Biologics Bioanalalysis Compendium

GUIDE TO INNOVATION
Biologics Bioanalysis


Biologics Bioanalysis – Guide to Innovation
Pharmaceutical companies have leveraged advancements in basic science perhaps more than any other industry. With the advent of whole genome sequencing, sophisticated analysis of metabolic pathways, and exponential improvements in computer processing, R&D organizations have expanded their drug portfolio focus on small molecules to encompass a new class of drugs — biotherapeutic compounds and biomarkers.
Helping customers by listening to their ideas, participating
in discussions, and creating cutting-edge solutions to
research challenges is top priority at AB SCIEX. The following compendium includes key solutions for peptide and protein bioanalysis — and, more importantly, describes in detail work done by, and in collaboration with, our customers. Your success is our success, and the AB SCIEX team will partner with you
to overcome the emerging challenges of bioanalysis, now and into the future.
Joe Fox
Senior Director – Pharmaceutical Business
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RUO-MKT-02-1486-A
Therapeutic Peptide Bioanalysis
The need for sensitive and selective quanti cation methods
Advances in the Bioanalysis of Protein and Peptide Therapeutics through 6-13 Innovations in Mass Spectrometry
Technology Drives High Performance in Biomolecular Mass Spectrometry 14-15 Achieving Low-Flow Sensitivities for Peptide Quantitation 16-19
Using Micro ow Rates on the QTRAP® 6500 System
A Sub-picogram Quanti cation Method for Desmopressin in Plasma 20-25 Using the AB SCIEX Triple QuadTM 6500 System
High-Sensitivity Quanti cation of the Triptorelin Decapeptide 26-29 Using the QTRAP® 6500 System
High-resolution accurate mass 30-55
High-Resolution Time-of-Flight for High-Quality Quantitative Analysis 30-31
Ultrasensitive Quantitation of Exenatide Using Micro ow Liquid 32-37 Chromatography Systems and High-Resolution Mass Spectrometry
Investigating Biological Variation of Liver Enzymes in Human Hepatocytes 38-43 Quanti cation of Large Oligonucleotides using High Resolution 44-49
MS/MS on the TripleTOF® 5600 System
Increasing LC/MS Assay Robustness through Increased Speci city 50-55 Using High Resolution MRM-like Analysis
Ultimate sensitivity
6-29 TTTContents


56-61
62-66
68-71
72-75
76-77 78-81
82-85 86-89
90-104
90-95 96-104
56-89
Orthogonal selectivity tools
Application of Differential Ion Mobility Mass Spectrometry to Peptide Quantitation
Improving Intact Peptide Quantitation with Differential Mobility Separation and Mass Spectrometry (DMS-MS)
Differential Mobility Separation Mass Spectrometry for Quantitation of Large Peptides in Biological Matrices
UGT Family of Enzymes: Quanti cation of Tryptic Peptides using SelexIONM Technology on the QTRAP® 6500 System
MRM3 Quantitation for Highest Selectivity in Complex Matrices
Improved Selectivity for the Low-Level Quanti cation of the Therapeutic Peptide Exenatide in Human Plasma
Quanti cation of Prostate Speci c Antigen (PSA) in Nondepleted Human Serum Using MRM3 Analysis
UGT Family of Enzymes: Quanti cation of Tryptic Peptides
Software tools
DiscoveryQuantM Software: Signature Peptide MRM Optimization Made Easy for Therapeutic Protein and Peptide Quanti cation
MultiQuantM Software 3.0: Peptide Bioanalysis for the Regulated Bioanalytical Laboratory
For Research Use Only. Not for use in diagnostic procedures.


6
Advances in the Bioanalysis of Protein and
Peptide Therapeutics through Innovations in
Mass Spectrometry
Overview of peptide and protein quantitation applications on the AB SCIEX QTRAP® System and the TripleTOF® System
Laura Baker1, Suma Ramagiri2
1Contract Technical Writer at AB Sciex, Pittsburgh, PA, 2AB SCIEX, Concord, Canada
Introduction
The importance of biotherapeutics as a class of drugs has increased signi cantly over recent years due to their enormous potential to treat a wide array of human diseases ranging from autoimmune and in ammatory diseases to cancer, cardiovascular diseases, and rare genetic disorders. These highly promising therapeutic agents, including very small peptide chains, such as insulin, up to much larger proteins, such as antibodies and novel Fc-like fusion proteins, are extremely attractive as drug candidates because of their low toxicity and high speci city, and these compounds continue to ll the pre-clinical and clinical pipelines of many pharmaceutical companies.
The rapid growth of biotherapeutics is a good indicator of its success, with the global market valued at around US$199.7 billion in 2013 and projected to grow by 13.5% through 2020. The number of clinically approved protein and peptide therapies has jumped to over 170 products with 350 antibody-based therapies currently awaiting clinical trials, making biotherapeutics the fastest growing class of drugs in the last decade. With increasing industry interest and investment and rising demand from the medical community for these unique, targeted therapies, there
is a growing requirement to develop high-throughput analytical techniques to expand biotherapeutic product lines.
To overcome regulatory hurdles and advance to clinical trials, biopharmaceutical drug development and discovery requires metabolic monitoring of a candidate drug, a process which necessitates accurate quantitation during pharmacokinetic (PK), toxicokinetic (TK), bioequivalence, and clinical drug monitoring studies—all of which are conducted in a complex biological matrices (blood, plasma, or urine). With this rapid growth in biotherapies comes increased demand for an analytical platform that is exible, robust, and is easily integrated into pre-existing drug development work ows. Widely used for small molecule drug development, liquid chromatography-tandem mass spectrometry (LC/MS/MS) has recently made a larger impact on bioanalysis applications due to recent technological developments in analyte detection. Presented here, we demonstrate how key mass spectrometry technologies from AB SCIEX can coalesce
into straightforward, accurate, extremely sensitive, and, most importantly, high throughput quantitative solutions. Already considered as the preferred choice for quantitation in other areas of bioanalytical quantitation such as proteomics, anti-doping, forensics, and clinical chemistry, LC/MS/MS is poised to replace and outperform other techniques for biotherapeutic analysis.
The current standard conventions for protein and peptide quantitation are based on ligand-binding assays (LBAs), such as the enzyme-linked immunosorbent assay (ELISA), or on UV
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Eksigent MicroLC and UHPLC System QTRAP® LC/MS/MS System TripleTOF® LC/MS/MS System DiscoveryQuantTM Software
MultiQuantTM Software
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of quantitative assays due to the narrow extraction widths
and high resolution TOF data. Lastly, Section 4 investigates the software tools available for robust peptide quantitation work ows that give researchers intuitive tools to automate the complex, multi-step calculations for peak area quantitation.
Each section and experiment featured in this resource includes an overview of the key challenges, bene ts, and features
of the bioanalytical technique presented. In this way, the technique of mass spectrometry can be put into context with other bioanalytical tools and help provide insight into its many advantages. LC/MS/MS analysis offers many attractive features for supporting biopharmaceutical drug development; however, establishing LC/MS/MS in the biopharmaceutical work ow
has been slow in spite of its dominance in the small-molecule laboratory. Widely accepted and easily validated, the LBA technique remains a popular method for protein and peptide bioanalysis due to its relatively lower investment in infrastructure and ease of implementation into the high-throughput environment. Yet, even LBA methods have their drawbacks,
and straightforward LC/MS/MS alternatives are sought that can support the operational challenges of accelerating the further development of biotherapies.
Key challenges of peptide bioanalysis
To understand why the pharmaceutical industry has been hesitant to fully embrace LC/MS/MS strategies for peptide quantitation, the complexities and challenges of the work ow must be fully appreciated. (For a summary of excellent reviews on LC/MS/MS protein and peptide quantitation, please see Table 1.) For both proteins and peptide quantitation, standard calibration curves are used to calculate concentration values for unknowns in biological samples; in addition the amassed data must be stringent enough to meet the rigorous benchmarks prescribed by the USFDA.
For therapeutic peptides, proteolysis is omitted, and the intact peptide can be directly quantitated by MS/MS after relatively limited sample preparation (Figure 1). There is appreciably much more complexity when evaluating larger molecular weight biotherapeutics (>10 kDa), which are not always suitable in their entirety for direct MS/MS analysis. Therefore, bioanalysis of larger proteins and antibodies is based on quantitation of a small portion of the protein, typically a tryptically digested signature peptide with a m/z ratio that is unique from all other peptides in the digest mixture. When coupled with a stable isotope-labeled (SIL) internal standard, the response ratio of the signature peptide to the SIL internal standard reveals a concentration representative
of the intact protein. To build this multifaceted process into the framework of regulated bioanalysis is extremely challenging in practice, which makes it easy to comprehend why LC/MS/MS quantitation of biopharmaceuticals has been slow to
gain acceptance in the GLP laboratory.
For Research Use Only. Not for use in diagnostic procedures.
Figure 1. Peptide and protein bioanalytical work ow strategies. Protein quantitation typically involves a tryptic digestion step, which is omitted during intact peptide bioanalysis, thereby simplifying the process.
detection of individual peptides using high pressure liquid chromatography (HPLC) separations. LBAs rely on immunoaf nity detection of a unique epitope on the protein or peptide
of interest, and the high speci city of the antibody-based interactions can track an analyte at high sensitivity, although
the dynamic range is narrowed to just one or two orders of magnitude. Because production of unique antibodies is lengthy, assay development can often be time-consuming and expensive; in addition, LBA results are often plagued by interferences and high background from antibody cross-reactivity. UV detection and quantitation of peptides is commonly used for peptide mapping, and this analytical method can be useful after extensive sample preparation and cleanup. UV detection with HPLC also does not require the expense and time commitment of antibody production, but the applicability of this method narrows as the complexity of the sample matrix increases.
Herein, we present an extensive resource on the quantitation of peptides using AB SCIEX mass spectrometry instruments, revealing how sensitive and selective detection can be achieved even in
the presence of high background noise. To meet bioanalytical quantitative standards and assay validation parameters, peptide bioanalysis must be sensitive enough to meet the standard benchmarks for excellency in accuracy and precision. In Section
1, we explore the highest level of sensitivity by evaluating experiments conducted on the AB SCIEX QTRAP® 6500 System and the AB SCIEX Triple QuadM 6500 System. Due to the inherent sample complexities, bioanalysis is often negatively impacted by high background noise and interfering peaks. Section 2 illustrates how realizing superb analyte selectivity—even in biological samples with numerous, highly abundant, endogenous proteins— is driven by innovative tools such as multiple reaction monitoring cubed (MRM3) work ow and SelexIONM Differential Ion Separation Technology. Advances in high resolution mass spectrometry are detailed in Section 3, which highlights targeted work ows on the TripleTOF® 5600+ System that extend the sensitivity and selectivity
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Title
Article Highlights
Citation
“Analysis of biopharmaceutical proteins in biological matrices by LC/MS/MS I. Sample preparation”
• Sample-preparation aspects for quantifying biopharmaceutical proteins in body-derived uids by LC/MS/MS
• Enrichment at the peptide level after proteolytic digestion
• Chemical derivatization of peptides for enhancing ionization ef ciency
• Automation of the entire analytical procedure for routine applications in pharmacokinetic and clinical studies
Bischoff R, Bronsema KJ, van de Merbel, NC. Trends in Analytical Chemistry. 2013; 48: 41-51.
“Analysis of biopharmaceutical proteins in biological matrices
by LC/MS/MS II. LC/MS/MS analysis”
• Overview of selected reaction monitoring (SRM) strategies for quantifying peptides in biological matrices
• Selection of signature peptides and internal standards
• Selectivity improvements using MS3 and differential mobility spectrometry (DMS)
• Quantitative LC/MS analysis with low-resolution and high-resolution MS
• Data-independent acquisition (DIA) for collection of all data in a single analysis
Hopfgartner G, Lesur A, Varesio E.
Trends in Analytical Chemistry. 2013; 48: 52-60.
“Bioanalytical LC/MS/MS of protein-based biopharmaceuticals”
• Overview of topics relating to the bioanalysis of biopharmaceutical proteins in biological matrices
• Compares alternative quantitative methodology, such as ligand binding assays (LBAs), to mass-spectrometry-based platforms
• Review of practical aspects of the seven “critical factors” for protein sample preparation
• Special focus on the quantitation of monoclonal antibodies in serum and plasma
• Advances in selectivity, including high-resolution mass spectrometry
van den Broek I, Niessen WMA, van Dongen WD, Journal of Chromatography B. 2013; 929: 161-179.
“Liquid chromatography coupled with tandem mass spectrometry for the bioanalysis of proteins in drug development: Practical considerations in assay development and validation”
• Approaches for overcoming operational challenges due to complex sample preparation
• Development and validation of a fast, simple, and reliable LC/MS/MS peptide quantitation method that ts into current pharmaceutical work ows
• Recommendations for validating quantitative methods based on surrogate peptides
Liu G, Ji QC, Dodge R, Sun H, Shuster D, Zhao Q, Arnold M. Journal of Chromatography A. 2013; 1284:155-162.
*These review articles were reprinted with permission in the rst 30 copies of this resource.
Table 1. Selected citations for further reading on protein and peptide LC/MS/MS methodologies
Evaluation of LC/MS/MS bioanalysis reveals that the major challenges for accurate and precise quantitation lie primarily
in the realm of sample preparation, which includes:
1) the lengthy and extensive work ows for producing signature peptides and 2) the diminishing accuracy of quantitative measurements in highly complex biological samples due to background interferences. Because the multi-step reduction/ alkylation/digestion process generates a more complex mixture than the starting sample, bioanalysis of low-level therapeutic
TripleTOF vs QTRAP for Protein/Peptide Bioanalysis
Figure 2. Comparison of mass spectrometric platforms for peptide quantitation.
peptides can be extremely challenging. Achieving LLOQs in
the low ng/mL range is highly dependent at this time on the optimization of sample preparation steps.9 The numerous competing background peptides are a major consideration in sample preparation, which typically requires enrichment and semi- puri cation of the analyte that introduce additional complexity
to the work ow. Of concern from a regulatory perspective is
the potential for variable peptide release during digestion of the target protein, and if digestion conditions are not well-controlled
TripleTOF® Work ows
• Versatile work ow for simultaneous qual and quant in drug discovery and development
• High resolution accurate mass work ows – SWATHTM Acquisition
• Characterization and comparability of biosimilar in research and development
• Bioanalysis and biotransformation work ows for PK/PD studies
• Retrospective data analysis and robust performance
QTRAP® Work ows
• When sensitivity is utmost importance – low bioavailability and high clearance
• IonDriveTM Technology for low LOQs and wider linear dynamic range
• Absolute quantitation of protein/peptide therapeutics
• GLP/non GLP bioanalysis in phase I and above
• High throughput and robust performance
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or compensated for, then irregular signature peptide release can have a lasting impact on the overall data quality.7 To overcome these drawbacks, strategies such as condensing sample prep steps and digestion optimization can lead to more straightforward method development with wider regulatory appeal. And to that end, as advances in technology deliver exceedingly more sensitive and selective mass spectrometry work ows for direct quantitation in the sub-picomolar range, sample prep protocols can be further streamlined and simpli ed, relying less on intricate sample enrichment and baseline reduction protocols, which will help propel this versatile and reliable MS methodology rmly into the domain of regulated biotherapeutic quantitation.
Summarized below are some of the current challenges of LC/MS/MS peptide quantitation:
• Limited quantitation range – Poor MS/MS sensitivity combined with often poor selectivity can compromise the desired lower limits of quantitation (LLOQ).
• Impaired sensitivity in complex matrices – Very low-level peptide detection (sub-pg/mL) can be suppressed by high background and competing ions in biological samples. The best, previously reported LOQ is 100 pg/mL, which is insuf cient for extended-release pharmacokinetic studies.
• Low speci city – Complex biological matrices hamper data resolution and require sophisticated sample preparation and/or advanced instrumentation.
• Co-eluting, multiply charged interferences limit accurate quanti cation and also peak integration at LOQ levels.
• Isobaric interferences will limit selectivity and speci city of the assay and cause issues for accurate identi cation during bioanalytical method development process.
• Reduced recovery, low sensitivity – The adsorptive properties and/or polarity of peptides can compromise recovery, and interferences from
biological matrices can negatively impact sensitivity and selectivity. • Challenging physicochemical proprieties of peptides such as non-
speci c binding, poor solubility, and complex charge state envelope
result can be problematic for the design of quantitation protocols. • Limited MRM selectivity – MRM approaches and ef cient UHPLC
separations may not provide adequate signal-to-noise ratios at LLOQ
due to isobaric interferences or high baseline noise.
• Systematic measurement errors – Especially for ultra-low-level
quantitation, errors in measurement have a signi cant effect on data
accuracy and precision.
• Poorly fragmenting peptides – Cyclic fragments often fragment poorly
resulting in few product ions for MRM analysis.
Key bene ts of the mass spectrometry based peptide quantitation work ow
While LBAs may be primarily used in industry at this time, LC/MS/MS techniques provide many potential bene ts that are grounded in the direct evaluation of the analyte’s chemical nature, rather than indirect signals stemming from an immunological interaction. Quantitative data obtained by LC/MS/MS methodology correlates well with LBA-derived concentrations.7 Unlike LBA assays that require speci c antibodies for each
analyte, mass spectrometry platforms have universal applicability, providing one technique for a large diversity of analytes. All
types of proteins and peptides can be evaluated by LC/MS/MS without exception, and a wide diversity of other biomolecules such as lipids and carbohydrates can also be identi ed, providing researchers with a exible platform for identifying non-protein impurities. LBAs are generally more limited in their applicability because of auto-antibody cross-reactivity and the lack of commercial kits for every protein of interest.11 Non-speci c
For Research Use Only. Not for use in diagnostic procedures.
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Multiple Orthogonal Selectivity Tools for Protein/Peptide Bioanalysis
Additional Selectivity
Sample Extraction HPLC Separation
Figure 3. Mass Spectrometry based additional orthogonal sample clean up tools such as SelexION (differential mobility spectrometry), MRM3 scan function on QTRAP LC/MS/MS System and Scheduled MRM for increase in duty cycle
QTRAP® 6500 System
SelexIONM MRM3 Scheduled MRMM
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10
binding and molecular class limitations are surpassed with LC/MS/MS, which can even quantify highly homologous isoforms that are impossible to distinguish using immunoaf nity techniques. Low-level biomolecule quantitation is analogous
to nding a needle in a haystack; yet LC/MS/MS is able to deliver quantitative data with excellent accuracy and precision over a wide linear dynamic range, often over 3 to 4 orders
of magnitude.7 Additionally, in contrast to the repeated
expense and time-consuming nature of antibody production, LC/MS/MS methods can be developed and validated within a relatively shorter amount of time for multiple targets all at once. All of these characteristics taken together, including its exibility, good data quality, and excellent selectivity, make LC/MS/MS
an attractive method for biopharmaceutical quantitation in
the regulated laboratory.
Key features of AB SCIEX instruments for MS/MS peptide quantitation
Ongoing optimization of sample preparation steps will continue to enhance the LC/MS/MS quantitation process, but the most signi cant gains in protein and peptide quantitation will be realized through technological innovations in mass spectrometry instrumentation. Focused on improving sensitivity and selectivity for the detection of very low levels of proteins and peptides in very complex backgrounds, AB SCIEX delivers high performance instruments that can rapidly and simultaneously measure multiple analytes—powering pharmaceutical discovery and development into the future (Figure 3).
1. Sensitivity. Biopharmaceuticals are very potent, highly targeted therapies that are administered in low concentration doses and exhibit a narrow therapeutic range. Often found
at circulating levels in the sub-ng/mL range, detection of biotherapies requires very highly sensitive methods, and the enhancement of ionization ef ciency and ion transmission have made it possible to detect drugs and metabolites in the sub-femtogram levels. New technologies such as the IonDriveTM QJet Ion Guide underpin the sensitivity enhancements in
the QTRAP 6500 System and the AB SCIEX Triple QuadTM
6500 System, bringing more ions to the detector through improved collisional focusing of ions. Heating and desolvation improvements in the IonDriveTM Turbo V Source and increased size and improved design of the aperture release more ions into the instrument. To fully detect the augmented signal, improvements to the dynamic range of the detector allow for accurate ion counting; the high energy conversion dynode (HED) detection system measures high ion signals without saturation to produce a linear dynamic range of over 6 orders of magnitude. These technologies are pivotal for providing continued improvements to sensitive bioanalysis.
2. Selectivity. Even if the pinnacle of sensitivity is reached, researchers will still be faced with the challenges of separating low levels of pharmaceutically active biomolecules from the highly complex biological matrix, where every endogenous compound can potentially interfere with the target signal. On the sample prep side, several strategies exist for the selective removal of competing background ions as well as enrichment of the analyte fraction. However, the required time and the potential for sample loss with additional cleanup steps makes this approach much less appealing. Currently, advances in MS selectivity are focused on methods that provide an additional degree of separation subsequent to the entrance to the MS
or post MS/MS selection to help improve separation capacity in highly complex biological matrices. To maximize instrument performance when detecting low-level analytes masked
by high background, AB SCIEX offers MRM3 scans and the SelexIONTM Differential Mobility Separation Device for improved peak shapes and signal-to-noise ratios during protein and peptide quantitation.
MRM3. Peak measurements obtained by multiple reaction monitoring (MRM) scans are occasionally challenged by interferences that cannot be removed without further, more elaborate sample clean-up. To provide additional speci city, the technique of MRM3 can be applied using the QTRAP
Series of instruments—extremely sensitive, hybrid triple quadrupole instruments with a linear ion trap for further fragmentation of the primary product ions. Quantitation of the secondary product ions is usually not affected by competing
or overlapping ions, which are ltered out in previous MRM selection steps. This reduction in baseline results in improved peak shape, higher signal-to-noise ratios, and superior LLOQs. The QTRAP® 5500 System and 6500 System are powered by eQTM Electronics for scan speeds that are fast enough to be compatible with fast LC ow rates; and these instruments
are equipped with single frequency excitation for highest selectivity of the product ion prior to secondary fragmentation. The Linear AcceleratorTM Trap Electrodes provide 100-fold more sensitivity for the detection of low-level secondary fragments resulting from the use of MRM3 to resolve issues of high background noise.
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3. Differential mobility spectrometry (DMS). In some cases, if secondary product ions are not speci c enough or are too low for MRM3 to be used, or method development time is too limited for prolonged MRM3 development, then additional selectivity can be gained through differential mobility spectrometry (DMS). This technique selects ions of interest based on their inherent mobility difference between a set of planar plates with high and low energy elds applied, where co-eluting interferences can be tuned out prior to analyte entrance into the mass spectrometer. AB SCIEX offers the SelexIONM Differential Mobility Separation Device for quickly resolving isobaric species and single and multiple charge
state interferences on a timescale compatible with UHPLC and multiple MRM acquisitions, thus providing an additional, orthogonal level of separation for dif cult-to-address overlapping peaks.
4. High resolution accurate mass spectrometry. Improvements to selectivity can also be gained through high resolution mass spectrometry on instruments such as the AB SCIEX TripleTOF® 5600+ System, which combines qualitative exploration and high resolution on a single platform. When using an MRMHR work ow, the TOF analyzer detects all the fragments from
the precursor at high resolution and high mass accuracy.
Using narrower extraction widths than the unit resolution of triple quadrupole-based experiments, dif cult separations between background peaks and analytes can now be achieved and improved to such an extent that minimal interferences
are observed. When fragment ions are extracted at these narrow extraction widths, analytes can be detected at higher speci city and at accurate mass in complex matrices.
5. Software. Evaluating the results of protein and peptide quantitation can often be time-consuming and repetitive, relying on manual peak identi cation and data integration—a process that does not lend itself well to the high-throughput environment. AB SCIEX has developed comprehensive, powerful, and easy-to-use solutions such as MultiQuantM Software and DiscoveryQuantM Software that simultaneously process multiple analytes. Not only do these software packages rapidly process MS scans and data, but they also support improved data integrity and security, combining unique audit trail functionality for improved regulatory compliance and an embedded digital link to the Watson LIMS system for increased con dence in data safety.
Advantages of the diversity of mass spectrometry systems
In this resource, we primarily focus on experiments conducted on two hybrid triple quadrupole instruments, the TripleTOF 5600+ System versus the QTRAP 6500 System. Each platform has distinct advantages (Figure 2): The TripleTOF is uniquely suited to qualitative discovery (as well as quantitation) due
to the underlying acquisition of a full spectrum of secondary fragments at high resolution, while the QTRAP System and its augmented ion generation, transmission and detection works best for applications requiring high sensitivity and expanded linear ranges. The AB SCIEX QTRAP 6500 System is fully accepted for regulated bioanalysis at the Phase 1 level and above, but the TripleTOF System dominates in ease of method development and non-targeted analysis during drug discovery protocols. In the event that one application demands the bene ts and strengths
of an alternative MS platform, transferring methods is easy and intuitive; the two MS systems have identical source and collision cell designs based on the innovative LINAC® Collision Cell, which allows for seamless coordination of quantitative data with qualitative analysis (Figure 4).
Perspectives for the future
As technological innovations surpass the limitations imposed
by biological sample complexity, LC/MS/MS biopharmaceutical quantitation will become more fully established as a routine methodology in the regulated laboratory. Time-consuming and complicated sample preparation steps will evolve to become better suited to the automated requirements of the MS-based bioanalytical work ow, and sample extraction procedures are likely to become more highly selective to achieve the sensitivities required for monitoring sub-picomolar concentrations of biotherapeutic agents. Working with highly sensitive methods based on the enhanced MS ionization ef ciency and transmission has yielded promising results on the QTRAP System, producing suf cient LLOQs for low-level biomolecule quantitation needed for PK and TK studies. Additionally, distinct gains using DMS and MRM3 are adding an additional layer of selectivity, removing hard- to-separate background and leading to better signal-to-noise parameters. The potential of high resolution mass spectrometry
to measure intact, high molecular weight biomolecules will gain increasing interest as technological advances push TOF sensitivities towards those of the hybrid linear ion trap instruments. By reducing the need for additional sample preparation steps with enhanced MS detection and selectivity capacities, LC/MS/MS techniques are becoming more closely aligned with the high- throughput work ows necessary for regulated bioanalysis.
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From Biologics Characterization to Biotransformation and Bioanalysis
TripleTOF® System
Discovery
SWATHTM Acquisition
m/z
High Res XICs
Development GLP Bioanalysis
Multiple Reaction Monitoring (MRM)
retention time
MRM
QTRAP® System
Figure 4. Continuity of work ows between TripleTOF to QTRAP. From product characterization during research and development process to biotransformation and bioanalysis during PK/PD analysis in preclinical and clinical studies
References
1“Biopharmaceuticals – A Global Market Overview.” November 2013. The abstract from a market research report from Reportbuyer.com. Accessed online at: http:// www.prweb.com/releases/2013/11/prweb11314067.htm
2 Zhong X, Neumann P, Corbo M, Loh E. “Recent Advances in Biotherapeutics Drug Discovery and Development.” Drug Discovery and Development – Present and Future, Dr. Izet Kapetanovic (Ed.) ISBN: 978-953-307-615-7, InTech. Accessed online at: http://www.intechopen.com/books/drug-discovery-and-development-present- and-future/recent-advances-in-biotherapeutics-drug-discovery-and-development
3 Reichert J. “Which are the antibodies to watch in 2013?” mAbs. Jan/Feb 2013; 5(1): 1-4.
4 Shi T, Su D, Liu T, Tang K, Camp DG 2nd, Qian WJ, Smith RD. “Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics.” Proteomics. Apr. 2012; 12(8): 1074-92.
5 Hopfgartner G and Gougongne E. “Quantitative high-throughput analysis of drugs in biological matrices by mass spectrometry.” Mass Spectrom Rev. May/Jun 2003; 22(3): 195-214.
6 Ezan E and Bisch F. “Critical comparison of MS and immunoassays for the bioanalysis of therapeutic antibodies.” Bioanalysis. Nov. 2009; 1(8): 1375-1388.
7 van den Broek I, Niessen WMA, van Dongen WD. “Bioanalytical LC/MS/MS of protein-based biopharmaceuticals.” Journal of Chromatography B. 2013; 929: 161-179.
8 Food and Drug Administration. “Guidance for Industry; Bioanalytical Method Validation.” US Department of Health and Human Services. FDA. Center for Drug Evaluation and Research, Rockville, MD, 2001.
9 Bischoff R, Bronsema K, van de Merbel NC. “Analysis of biopharmaceutical proteins in biological matrices by LC/MS/MS 1. Sample preparation.” Trends in Analytical Chemistry. 2013; 48:41-51.
10 Liu G, Ji QC, Sun H, Shuster D, Zhao Q, Arnold M. “Liquid chromatography coupled with tandem mass spectrometry for the bioanalysis of proteins in drug development: Practical considerations in assay development and validation.” Journal of Chromatography A. 2013;1284: 155-162.
11 Hopfgartner G, Lesur A, Varesio E. “Analysis of biopharmaceutical proteins in biological matrices by LC/MS/MS II. LC/MS/MS analysis.” Trends in Analytical Chemistry. 2013; 48:52-61.
12 Thomson B. “Driving high sensitivity in biomolecular MS.” Genetic Engineering and Biotechnology News. Nov 2012; 32(20). Accessed at: http://www.genengnews. com/gen-articles/driving-high-sensitivity-in-biomolecular-ms/4603/?kwrd=high%20 sensitivity%20in%20biomolecular%20MS.
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Technology Drives High Performance in Biomolecular Mass Spectrometry
Enhancing the sensitivity and dynamic range of the AB SCIEX QTRAP® 6500 with IonDriveTM System Technology
Bruce Thomson and Bruce Collings AB SCIEX, Concord, ON, Canada
In applications that range from proteomics to biomarker discovery to drug development, mass spectrometry has become the tool that provides the high accuracy and speci city in trace chemical analysis. While there are many important metrics of analytical performance (accuracy, precision, limit of quantitation), they all rely heavily on two key instrumental performance characteristics
– sensitivity and dynamic range. In mass spectrometry, instrument sensitivity can best be de ned as the number of ions detected per molecule of analyte injected, thus accounting for all losses in ionization, transmission, and detection. Dynamic range is usually de ned as the range of linear response of the instrument, limited at the low end by absolute sensitivity and, at the high end, by detector or other instrument-related saturation effects.
Over the last thirty years or more of development at AB SCIEX, enormous strides have been made in improving both the instrument sensitivity and the dynamic range. This improved performance has enabled new applications to be addressed by mass spectrometry, and allowed analyses to be performed more rapidly and with greater con dence and higher precision. Higher sensitivity has also enabled the use of additional capabilities and techniques that provide improved analytical speci city – such as higher mass resolution, faster scans speeds, and shorter reaction monitoring times, and techniques such as ion mobility/mass spectrometry combinations, or added levels of tandem mass spectrometry (MS/MS/MS). The growth curve of sensitivity in
AB SCIEX triple quadrupole mass spectrometers over this time period is plotted in Figure 1, which shows a growth of nearly six orders of magnitude in absolute sensitivity since our rst LC/MS/MS product, the TAGA 6000. The AB SCIEX QTRAP® 6500 System, our newest and highest-performance instrument, reaches new levels in both sensitivity and dynamic range. New technologies in both the ion optics and ion counting detection system have driven these performance increases.
A key step in achieving higher sensitivity is to create more ions in the source. Over the years, improvements in ionization ef ciency have been achieved by increasing the ef ciency of desolvation and declustering in the source. The new IonDriveTM Turbo V Source of the QTRAP 6500 System has reached a new level.
Bruce Collings
Bruce Thomson
RUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS www.absciex.com
1E+6 1E+5 1E+4 1E+3 1E+2 1E+1 1E+0
Driving Sensitivity by Orders of Magnitude
Figure 1: The growth in sensitivity of high- ow LC/MS/MS mass spectrometer systems over the last thirty years at AB SCIEX.
By optimizing the design of the IonDrive Turbo V Source heaters for better and more uniform distribution of heat in the region of droplet evaporation, the ef ciency of creating ions in front of the sampling ori ce has been improved, especially at higher liquid ow rates and with less volatile compounds.
However the sampling aperture into the vacuum still typically represents the largest area of ion losses. We have, therefore, increased the size of the ori ce in order to sample more ions. Improved pumping in the interface helps maintain an acceptable core vacuum pressure without increasing the size of the turbo pumps. The gas expanding through the ori ce forms a supersonic free jet with a characteristic barrel shock structure as shown
in Figure 2. The high gas ow and pressure provide a strong drag force on the ions that are entrained in this jet, making it more challenging to effectively focus the ions through the next aperture. The new IonDriveTM QJet Ion Guide optics employs a two-stage RF quadrupole to capture and focus the ions to
the center-line of the optics using the technique of collisional focusing, allowing the majority of gas to be pumped away. The rst section is a large-diameter, RF-only quadrupole with narrow gaps between the rods in order to contain the ions. The narrow gaps minimize the radial out ow of gas, and, therefore, ion losses, while allowing the entrained ions to become collisionally
TT1980 1990 2000 2010 2020
Year
Relative Sensitivity
ULTIMATE SENSITIVITY


For Research Use Only. Not for use in diagnostic procedures.
www.absciex.com THERAPEUTIC PEPTIDE BIOANALYSIS
Figure 2: Cross-sectional view of the IonDriveM QJet showing the supersonic free jet (supersonic ow region in red) and the gas ow along the axis.
focused. The second section is a smaller diameter quadrupole that provides the nal stage of ion-beam focusing while the gas escapes. The transmission ef ciency of the ions into the next chamber is approximately 50%, an impressive gure of merit considering the larger ori ce diameter, and higher pressure and higher gas velocity.
The increased rate of ion generation in the source, and improved transmission ef ciency in the ion optics results in a higher ion
ux reaching the detector for a given amount of sample injected. At the detector, ions are detected and registered with very high ef ciency using a pulse counting system with a very low noise level. The challenge with pulse counting has always been to be able to measure high ion signals without saturation. The new high energy conversion dynode (HED) detection system powered by IonDriveM Technology provides a very signi cant improvement in this area, extending the upper level of ion counting while maintaining the ability to register single ion events for the best signal-to-noise ratios at the detection limit. The improved dynamic range can be seen in Figure 3, which compares the new HED detection system to the standard CEM detection system.
In Figure 3, the measured count rate of the rst isotope of reserpine is plotted against the true count rate as determined from the known ratio and intensity of its fourth isotope. The new system uses high-energy ion-to-electron conversion and a low impedance multi-channel continuous dynode detector with a closely coupled transimpedance ampli er system that allows high count rates to be sustained without loss of signal. Arrival rates
of up to 200 million ions per second can be achieved resulting
in a detector linear dynamic range of more than six orders of magnitude. With the sensitivity and dynamic range improvement described above, the QTRAP® 6500 System provides a new level of analytical performance, as evidenced by the ability to detect and quantify sub-femtogram amounts of biomolecules injected
Figure 3: Dynamic range of the high energy conversion dynode (HED) detection system compared to the standard CEM detection system.
on-column as shown in Figure 4. Demands for ever decreasing detection limits will continue to drive the need for newer and better methods of ionization, transmission, and detection in the future. However, the growth curve of sensitivity will become more and more dif cult to maintain as we approach the limit of measuring and detecting nearly every ion injected.
Figure 4: Signal from 500 attograms of verapamil injected on-column monitored in MRM mode using the transition 455/165.
True Count Rate (cps)
15
Measured Count Rate (cps)
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16
Achieving Low-Flow Sensitivities for Peptide Quantitation using Micro ow Rates on the
QTRAP® 6500 System
High-throughput, sensitive, micro ow analysis of bradykinin and other peptide standards with a hybrid triple quadrupole linear ion trap coupled with the Eksigent ekspertTM nanoLC 425 System
Kelli Jonakin and Christie Hunter AB SCIEX, Redwood City, CA, USA
Key challenges of nano ow quantitation
Reduced throughput – Nano ow platforms lack the robustness and high-throughput required for multi-sample drug metabolism studies.
In exible and complicated interface assembly – Installing and troubleshooting nano ow ttings is time-consuming, making variable-rate method development cumbersome.
Limited options for sensitive peptide quantitation – Easier and faster peptide quantitation methods that meet nano ow standards for sensitivity are needed for pharmaceutical applications.
Key bene ts of micro ow peptide analysis on the QTRAP® 6500 System
Ultra-sensitive peptide quantitation – LLOQs obtained on the robust micro ow platform meet or exceed nano ow standards by 2-fold – even in complex matrices.
Accelerated throughput – Easy-to-use micro ow
work ow provides >2-fold faster run times, suitable for high- throughput analysis.
Easy hardware assembly – Micro ow components take only a few minutes to interchange – realizing a single, adaptable LC platform for peptide quantitation.
Method portability – No loss of sensitivity is observed when transferring nano ow regimens to a micro ow platform on the QTRAP® 6500 System.
Key features of the micro ow work ow on QTRAP® 6500 System
IonDriveTM Technology – Increased detector dynamic range and signal-to-noise improvements are due to ionization ef ciency, ion sampling, and ion transmission enhancements.
Mass range of m/z 5–2,000 – Comprehensive mass range provides the versatility needed for peptide quantitation.
Flexible and reproducible chromatography – The nanoLC 425 System supports a wide range of rates – nano to micro ow – providing unparalleled method exibility.
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Introduction
To obtain the best sensitivities and ionization ef ciencies for peptide analysis, nano ow chromatography is often used
in combination with hybrid triple quadrupole/linear ion trap
mass spectrometry to provide an established, highly-sensitive quantitation method. However, sample processing times are typically slowed by the extended chromatography run times obtained at sluggish, nL/min ow rates. Shifting to quicker micro ow rates (3–50 μL/min) has improved sample run time, but the higher ow rates cause dampening of the ionization ef ciency compared to nano ow, diminishing sensitivity and driving up LLOQs.
To nd a good balance between sensitivity, robustness, and throughput, we compared the LLOQs of various tryptic peptides using nano- or micro ow rates – including bradykinin, a nine amino-acid peptide involved in vasodilation. Recent evaluation
of peptides on the QTRAP® 5500 System indicated a 3-fold loss
in sensitivity when transitioning from nano ow to micro ow chromatography (4 μL/min).1 However, other peptide quantitation experiments have demonstrated a roughly 3–5-fold improvement in sensitivity when moving to the QTRAP® 6500 System from the
TTULTIMATE SENSITIVITY


Chromatography
Column:
Injection: Flow rate:
Eksigent ChromXPM C18 cHiPLC, (75 μm × 15cm)
2–5 μL 300 nL/min
LC system:
Eksigent ekspertM nanoLC 425 System with 0.1–1 μL/min or 1–10 μL/min ow module in
For Research Use Only. Not for use in diagnostic procedures.
Nano ow settings
Micro ow settings
Column: Injection: Flow rate:
Eksigent ChromXPM C18 (300 μm × 15 cm) 2–5 μL
4 μL/min
® combination with the Eksigent cHiPLC
in trap and elute mode
System
Figure 1: Comparing signal intensities of standard protein digest. A beta- galactosidase digest was analyzed by nano ow LC on a QTRAP® 5500 System (top) and compared to micro ow LC on the QTRAP® 6500 System (bottom). Similar signal intensities were observed with similar separation quality but with >2-fold faster total run times with micro ow LC.
5500. From these two studies, we hypothesized that nano ow peptide assays currently performed on the QTRAP® 5500 System could be upgraded to micro ow rates on the QTRAP® 6500 System, resulting in similar sensitivities, but with improved robustness and increased throughput.
To realize the most ef cient strategy for large-scale peptide analysis, this study explores the LLOQs and the speed of analysis for a range of peptide standards, including bradykinin, using
two, hybrid triple quadrupole/linear ion trap systems operating
at different ow rates. Variable chromatography was executed using an ekspertM nanoLC 425 system, which has the exibility
to support micro and nano ow rates in a single system. Coupled with recent advances in IonDriveM Technology for higher-sensitivity detection, micro ow chromatography provides a step forward
in productivity and ease-of-use, meeting or exceeding sensitivity levels established for nano ow peptide analysis.
Methods
Sample preparation
The beta-galactosidase digest mixture and the 6-peptide mixture containing the bradykinin 2–9 fragment (monoisotopic mass 904.9681) were obtained from AB SCIEX. The six protein digest containing carbonic anhydrase and ve other proteins was obtained from Michrom BioResources. Protein-precipitated (crashed) plasma matrix was prepared by mixing equal volumes of plasma and acetonitrile, followed by centrifugation.
System: Interface:
QTRAP® 5500 System NanoSpray® Ion Source
Mass spectrometry
Nano ow settings
Micro ow settings
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Figure 2: IonDriveM Turbo V Source. The optimized geometry and larger diameter heaters provide more ef cient heat transfer to a larger cross section of the spray cone. Equipped with the lower inner diameter hybrid electrodes from Eksigent, this source provides a robust, easy-to-optimize solution for micro ow chromatography.
Data processing
MRM transitions were optimized for each peptide and used on both instruments. Standard concentration curves were performed to evaluate impact of ow rates and separation times on the two
System: Interface:
QTRAP® 6500 System
IonDriveM Turbo V Source with 25 μm ID electrodes (Figure 2)
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18
Peptide
Fragment Ions Summed for Quant
QTRAP® 6500 System – 4 μL/min LLOQ (amol on column)
QTRAP® 5500 System – 300 nL/min LLOQ (amol on column)
Sensitivity Improvement on Micro ow QTRAP® 6500 System
IDALNENK 2y4, 2y6, 2y7 .48 1.9 4.0
TPEVDDEALEK 2y102+, 2y7, 2y8 3.8 3.8 1.0
VLVLDTDYK 2y5, 2y6, 2y7 1.9 3.8 2.0
AEFVEVTK 2y5, 2y6 3.8 3.8 1.0
ATEEQLK 2y5 7.6 3.8 0.5
DGPLTGTYR 2y5 1.9 3.8 2.0
VGDANPALQK 2y5, 2y6, 2y7 .95 3.8 4.0
VLDALDSIK 2y6, 2y7, 2y8 1.9 3.8 2.0
Average Difference: 2.1
Standard concentration curves in simple matrix were generated and the LLOQs were determined using both nano ow on QTRAP® 5500 System and micro ow on QTRAP® 6500 System. The results for the peptides show some variation observed across peptides but on average a 2-fold lower LLOQ was seen on the micro ow QTRAP® 6500 System.
Table 1: Lower limits of quanti cation (LLOQ) obtained for eight tryptic peptides on the two LC/MS systems Standard concentration curves in simple matrix were generated and the LLOQs were determined using both nano ow on QTRAP® 5500 System and micro ow on QTRAP® 6500 System. The results for the peptides show some variation observed across peptides but on average a 2-fold lower LLOQ was seen on the micro ow QTRAP 6500 System.
different MS systems. All samples were analyzed in triplicate. Lower limits of quanti cation (LLOQ) were determined using MultiQuantTM Software.
Results and discussion
Micro ow LC for peptide quantitation
Previous peptide studies that used mass spectrometry for quantitation demonstrated lowered ionization ef ciencies during micro ow analysis.2 To improve sensitivities for chromatographic runs conducted at 4–50 μL/min on the QTRAP® 6500 System,
the IonDriveTM Turbo V Source was used to provide high-ef ciency ionization and increased ruggedness (Figure 2). For best performance at micro ow rates, the sources were plumbed
with the hybrid electrodes speci cally designed for micro ow.2 These electrodes signi cantly reduced post-column dead volumes for minimized dispersion and sharper peak widths. In this work, we used the 25 μm ID electrode, ideal for 300 μm ID columns and 3–25 μL/min ow rates.
Comparing the sensitivity differences across LC/MS platforms
To better understand the sensitivity of peptide detection at variable ow rates, a series of experiments were performed to compare LLOQs of peptide standards obtained using a nano ow platform on the QTRAP® 5500 System versus a micro ow platform on the QTRAP® 6500 System. First, the magnitude of signal intensities of beta-galactosidase peptides (10 fmol on column) were compared, and analysis of the resulting spectra indicated that similar intensities were obtained for peptides analyzed by micro ow versus nano ow (Figure 1). Total run time was reduced 2-fold in the micro ow experiment, while preserving good peak resolution. Preliminary data indicate that higher LC ow rates
do not impede the detection and resolution of peptide peaks analyzed using the QTRAP® 6500 System, laying the groundwork for further micro ow studies.
Tryptic peptides from the six protein digest were evaluated under micro ow and nano ow conditions in a simple matrix to assess
sensitivity. Peak areas were calculated for increasing protein concentrations and were assembled into concentration curves. LLOQs determined for peptides analyzed under micro ow and nano ow conditions were compared for individual peptides within the mixture (Table 1). When using the micro ow con guration on the QTRAP® 6500 System, a 2-fold lower LLOQ was observed on average for each individual peptide with some variation across the group.
To establish if signal intensity improves under micro ow conditions for a more complex mixture of peptides, the MRM signals for eight peptides from the six protein mixture were assessed (using an on-column concentration just above the LLOQ for the group of peptides). Signal intensities for peptides from the mixture under micro ow conditions were elevated over those obtained under nano ow conditions (Figure 3). Additionally, chromatographic
run times, shortened by 25 min. under micro ow conditions, allowed for more rapid peak elution while preserving good peak resolution (Figure 3). Focusing on peak intensity for one particular peptide (2y5 from carbonic anhydrase) within the peptide mixture from the six peptide mixture reveals improving LLOQs (Figure 4) on the micro ow platform. The LLOQ achieved under micro ow conditions (1.9 amol on column) was ~2-fold more sensitive than that obtained by nano ow (3.8 amol on column).
To evaluate peptide response in a more complex matrix system – protein-precipitated or crashed plasma – peak intensities and elution times for the bradykinin peptide in the AB SCIEX six- peptide mixture were assessed for both LC/MS/MS platforms. The introduction of competing ions and background noise from the plasma did not impact the intensity, elution times or resolution of microflow peaks over the nanoflow peaks. LLOQs from the microflow experiments (6.3 amol on column, Figure 5) were lower than those achieved under nanoflow conditions (12.5 amol on column) and were indicative of equivalent or slightly better sensitivity when using microflow on the QTRAP® 6500 System.
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ULTIMATE SENSITIVITY


For Research Use Only. Not for use in diagnostic procedures.
Figure 4: LLOQ comparisons for carbonic anhydrase peptide DGPLTGTYR from the six protein digest. The MRM signal at LLOQ for the carbonic anhydrase peptide
with micro ow (left) and nano ow (right) chromatography. An approximate 2-fold improvement in sensitivity is obtained when using the micro ow chromatography on the QTRAP® 6500 System.
Analysis of peak areas for various concentrations of bradykinin 2y7 peptide on the QTRAP® 6500 System
Figure 3: Signal intensity comparison of eight tryptic peptides from the six protein mixture at concentrations near LLOQ. Peptide signals from the six-protein digest (7.6 amol on column) are shown for (top) the QTRAP® 5500 System under nano ow conditions and (bottom) the QTRAP® 6500 System under micro ow conditions. Micro ow signal intensities showed a small improvement over the nano ow data.
Conclusions
An easy-to-use, high-throughput, and highly-sensitive work ow was established on the QTRAP® 6500 System using micro ow chromatography for performing targeted peptide quantitation. The exibility and reproducibility of the Eksigent ekspert nanoLC 425 makes it an ideal LC system for labs performing a broad range of proteomics work ows, including both nano and micro ow rate applications. Assays currently performed using nano ow LC on the QTRAP® 5500 System can be easily translated to the micro ow QTRAP® 6500 System for accelerated sample analysis with similar sensitivities.
High-throughput capacities were realized with decreased peak retention times under micro ow conditions – while preserving peak resolution and intensity.
Similar or lower LLOQs attained using micro ow chromatography on the QTRAP® 6500 System met or exceeded nano ow sensitivity standards for robust peptide quantitation.
Highly reproducible micro ow chromatography on the Eksigent ekspertM nanoLC 425 ensured accurate peptide quantitation.
References
1 Exploring the Sensitivity Differences for Peptide Quanti cation in the Low Flow Rate Regime – Eksigent ekspertM nanoLC 400 System. AB SCIEX technical note 6560212_02. Poster # TP08 – 151
2 Higher Sensitivity and Improved Resolution Micro ow UHPLC with Small Diameter Turbo VM Source Electrodes. AB SCIEX technical note 4590211-01
Figure 5: Quanti cation of bradykinin using micro ow LC on a QTRAP® 6500 System. MRM signals for bradykinin at LLOQ (top gure) were obtained using micro ow conditions. The standard concentration curve (bottom gure) of bradykinin in protein- precipitated plasma provided an LLOQ of 6.3 amol on column. Linearity was very good across the limited dynamic range interrogated (>4 orders of magnitude in this example). The equivalent experiment using nano ow on the QTRAP® 5500 resulted
in an LLOQ of 12.5 amol on column for the same y7 fragment ion. Assay performance metrics are listed in the table for the bradykinin standard curve.
Conc. (ng/mL)
Mean (n=3)
Std. Dev.
%CV
Accuracy (%)
6.3 5.411 0.8429 15.58 85.88
12.5 12.63 1.120 8.87 101.01
25 25.55 2.747 10.75 102.18
50 46.26 5.584 12.07 92.52
100 106.8 6.212 5.81 106.83
200 194.2 4.001 2.06 97.09
400 427.9 8.055 1.88 106.98
800 841.3 9.923 1.18 105.16
4,000 4,000.7 84.15 2.10 100.19
20,000 20,560 236.3 1.15 102.78
100,000 99,370 1641 1.65 99.37
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THERAPEUTIC PEPTIDE BIOANALYSIS
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20
A Sub-picogram Quanti cation Method for Desmopressin in Plasma using the AB SCIEX
Triple QuadTM 6500 System
A high-throughput method for detecting ultra-low levels (0.5 pg/mL) of a therapeutic peptide in human plasma using an AB SCIEX Triple QuadTM 6500 LC/MS/MS System and UHPLC Chromatography
Rahul Baghla1, Swati Guttikar2, Dharmesh Patel2, Abhishek Gandhi2, Anoop Kumar1, and Manoj Pillai1 1AB SCIEX, 121, Udyog Vihar, Phase IV, Gurgaon, Haryana, India
2 Veeda Clinical Research India, Ahmadabad, India
TTKey challenges of desmopressin quantitation
Impaired sensitivity in complex matrices – Very low-level peptide detection (sub-pg/mL) can be suppressed by high background and competing ions in biological samples.
Poor data quality – Precision and accuracy can be compromised at low peptide levels, giving results below accepted bioanalytical standards.
Key bene ts of peptide quantitation on the Triple QuadTM 6500 LC/MS/MS System
High sensitivity – Very low level peptide detection in human plasma (at sub pg/mL concentrations) is enabled by IonDriveTM Technology.
Excellent precision and accuracy at the LOQ level – Data quality (for LOQ, LQC, MQC and HQC levels) met or exceeded USFDA bioanalytical method validation criteria.
High throughput – High sensitivity was achieved under high- ow conditions (0.750 mL/min), optimal for multi-sample analysis.
Unique features of the Triple QuadTM 6500 System for low-level peptide detection
IonDriveTM Turbo V Source – Increased ionization ef ciency and heat transfer contribute to sensitivity enhancements, including improved signal-to-noise.
IonDriveTM QJet Ion Guide – Increased ion sampling improves method ef ciency and ruggedness.
IonDriveTM High Energy Detector – Innovative detector technology boosts dynamic range and sensitivity.
Introduction
Low-level peptide detection has a number of applications
in clinical studies and in the pharmaceutical discovery and development processes, highlighting the increasing relevance of sensitive and selective mass spectrometric platforms in the
Abhishek Ghandi
Figure 2: Unique features of Triple QuadTM 6500 System.
bioanalytical laboratory. Regulatory requirements demand
intensive and rigorous quantitation of therapeutic peptides during pharmacokinetic, bioequivalence, and metabolic studies. In addition, drug discovery and development strategies seek
RUO-MKT-02-1486-A THERAPEUTIC PEPTIDE BIOANALYSIS www.absciex.com
TTULTIMATE SENSITIVITY


Figure 3: Structure of desmopressin.
to monitor and quantitate peptide biomarkers in complex
biological samples, necessitating highly-selective separations of low concentration analytes from high background noise and prominent levels of competing ions. The AB SCIEX Triple QuadM 6500 LC/MS/MS System, equipped with IonDriveM Technology
for enhanced detector performance, has demonstrated particular strength in the detection of low-level amounts of small molecules, and in this study, we extend the augmented signal-to-noise, broad dynamic range, and the ef cient method development capacities of the Triple Quad 6500 System to the detection of sub-picogram levels of a therapeutic peptide under high-throughput conditions.
We have developed a reliable, fast, and sensitive method for the detection of a nine-amino-acid peptide, desmopressin
(1 desamino-8-D-arginine, vasopressin), which is structurally similar to the hormone arginine vasopressin, but contains a deaminated rst amino acid and dextro-arginine (rather than levo-) in the eighth position. Therapeutically, desmopressin reduces urine production, restricting water elimination from the kidneys by binding to the V2 receptors in renal-collecting ducts, thereby facilitating increased reabsorption. The longer half-
life of desmopressin over vasopressin offers some therapeutic advantages, and typical doses of desmopressin to treat diabetes insipidus and bedwetting range between 0.200 to 1.20 mg
per day, resulting in very low plasma concentrations. In this bioanalytical study, we have established a sensitive and selective LC/MS/MS method for the quantitation of desmopressin in human plasma, detecting peptide levels as low as 0.500 pg/mL with excellent accuracy and precision. This technique should facilitate additional mass spectrometric method development for accurate quantitation of a range of therapeutic peptides in biological matrices on the Triple Quad 6500 System.
Materials and methods
Sample preparation
Plasma samples (1000 μL) containing 2% desmopressin standard and 50μL internal standard were vortexed and spiked with 50μL of orthophosphoric acid (OPA). Samples were extracted on weak
Figure 4: Structure of internal standard, desmopressin-d5..
cation exchange cartridges conditioned with methanol followed by 100mM ammonium acetate solution. After loading, samples were washed in three steps: 1) 2% OPA:methanol (80:20 v/v); 2) 2% NaOH:Methanol (60:40 v/v); and 3) water:methanol (60:40 v/v). Analytes were eluted with 5% acetic acid in methanol, dried under nitrogen at 40 oC, and reconstituted with 0.1% acetic acid (150 μL) prior to analysis by mass spectrometry.
Chromatography
For Research Use Only. Not for use in diagnostic procedures.
LC system: Column:
Column temp.: Injection:
Flow rate: Mobile phase:
Gradient:
GL Sciences LC 800 System
Agilent 300 Extend C18 (150 x 2.1 mm, 3.5 μm)
40 °C
50 μL
0.750 ml/min
A) water, 0.1% acetic acid
B) acetonitrile, 0.1% acetic acid
Time/min A% B%
0 85 15 1.5 85 15 3.5 50 50
3.51 85 15 5 85 15
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Mass spectrometry
Analysis of desmopressin and desmopressin-d5 required
different mass spectrometry settings (Table 1). The MRM transition monitored for desmopressin was m/z 525.4/328.0 and 537.9/328.0 at a dwell time of 50 msec. Five replicate injections were performed at all concentrations.
Data system: Interface:
Triple QuadM 6500 System
IonDriveM Turbo V Source in positive ion mode
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22
Instrument Parameter
Desmopressin
Desmopressin-d5
CUR 40 40
TEM 600 °C 600°C
ISV 5500 5500
GS1 50 50
GS2 60 60
CAD 10 10
DP 50 71
EP 10 10
CE 23 23
CXP 12 12
Table 1: Compound-dependent parameters for desmopressin and desmopressin-d5 on the Triple QuadTM 6500 System.
Figure 6: High signal-to-noise ratio for desmopressin. The signal-to-noise ratio was calculated for desmopressin extracted from plasma at LLOQ level (0.500 pg/mL in plasma, S/N = 60.7).
Figure 7: Desmopressin technical replicates. Chromatograms of six LLOQ quality control samples (0.502 pg/mL) for precision and accuracy calculations are shown (Table 2).
Figure 5: Desmopressin MRM signal (shown in left side) panes for multiple concentrations and desmopressin D5 MRM signal (shown in right side panes).
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For Research Use Only. Not for use in diagnostic procedures.
Sample Type
Analyte Retention Time (min)
Analyte Peak Area
IS Retention Time (min)
IS Peak Area
Area Ratio
Analyte Conc. (pg/mL)
Calculated Conc. (pg/mL)
% Accuracy
Blank
0
0
0
0
#DIV/0!
N/A
#DIV/0!
Standard
2.60
168,629
0.049
2.498
2.544
101.85
Standard
2.60
51,086
2.57
187,669
0.272
16.122
16.167
100.28
Sample ID
AQM 18122013 BLANK 01 BLANK+IS 01 STD A 01 STD B 01 STD C 01 STD D 01 STD E 01 STD F 01 STD G 01 STD H 01 LLOQ QC 01 LQC 01 MQC 01 HQC 01 LLOQ QC 02 LQC 02 MQC 02 HQC 02 LLOQ QC 03 LQC 03 MQC 03 HQC 03 LLOQ QC 04 LQC 04 MQC 04 HQC 04 LLOQ QC 05 LQC 05 MQC 05 HQC 05 LLOQ QC 06 LQC 06 MQC 06 HQC 06
Unknown 2.60 Unknown 0
Standard 2.60 Standard 2.60 Standard 2.60 Standard 2.60
Qual. Control 2.60 Qual. Control 2.60 Qual. Control 2.6 Qual. Control 2.60 Qual. Control 2.61 Qual. Control 2.60 Qual. Control 2.61 Qual. Control 2.61 Qual. Control 2.61 Qual. Control 2.60 Qual. Control 2.61
Qual. Control 2.60
119046 2.57 0 2.57 3,567 2.57 20,050 2.57 94,706 2.57 321,985 2.57 5,296 2.57 244,061 2.57 5,433 2.57 227,932 2.57 5,266 2.58 228,311 2.58 4,940 2.58 222,646 2.58 5,299 2.58 225,914 2.58 5,414 2.58 223,129 2.58
119,422 0.997 120,418 0 157,232 0.023 180,711 0.111 145,728 0.65 187,366 1.718 160,710 0.033 183,047 1.333 154,564 0.035 179,913 1.267 153,330 0.034 175,270 1.303 155,534 0.032 167,585 1.329 161,308 0.033 169,163 1.335 161,549 0.034 168,042 1.328
N/A
N/A 1.000 6.448 40.304 100.760 1.492 81.384 1.492 81.384 1.492 81.384 1.492 81.384 1.492 81.384 1.492 81.384
60.309 No Peak 0.967 6.344 39.173 104.268 1.593 80.806 1.727 76.760 1.678 78.936 1.520 80.516 1.587 80.937 1.627 80.471
N/A
N/A 96.73 98.39 97.19 103.48 106.75 99.29 115.74 94.32 112.44 96.99 101.90 98.93 106.33 99.45 109.05 98.88
Standard
2.60
218,389
2.57
163,086
1.339
80.608
81.158
100.68
Qual. Control
2.62
2,395
2.58
153,948
0.016
0.502
0.533
106.19
Qual. Control
2.60
114,782
2.57
180,711
0.635
40.692
38.277
94.07
2,379
Table 2: Full analysis of precision and accuracy measurements for desmopressin (batch 1 samples) in human plasma.
Data processing
All Triple Quad 6500 System data was processed using MultiQuantM Software. The concentration curves were analyzed using a linear t with a 1/x2 weighting. Data acquired on the Triple Quad 6500 System was processed using the quantitation tools within Analyst® 1.6 Software.
Results and discussion
Method analysis and data quality
The desmopressin quantitative assay was validated by generating an internal standard curve using standards alone and standards spiked into human plasma. Left side pane of Figure 5 shows representative peaks for A) blank extract, B) plasma spiked with
0.5 pg/mL desmopressin and the right side pane of Figure 5 shows the MRM response from the internal standard. Standard concentrations varied from 0.5 to 100 pg/mL generating an LLOQ in plasma of 0.5 pg/mL resulting in a signal to noise ratio of 60.7 (Figure 6). Reproducibility of the assay was assessed by multiple technical replicates of the same sample (n = 6, Figure 7) on an LLOQ quality control sample of 0.5 pg/mL. The calibration curve for desmopressin in plasma shows excellent linearity over 2.5 orders of magnitude concentration range with an r value of >0.99 (Figure 8).
The data collected for a single calibration curve are presented in Table 2. Analyte retention time and internal standard
peak retention times were consistent, with both eluting at approximately 2.6 min. The calculated concentration correlates
www.absciex.com THERAPEUTIC PEPTIDE BIOANALYSIS
N/A
Standard
2.61
2,148
2.58
141,964
0.015
0.500
0.507
101.40
8,190
2.57
Qual. Control
2.63
2.58
152,201
0.016
0.502
0.538
107.11
Qual. Control
2.60
119,912
2.57
183,163
0.655
40.692
39.465
96.99
Qual. Control
2.61
2,387
2.57
145,187
0.016
0.502
0.587
116.93
Qual. Control
2.60
117,712
2.57
178,847
0.658
40.692
39.678
97.51
Qual. Control
2.61
2,203
2.57
142,754
0.015
0.502
0.526
104.70
Qual. Control
2.60
102,226
2.58
156,822
0.652
40.692
39.294
96.56
Qual. Control
2.62
2,323
2.58
144,891
0.016
0.502
0.562
111.96
Qual. Control
2.60
101,960
2.58
157,530
0.647
40.692
39.013
95.87
Qual. Control
2.62
2,309
2.58
144,368
0.016
0.502
0.560
111.50
Qual. Control
2.61
103,636
2.58
159,865
0.648
40.692
39.076
96.03
23
ULTIMATE SENSITIVITY


24
well with the actual spiked-analyte concentration in plasma matrix with a percent accuracy of the standard curve very close to 100% for all concentrations of standard, and the quality control samples had a percent accuracy of 110%. Table 3 shows the individual statistics for three separate batch runs of desmopressin. Data from Table 2 are taken from Batch 3. Table 4 shows the mean values for the percent accuracy and % CV for three separate batch runs. For the LLOQ quality control, the mean accuracy was calculated to be 108% with a %CV of 10.5%.
PA BATCH 01
Nominal Concentration (pg/mL)
Desmopressin
LLOQ QC
LQC
MQC
HQC
0.502 1.492 40.692 81.384
1 0.533 1.593 38.277 80.806
2 0.538 1.727 39.465 76.760
3 0.587 1.678 39.678 78.936
4 0.526 1.520 39.294 80.516
5 0.562 1.587 39.013 80.937
6 0.560 1.627 39.076 80.471
Mean
0.5510
1.6220
39.1338
79.7377
S.D (+/-) 0.02287 0.07302 0.48653 1.62681
C.V. (%)
4.15
4.50
1.24
2.04
% Nominal 109.76 108.71 96.17 97.98
N
6
6
6
6
PA BATCH 02
7 0.519 1.590 39.191 79.548
8 0.491 1.283 40.359 82.140
9 0.490 1.509 39.486 78.094
10 0.571 1.436 39.828 78.854
11 0.526 1.387 40.624 78.472
12 0.680 1.319 40.355 79.635
Mean
0.5462
1.4207
39.9738
79.4572
S.D (+/-) 0.0719 0.1159 0.5636 1.4443
C.V. (%)
13.17
8.16
1.41
1.82
% Nominal 108.80 95.22 98.24 97.63
N
6
6
6
6
PA BATCH 03
13 0.418 1.364 41.098 79.992
14 0.602 1.446 39.814 80.103
15 0.520 1.399 40.988 79.854
16 0.463 1.350 39.391 80.937
17 0.563 1.274 39.960 80.577
18 0.528 1.332 39.188 82.162
Mean
0.5157
1.3608
40.0732
80.6042
S.D (+/-) 0.05867 0.05867 0.80200 0.86337
C.V. (%)
11.38
4.31
2.00
1.07
% Nominal 102.73 91.21 98.48 99.04
N
6
0
6
6
Figure 8: Calibration curve of desmopressin in plasma from 0.500 pg/mL to 100.760 pg/mL. The method has shown excellent linearity over the concentration range with r = 0.9996.
TNominal Concentration (pg/mL)
Desmopressin Sample
LLOQ QC
LQC
MQC
HQC
0.502 1.492 40.692 81.384
1 0.533 1.593 38.277 80.806
2 0.538 1.727 39.465 76.760
3 0.587 1.678 39.678 78.936
4 0.526 1.520 39.294 80.516
5 0.562 1.587 39.013 80.937
6 0.560 1.627 39.076 80.471
7 0.519 1.590 39.191 79.548
8 0.491 1.283 40.359 82.140
9 0.490 1.509 39.486 78.094
10 0.571 1.436 39.828 78.854
11 0.526 1.387 40.624 78.472
12 0.680 1.319 40.355 79.635
13 0.418 1.364 41.098 79.992
14 0.602 1.446 39.814 80.103
15 0.520 1.399 40.988 79.854
16 0.463 1.350 39.391 80.937
17 0.563 1.274 39.960 80.577
18 0.528 1.332 39.188 82.162
Mean
0.5376
1.4678
39.7269
79.9330
S.D (+/-) 0.05691 0.14051 0.73499 1.36511
C.V. (%)
10.56
9.59
1.85
1.71
% Nominal 107.57 98.53 97.64 98.21
N
18
18
18
18
Table 3: Precision and accuracy calculations for individual batches of desmopressin samples.
Table 4: Mean precision and accuracy calculations for desmopressin for three batches of measurements from different days.
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For Research Use Only. Not for use in diagnostic procedures.
LQC RESPONSE
MQC RESPONSE
HQC RESPONSE
Sample ID
Extracted
Unextracted
Extracted
Unextracted
Extracted
Unextracted
01, 013
5,923 6,282
145,169 148,017
292,234 290,315
02, 014
6,645 6,157
135,153 145,270
267,547 291,138
03, 015
6,572 6,319
145,616 153,630
302,953 288,730
04, 016
5,879 6,446
141,823 156,298
270,214 318,201
05, 017
5,823 6,072
122,242 154,238
240,994 304,383
06, 018
5,263 5,825
114,853 146,425
260,462 287,647
Mean
6,017.5 6,183.5
134142.7 150,646.3
272,400.7 296,735.7
S.D
517.00
218.46
12860.35
4,634.26
22,289.26
12,153.15
C.V
8.59 3.53
9.59 3.08
8.18 4.10
N
6
6
6
6
6
6
% Recovery
97.32
89.04
91.80
Mean
92.72
SD (+/-)
4.216
CV (%)
4.547
N
3
Table 5: Recovery of desmopressin from plasma at three different concentrations, LQC, MQC and HQC, was 92.72%.
The percent recovery and plasma matrix effect were evaluated by comparing the peak areas for standard curve samples with and without plasma (Table 5). The mean percent recovery was calculated to be 93%. The recovery of the internal standard was calculated to be 78% (Table 6).
Conclusions
A highly sensitive and high-throughput bioanalytical method was developed and validated for the detection of ultra-low-levels of the therapeutic peptide, desmopressin, in human plasma on the AB SCIEX Triple QuadM 6500 LC/MS/MS System.
Method sensitivity for desmopressin detection was exceptional (0.5 pg/ml or 2.5 fg on column), and demonstrated high- reproducibility and cost effectiveness with good precision
and accuracy.
Analyte recovery is 92.7%, even under high-throughput conditions.
Total run time for each sample was only 5 min, using a ow rate rapid enough for high-throughput analysis in the bioanalytical laboratory.
Acknowledgements
The authors are indebted to Dr. Venu Madhav, Chief Operating Of cer (COO), Veeda Clinical Research, India, for his encouragement and support for the successful completion of the work.
Table 6: Recovery for desmopressin-d5 from plasma at the MQC level was 77.89%. References
1 Friedman, F and Weiss JP. “Desmopressin in the treatment of nocturia: clinical evidence and experience.”Therapeutic Advances in Urology. 2013; 5(6): 310-317.
2 Neudert, L, Zaugg, M, Wood, S, Struwe, P. “A high sensitivity dual solid phase extraction LC/ MS/MS assay for the determination of the therapeutic peptide desmopressin in human plasma.” Celerion white paper.
PA Batch No. 03
Sample ID
Extracted (MQC)
Unextracted
01, 013 251,778 301,602
02, 014 241,864 297,780
03, 015 253,224 305,778
04, 016 256,487 313,985
05, 017 217,971 316,703
06, 018 208,773 300,178
Mean 238,349.5 306,004.3
S.D 20,164.07 773,5.25
% C.V. 8.46 2.53
N66
% Recovery 77.89
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25
ULTIMATE SENSITIVITY


26
High-Sensitivity Quanti cation of the Triptorelin Decapeptide using the
QTRAP® 6500 System
IonDriveTM Technology delivers improved sensitivity for peptide detection under high- ow conditions on a hybrid triple quadrupole/linear ion trap mass spectrometer
Kelli Jonakin1, Simon Wood2, Laurence Meunier2
1AB SCIEX, USA, 2Celerion, Switzerland
Key challenges of high-throughput peptide quanti cation in plasma
Low accuracy – Detection of triptorelin in plasma at higher accuracy is needed for drug development regulatory requirements and therapeutic monitoring.
Diminished sensitivity – Detection at low pg/mL levels is challenging in complex matrices.
Substandard data quality – Precision and accuracy are compromised at low peptide levels, giving results below accepted bioanalytical standards.
Key bene ts of IonDriveTM Technology for high-throughput peptide quanti cation
Excellent linearity – Dynamic range was linear over a wide peptide concentration in a complex matrix.
Ultrasensitive method – Triptorelin LLOQs of 5 pg/mL on the QTRAP® 6500 System were improved 8-fold over those obtained on the 5500.
Accurate and precise measurements – Data quality met or exceeded validation criteria over the standard curve range.
Key features of IonDriveTM Technology for high-throughput peptide quanti cation
IonDriveTM QJet Ion Guide – Increased ion sampling improves method ef ciency and ruggedness.
IonDriveTM High Energy Detector – New detector technology boosts dynamic range and sensitivity.
Mass range of m/z 5 – 2,000 – Comprehensive mass range provides the versatility needed for peptide quant.
Ion DriveTM Turbo V Source – Increased ionization ef ciency and heat transfer contribute to sensitivity enhancements, including improved signal-to-noise.
Laurence Meunier
Simon Wood
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Figure 1: IonDriveTM Turbo V Source and IonDriveTM QJet Ion Guide. High ef ciency heaters provide ef cient desolvation and ion production for high sensitivity at high ow rates. Dual RF stages maximize ion sampling from a large ori ce while increasing ion transfer ef ciency into the Q0 region – without increasing vacuum load in the analyzer region.
TTTTTULTIMATE SENSITIVITY


Materials and methods
Sample preparation
A standard curve for the triptorelin peptide in rat plasma was prepared over a concentration range of 0.005–4.0 ng/mL. Samples underwent solid phase extraction (SPE), were dried down to completion, and then were reconstituted in H2O/10% MeOH/0.02% acetic acid (100 μL).
Chromatography
Direct injection work ow
LC system: Column:
Guard column: Column temp.: Injection:
Flow rate: Mobile phase:
Gradient:
Shimadzu LC20AD LC System
Ascentis Express Peptide ES C18, 2.7 μm (2.1 x 50 mm, Sigma Aldrich)
25 °C
20 μL
300 μL/min
A) water
B) methanol, 0.02% acetic acid
Time %B 0 15 1.2 60 1.8 60 1.81 90 2.3 90 2.31 15 4 15
For Research Use Only. Not for use in diagnostic procedures.
www.absciex.com THERAPEUTIC PEPTIDE BIOANALYSIS
Figure 2: Chemical structure of triptorelin (MW = 1311.5 g/mol).
Introduction
Very high levels of sensitivity at high- ow rates have been achieved for peptide analysis on the AB SCIEX 6500 Series of mass spectrometers, and new methods on these instruments have demonstrated long-term robustness during multi-sample runs. Integral to the enhanced levels of sensitivity obtained
on the 6500 Series, the new IonDriveM Technology (Figure 1) boosts ionization ef ciency, sampling, and transmission of ions, driving up the detector dynamic range, improving signal-to-noise measurements, and accelerating scan speeds. The new heater design of the IonDriveM Turbo V Source improves desolvation and ion production, allowing for high-levels of sensitivity even at high ow rates. The innovative, dual QJet® Ion Guide increases the ef ciency of ion transmission while maintaining simplicity and robustness. Together, these advances in detector technology and ion transmission augment the capacity for sensitivity in any peptide quantitation method.
Triptorelin, a ten-amino-acid synthetic peptide, is a gonadotropin- releasing hormone agonist (GnRH agonist) used in the treatment of hormone-responsive cancers such as prostate cancer and breast cancer (Figure 2). Used in men, triptorelin reduces the amount
of testosterone in the blood, which limits the growth of prostate cancer. When given to women, triptorelin reduces the production of estrogen. Many clinical and pharmaceutical applications
require low-level detection of triptorelin, and the demands of processing multiple biological samples requires a robust and high- throughput strategy. Here, we demonstrate a LC/MS/MS detection strategy for monitoring low levels of triptorelin in rat and human plasma at pg/mL levels using a QTRAP® 6500 System and fast chromatographic ow rates for sample run times of <5 minutes.
Table 1. Chromatography gradient
Mass spectrometry
A QTRAP 6500 System equipped with an IonDriveM Turbo V Source was operated in positive ESI mode. The MRM transition monitored for triptorelin was m/z 656.5/249.1 at a dwell time of 50 ms. Source and compound dependent parameters are shown in Table 2. Five replicate injections were performed at all concentrations.
Sensitivity was compared to previously obtained data from the QTRAP® 5500 System (courtesy of Celerion) using identical chromatography and MS methods. Source conditions on the IonDriveM Turbo V Source were slightly altered compared to those optimized for the Turbo VM Source on the 5500 System (Table 2).
27
ULTIMATE SENSITIVITY


28
Instrument Parameter
QTRAP® 6500 System
QTRAP® 5500 System
CUR 25 30
TEM 500 700
ISV 5500 5000
GS1 50 50
GS2 60 60
CAD 11 High
DP 90 180
EP 10 10
CE 39 39
CXP 12 12
QTRAP® 6500 System
QTRAP® 5500 System
Conc. (ng/mL)
On-Column (fmol)
Accuracy (%)
% Coef cient of Variation
Accuracy (%)
% Coef cient of Variation
0.005 0.038 97.1 12.2 – –
0.01 0.076 106.9 6.7 – –
0.02 0.152 98.9 7.0 – –
0.04 0.305 93.4 9.1 102.8 9.7
0.08 0.61 106.9 0.9 93.8 2.1
0.2 1.52 106.5 0.5 96.7 4.0
0.8 3.05 98.6 1.2 99.8 1.3
1.6 12.2 97.3 1.2 99 0.15
4 30.5 94.4 3.3 103.4 4.0
Table 2: Source and compound-dependent parameters for the QTRAP® 6500 System and the QTRAP® 5500 System.
Data processing
All QTRAP 6500 System data was processed using MultiQuantTM Software and the SignalFinderTM Algorithm. The concentration curves were analyzed using a linear t with a 1/x2 weighting. Data obtained on the QTRAP 5500 System was processed using the quantitation tools within Analyst® Software.
Measuring triptorelin sensitivity in rat plasma
This triptorelin quanti cation method was initially optimized and validated on the QTRAP® 5500 System. A concentration curve
was constructed from peak area measurements of peptide in rat plasma matrix. An LLOQ of 40 pg/mL was obtained for triptorelin (on column) on the QTRAP 5500 System; assessments of the data quality demonstrated a %CV of 9.7% and an accuracy of 102.8%. Figure 3 shows a representative chromatogram at the LLOQ of 40 pg/mL (0.305 fmol on column) from the QTRAP 5500 System.
A comparable chromatographic strategy was translated to the QTRAP 6500 System to evaluate sensitivities obtained on both instruments for peptide quantitation. The signal at the lower limit of quanti cation for triptorelin in plasma (n = 5) is shown in Figure 4, with very good reproducibility and S/N. The LLOQ obtained with this method was 5 pg/mL (0.038 fmol of triptorelin on column). The matrix blank and signal at the determined LLOQ (5 pg/mL) can be found in Figure 5.
The data (Figure 4) showed excellent linearity across the concentration curve range analyzed, from 5 pg/mL to 4 ng/mL on column. The statistics for this analysis are shown in Table 3. The coef cients of variation (%CV) and the accuracies of the curve fall well within commonly accepted bioanalytical validation criteria throughout the range of concentrations measured.
The observed sensitivity increase for this assay in rat plasma on the QTRAP 6500 System (5 pg/mL) was found to be ~8x improved over the QTRAP 5500 System (40 pg/mL) (Table 3).
Table 3: Statistics for the quanti cation of triptorelin in buffer. Replicates (n = 5) were run at every concentration, and the statistics for accuracy and precision were computed. The LLOQ for triptorelin on the QTRAP® 6500 System (bold) was 0.038 fmol of the molecule on column (5 pg/ml), 8-fold improved over the LLOQ obtained on the QTRAP® 5500 System at 0.305 fmol on column (40 pg/mL).
Figure 4: Quanti cation of triptorelin on the QTRAP® 6500 System. (Top) The signal measured at the LLOQ (5 pg/mL) in buffer is shown (one replicate, 0.038 fmol on column) with a CV of 12.2%. (Bottom) Using linear (1/x2) regression, good accuracy was achieved across the range of concentrations analyzed. Replicate triptorelin measurements (n= 5) were well within accepted bioanalytical method validation criteria (statistics are shown in Table 3).
TTRUO-MKT-02-1486-A
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ULTIMATE SENSITIVITY


Figure 5: Detection of triptorelin on the QTRAP® 6500 System. Peak areas for triptorelin present in the matrix at 0 pg/mL (left) and at the LLOQ of 5 pg/mL (right).
Conclusions
High-throughput peptide quantitation at high sensitivity was performed on the AB SCIEX QTRAP® 6500 System equipped with IonDriveM Technology.
Ultra-low levels of triptorelin were detected in plasma on the QTRAP® 6500 System under high- ow conditions – giving an LLOQ of 5 pg/mL (0.038 fmol of peptide on column).
The high- ow data obtained on the QTRAP 6500 System provided detection limits for triptorelin of 5 pg/mL – an ~8x improvement in LLOQs previously developed on the QTRAP 5500 System.
References
1 The AB SCIEX Triple QuadM 6500 and QTRAP® 6500 Systems for Bioanalysis – A New Level of Sensitivity, AB SCIEX Technical Note, Publication 5780212-0
For Research Use Only. Not for use in diagnostic procedures.
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THERAPEUTIC PEPTIDE BIOANALYSIS
29
ULTIMATE SENSITIVITY


30
High Resolution Time-of-Flight for High Quality Quantitative Analysis
Yves Leblanc
1AB SCIEX, Concord, ON, Canada
For many years, quantitative analysis were carried on triple quadrupole (QqQ) MS instruments, while qualitative analysis would be carried on instruments with high resolution and mass accuracy, such as Q-ToF MS systems. As laboratories are looking into improving ef ciency in streamlining their decision making process, research ranging from drug metabolism to proteomics are looking at instrument that would provide both qualitative
and quantitative data simultaneously. As we looked at the attributes of MS system that deliver typical quantitative analysis, their main features are: high duty cycle, high sensitivity, high selectivity and wide dynamic range. In addition to all of the front end development associated with QqQ systems such as the
Turbo VTM source and the QJET® ion optics, key technologies were integrated into the TripleTOF® 5600 system that enabled high performance quantitative analysis. First, operating the accelerator/ pulser at 30kHz provides high duty cycle extraction of the ion beam exiting the collision cell. To match this capability, a 40-GHz multichannel TDC detection system that ensures high rate data collection was integrated into the system. Secondly, operating
at 15 kV TOF acceleration voltage with high transmission grids (~92% transparency) assisted in maintaining a great deal of
the sensitivity gains from the front end changes. Both of these technologies ensure high ef ciency extraction of the ion beam
to provide high sensitivity. On the qualitative front, it was also important to improve the performance of the system in terms of resolution as well as mass accuracy. To improve resolution above 30,000 resolution, the ion optic was optimized to transfer ions with coherent ion trajectories in the pulser region over a distance of 2.5m. And nally, the last key attribute of the system was to ensure to maintain mass accuracy <2ppm RMS over long periods of analysis. In the case of the TripleTOF® 5600 system, this is done in a 2 step fashion: rst the mass accuracy is established
via scheduled introduction of calibrant as part of batch, and secondly the mass precision is maintained by dynamic monitoring of background ions that are determined adaptively to analysis conditions.
Yves Leblanc
Figure 1: Analysis of verapamil in diluted urine samples (2x). Data was collected at 30K resolution in MS mode. The width of the XIC was set to mimic single quadrupole analysis (XIC = 0.7 Da) and high resolution mode (XIC = 10mDa)
With the ability to collect data at rates as high as 50 Hz in either MS or MSMS mode, the TripleTOF® 5600 system offers unique way to support both qualitative and quantitative analysis. First and foremost, compatibility with UPLC separation where the system can easily be set to collect more than 12 data points across most LC peaks. Secondly, the high resolution (>30K) and the high mass stability (<2ppm rms) provides the ability to extract narrow ion chromatogram (<10mDa) to achieve selectivity in MS mode that
is comparable to MRM analysis on QqQ systems. The advantage of this approach is that generic data acquisition can be used
1.0 2.0
Time, min
1.0 2.0
Time, min
XIC Peak Width
0.7 Da (Unit resolution)
3.0 4.0
10 mDa (High resolution)
3.0 4.0
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HIGH RESOLUTION ACCURATE MASS


Figure 2: Isotope distribution of neuromedin U for the dominant charge state (Z=+3 and Z=+4). When the highlighted isotopes are extracted and combined, then 91% of the signal association with the peptide, thus improving the overall sensitivity of the system.
for analysis, thus the need for tuning. Equally important is the ability to obtained reliable peak area from narrow-XIC which is indicative of the instrument stability in terms of mass accuracy. Figure 1 shows the bene t of reducing the XIC width in order to gain selectivity in the detection of verapamil in diluted urine. As the XIC width is reduced from unit resolution (0.7Da to mimic quadrupole isolation) to 10mDa, all interferences are eliminated and a single LC peak is detected. This approach can be further extended to peptide analysis by summing the signal associated with both the charge state distribution as well as the isotope distribution for each ion. Figure 2 shows the observed isotope distribution associated with the +3 an +4 ions of neuromedin U (NMU, seq.: YKVNEYQGPVAPSGGFFLFRPRN). As can be seen here, the isotope contributes to a large portion of the ion signal and each one of them can be combined to give the appropriate S/N for selectivity and proper detection of LC peaks. For neuromedin U spiked in protein precipitated, considering the top 4 isotopes, more than 80% of the signal can be captured for detection. This approach was shown to improve linearity and precision for the detection of NMU.
An additional bene t of the TripleTOF® 5600 system is the ability to record MSMS spectra that can also be processed post-LC.
This mode of operation is referred to as MRM-HR from which selective fragment(s) can be used as representative of the peptide LC peak. Figure 4 shows an example associated with the ability to extract multiple fragment ions associated a given peptide, in this particular case a phosphorylated peptide. This also provides the ability to compare the experimental mass spectrum to library entries to ensure the proper peptide was detected.
Figure 3: Neuromedin U spiked into protein precipitated plasma. For each charge states, the top 4 isotopes were extracted and summed for each charge states. The 3 charge states can be also be combined to further improve the sensitivity as little noise is captured in the process.
Figure 4: Narrow XIC associated with dominant fragment masses of the illustrated peptides. The full scan mass spectrum can also be compared to library spectrum to gain further con dence in the detection of the peptide.
For Research Use Only. Not for use in diagnostic procedures.
Z = +4
Z = +3
+4
1388 1000 500
0 0
SUMMED top 4 Isotope
1.0 2.0 3.0 4.0 5.0 Time, min
+3
565 400 200
0 0
1.0 2.0 3.0 4.0 5.0 Time, min
+2
51 40
20 0
0 1.0
2.0 3.0 4.0 5.0 Time, min
[PGQ]-QSPASPPPLGGGAPVR [email protected]
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31
HIGH RESOLUTION ACCURATE MASS


32
Ultrasensitive Quantitation of Exenatide Using
Micro ow Liquid Chromatography Systems
and High-Resolution Mass Spectrometry
A high resolution multiple reaction monitoring (MRMHR) method for low-level peptide quantitation developed on an AB SCIEX TripleTOF® 5600 LC/MS/MS System coupled with Eksigent ekspertTM micro ow ultrahigh pressure liquid chromatography (μUHPLC) systems
Leo Jinyuan Wang1, Daniel Warren2, and Anthony Romanelli2
1AB SCIEX, Redwood City, CA; 2AB SCIEX, Framingham, MA;
Key challenges of exenatide quantitation in biological samples
Insuf cient sensitivity – The best, previously reported LOQ is 100 pg/mL; extended-release pharmacokinetic studies demand lower levels of detection.
Limited quantitation range – Analytical range of ELISA-based method is <2 orders of magnitude; at least 3 orders of magnitude is desired in bioanalysis.
Low speci city – Complex biological matrices hamper data resolution and require sophisticated sample preparation and/or advanced instrumentation.
Systematic measurement errors – Especially for ultra-low level quantitation, measurement errors have a signi cant effect on data accuracy and precision.
Key bene ts of MicroLC and MRMHR work ow
Robust sensitivity – Up to 10-fold better than the best reported result (LLOQ of 10 pg/mL using the nanoLC trap-and-elute method) over a linear range 3 orders in magnitude.
Improved speci city – Background noise is signi cantly reduced with MRMHR offering the high selectivity.
Excellent accuracy and precision – %CV <14% across the whole analytical range; deviation of accuracy <18% including LOQ.
Reduced operating costs – MicroLC usage results in >90% savings on solvents and waste disposal.
Key features of MicroLC and MRMHR work ow
Dedicated micro ow UHPLC system Seamlessly integrated microLC ESI interface
MRM-like quantitation – with high resolution fragment ion spectrum.
Fast cycle times – compatible with UHPLC speed
Wider linear dynamic range of 4 orders – with TripleTOF®
5600 system
RUO-MKT-02-1486-A
THERAPEUTIC PEPTIDE BIOANALYSIS
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TripleTOF® 5600 System Eksigent ekspertTM microLC μUHPLC Optimized Micro Flow ESI Source
HIGH RESOLUTION ACCURATE MASS


For Research Use Only. Not for use in diagnostic procedures.
Introduction
Exenatide, marketed as Byetta and Bydureon, is an effective medication for treating type 2 diabetes. This 39-amino-acid peptide is a synthetic version of exendin-4, a hormone excreted in Gila monster saliva. Exenatide displays biological properties similar to human glucagon-like peptide-1 (GLP-1), a regulator of glucose metabolism and insulin secretion. While exenatide shares 50% amino acid homology with GLP-1, exenatide is more resistant to metabolic degradation and, thus, has a therapeutic advantage due to its longer pharmacological half-life.
Reported methods for quantifying exenatide in plasma include immunoassay (ELISA)1,2 and liquid chromatography mass spectrometry (LC/MS).3,4 Quantitation by immunoassay suffers from limited analytical range, endogenous interferences, lack
of reproducibility and speci city, and a requirement for expensive antibodies. Alternatively, LC/MS detection methods have excellent selectivity (when using MS/MS or high resolution MS/MS (MS/MSHR)), high sensitivity, a wide analytical range (usually greater than 3 orders of magnitude), and good reproducibility. Currently published MS/MS methods report adequate sensitivity for exenatide quantitation (LLOQs of 100 pg/mL).3 These levels may be suf cient to quantify quick-
release exenatide formulations in plasma (i.e., Byetta); however, quantifying ultra-low levels of the slow-release formulation in plasma remains challenging.
Micro ow ultra-high-performance liquid chromatography (μUHPLC) has gained substantial popularity for peptide detection, where low sensitivity, slower throughput, and rising operational costs have been constant challenges. The advantages
of μUHPLC/MS methods over high ow LC include approximately 14-fold sensitivity gain5, fast gradient separation, reduced source contamination, and shrinking solvent consumption and
disposal costs.6
We developed an ultra-sensitive μUHPLC-MRMHR method suitable for quantitating low-level exenatide in plasma, resulting in an LLOQ of 10 pg/mL that gives excellent linearity, accuracy, and precision. In addition, two injection work ows were compared to determine the impact of sample handling on sensitivity:
1) a direct injection method on the ekspert microLC 200 System and 2) a large-volume injection method combined with a trap- and-elute protocol on the ekspert nanoLC 425 System.
Materials and methods
Sample preparation
Proteins were precipitated from of plasma by mixing plasma (1 mL), acetonitrile (3 mL), and formic acid (0.4 mL). After vortexing for 15 sec, the mixture was centrifuged (4000 rpm for 15 min.). The supernatant was transferred and stored at -20 °C.
Exenatide stocks and isotope-labeled internal standard were kindly supplied by GlaxoSmithKline. Stock solutions were prepared in 20% acetonitrile and diluted in rat plasma in series from 1–100 ng/mL. For the trap and elute work ow, multiple concentrations of calibration standards were prepared as follows: 10, 20, 50, 100, 200, 500, 1000, and 2000 pg/mL. Dilutions were spiked with internal standard (1 ng/ml) and 20% rat plasma. For the direct injection work ow, calibration standards were prepared in rat plasma from 50–50,000 pg/mL (10 concentrations).
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34
Trap-and-elute work ow
To analyze samples using the trap-and-elute work ow, two gradient pumps (set for 5-50 μL/min ow rates) and two valves (6-port, 2-position injection valve and 10-port, 2-position switching valve) were con gured for synchronized sample loading, transferring, injection, and elution. The detailed ow diagram is illustrated in Figure 1.
TLC system:
Eksigent ekspert nanoLC 425 μUHPLC with 5–50 μL/min ow modules (2x)
Eksigent ChromXP C18CL, 0.5 × 50 mm, 3 μm C18, 0.5 × 5 mm, 5 μm
ambient
50 μL (50 μL loop with 60 μL loading volume)
Figure 1: Trap-and-elute work ow ow diagram
μUHPLC-MRMHR analysis
Direct injection analysis was performed using an Eksigent ekspertTM microLC 200 System; and the large volume injection analysis
with the trap-and-elute work ow was performed on an ekspert nanoLC 425 System. A high-resolution quadrupole time-of- ight mass spectrometer, the AB SCIEX TripleTOF® 5600 System, was employed to capitalize on the instrument’s high speci city with analytes in biological samples. The parameters and instrument con gurations are listed as follows:
Chromatography
Direct injection work ow
Column:
Trap column:
Column temp.:
Injection:
Gradient 1: Loading pump
Flow rate: Mobile phase:
Gradient 1:
50 μL
A) water, 0.1% formic acid
B) acetonitrile/isopropanol (1:1),
1% tri uoroethanol, 0.1% formic acid
Gradient 2: Eluting pump
Time/min A%
0 100 0
B% 1.5 100 0
1.6 0
5.5 0 100
100
5.6 100
6.0 100
0
0
LC system:
Column: Guard column: Column temp.: Injection:
Flow rate: Mobile phase:
Gradient:
Eksigent ekspert microLC 200 μUHPLC with 20–200 μL/min ow module
Eksigent ChromXPTM C18CL, 1 × 50 mm, 3 μm C18, 1 × 5 mm, 5 μm
40 °C
10 μL (10 μL loop with 20 μL loading volume) 150 μL
A) water, 0.1% formic acid
B) acetonitrile, 0.1% formic acid
Time/min A% B% 0 95 5 0.5 95 5
2 10 90
4 10 90 4.1 95 5 5.0 95 5
In-line lter:
Micro lter with 1 μm titanium frit for 1/32” PEEKsil tubing
Gradient 2:
Time/min A%
0 95 5
0.5 95 5 2 10 90
B%
4 10 4.1 95 5.0 95
40 μL
90 5 5
Flow rate: Mobile phase:
A) water, 0.1% formic acid
B) acetonitrile, 0.1% formic acid
Chromatography and MS synchronization was achieved with the nanoLC 400 System autosampler method detailed below:
Initialize Valve
Autosampler device
Switch injection valve to load
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Valve
Wait
Needle wash Wait
Wait
Get sample
Wait
Start
Valve
Wait
Start
Valve
Wait
Valve
Needle wash
Needle wash
Mass spectrometry
System: Interface:
Ion source gas 1 (GS1): Ion source gas 2 (GS2): Curtain gas (CUR): Temperature (TEM): Ion spray voltage Floating (ISVF):
Scan type:
Scan experiment 1:
Scan experiment 2:
Accumulation time: Declustering potential: Collision energy: Collision energy spread: Calibration:
Switch ISS-A valve to load (10-1) for 1 sec
Pre-wash 1x using wash solvent 1 for gradient 2 to be ready
for gradient 1 to be ready
Fill loop 60 μL: 2 mm from bottom at 2 μL/s
for 5 sec
Start gradient 1
Injector inject
for 1 min 30 sec
Gradient pump 2
Switch ISS-A valve to inject (1-2)
for 1 min 40 sec
Switch ISS-A valve to load (10-1)
Wash 2x cycles, inner wash solvent 2, outer wash solvent 2
Wash 2x cycles, inner wash solvent 1, outer wash solvent 1
TripleTOF 5600+ System
DuoSprayM Ion Source with 65 μm electrode
55
60
20 650 °C 5500
TOF Product Ion Scan of 833 and 844
220–420 with high sensitivity mode and enhanced mass 396.2
220–420 with high sensitivity mode and enhanced mass 402.2
0.15 sec for each scan experiment 141
30
5
Automated calibration with CDS
For Research Use Only. Not for use in diagnostic procedures.
Figure 2: Typical chromatograms and TOF MS/MS spectra at 100 pg/mL exenatide in plasma.
Figure 3A: Chromatograms of low-level exenatide in plasma using the nano ow- based, trap-and-elute work ow.
Figure 3B: Chromatograms of low-level exenatide in plasma using the micro ow- based direct injection work ow.
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36
Effective System Cleaning
Injection #2: Exenatide 3333 pg/mL in Matrix
Injection #3: 1st Wash Solution (autosampler strong wash solvent) Injection #4: 2nd Wash Solution
Injection #5: 1st Blank Matrix
Injection #6: 2nd Blank Matrix
Injection #7: 3rd Blank Matrix
Figure 4: Effectiveness of the system cleaning protocol between sample runs. Data processing
The MRMHR transitions for exenatide quantitation were 838/396 and 838/299. These transitions were determined to be optimal based on MS/MS analysis (not shown). Data were quantitated using MultiQuantTM Software.
Results and discussion
μUHPLC-MRMHR method development
An ultrasensitive method for quantitating ultra-low levels of exenatide in plasma was developed using high-resolution mass spectrometry, relying on the quantitation of peaks obtained from MRM scans speci c to the intact peptide (or fragment of the peptide?). To evaluate the effects of different sample injection methods and ow rates on the sensitivity of the exenatide detection method, two chromatography work ows were
created: 1) a simple, direct-injection, high-throughput work ow was developed on a more affordable microLC 200 system that produced excellent sensitivity (LLOQ of 50 pg/mL); and 2) a sophisticated, exible, large-volume-injection, trap-and-elute work ow was established on a nanoLC 425 system that produced the best sensitivity between the two systems (LLOQ of 10 pg/mL).
A representative total ion chromatogram (TIC) of exenatide (100 pg/mL) in plasma (analyzed using the nano ow trap-and- elute work ow) is shown in Figure 2. Peaks eluting at 1.3 min were extracted for MS/MS analysis, and MRMHR transitions
of 838/396 and 838/299 were followed, providing excellent speci city. To ensure high quality data, it was only necessary to acquire only two MRMHR experiments due to the high resolution achieved using the narrow MS/MS scan windows.
Ultra-low level amounts of exenatide can be reliably quantitated by the nano ow-based, trap-and-elute work ow (Figure 3A) and by the micro ow-based, direct-injection work ow (Figure 3B) using the selected MRMHR transitions. Exenatide detection in plasma using both the nano ow- and micro ow-based protocols reveals the maintenance of peak shape over a wide range of concentrations. Additionally, the detector response increases
Figure 5A: Calibration curve with exenatide concentration data obtained from the direct injection work ow.
Figure 5B: Calibration curve developed from exenatide concentration data obtained using the trap-and-elute work ow.
proportionally with increasing concentrations of exenatide for both chromatography methods.
Peptide residue removal between sample runs
Ensuring an uncontaminated system after repeated sample exposure is critical for accurate quantitation of ultra-low levels of peptides. An exceptionally hydrophobic peptide, exenatide sticks persistently to the sampling path and the column’s stationary phase, and a strong, organic wash (see method) is required to effectively remove exenatide from needle surfaces and sampling paths. Following the injection of a high concentration of sample (3333 pg/mL, Figure 4), the exenatide residue was quickly reduced to a very low level after three injections of wash solution (<2 pg/ mL based on estimated peak height) and eliminated completely after ve injections.
Injection #1: Blank Matrix
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For Research Use Only. Not for use in diagnostic procedures.
Direct Injection Work ow
Trap-and-Elute Work ow
Concentration (ng/mL)
Mean
%CV
%Accuracy
Concentration (pg/mL)
Mean
%CV
%Accuracy
0.05 0.045 5.8 90.3
10 10.6 12.2 106
0.1 0.086 10.1 86
20 18.2 13.2 91.2
0.25 0.23 6.62 90.7
50 45 6.84 90
0.5 4.33 1.18 86.6
100 103 6.6 103
1 1.18 1.55 118
200 196 6.47 98
2.5 2.59 5.45 103
500 447 8.84 89.5
5 5.17 3.96 103
1000 1124 6.23 112
10 11.6 2.72 116
2000 2190 4.71 109
25 27.1 1.3 108
50 47.9 2.33 95.8
Direct Injection
Trap-and-Elute
LLOQ (pg/mL) 50 10
Injection Volume (μL) 10 50
Linear Range (pg/mL) 50–50,000 10–2,000
%CV <11% <14%
Accuracy 86%–118% 89.5%–112%
Run Time 6 min 10 min
Table 1: Summary of Quantitation Performance. Assessment of quantitative results
Exenatide quantitation methodology was evaluated for sensitivity, linearity, accuracy, and precision for both the direct injection and trap-and-elute chromatography work ows.
Sensitivity was de ned by the LLOQ achieved – the lowest concentration in calibration standards that satis es both signal- to-noise (S/N >10) and statistical requirements (precision and accuracy with <20% deviation). The LLOQ from the direct- injection method was determined to be 50 pg/mL with a dynamic range of 50–50,000 pg/mL (Figure 5A). The trap-and-elute work ow signi cantly reduced the LLOQ to 10 pg/mL (Figure 5B), enabling exceptionally low-level analysis of exenatide in plasma within a dynamic range of 10–2,000 pg/mL. An excellent linear response across a wide concentration range was demonstrated for both chromatography work ows (Figure 5A and 5B).
Both micro ow methods demonstrated good data reproducibility and accuracy within an acceptable experimental range, and
peak areas were calculated from multiple injections at each concentration (n>3). Method precision and accuracy are summarized in Table 1. For the direct-injection work ow, the %CV was observed within 11%, and data accuracy ranged from 86–118%. For trap-and-elute work ow, %CV was observed within 14%, and accuracy was from 89.5–112%.
Table 2: Comparison of microLC work ow features for exenatide quantitation
Conclusions
A high-resolution method for peptide quantitation was developed for the TripleTOF® 5600 system using a trap-and-elute, micro ow work ow (on an Eksigent ekspert nanoLC 425 System) that provided an extremely reproducible and highly-sensitive method for the ultra-low-level quantitation of exenatide
(LLOQ = 10 pg/mL). An additional micro ow direct injection work ow (on the ekspert microLC 200) was created for a higher-throughput, but less sensitive method of exenatide quantitation (LLOQ = 50 pg/mL).
References
1 Fineman, M.; Flanagan, S.; Taylor, K.; Aisporna, M.; Shen, L.; Mace, K.; Walsh, B.; Diamant, M.; Cirincione, B.; Kothare, P.; Li, W.-I.; MacConell, L. Pharmacokinetics and pharmacodynamics of exenatide extended-release after single and multiple dosing. Clin Pharmacokinet. 2011, 50 (1), 65-74.
2 Lin, Y.-Q.; Khetarpal, R.; Zhang, Y.; Song, H.; Li, S. S. Combination of ELISA and dried blood spot technique for the quanti cation of large molecules using exenatide as a model. Journal of Pharmacological and Toxicological Methods. 2011, 64 (2), 124-128.
3 Zhang, J.-F.; Sha, C.-J.; Sun, Y.; Gai, Y.-Y.; Sun, J.-Y.; Han, J.-B.; Shao, X.; Sha, C.-N.; Li, Y.-X.; Liu, W.-H., Ultra-high-performance liquid chromatography for the determination of exenatide in monkey plasma by tandem quadrupole mass spectrometry. Journal of Pharmaceutical Analysis. 2013, 3 (4), 235-240.
4 Kehler, J. R.; Bowen, C. L.; Boram, S. L.; Evans, C. A. Application of DBS for quantitative assessment of the peptide Exendin-4; comparison of plasma and DBS method by UHPLC–MS/MS. Bioanalysis. 2010, 2 (8), 1461-1468.
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THERAPEUTIC PEPTIDE BIOANALYSIS
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Christianson, C. C.; Johnson, C. J. L.; Needham, S. R. The advantages of micro ow LC–MS/MS compared with conventional HPLC–MS/MS for the analysis of methotrexate from human plasma. Bioanalysis. 2013, 5 (11), 1387-1396.
Yang, M.; Gong, X.; Schafer, W.; Arnold, D.; Welch, C. J. Evaluation of micro ultrahigh pressure
6
liquid chromatography for pharmaceutical analysis. Analytical Methods. 2013, 5 (9), 2178.
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Investigating Biological Variation of Liver Enzymes in Human Hepatocytes
MS/MSALL with SWATHTM Acquisition on the TripleTOF® Systems
Xu Wang1, Hui Zhang2, Christie Hunter1 1AB SCIEX, USA, 2Pfizer, USA
MS/MSALL with SWATHTM Acquisition is a data independent work ow, performed on the TripleTOF® 5600+ system, which is of great interest today as it provides a higher level of reproducibility and comprehensiveness in proteomics data1. In a SWATH acquisition experiment, a wide Q1 isolation window is stepped through the precursor mass range, transmitting multiple analytes into the collision cell. The transmitted ions from each step are fragmented and a composite MS/MS spectrum is measured in
the TOF MS Analyzer at high speed and high resolution. Post- acquisition, the peptides of interest are quanti ed by generating fragment ion extracted ion chromatograms (XICs) and measuring their peak areas.
For drug development, understanding the protein expression levels of drug metabolizing enzymes responsible for phase I
and II bio-transformations (Figure 1) is a fundamental aspect of assessing drug-drug interactions, and evaluating drug safety and ef cacy. Targeted quantitation using multiple-reaction-monitoring (MRM) has been used to quantitatively pro le these enzymes in liver hepatocytes or microsomes. However, an MRM experiment normally focuses on a limited set of selected proteins for quantitation and requires signi cant upfront assay development work. In this work, the MS/MSALL with SWATHTM Acquisition method was used to analyze large numbers of proteins and multiple enzyme families involved in drug metabolism.
Key Advantages of Targeted Quantitation using MS/MSALL with SWATHTM Acquisition
• High quality protein quantitation strategy for biological samples
• ‘MRM-like’ quality quantitation obtained on large numbers of proteins
and peptides
• No method development required to target and quantify large numbers
of proteins and peptides in a single run
• Easy transition to future MRM assays
• Data independent acquisition using the TripleTOF® 5600+ Systems and
MS/MSALL with SWATHTM Acquisition
• High sensitivity and speed of MS/MS acquisition
• Easy data processing using the SWATHTM Acquisition MicroApp in
PeakView® Software
• Post-acquisition extraction of large numbers of high resolution
sequence speci c fragment ions of the targeted peptides and proteins to generate peak areas with high speci city
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ff

Figure 1. Phases of Detoxi cation. Drug metabolism is the biochemical modi cation of xenobiotics or drugs using a specialized enzymatic system. These metabolic pathways modify the chemical structure of the foreign compounds to detoxify and prepare for excretion. Understanding the biological variation across individuals as well as the protein expression changes in response to drug is critical during drug discovery. © Wikipedia.
Figure 2. Experimental Design. Protein extracts from thirteen liver samples were digested in parallel. The pooled sample was created and analyzed using an IDA experiment in order to generate a spectral ion library. The individual samples were analyzed in triplicate using SWATHM Acquisition. Resulting data was then analyzed using targeted data extraction as guided by the ion library.
• Targeted multiple peptides per protein to con rm differential quantitation on proteins from key protein families that share high sequence homology
• High quality, reproducibility LCMS using Eksigent ekspert nanoLC 425 System with cHiPLC® System.
Methods
Sample Preparation: Tissue cells (0.625 million) were resuspended in 1mL extraction buffer from ProteoExtract Native Membrane Protein Extraction Kit (EMD, Billerica, MA) with protease inhibitor. The cells were then lysed for 10 min at 4°C. The lysate was mixed with 250μL 100mM NH4HCO3 /3.6% DOC buffer and shaken for 20 minutes. Reduction agent (50μL of 100 mM DTT) was added and incubated for 10 min at 95 °C to disrupt disul de bonds, followed by alkylation of free sulfhydryl groups with 50μL of 5mM iodoacetamide at room temperature in the dark for 30 min with continuous shaking. Extracted proteins were digested with trypsin (1:50) at 37 °C for 18 hrs. The digestion was stopped with addition of 0.2% formic acid/H2O solution, then
Figure 3. Reproducible Quantitation of Liver Proteins. Quantitation for individual proteins of interest can be easily extracted from the SWATHM acquisition dataset. Shown here are two different esterases, EST1 - liver carboxylesterase 1 and EST2 - cocaine esterase. There is very good agreement between the multiple peptides per protein highlighting the reproducibility of quantitation.
vortexed and centrifuged at 10,000 g for 5 min. The supernatant was transferred to a new eppendorf tube and dried down in speed vacuum for 3hrs at 50°C.
Chromatography: Tryptic digests were separated using an Eksigent ekspert nanoLC 425 with a cHiPLC® column (75 μm x 150 mm, 300 Å pore size ChromXPM column) running a ow
rate of 300 nL/min. The gradient was 90 mins as follows: 5 % B for 2 min, from 5 % B to 50 % B in 60 min, from 50% B to 90
% B in 8 min, 98% B for 5 min, from 98 % B to 5 % B in 5 min, and 5 % B for 10 min. (10 μL sample) Mobile phase A consisted of H2O and 0.1% formic acid, and mobile phase B consisted of acetonitrile and 0.1% formic acid. The column oven was operated at 35 ˚C. Sample injection volume was 10 μL.
For Research Use Only. Not for use in diagnostic procedures.
140 120 100
80 60 40 20
0
EST1FLSDLOGDPR EST1AGQLLSELFTNR EST1EVAFWTNL EST1YLGGTDDTVK
091 098 DAD I2G IDE KMI MRS NQT RML ROE SED VCM YAA
Hepatocyte Samples
140 120 100
80 60 40 20
0
EST2IQELEEPEER EST2FTEEEEQLSR
091 098 DAD I2G IDE KMI MRS NQT RML ROE SED VCM YAA
Hepatocyte Samples
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Lipophilic
Electrophiles R R R O
R — SG Hydrophilic
R — SO3H
R — Ac
R — GI
O R Phase I
Glutathione conjugation
Oxidation
Phase II
Hydrolysis Reduction
R — OH R—SH R—NH2 Nucleophiles
Sulfation Acetylation
Glucuronidation
123
4 9899
...
13 Liver Hepatocyte Samples
Apply Ion Library
39
Parallel Denature & Digest
IDA SWATHM Acquisition
Relative Quantiication (%) Relative Quantiication (%)
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40
Scores for PC1 (27.7%) versus PC2 (19.5%), Pareto MRS
091 091
Loadings for PC1 (27.7%) versus PC2 (19.5%), Pareto CPS1
12000 8000 4000
MRS MRS
0.16
0.12
0.08
0.04
0.00
-0.04
-0.08
-0.12
EPHX1 AGXT
I2G
091
NQT NQT
ACAA2
CYP3A4 ECHS1
GLUD1
PCK2 ALDH1B1
I2G
KMI KMI
KMI
YAA 098
DAD DAD
098
CES1
098 VCM YAA
VCM
VCM
I2G 0
-4000
-8000 -12000 -16000
100
80
60
40
20
0
SED
NQT
IDE IDE IDE
SED SED RML
ARG1
ALDH1A1
ADH1B
Sample (by index)
Figure 4. Analyzing Global Protein Expression Differences Across the Samples. After the data extraction was performed on the SWATH acquisition data, the protein areas were loaded into MarkerViewTM Software for statistical analysis. PCA was performed to obtain the Scores and Loadings plots (top) for easy visualization of the proteins that are showing the highest degree of change across the multiple samples. As an example, the protein expression differences across the different liver hepatocyte samples for the two cytochrome P450 proteins, CYP2C8 and CYP 2A6, are shown.
TTMass Spectrometry: Eluant from the column was sprayed
using the NanoSpray® Source into a TripleTOF® 5600+ system
(AB SCIEX). Data were acquired using an MS/MSALL with SWATHTM acquisition method with a Q1 window size of 25 Da and a
mass range of 400-1000 m/z (cycle time 2.5 sec). Information dependent acquisition (IDA) experiment was performed on the pooled sample to obtain peptide identi cations to generate ion library. Thirteen individual hepatocytes samples (labeled 098, 091, DAD, I2G, IDE, KMI, MRS, NQT, RML, ROE, SED, VCM, YAA) were analyzed in triplicate by SWATH acquisition (Figure
2). Protein/peptide data were loaded into Skyline for MRM assay development and samples were also run in triplicate by MRM on the QTRAP® 6500 System.
Data Processing: The pooled sample was analyzed with ProteinPilotTM Software 4.5 beta to create a spectral library of proteins and peptides in the sample. The SWATH Acquisition data was processed using the SWATHTM Acquisition MicroApp 1.0 in PeakView® Software. Only proteins that were identi ed at a 1% global FDR were used in SWATH acquisition processing. Fragment ion XICs were summed to obtain peptide peak areas, and the areas for multiple peptides per protein were summed to obtain protein areas. Statistical analysis including principal component analysis (PCA) and t-tests were conducted with MarkerViewTM Software 1.2. Data analysis of the MRM data was performed using MultiQuantTM Software 2.1.
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TDAD
RML
RML ROE
ROE
HLO17B13
-0.05 0.00 0.05 0.10 0.15
PC1 Loading
CYP2C8 CYP2A6
SED
GSTA2 ALDOB
ROE
-10000 0 10000
PC1 Score
DAD
MRS
091
098
RML
KMI
I2G
VCM YAA
IDE
ROE
NQT
091 098 098 DAD I2G I2G IDE KMI KMI MRS NQT NQT RML ROE ROE SED VCM VCM YAA
% Response
PC2 Score
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PC2 Loading


High Quality Quanti cation on Large Number of Proteins
The pooled sample was used to generate a spectral ion library containing more than 2000 proteins. From the SWATHM acquisition, an average of 1987 proteins was quanti able across the 13 samples. Highly reproducible results were obtained for enzyme families of interest, such as 19 CYP proteins, 12 UGT proteins, and 7 GST proteins. The reproducibility was assessed across three technical replicates. The quantitative pro ling of
the key metabolizing enzymes potentially helps to discover the expression variations of these enzymes, as well as their correlation/ anti-correlation properties across the sample set and ultimately within populations when larger sample sizes are available. This systematic understanding of protein expression is essential during the drug development process.
Figure 3 (previous page) shows the relative comparison of the
two phase II metabolism enzymes (liver carboxylase EST1 and EST2) across 13 samples. For comparison, the peptide signal was normalized against sample 091. A good correlation of quantitative differences was observed across multiple peptides of each of the two proteins, demonstrating that good reliability was observed in the SWATHM Acquisition data.
Because of the comprehensive nature of SWATH acquisition,
it overcomes some common limitations existing in the MRM based targeted methods, such as limited multiplexing capabilities and the fact that only targeted analytes will be detected and quanti ed. Unlike a targeted method, SWATH acquisition creates a permanent record of MS and MS/MS spectra of all detectable species in the sample, and all these information can be extracted from the data. In addition, the data can be further interrogated
Figure 5. Speci c Quantitation of Closely Related Protein Isoforms. Three peptides that uniquely distinguish the closely related 3A4 and 3A5 CYP450 enzymes were summed to represent protein intensity. The absolute signal intensities were used to compare protein expression across the samples.
when more information arises and a researcher wants to ask more questions of the study.
Also because of the high information content of the technique, statistical analysis and visualization strategies are important. PCA analysis can provide one way of quickly detecting proteins that
are differing between the samples. The scores plot (Figure 4, top left) shows how the samples are different from each other and the loadings plot (Figure 4, top right) shows which speci c features are responsible for this difference. Proteins of interest can then be selected and viewed for their changes across the whole sample set (Figure 4, bottom). Here, the expression differences between two members of the Cytochrome P450 enzyme family are displayed. The protein expression of these two proteins track each other fairly closely across the samples except for a few individuals were differences are observed.
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100 80 60 40 20
CYP3A4 HSD17B13
100
80 091
098
KMI
NQT
YAA
VCM
091
MRS
RML SED ROE
MRS
60 YAA 40
20
NQT
RML
GSTK1
Figure 6. Visualize Protein Pro les for Different Expression Behaviors. Using the PCA analysis in MarkerViewM Software, proteins that show similar correlated expression patterns as well as anti-correlated patterns can be readily found (Left). Using the pattern of CYP3A4 enzyme for comparison, the UGT1A6 enzyme was found to show a similar protein expression pattern while the HSD17B13 protein pattern was found to have a more opposite behavior to CYP3A4. On the right of the gure, two proteins UGT2B7 and UGT2B15 show very similar patterns to each other and also high variation across the individual samples. In contrast, the GSTK1 protein shows very minimal variation across individual samples.
100
091 80
60 40 20
CYP3A4 CYP3A5
098 DAD I2G IDE KMI
MRS
For Research Use Only. Not for use in diagnostic procedures.
0
091 098 098 DAD I2G I2G IDE KMI KMI MRS NQT NQT RML ROE ROE SED VCM VCM YAA
Sample (by index)
DAD
I2G IDE
RML NQT
YAA
ROE SED VCM
100 80 60 40 20
CYP3A4 UGT1A6
100 80 60 YAA 40 20
VCM
SED RML
ROE
YAA
091
NQT
MRS
KMI
MRS
091
098
I2G
DAD
IDE
VCM
098
NQT RML
SED
UGT2B7 UGT2B15
DAD
I2G
IDE
KMI
ROE
00 091 098 098 DAD I2G I2G IDE KMI KMI MRS NQT NQT RML ROE ROE SED VCM VCM YAA
Sample (by index)
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Mining Data for Interesting Protein Expression Patterns
The quantitative pro les of the phase I and II enzymes are needed to facilitate drug development, understanding which proteins show higher or lower biological variation is important to understand and therefore a straightforward assay for measuring this is an advantage. Correlation as well as variation in protein expression can be easily assessed after PCA analysis (Figure 4).
In the results, multiple proteins were found to be correlated,
such as CYP3A4 and CYP3A5, which are two major phase I drug metabolizing enzymes in cytochrome P450 superfamily (Figure 5). As their sequence homology is very high, >80%, the quantitation from the SWATH acquisition data was performed using unique peptides to each protein isoform. Previously published data
also demonstrated the positive correlation between these
two proteins2. In addition, a higher correlation between UDP glucuronosyltransferases UGT2B7 and UGT2B15, and between CYP3A4 and UGT1A6 was observed (Figure 6, top left and right).
As PCA is a multivariate statistical analysis, the anti-correlation of proteins can also be discovered. Figure 6 (bottom left) displays an anti-correlated behavior between CYP3A4 and HSD17B13. At this moment, it is not clearly understood what biological events are behind this observation. But either anti-correlated or correlated behavior of inter- and intra- protein family isoforms may indicate their networking relations or metabolic activities.
In addition to the proteins discussed above, proteins were observed with minimum population variation. As shown in Figure 6 (bottom right), Glutathione S-transferase kappa1 (GSTK1)
was consistently expressed across the 13 samples (with ~10% variation). These examples illustrate how much information is present within the SWATH acquisition data for mining and also demonstrate how valuable the quantitative protein expression information is for the evaluation of drug biotransformation during the early drug development.
Good Correlation with MRM Assay Results
After quantitative pro ling with SWATHTM Acquisition during the early stages of research, MRM assays to proteins of interest can be quickly developed for better sensitivity and throughput3. As the MRM strategy is the well accepted approach for this type
of assay, it was of interest to compare the quantitative results. Skyline software was used to develop an MRM assay for a selected set of proteins and the assay was run on the same set of samples. Comparable results were observed between the MRM results generated with QTRAP® 6500 system and the SWATHTM Acquisition generated with the TripleTOF® 5600+ system as illustrated by a selected peptide for the CYP1A2 protein (Figure 7).
Conclusions
MS/MSALL with SWATHTM Acquisition provides a powerful acquisition strategy for the quantitative pro ling of a large number of proteins key in the investigation of drug metabolism.
• Multiple drug metabolizing enzymes were quantitatively pro led in a single assay across multiple samples.
• As the SWATHTM Acquisition requires very little method development, it is easy to establish and use.
• Principal Component Analysis (PCA) provides one method of statistical analysis that makes data interpretation easier.
• With the SWATH acquisition approach, comparable quantitation results were obtained as with the MRM strategy.
References
1. MS/MSALL with SWATHTM Acquisition - Comprehensive Quanti cation with Qualitative Con rmation using the TripleTOF® 5600+ System. AB SCIEX Technical Note 3330111-03.
2. Lin Y. S. et al., (2002) Co-Regulation of CYP3A4 and CYP3A5 and Contribution to Hepatic and Intestinal Midazolam Metabolism. Mol. Pharmacol. 62: 162-172.
3. Discovery to Validation: Transition from SWATHTM Acquisition to Targeted MRM Analysis for Quantitative Proteomics Pipeline - Using the TripleTOF® 5600+ System and QTRAP® 6500 System. AB SCIEX Technical Note 7940213-01.
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Quanti cation of Large Oligonucleotides Using High Resolution MS/MS on the
TripleTOF® 5600 System
Thomas Knapman1, Vicki Gallant1 and Martyn Hemsley2 1AB SCIEX, Warrington, UK, 2Covance, Harrogate, U
Key Challenges of Oligonucleotide Bioanalytical Assay
1. The bioanalysis of oligonucleotides as therapeutics requires sensitive, speci c and robust analysis.
2. Many ELISA-based oligonucleotide measurements do not accurately distinguish large metabolites from the full-length oligonucleotide of interest.
3. ELISA- and UV-based measurements have limited dynamic range which complicates quantitative analysis of oligonucleotides in complex matrices.
Key Bene ts of MRMHR Work ow for Oligonucleotide Bioanalytical Assay
1. High sensitivity MS/MS enables the quantitative MRMHR work ow, providing high selectivity in biological matrices.
2. High resolution, accurate mass MS/MS spectra enable qualitative veri cation of oligonucleotide sequences.
3. The MRMHR work ow offers a dynamic range of two to three orders of magnitude.
Unique Features of MRMHR Work ow on TripleTOF® 5600 System
1. Summing of multiple ion transitions to increase both sensitivity and selectivity of quantitation.
2. Accelerated method development times, since ion transitions can be selected post-acquisition to eliminate background interferences.
3. High multiplexing due to high acquisition speeds (up to 100 spectra per second) for simultaneous quantitation of multiple species, including multiple oligonucleotide sequences and/or their metabolites.
Introduction
Quantitative analysis of synthetic oligonucleotides in biological matrices is an important aspect of pharmacokinetic (PK), toxicokinetic (TK) and metabolic pathway studies in drug development1. With an increasing number of oligonucleotide based drugs in research pipelines, the acceleration of the drug development process by reducing the time spent on method
TTFigure 1: The TripleTOF® 5600 System has the speed and sensitivity to deliver high-throughput targeted quantitation of many species in a single run. This example focuses on using MRMHR work ow to quantify a synthetic oligonucleotide from human plasma, demonstrating post-acquisition fragment ion selection and summing product ions to achieve the highest possible sensitivity and selectivity.
Figure 2: Principle of MRMHR Work ow Quantitation. Looped full scan MS/MS spectra are acquired for each precursor. Selected fragments are then extracted post- acquisition using narrow extraction widths (in this case 50 mDa) to produce high resolution XICs. These XICs can be used individually or summed, depending on which provides the best selectivity and sensitivity for quantitation.
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Figure 3: TOFMS Analysis of Intact Oligonucleotide 1 acquired on the TripleTOF® 5600 system (top). Isotope resolution of higher charge states is achieved with a resolution of 30,000-40,000, as illustrated by zooming in on the 6- charge state (bottom). This resolution is achieved at TOF MS scan speeds of 10 MS spectra per second.
development, and by performing simultaneous qualitative structural analysis with quantitative analysis are crucial advantages in any potential quantitation approach1.
Current LC/MS approaches to oligonucleotide quantitation predominantly use multiple reaction monitoring (MRM), however the complex fragmentation pathways of oligonucleotide species coupled with the variability of matrix effects mean that it can be dif cult to predict the sensitivity and selectivity of a given MRM transition without signi cant optimization1. These effects limit the utility of low resolution quantitation methods both in terms of the achievable limits of quantitation and in sample throughput, particularly when quantifying large numbers of potential drug candidates of different sequences, and their metabolites.
Materials and Methods
Sample Preparation
The synthetic DNA Oligonucleotide 1 was spiked into human plasma over a concentration range of 0.025 to 10 nM. Oligonucleotide 2 was used as an internal standard.
MS Conditions
MS System
Ionization Mode TOF MS range
MRMHR
Collision energy spread Source temperature
Software
Data acquisition Data review Deconvolution Quantitation
Results and Discussion
TripleTOF® 5600+ system with a DuoSprayM Source
ESI with Negative Mode
m/z 100-2500 at 250 msec accumulation time
2 product ions each 250 msec -40 ± 4 eV
550°C
Analyst TF® 1.5.1 Software PeakView® 1.2 Software BioAnalyst® Software MultiQuantM 2.1 Software
MS and MS/MS Analysis of Oligonucleotides. TOFMS analysis of Oligonucleotide 1 showed a charge state envelope consisting of [M-5H]5-, [M-6H]6- and [M-7H]7- ions (Figure 3) with a resolution of approximately 36,000. Inspection of the TOFMS spectrum showed that the system passivation process had reduced adduct formation to less than 5% relative to the fully protonated form, thus facilitating quantitation from the [M-6H]6- peak (data not shown).
The principle of the MRMHR work ow for quantitation is to acquire full scan TOF MS/MS spectra for each species of interest, and to use high resolution extracted ion chromatograms (XICs) for quantitation, summing multiple transitions where appropriate to achieve optimum sensitivity and selectivity (Figure 2). To develop an MRMHR work ow assay for oligonucleotides 1 and 2, full scan MS/MS spectra were acquired for m/z 761.9 for Oligonucleotide 1, and m/z 745.6 for Oligonucleotide 2. The full scan MS/MS spectra were also used to verify the sequence of Oligonucleotide 1 and 2 (Figure 4). The MS/MS spectra were deconvoluted using BioAnalyst® Software to enable the singly and multiply-charged fragment ions to be plotted on a mass scale. The sequences were subsequently veri ed by matching theoretical sequence ions to the fragment ions observed in the deconvoluted spectra.
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THERAPEUTIC PEPTIDE BIOANALYSIS
LC Conditions
LC System Analytical column
Analytical ow Mobile Phase A Mobile Phase B
Shimadzu Prominence XR UFLC Waters Acquity BEH, 50 x 2.1 mm, 1.7 μm, temp.= 60 oC
0.40 ml/min (initial 24 hour ush) Water (15 mM TEA, 400 mM HFIP) 50:50 Methanol:Water (15 mM TEA, 400 mM HFIP)
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Figure 4: High Quality MS/MS of Oligonucleotide 1 for Characterization and Quantitation Method Development. (top) The full scan MS/MS approach of MRMHR work ow enables full qualitative analysis of any targeted analyte. Full sequencing of Oligonucleotide 1 was achieved on the mass reconstructed MS/MS spectrum (bottom) for fragment ion selection.
MRMHR Work ow Assay Development. Having acquired full scan TOF MS/MS spectra, fragment ions can be selected and extracted post-acquisition for use in quantitation. Figure 2 shows the stepwise process of selecting fragment ions from a full scan MS/MS spectrum, generating multiple high resolution XICs from the selected fragment ions, and summing the XICs to optimize the signal-to-noise. For transitions arising from Oligonucleotides 1 and 2, an extraction width of 50 mDa was used to generate XICs; however it is possible to collect the MS/MS spectra in high resolution mode (>30,000 resolution) and extract with narrower windows to improve selectivity in any given assay if required.
The post-acquisition selection of fragments is a signi cant advantage of the MRMHR work ow, since the selectivity of speci c fragment ions in matrix cannot necessarily be predicted prior
to data acquisition. In the case of Oligonucleotide 1, the three most intense fragment ions in the MS/MS spectrum gave poor selectivity when extracted (Figure 5), and therefore could not be used for quantitation. In contrast, other less intense transitions showed excellent selectivity, and were subsequently included in the assay. The nal XIC trace was achieved by summing
25 different fragment ion XICs (Figure 5). The nal data processing using the summed XICs was performed using MultiQuantTM Software.
Figure 5: Post-Acquisition Extraction of Structurally Speci c Ions. In the case of Oligonucleotide 1, the three most intense fragment ions (top left) are non-selective in plasma at low concentrations and therefore summing of these XICs does not provide a good assay (top right). Because the full scan MS/MS spectrum is acquired in the MRMHR work ow, this allows different fragment ions to be selected and extracted
for quantitation post-acquisition (bottom left, summed bottom right), and therefore requires signi cantly less method development than traditional MRM approaches.
Figure 6: Selectivity of MRMHR Work ow in Complex Matrices Allows Better LLOQs to be Obtained. In the case of Oligonucleotide 1, background interferences in full scan TOF MS result in higher limits of detection and quantitation (0.5 nM), while the selectivity of the MRMHR work ow allows quantitation of concentrations less than 0.1 nM.
TOF MS vs. MS/MS Quanti cation Strategies. The MRMHR work ow for quantitation from complex matrices offers signi cant advantages over the full scan TOF MS approach to quantitation, due to both the high selectivity of the MS/MS based XICs and the ability to remove fragment ions with background interferences, which can impact signi cantly on the achievable limits of detection and quanti cation.
TTOF MS MRMHR Workflow
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For Research Use Only. Not for use in diagnostic procedures.
Calibration for Oligo 1: y= 0.09896 x + 0.00175 (r=0.99943) (weighting: 1/x)
Figure 7: Standard Concentration Curve for Oligonucleotide 1 in Matrix using MRMHR Work ow. Concentration curve for Oligonucleotide 1 in plasma, using Oligo- nucleotide 2 as an internal standard. Using MRMHR, excellent linearity was observed, with a lower limit of quantitation of 0.05 nM.
Figure 6 shows a comparison of the TOF MS and MRMHR work ows applied to the analysis of Oligonucleotide 1. In the case of the TOF MS quantitation, each peak in the isotopic envelope of the [M-6H]6- charge state was extracted using
a 10 mDa extraction window (Figure 6, left). The extracted ion chromatograms from the TOF MS approach show signi cant matrix interferences that seriously impact the limit of quanti cation, which is approximately 0.5 nM using this approach.
In contrast, the speci city of the MRMHR work ow produced signi cantly lower limits of detection and quanti cation (Figure 6, right). This improvement is due to the signi cant reduction in noise from the complex matrix.
Figure 7 shows the calibration plot of Oligonucleotide 1 using
the MRMHR work ow with Oligonucleotide 2 used as an internal standard. The %CV values for each concentration analyzed are shown in the embedded table. The correlation coef cient of the response is in excess of 0.99 over ~2 orders of magnitude; the linearity of response at concentrations in excess of 10 nM was not investigated. The lower limit of quanti cation (LLOQ) using the MRMHR work ow was 0.05 nM (Figure 7), while the LLOQ was 10 fold higher when the same oligonucleotide was analyzed by full scan TOF MS work ow (Figure 6).
Conclusions
1. The TripleTOF® 5600 system offers sensitive, high-resolution analysis of large oligonucleotides, with the opportunity to perform both qualitative and quantitative analysis in a single run.
2. Using a targeted MRMHR work ow, looped full scan TOF MS/MS spectra of a ~4.5 kDa synthetic oligonucleotide were acquired with a resolution of ~16,000 and XICs were generated from speci c fragment ions to achieve a highly sensitive and selective quantitative assay.
3. The ability to quantify oligonucleotides from complex matrices with minimal assay optimization offers the opportunity for high-throughput analysis of potential oligonucleotide-based therapeutics. Upfront method development is highly simpli ed and consists of specifying a theoretical oligonucleotide m/z and a collision energy, the remainder of the analysis being done post-acquisition.
References
1. Z. J. Lin, W. Li and G. Dai, J. Pharm. Biomed. Anal. 44 (2007) 330-341.
Note: Thanks to Randy J. Arnold, AB SCIEX help write and organize this material.
For Research Use Only. Not for use in diagnostic procedures.
© 2014 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEXM is being used under license.
Publication number: : 9480114-01
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THERAPEUTIC PEPTIDE BIOANALYSIS
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Considerations for Handling Therapeutic
Oligonucleotide Reference Standard
and Sample Extraction Tips
Cindy Sanderson
AB SCIEX, Framingham, USA
Handling Reference Standard Material
The Oligonucleotide API (active pharmaceutical ingredient in
GMP Studies) or test article (in GLP Studies) is highly hygroscopic and the water content of an oligonucleotide is related to its environment. Additionally, concentrated solution such as those observed in a clinical setting can be very viscous; both of these traits can lead to inaccuracies in the Assay. To prevent inaccuracies due to the hygroscopic nature of the API, one of two options are presented. The drug substance can be handled only in a room that enables precise temperature and humidity control. The glaring limitation of this approach is that not every site has this available. The alternate option is to equilibrate the API to ambient temperature and humidity prior to handling and using the UV absorbance (typically at 260 nm) and purity to determine the actual concentration. The latter approach assumes knowledge
of the oligonucleotide purity. The viscosity issue can be overcome by relying on a gravimetric technique as opposed to a
volumetric one.
Dealing with Non-Speci c Binding
Oligonucleotides are prone to non-speci c binding with the container/closure system, components in biological matrices and components of the LCMS system used for analysis. Non-speci c binding to sample containers are more pronounced at lower concentrations and can be helped by either storing the solutions in Type I glass containers or using EDTA or other preservative with plastic container. Regardless of oligonucleotide concentration EDTA is a useful preservative as it will chelate divalent cations which are required for nuclease activity. To prevent non-speci c binding to components of the LCMS system, one option is to replace as much stainless steel as possible and replace with PEEK or similar tubing; of course this is only applicable to relatively
low pressure systems as the PEEK tubing will not tolerate as high a pressure. Another little trick is to add a minute amount (μM quantities) of EDTA to the mobile phase again to chelate any divalent cations present. Dealing with the non-speci c binding of oligonucleotides to components in the biological matrices is more involved and intricate given that oligonucleotides carry lots of negative charge on their phosphodiester-based backbone.
The negative charge imparts a strong af nity for ubiquitous cations such as Na+ and K+ as well as other matrix components.
Sample Extraction Tips
There are many ways to separate these matrix components from the therapeutic oligonucleotide including protein precipitation, liquid-liquid extraction, solid phase extraction and various combinations of these procedures. Simple protein precipitations with organic solvents such as acetonitrile are met with limited success as they are prone to low recoveries and the ubiquitous cations are not necessarily removed.
Several liquid-liquid extraction techniques have met with some success, particularly using a phenol/chloroform solution for the LLE. Often, the LLE will include a step that adds a detergent
or other modi er to break up any complexes between the oligonucleotide and matrix components. The proteins will partition into the organic layer while the oligonucleotide is
left in the aqueous portion along with other polar matrix components. These remaining matrix components can and do interfere with LCMS. For this reason, the liquid-liquid extract can be further treated by solid phase extraction. Adsorption of the oligonucleotide to a reverse phase solid packing material can be enhanced by the addition of a modi er to both the loading and elution solvent; typically an ion pairing agent and/or ammonium hydroxide are used. Once the oligonucleotide is adsorbed onto the solid phase, washing and elution. This combination of LLE followed by SPE is laborious, time consuming and the ability to automate for a larger number of samples is limited. A few years ago Phenomenex came out with their Clarity OTX system which is a mixed-mode (weak anion exchange and reverse phase) SPE cartridge along with buffers designed to work with the cartridge to clean up oligonucleotide samples. The Clarity OTX offers a quicker method for sample preparation from biological matrices with adequate recoveries.
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Increasing LCMS Assay Robustness through
Increased Speci city using High Resolution
MRM-like Analysis
MRMHR Analysis using the TripleTOF® 5600 System
Christie L Hunter1, Hasmik Keshishian2, Steven A. Carr2 1AB SCIEX, CA, USA; 2Broad Institute, MA, USA
Interference in targeted quantitative assays can come from different sources and can degrade the quality of the quantitative data and ultimately the quanti cation limits. Many assays are developed using a single sample or a small pool, however
when the assay is used on a larger sample set, interferences
are discovered that confound the results. When running MRM assays on human biological uids or tissue, it is imperative
that MRM assays are developed to be robust to the presence
of interferences that are possible and likely. Robustness to interference can come in two forms: the assay can be robust
to the detection of interferences or the assay can be robust
by avoiding interferences. Robustness to the detection of interferences can be achieved by designing assays to contain multiple peptides per protein and multiple transitions per peptide so that any interfered peaks can be removed from the nal dataset. Robustness to interference through avoidance can
be achieved by increasing the assay speci city.
Figure 1. Looped High Resolution MS/MS – MRMHR. In this analysis, the instrument is set up to acquire full scan MS/MS data on a xed precursor, over and over again across the LC run. The Q1 is xed, the peptide is fragmented in the collision cell
and the full scan TOF MS/MS is acquired. After data acquisition, extracted ion chromatograms on sequence speci c ions are generated. Multiple fragment ions
can be monitored or even summed together because the full scan MS/MS is always acquired. After this extraction, the processing for quanti cation is now very similar to how one would handle MRM data acquired on a triple quad or QTRAP® system.
Recent innovations on the TripleTOF® 5600 System have enabled a new acquisition strategy, where looped MS/MS spectra are collected at high resolution and then fragment ions are extracted post-acquisition to generate MRM-like data. The technique is sensitive and fast enough to enable quantitative performance similar to high end triple quadrupole instruments. But the resolution capabilities are far greater, thereby enabling a degree of selectivity that cannot be reached using standard MRM on
a triple quadrupole platform. This work ow on the TripleTOF® 5600 System is termed the MRMHR work ow.
To understand the ultimate utility of this type of acquisition strategy on complex biological samples, a set of peptides dosed into depleted human plasma. Injections were performed in triplicate on the TripleTOF® 5600 System and the lower limits of quanti cation (LLOQ) using various fragment ions to the peptides were measured. Post-acquisition extractions of varying width were analyzed and the impact on speci city and sensitivity
were examined.
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