromatography combined with electrospray ionization is widely used for direct analysis of polar and labile molecules by LCMS. The on-line coupling in LCMS is a major strength but also causes a principal limitation that each eluting analyte has to be analyzed immediately and is not available for detailed interrogation after the LCMS run. Here we developed a new chromatographic strategy, which removes this limitation. After column separation the flow is split, one portion is analyzed directly, and the other is diverted to a capture capillary. After the direct LCMS run, the flow is switched, and the portion stored in the capillary is analyzed (“replay run”). We describe a setup consisting of an analytical column, a splitting valve, and a focusing column, which performs at full sensitivity and undiminished chromatographic resolution. We demonstrate three principal advantages of this system: nearly continuous MS utilization, duplicate analysis without requirement for additional sample, and targeting of important but undersampled features in the replay run.

Quantification of gemcitabine incorporation into human DNA by LC/MS/MS as a surrogate measure for target engagement.

In this study, we report a method for direct determination of gemcitabine incorporation into human DNA. Gemcitabine (dFdC), a structural analog of the nucleoside deoxycytidine (dC), derives its primary antitumor activity through interruption of DNA synthesis. Unlike other surrogate measures, DNA incorporation provides a mechanistic end point useful for dose optimization. DNA samples (ca. 25 microg) were hydrolyzed using a two-step enzymatic procedure to release dFdC which was subsequently quantified by LC-ESI-MS/MS using stable isotope labeled internal standards and selected reaction monitoring (SRM). dFdC was quantitated and reported relative to deoxyguanosine (dG) since dG is the complementary base for both dFdC and dC. The SRM channel for dG was detuned using collision energy as the attenuating parameter in order to accommodate the difference in relative abundance for these two analytes (>104) and enable simultaneous quantification from the same injection. The assay was shown to be independent of the amount of DNA analyzed. The method was validated for clinical use using a 3 day procedure assessing precision, accuracy, stability, selectivity, and robustness. The validated ranges for dFdC and dG were 5-7500 pg/mL and 0.1-150 microg/mL, respectively. Results are presented which confirm that the ratio of dFdC to dG in DNA isolated from tumor cells incubated with dFdC increases with increased exposure to the drug and that dFdC can also be quantified from DNA extracted from blood.

Large-scale implementation of sequential protein and peptide immunoaffinity enrichment LC/nanoLC-MS/MS for human β-nerve growth factor

BACKGROUND A previously described immunoaffinity (IA)-LC-MS/MS assay for human β-nerve growth factor (β-NGF) was implemented to support large-scale sample testing for multiple clinical trials. Methodology & results: The procedure was modified to increase throughput by simultaneous preparation of two 96-well plates and LC duty-cycle reduction. Robustness of the LC method and nano-ESI was ensured during large-scale assay execution by closely monitoring and, if needed, replacing system components prior to failure. Following validation, the assay was used to analyze approximately 19,000 samples from multiple clinical studies over several years. CONCLUSION Routine implementation of the β-NGF IA-LC-MS/MS assay supported drug development programs. This optimized assay format now serves as a template for other clinical protein biomarker assays.

8th GCC: Consolidated feedback to US FDA on the 2013 Draft FDA Guidance on Bioanalytical Method Validation

The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.

Integrated microchip-based nanoelectrospray device for high-throughput mass spectrometry

The emerging field of microfluidics may provide for the rapid, automated analysis of samples. Here we describe the microfabrication and operation of a nanoelectrospray device formed from the planar surface of a monolithic silicon substrate for electrospray mass spectrometry sample analysis at low nanoliter per minute flow rates. To generate a useful electrospray from a microchip, a high aspect ratio nozzle structure of small dimensions is required. Deep reactive ion etching technologies allow these high aspect ratio structures to be fabricated in parallel and are widely available for the etching of silicon.

Fundamentals of LC-MS/MS for Regulated Bioanalysis

The development of liquid chromatography-mass spectrometry technologies in the 1970s and 1980s had fundamentally changed the development of new drugs and the pharmacokinetic data quality generated during bioanalysis. A brief review of these historical developments of the interfacing of liquid chromatography with mass spectrometry transitions into the best use practices for those new to the field of bioanalysis. The chapter highlights the benefits of implementing ultra high-pressure liquid chromatography to provide improved sensitivity, selectivity, and analysis speed combined with triple quadrupole mass spectrometry . The use of appropriate solvents and solvent modifiers combined with the stationary phase appropriate for the analyte’s structure is discussed and provided in an easy to read and understand manner. The introduction of the eluate to the mass spectrometer ion source is described for the two most commonly used atmospheric pressure ionization techniques in use today. The process of electrospray ionization and atmospheric pressure chemical ionization are used to create charged molecules which are directed into the vacuum region of the mass spectrometer. A step-by-step guide is provided on the optimization of the mass spectrometer settings which provides selective and sensitive selected reaction monitoring signals that can be used to quantify compounds. Best practices are discussed for system maintenance, calibration, and tuning and appropriate documentation needed to support regulatory requirements.