XEVO TQ MS PHARMACEUTICAL
APPLICATION NOTEBOOK
The Changing Face of LC/MS: From Experts to Users ...
3
Improving MS/MS Sensitivity using Xevo TQ MS with ScanWave ...
11
Simultaneous Confirmation and Quantification using Xevo TQ MS:
Product Ion Confirmation (PIC) ...
15
Novel Dual-Scan MRM Mode Mass Spectrometry for the Detection of
Metabolites during Drug Quantification ...
19
Data-Directed Detection and Confirmation of Drug Metabolites
in Bioanalytical Studies ...
23
Improving Qualitative Confirmation using Xevo TQ MS with Survey Scanning ...
27
A Novel Method for Monitoring Matrix Interferences in Biological Samples
using Dual-Scan MRM Mode Mass Spectrometry ...
31
Rapid, Simple Impurity Characterization with the Xevo TQ Mass Spectrometer ...
35
THE CHANgINg FACE OF LC/MS:
FROM EXPERTS TO USERS
Robert S. Plumb
Senior Applications Manager, Pharmaceutical Business
Operations, Waters Corporation
Michael P. Balogh
Principal Scientist, MS Technology Development,
Waters Corporation
Researchers and practitioners from various
disciplines and sub-disciplines within chemistry,
biochemistry, and physics regularly depend on
mass spectrometric analysis. Pharmaceutical
industry workers involved in drug discovery and
development rely on the specificity, dynamic
range, and sensitivity of mass spectrometry
(MS). Particularly in drug discovery, where
compound identification and purity from
synthesis and early pharmacokinetics are
determined, MS has proved indispensable.
Biochemists expand the use of MS to protein, peptide, and oligonucleotide analysis. Using mass spectrometers, they monitor enzyme reactions, confirm amino acid sequences, and identify large proteins from databases that include samples derived from proteolytic fragments. They also monitor protein folding, carried out by means of hydrogen-deuterium exchange studies, and important protein-ligand complex formation under physiological conditions.
Clinical chemists, too, are adopting MS, replacing the less-certain results of immunoassays for drug testing and neonatal screening. So are food safety and environmental researchers. They and their allied industrial counterparts have turned to MS for some of the same reasons: PAH
and PCB analysis, water quality studies, and to measure pesticide residues in foods. Determining oil composition, a complex and costly prospect, fueled the development of some of the earliest mass spectrometers and continues to drive significant advances in the technology.
Today, the MS practitioner can choose among a range of ionization techniques that have become robust and trustworthy on a variety of instruments with demon-strated capabilities.
Two decades ago, mass spectrometry was the preserve of experts and skilled technicians: the instrumentation required constant attention and adjustment. At this time LC/MS was in it infancy and atmospheric pressure ioniza-tion (API) source interfacing was just beginning. Samples
Advances in chromatography
Interfacing Liquid Chromatography with Mass Spectrometry (LC/MS) allows analytical chemists access to about 80 percent of the chemical universe unreachable by Gas Chromatography (GC); it is also responsible for the phe-nomenal growth and interest in mass spectrometry in recent decades.
A few individuals can be singled out for coupling LC with MS. Beginning arguably in the 1970s, LC/MS as we know it today reached maturation in the early 1990s. Many of the devices and techniques we use today in practice are drawn directly from that time.
In its simplest form, liquid chromatography relies on the ability to predict and reproduce – with great precision – competing interactions between analytes in solution (the mobile or condensed phase) being passed over a bed of packed particles (the stationary phase). The development of columns, packed with a variety of functional moieties in recent years, and of the solvent delivery systems, able to precisely deliver the mobile phase, has enabled LC to become the analytical backbone for many industries. Continued advances in performance since then, including development of smaller particles and greater selectivity, also saw the meaning of the acronym change to high-performance liquid chromatography (HPLC). In 2004, further advances in instrumentation and column technol-ogy achieved significant increases in resolution, speed, and sensitivity in liquid chromatography. Columns packed with smaller particles – 1.7 µm – and instrumentation with specialized capabilities designed to deliver the mobile phase at pressures up to 15,000 psi (1,000 bar) came to be known as UltraPerformance® (UPLC®) technology. Much of what is embodied in this current technology was predicted by investigators such as Prof. John Knox in the 1970s. requiring analysis were passed from the requesting
scien-tist to these “experts for analysis,” the samples would be analyzed, processed, interpreted and the results returned via a written report.
Two decades later, both the users and the capabilities of LC/MS have changed significantly. Now mass spectrom-eters and LC/MS systems are ubiquitous in the analytical laboratory, especially in the pharmaceutical industry. These instruments are used by a wide variety of scientists for a diverse range of tasks, from purity screening in medicinal chemistry, to the quantification of drugs in blood, and the identification of proteins for biomarker discovery.
The usability of current mass spectrometry platforms has improved dramatically – scientists are now able to oper-ate the systems remotely via the Internet; they can carry out complex, data-dependent tasks such as purification and peptide fragmentation; they are able to use to open access systems where a non-analytical chemist can queue samples for analysis and have the results emailed to them without ever having to know or concern themselves about the LC/MS process.
Recent reports put the number of LC/MS systems sold per year in excess of 2500 units. This large number of units sold each year is also reflected in the increased number of users. In 1980, the number of scientists attending ASMS was around 1250; by 2002 this had risen to greater than 4000 with a growth rate of 10 percent per year. This growth in LC/MS users occurred because of the increase in the number of samples analyzed each year per user, creating larger and larger amounts of high-quality data. More and more, this data is being turned directly into information or knowledge so that decisions are made in real-time. Many of these new users have little interest in becoming expert mass spectroscopists and are instead looking for the instrumentation itself to decide the appro-priate experiments to be performed as well as to interpret the data automatically and recommend a course of action (pass/fail, pure/impure).
Advances in mass spectrometry
Mass spectrometers can be smaller than a coin, or they can fill very large rooms. Although the various instrument types serve in vastly different applications, they neverthe-less share certain operating fundamentals. The unit of measure has become the dalton (Da), displacing other terms such as amu. 1 Da = 1/12 of the mass of a single atom of the isotope of carbon-12 (12C).
Once employed strictly as qualitative devices – adjuncts in determining compound identity – mass spectrometers were once considered incapable of rigorous quantification. But in more recent times, they have proven themselves as both qualitative and quantitative instruments. A mass spectrometer can measure the mass of a molecule only after it converts the molecule to a gas-phase ion. To do so, it imparts an electrical charge to molecules and converts the resultant flux of electrically-charged ions into a pro-portional electrical current that a data system then reads. The data system converts the current to digital informa-tion, displaying it as a mass spectrum.
The ions required in mass spectrometry can be created in a number of ways suited to the target analyte in question: by laser ablation of a compound dissolved in a matrix on a planar surface such as by MALDI; by interaction with an energized particle or electron such as in electron ioniza-tion (EI); or as part of the transport process itself, as we have come to know electrospray ionization (ESI), where the eluent from a liquid chromatograph receives a high voltage resulting in ions from an aerosol.
The ions are separated, detected, and measured according to their mass-to-charge ratios (m/z). Relative ion current (signal) is plotted versus m/z producing a mass spectrum. Small molecules typically exhibit only a single charge: the m/z is therefore some mass (m) over 1, with the “1” being a proton added in the ionization process [represented by M+H+ or M-H+ if formed by the loss of a proton], or, if the ion is formed by loss of an electron, it is represented as the radical cation [M+.]. Larger molecules can capture charges in more than one location within their structure.
Small peptides typically may have two charges [M+2H+], while very large molecules have numerous sites, allowing simple algorithms to deduce the mass of the ion repre-sented in the spectrum.
The general term atmospheric pressure ionization (API) includes the most notable technique, electrospray ioniza-tion (ESI), which itself provides the basis for various related techniques capable of creating ions at atmospheric pressure rather than in a vacuum. The sample is dissolved in a polar solvent (typically less volatile than that used with GC) and pumped through a stainless steel capillary that carries between 500 and 4000 V. The liquid forms an aerosol as it exits the capillary at atmospheric pressure, and the desolvating droplets shed ions that flow into the mass spectrometer, induced by the combined effects of electrostatic attraction and vacuum.
The mechanism by which potential transfers from the liquid to the analyte, creating ions, remains a topic of con-troversy. In 1968, Malcolm Dole first proposed the charge residue mechanism, in which he hypothesized that as a droplet evaporates, its charge remains unchanged. The droplet’s surface tension, ultimately unable to oppose the repulsive forces from the imposed charge, explodes into many smaller droplets. These Coulombic fissions occur until droplets containing a single analyte ion remain. As the solvent evaporates from the last droplet in the reduc-tion series, a gas-phase ion forms. In 1976, Iribarne and Thomson proposed a different model, the ion evaporation mechanism, in which small droplets form by Coulombic fission, similar to the way they form in Dole’s model. It is possible that the two mechanisms may actually work in concert: the charge residue mechanism dominant for masses higher than 3000 Da while ion evaporation domi-nant for lower masses.
The mass analyzer is the heart of the instrument and is a means of separating or differentiating introduced ions. Both positive and negative ions (as well as uncharged, neutral species) form in the ion source. However, only one polarity is recorded at a given moment.
The modern mass spectrometer
Modern instruments can switch polarities in milliseconds, yielding high fidelity records. As well as separating the ions, modern mass spectrometers can trap and fragment ions (MS/MS or MSn) to produce a wealth of information
about the molecule’s structure. Other instruments such as magnetic sector instruments, hybrid quadrupole time-of-flight (Q-ToF), and ion cyclotron (ICR) mass spectrometers can record the mass of a compound to 1 ppm, allowing for the elemental composition of a molecular ion or fragment ion to be deduced.
The increased sensitivity afforded by modern mass spectrometry over other forms of detection, such as UV and fluorescence, comes from the selectivity and specificity of the MS and MS/MS process. During these experiments, specific ions are allowed to pass through the analyzer and reach the detector. During a multiple reac-tion monitoring (MRM) MS/MS experiment, only ions that undergo a specific fragmentation are allowed to reach the detector; while this reduces the number of ions reaching
superior signal-to-noise ratio. This dramatically improves assay sensitivity and specificity. The vast majority of quantitative experiments are performed on quadrupole-based instruments; whereas ion traps and accurate mass instruments are preferred for structural elucidation experiments.
Single quadrupole mass spectrometers require a clean matrix to avoid the interference of unwanted ions, and they exhibit very good sensitivity. Triple quadrupole, or tandem, mass spectrometers (MS/MS) add to a single quadrupole instrument an additional quadrupole, which can act in various ways. One way is simply to separate and detect the ions of interest in a complex mixture by the ions’ unique mass-to-charge (m/z) ratio. Another way that an additional quadrupole proves useful is when used in conjunction with controlled fragmentation experiments. Such experiments involve colliding ions of interest with another molecule (typically a gas like argon). In such an application, a precursor ion fragments into product ions, and the MS/MS instrument identifies the compound of interest by its unique constituent parts.
An ion trap instrument operates on principles similar to those of a quadrupole instrument. Unlike the quadrupole instrument, which filters streaming ions, both the ion trap and more-capable ion cyclotron instruments store ions in a three-dimensional space. Before saturation occurs, the trap or cyclotron allows selected ions to be ejected, according to their masses, for detection. A series of experiments can be performed within the confines of the trap, fragmenting an ion of interest to better define the precursor by its fragments. Dynamic range is sometimes limited in ion trap instruments and the finite volume/ capacity for ions limits the instrument’s range, especially for samples in complex matrices.
The tandem quadrupole mass spectrometer
Tandem quadrupole and ion trap instruments have become the workhorses of modern analytical LC/MS(MS). The two capabilities have been incorporated into one instrument platform to produce a linear ion-trapment that has all the structural characterization benefits of ion trap mass spectrometers, with the quantitative capabilities of tandem MS instrumentation.
These instruments have become popular with scientists who are required to perform more than one type of exper-iment (quantitative and qualitative) during the course of their work and require the flexibility to perform it on the same analytical platform. These tasks include impurity identification and quantification and discovery DMPK, where both dosed parent concentration and metabolite characterization are required.
These instruments, while sounding ideal, do have some drawbacks especially when using modern high-resolution chromatography such as UPLC. Here, the chromato-graphic peak widths are so narrow (1 to 2 seconds) that there is not sufficient time for these ion trap mass spectrometers to select the ions for trapping, fragment the ion, and measure them to produce enough data points to accurately define the peak. Although the collection of MS/MS spectra can be performed with a standard tandem quadrupole MS instrument while still correctly defining the LC peak, sensitivity is compromised due to the low duty cycle of the instrument.
A new direction for tandem quadrupole MS: The Xevo TQ
Along with the need to improve the utility and flex-ibility of tandem quadrupole MS instrumentation are the requirements to improve its data processing and monitoring capabilities. The recent introduction of a new iteration of the tandem quadrupole mass spectrometer, using traveling-wave technology1, holds the potential toresolve many of these issues. The Waters® Xevo™ TQ
Mass Spectrometer employs traveling-wave technology that improves MS capabilities by performing simultane-ous, multifunctional data acquisition, such as MRM and product ion acquisition, all within a timescale compatible with sub-2 µm UPLC. The instrument is equipped with a modern tool-free source that simplifies the process of routine maintenance and cleaning; instrument workflow is also simplified with automated tuning, method generation
wizards, as well as real-time data checking functionality that prevents sample waste if the analytical run fails for any reason. The Xevo TQ MS’s software also features an interactive LC/MS method database, QuanPedia™, that
ensures that the analyst selects and uses the correct method parameters.
The T-Wave™ collision cell, originally introduced by Waters
in the Quattro Premier™ and used in the SYNAPT™ family
mass spectrometers, is employed to allow functionality such as rapid MRM switching, fast 20-msec positive ion/ negative ion switching, and minimized crosstalk. This functionality makes the instrument ideal for rapid method development or use in a drug discovery environment for development of multi-component assays.
Figure 3. MS software advancements such as QuanPedia allow users to choose pre-defined tasks for ease of operation.
Xevo TQ MS: Benefits for pharmaceutical laboratories
In drug discovery, mass spectrometers often serve a dual purpose as both a quantitative and a qualitative instrument in DMPK departments. The Xevo TQ MS provides the flexibility to not only perform both of these tasks, but also to achieve them in the same analytical run or even with the same eluting peak. This ScanWave™ functionality isachieved by maximizing the duty cycle of the instrument. In conventional mass spectrometers, the selected ions enter from the first quadrupole (Q
1
) into the collision cell where they are fragmented. The resulting fragmented ions exit the collision cell and are transferred through the third quadrupole (Q3) to the detector. This quadrupole (Q3) can act either as a selective filter, as in MRM mode, only allow-ing ions with a specific m/z value to pass through to the detector, or it can scan across the entire m/z value range providing a full spectrum.This full-scan mode is particularly useful when performing structural analysis; unfortunately, conventional instrumen-tation suffers from poor duty cycle. This is because the ions exit the collision cell simultaneously regardless of their m/z value; as the third quadrupole scans, it can only measure or detect one m/z value at a time. Therefore, for a scan speed of 1000 m/z per second over a mass range of 1000 Da and a 2-second-wide peak, the instrument will only spend 2 x 1/1000 second measuring each m/z value.
The Xevo TQ MS uses a novel collision cell design to improve full scan sensitivity. In the last third of the colli-sion cell, the fragmented ions are accumulated behind a DC barrier to effect ion enrichment. These ions are then released and contained between the DC barrier and an RF barrier at the end of the collision cell. The RF barrier is gradually reduced, ejecting the ions from the collision cell to the third resolving quadrupole. These ions are ejected according to their m/z ratio, with the heavier ions being ejected first. To improve the duty cycle of the instrument, the final quadrupole (Q3) is scanned in synchronization with the ejection of the ions from the collision cell, thus
This increased scan sensitivity can be used to address several business and scientific needs in pharmaceutical analysis. The acquisition of this high-duty-cycle acquisition scan can be triggered from a standard MS experiment to provide structural information on the identity of an LC peak.
n In the field of bioanalytical analysis, the
functional-ity can be employed to confirm identfunctional-ity of a peak by Product Ion Confirmation (PIC), which is carried out within an MRM analysis. PIC works by taking one high quality, high sensitivity spectra after the apex of a peak and before the “touch-down” of a chromatographic peak. This does not affect the fidelity or accuracy of the peak quantification but allows for the acquisition of a product ion spectra to confirm the identity of the peak.
n In the disciplines of metabolite identification and
impu-rity analysis, MS experiments such as constant neutral loss or common fragment ion analysis are often em-ployed to detect ions that are related to the parent API molecule, or to look for particular metabolites that may be toxic. Once the peaks of interest have been detected, a second analytical run is often required to obtain MS/MS structural information. The Xevo TQ MS can utilize its ability to perform either constant neutral loss or common fragment ion analysis and then rapidly switch to high-sensitivity MS/MS to obtain structural information. This capability removes the need for a
sec-Figure 4. Daughter ion scans obtained with ScanWave, top, and without, bottom. ScanWave DS DS O O O O O H O H O H O O C H3 O H N H2 C H3 Cl Cl N H N H O N H O O H NH O NH C H3 C H3 C H3 O H O H O H N H O N H O O NH2 O H O CO O H Fragmentation 0 1306
Daughter Ion Scan ScanWave Daughter Ion Scan
O O O O O H O H O H O O C H3 O H N H2 C H3 Cl Cl N H N H O N H O O H NH O NH C H3 C H3 C H3 O H O H O H N H O N H O O NH2 O H O CO O H O O O O O H O H O H O O C H3 O H N H2 C H3 Cl Cl N H N H O N H O O H NH O NH C H3 C H3 C H3 O H O H O H N H O N H O O NH2 O H O CO O H 1144 1332 100 m/z 200 400 600 800 1000 1200 % 100 % 0 144 170 725 371 144 1306 725
Recent regulatory guidelines on bioanalysis have placed greater emphasis on the measurement of ion suppression and control of the matrix, and measurement of drug-related metabolites. The functionality of the Xevo TQ MS facilitates rapid switching between matrix molecules and analytes of interest. Phospholipids are a class of endogenous molecules that have been associated with ion suppression. These molecules can be monitored by measuring the parents of the common fragment ion m/z 184, which is associated with the choline polar head group. The Xevo TQ MS allows the simultaneous MRM monitoring of the compound(s) of interest and precursors of m/z 184, allowing rapid and reliable method develop-ment. This capability can also be employed to monitor the background ions during the course of a clinical trial, to evaluate any differences between patients due to pheno-type, gender, age, or diet. This information provides extra confidence in the results and allows anomalies to be explained.
Ease-of-use and performance extends the utility of LC/MS
The usability and functionality of mass spectrometers have improved greatly over the last 15 years. Not only have these instruments become more sensitive and capable of performing multiple experiments simultane-ously, they have also become easier to use – thus improving instrument up-time and laboratory productiv-ity. The advent of fast electronics and novel collision cell designs has allowed high-sensitivity, full-scan MS/MS experiments to be performed at the same time as high-sensitivity MRM quantitative experiments. Concurrent with an increase in MS capabilities has been the move from analysis by an expert MS scientist to analysis by a user who is tasked with answering a specific question, such as to determine whether a product can be shipped or to monitor food safety. Improvements in ease-of-use and intelligent software features that help the general user be successful in their task will continue to push LC/MS adop-tion even wider into the general analytical community.Figure 5. Monitoring of a model drug, alprazolam, and matrix effects of phospholipids, in a single analysis.
Acknowledgements
The authors would like to thank Paul Rainville and Marian Twohig for their scientific contributions.
Reference
IM P ROV INg MS / MS S ENSIT IV IT y USINg X E VO T Q MS w IT H S C A N wAV E
Marian Twohig, Peter Alden, Gordon Fujimoto, Daniel Kenny, and Robert S. Plumb
Waters Corporation, Milford, MA, U.S.
INT RODUCTION
Tandem quadrupole mass spectrometry (MS) combined with liquid chromatography (LC) – and, in particular, UltraPerformance LC®
(UPLC®) – has become the technology of choice for high sensitivity
quantitative analyses such as bioanalysis in the pharmaceutical industry. The high selectivity and specificity of multiple reaction monitoring (MRM) analysis gives rise to excellent signal-to-noise ratios for the analysis of compounds in complex matrices. Full-scan acquisitions are also used to provide useful information for struc-tural elucidation in MS and MS/MS modes.
Conventional tandem quadrupole MS instruments have limited sensitivity in full-scan mode due to poor duty cycle. The Waters®
Xevo™ TQ Mass Spectrometer with ScanWave™ functionality delivers significant duty cycle improvements that provide enhanced sensitivity in scanning acquisition modes.
ScanWave experiments are performed at up to 10,000 amu/sec, making it possible to characterize narrow chromatographic peaks better. This has become a necessity since the advent of sub-2 µm column particle technology where narrow chromatographic peaks can be 2 seconds wide or less.
ScanWave defined
The Xevo TQ MS employs a unique concept in collision cell technology. Based on a novel use of Waters’ proven T-Wave™1 collision cell, the new
ScanWave™ mode of operation enhances both MS scan and product ion data. ScanWave operation is based upon two concepts (Figure 2). The first is that the front and back of the collision cell are indepen-dently controlled, which allows fragmentation and accumulation of ions to occur in the front of the gas cell while previously-accumulated ions are simultaneously ejected from the back of the gas cell. This provides 100 percent sampling efficiency.
Ejection of ions from the gas cell is mass dependent, although low resolution. This low-resolution behavior allows for high space-charge capacity without degradation of performance. The second concept behind ScanWave is that it links the low-resolution ion ejection from the gas cell with scanning of the final-resolving quadrupole (MS2). This enables an intelligent ion delivery where ions are presented to the final quadrupole when they are actually needed, rather than continuously as in traditional tandem quadrupole instruments.
This novel ion delivery technique provides significant duty cycle improvements that in turn result in enhanced signal in scanning acquisition modes. Since the scanning quadrupole (MS2) is the device performing the mass analysis, it is not necessary to perform a separate calibration. Scan rates, mass accuracy, and mass resolu-tion are all identical to that for operaresolu-tion in tradiresolu-tional scanning acquisition modes.
Figure 1. Unique T-Wave and ScanWave-enabled collision cell technology for the very best MS/MS data.
Figure 2. Schematic depicting a ScanWave experiment, where ions are accumulated before being sequentially ejected.
Significant increases in sensitivity using ScanWave
The data shown in Figure 3A are chromatograms for the conven-tional product ion scan, DS, and for the enhanced product ion scan, using ScanWave DS, produced from the UPLC/MS/MS analysis of vancomycin, a glycopeptide antibiotic, with m/z 725 for the [M+2H]2+ in positive ion electrospray mode. The chromatograms
have been superimposed and the vertical axes are displayed on the same scale.
A factor of 6X signal enhancement is observed for the largest chromatographic peak, number 5, when ScanWave DS is used.
Figure 3. Chromatogram A shows ScanWave product ion scan (ScanWave DS, green trace) versus the regular product ion scan (DS, red trace) of vancomycin, [M+2H]2+ m/z 725. In
B, the ScanWave DS chromatogram is shown with the x-axis plotted in scan number.
In the conventional product ion scan mode, peaks 1, 2, 4, and 5 are detected. When the same sample is analyzed using ScanWave DS, the resulting signal enhancement improved the level of sensitivity and the total number of peaks detected. In addition to the peaks that were found in this sample using the conventional product ion scan, spectra can be obtained for peaks 3, 6, 7, and 8.
Modern high resolution chromatography using sub-2 µm column particles produces peaks with widths of 1 to 3 seconds at the base. To accurately define these peaks, a high duty cycle/scan speed mass spectrometer is required.
Figure 3B shows the same chromatogram plotted with scan number as the x-axis. The scan speed of both the ScanWave DS and the conven-tional product ion scan experiments was 5000 amu/sec. This allowed more than 10 data points for the mass range 90 to 1455 amu to be collected across chromatographic peaks which were 3 seconds wide. Ejection Region DC Barrier RF Barrier Storage Region Potential To Scanning Quadrupole (MS2) Traveling Wave Traveling Wave Traveling Wave Traveling Wave Low m/z Ion High m/z Ion Intermediate m/z Ion Time 2.80 3.00 3.20 3.40 3.60 3.80 % 0 100 2, 3 4 5 8 7 1 6 Scan 550 600 650 700 750 800 850 % 0 100 ScanWave DS of 726ES+ TIC 1.25e8 2.88 3.73 A B
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com Figure 4 shows a mass spectrum of the largest chromatographic peak
(number 5) shown in Figure 3A. ScanWave DS of the doubly-charged ion m/z 725 resulted in the major singly-charged fragments m/z 100, m/z 144, and m/z 1306.
The data illustrates that the Xevo TQ MS is capable of acquiring high-quality spectral data while operating at the high scan speeds required to characterize narrow UPLC peaks.
Figure 4. ScanWave DS spectrum for vancomycin, [M+2H]2+ m/z 725.
CONCLUSION
The enhanced sensitivity of the Xevo TQ MS in ScanWave mode allows users to better characterize low-level components in their samples. ScanWave technology allows ions to be accumulated, separated, and ejected according to their m/z. The final quadrupole scanning is synchronized with ion ejection from the collision cell such that the ions of a given mass-to-charge ratio are delivered to the quadrupole when it is ready to scan this m/z value. This results in a more efficient instrument duty cycle and better sensitivity in scanning acquisitions. In this application note, ScanWave technology has allowed the peak detection of the vancomycin sample in MS/MS mode to be significantly improved. When ScanWave DS mode was used, spectra could be obtained for chromatographic peaks that were previously not detected by the conventional product ion scan.
Reference
1. The traveling wave device described here is similar to that described by Kirchner in U.S. Patent 5,206,506 (1993). 100 % 0 200 400 600 800 1000 1200 1400 1306 144 217 1143 725 m/z ScanWave DS of 725ES+ 3.64e6
Waters, UPLC, and ACQUITY UPLC are registered trademarks of Waters Corporation. Xevo, ScanWave, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2008 Waters Corporation. Printed in the U.S.A. October 2008 720002828EN LB-CP
SIMU LTA N EOUS CON F I RMAT ION A N d QUA NT I F IC AT ION USINg X E VO T Q MS:
P RO dU C T ION CON F I RMAT ION ( P IC )
Marian Twohig, Gordon Fujimoto, Joanne Mather, and Robert S. Plumb
Waters Corporation, Milford, MA, U.S.
INT RODUCTION
Tandem quadrupole mass spectrometers are used extensively in the pharmaceutical industry for analyte quantification. This is primarily performed by multiple reaction monitoring (MRM) as the matrices are complex and the specificity of MRM gives the best signal-to-noise ratios.
As well as performing quantification, these instruments are often used for initial qualitative information, with the instrument operated in scan mode. This information is used to confirm the identity of the peak of interest that is being quantified.
In complex matrices, situations can arise where closely-related compounds, e.g., metabolites or matrix interferences, can give rise to signals even in MRM mode. This can lead to ambiguity and may require a second qualitative experiment. Product ion confirmation provides a means of verifying that the signal from the MRM peak is from the compound of interest.
With conventional instrumentation, these experiments require separate full-scan analyses. Many conventional tandem quadrupole MS instruments are unable to perform MRM and scan experiments simultaneously, in the timeframe of an LC peak, while maintaining data quality. The Waters® Xevo™ TQ Mass Spectrometer is equipped
with a novel collision cell design. The collision gas is always on, allowing both quantification (MRM) and characterization to be performed simultaneously on the peak as it elutes from the LC or UPLC® column while maintaining good data quality.
The new ScanWave™ mode of operation allows ions within the collision cell to be accumulated and then separated according to their mass-to-charge (m/z) ratio. Synchronizing the release of these ions with the scanning of the second quadrupole mass analyzer greatly improves duty cycle, which significantly enhances the signal intensity of full-scan spectra for both MS and product ions.
Figure 1. Xevo TQ Mass Spectrometer with the ACQUITY UPLC® System.
EXPERIMENTAL
Product ion confirmation on Xevo TQ MS
The Xevo TQ MS can simultaneously acquire a product ion con-firmation (PIC) scan along with an MRM chromatogram to obtain additional information about an eluting peak. A PIC scan is enabled in the MRM method, where a scan is used to collect either:
n MS scan
n Enhanced MS scan using ScanWave mode n Product ion scan
n Enhanced product ion scan using ScanWave DS mode
In PIC mode, the Xevo TQ MS will switch from MRM to scan after the apex of an LC peak as long as a minimum intensity threshold is achieved. The trigger to start will occur after four consecutive downward scans have been detected. If the minimum intensity criteria is met, an MS or MS/MS spectrum is acquired using the final resolving quadrupole (MS2) to perform the scan before switching back to MRM mode (Figure 2). The threshold ensures that the PIC scan is of sufficient quality to be beneficial to the user.
The high data collection rate of the Xevo TQ MS is such that the area of the MRM peak can still be accurately determined, since PIC is triggered after the peak top is detected and the definition of the peak itself is not affected. Consequently, quantitative and qualita-tive data are acquired simultaneously.
Figure 2. Schematic showing Product Ion Confirmation (PIC) switching after the peak top.
Figure 3 shows an example of an MRM chromatogram (3A) obtained from the quantification of the corticosteroid fluticasone, m/z 501. Qualitative confirmation of the peak of interest is provided by the resulting PIC spectrum operated in ScanWave DS mode (3B). The scan range for the PIC is selected by the software, in this case m/z 40 to 511.
Figure 3. Chromatogram from the analysis of fluticasone, with MRM 501 > 293, and an example of the ScanWave DS PIC spectrum.
A PIC spectrum using ScanWave DS is displayed in Figure 4A. Here it is been compared with a PIC spectrum using conventional product ion scan (DS), 4B, and a combined spectrum (20 scans) from a ScanWave DS of fluticasone, 4C. The spectral quality is maintained when a PIC spectrum in ScanWave DS mode (4A) is compared to a combined ScanWave DS spectrum (4C).
The data show that a four-fold signal enhancement was observed when ScanWave DS mode (4A) is used to collect the PIC spectrum compared to a conventional product ion spectrum (4B). This is due to the more efficient duty cycle that is achieved in ScanWave mode. This extra sensitivity available with ScanWave mode allows for high quality spectra to be obtained even at low levels.
Switches here and acquires PIC Scan
Switches back to MRM data acquisition MRM Trace
B
m/z 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 % 0 100 293 205 109 185 155 121 147 275 265 251 217235 313 501 361 333 389 481 Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 % 0Flu_1_9_015d 1: MRM of1 Channel ES+
501.3 > 293.2 (Fluticasone) 4.09e7 MRM for Fluticasone PIC spectrum from MRM peak at Rt = 1.80 min O O O H C H3 H CH3 F F H CH3 O S F O CH3
A
100Figure 4. Spectrum shows a comparison of a PIC spectrum for ScanWave DS, a regular product ion PIC spectrum and a combined spectrum acquired by ScanWave DS for fluticasone m/z 501 (Vertical axis linked).
CONCLUSION
The Xevo TQ MS can be used to perform quantification of fluti-casone with simultaneous characterization of the MRM peak as it elutes from the chromatographic system. This eliminates the need for separate injections when qualitative confirmation of MRM peaks is required and reduces the total analysis time in these situations. When used routinely, product ion confirmation increases user confidence in qualitative results from complex matrixes, and thus reduces the need for re-analysis.
m/z 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 % 0 100 % 0 100 % 0 100Flu_1_9_015d 2 (1.813)
1: Product Ions of 501 ES+ 501.3 > 293.2 (Fluticasone) 5.26e7 293 205 109 185 155 135 275 251 217 313 501 361 333 389 481 Flu_1_9_014d 2 (1.814)
1: Product Ions of 501 ES+ 501.3 > 293.2 (Fluticasone) 5.26e7 293 205 109 155 251275 313 359 333 389
Flu_1_9_017d 961 (1.796) 293 ScanWave DS of 501ES+ 5.26e7
205 109 95 121 155 185 275 251 217 313 501 361 333 389 481 PIC spectrum ScanWave DS PIC spectrum DS Spectrum ScanWave DS A B C Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
Waters, UPLC, and ACQUITY UPLC are registered trademarks of Waters Corporation. Xevo, ScanWave, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2008 Waters Corporation. Printed in the U.S.A. October 2008 720002829EN LB-CP
NOV E L dUA L-S C A N M RM MO d E MA S S S P EC T ROM E T Ry FO R T H E d E T EC T ION
O F M E TA BO L IT E S dU RINg d RUg QUA NT I F IC AT ION
Paul D. Rainville, Jose Castro-Perez, Joanne Mather, and Robert S. Plumb
Waters Corporation, Milford, MA, U.S.
INT RODUCTION
The measurement of the levels of circulating drugs and their metab-olites is important information in the development of new therapies. Drug levels in biofluids are used to determine the bioavailability of a drug. Additionally, elucidation of drug metabolite information is vital due to the fact that they can often be toxic at certain levels, have a greater pharmacodynamic effect than the parent drug, inter-fere with concomitant medication, and impact liver function. These two different pieces of information are normally acquired in separate analytical experiments, resulting in increased laboratory workload and reduced efficiency. Therefore the ability to determine drug concentration and obtain metabolite structural information during a single analysis is not only faster but more cost effective. In the case of low sample volumes, e.g., pediatric studies, this capability is critical for laboratories to obtain required quantitative and qualitative data.
The Waters® Xevo™ TQ Mass Spectrometer is a tandem quadrupole
system equipped with a novel collision cell design that allows full-scan MS and quantitative multiple reaction monitoring (MRM) data to be acquired in a single analytical run.
Here, we present a method whereby full-scan MS and MRM data can be acquired in a single run to determine the levels of a model pharmaceutical in urine and utilize the associated full-scan data to determine its related metabolites.
EXPERIMENTAL
Human urine was collected from volunteer individuals eight hours after dosing with 400 mg of ibuprofen. The samples were stored frozen prior to analysis. Samples were prepared by centrifugation at 13,000 RCF for 5 minutes and diluted with water. Samples were then injected onto the UPLC®/MS/MS system.
LC conditions
LC system: Waters ACQUITY UPLC® System
Column: ACQUITY UPLC BEH C18 Column
2.1 x 50 mm, 1.7 µm Column temp.: 40 °C
Flow rate: 600 µL/min
Mobile phase A: 0.1% NH4OH Mobile phase B: ACN
MS conditions
MS system: Waters Xevo TQ MS
Ionization mode: ESI negative Capillary voltage: 2000 V Cone voltage: 15 V Desolvation temp.: 550 °C Desolvation gas: 1000 L/Hr Source temp.: 150 °C Scan range: m/z 100 to 500
Collision energies: MRM data 7 V, full-scan data 3 V MRM transition: m/z 205 > 161
RESULTS
Determining drug concentration and drug metabolites are both important aspects in developing a new medicine. This experiment was designed such that the levels of ibuprofen in urine were measured by MRM mass spectrometry and full-scan MS data was collected to detect the associated metabolites during a single injection.
The unique collision cell design of the Xevo TQ MS, which is continu-ously filled with collision gas, enables it to operate with rapid switching between MS and MS/MS data acquisition modes. This occurs in timeframe that is compatible with the fast chromatography and narrow peaks generated by the ACQUITY UPLC System: the Xevo TQ MS is capable of operating at up to 10,000 Da/sec and can correctly define the very sharp peaks produced by UPLC.
In this dataset, greater than 12 scans were acquired for the MRM channel of ibuprofen while also obtaining full scan MS data. Peaks widths were on the order of 2.4 seconds measured at peak base (data not shown). Figure 2 shows the chemical structure of ibuprofen and some of its major in vivo metabolites. Figure 3 displays the MRM transition data for ibuprofen in the urine samples and also the simultaneously acquired full-scan data. The full-scan data were then mined for potential metabolites resulting from ibuprofen. Figure 4 shows extracted ion chromatograms (XIC) that were generated relating to the ketone glucuronide (m/z 411), glucuronide (m/z 381), and hydroxy glucuronide (m/z 397) metabolites.
Figure 2. Ibuprofen and some of its associated metabolites.
Figure 3. MRM of ibuprofen and full-MS scan data acquired from subject urine. O CH2 OH CH3 C H3 CH3 O O-Gluc CH3 CH3 HO O O-Gluc CH3 C H3 CH3 OH O O-Gluc CH3 C H3 CH3 OH O O-G luc CH3 C H3 CH3 1-Hydroxy Ibuprofen Glucuronide 3-Hydroxy Ibuprofen Glucuronide 2-Hydroxy Ibuprofen Glucuronide Ibuprofen Glucuronide Ibuprofen MRM
Diluted urine patient 1 Full scan
Diluted urine patient 2 Full scan
Figure 4. XIC of ibuprofen metabolites and full-scan data.
Further confirmatory product ion MS experiments revealed several diagnostic fragment ions, such as m/z 193 and 175 for the glucuronide acid moieties, m/z 221 for the aglycone, and m/z 113 for ibuprofen itself (Figure 5).1
Figure 5. Product ion spectra of ibuprofen metabolites.
A further advantage to this acquisition approach is that it provides the scientist with the ability to visualize the differences between subject matrix using the full-scan MS. These differences may be related to several factors: diet, sex, age, or the state of an individual’s health. Thus the full scan data could be additionally utilized for the detection of biomarkers. Further, the full-scan MS data could be interrogated in the future if new information is required about the metabolism of the compound – without the need to re-run the samples.
Ketone XIC m/z 411 Glucuronides XIC m/z 381 Hydroxy glucuronides XIC m/z 397 Full scan Ketone Glucuronides Hydroxy glucuronides
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990
Waters, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. Xevo, ScanWave, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2008 Waters Corporation. Printed in the U.S.A.
CONCLUSION
In this application note, we have demonstrated that the Xevo TQ MS can acquire full-scan and MRM channel data to determine the level of a model pharmaceutical compound in urine and its metabolite information in a single analysis. The speed of the Xevo TQ MS proves to be highly compatible with the high resolving power of the ACQUITY UPLC System.
The benefits of this technique are realized in several ways. First, the ability to gather full-scan data along with MRM channel data enables scientists to collect multiple dimensions of information about a sample in a single run – maximizing the resource utilization of a laboratory that otherwise would have been performing multiple experiments to gain the same information. Second, coupling this MS technique with UPLC ensures a faster analysis. Finally, the richness of the data acquired by full-scan MS allows that information to be mined in multiple ways, giving researchers more confidence in their deci-sions as they direct their drug discovery and development studies.
Reference
1. Plumb R, Rainville P., et al. Rapid Communications in Mass Spectrometry. 2007; 21: 4079-85.
dATA- dI R EC T E d d E T EC T ION A N d CON F I RMAT ION O F d RUg M E TA BO L IT E S
IN BIOA NA Ly T IC A L S T U dI E S
Robert S. Plumb and Paul D. Rainville
Waters Corporation, Milford, MA, U.S.
INT RODUCTION
LC/MS/MS analysis has become the analytical method of choice for the accurate quantification of pharmaceutical compounds or active metabolites in biological fluids. The specificity and selectivity provided by tandem quadrupole MS in multiple reaction monitoring (MRM) mode allows for rapid high-sensitivity analysis, often in the pg/mL range. The data produced by LC/MS/MS analysis provides drug concentration data that is critical to successful drug discovery and development.
Recent U.S. FDA Guidance, “Industry Safety Testing of Drug Metabolites,” provides recommendations to industry on when and how to identify and characterize drug metabolites whose non-clinical toxicity needs to be evaluated. The aim of these guidelines is to ensure that variations in metabolic profiles across species are both quantitatively and qualitatively measured.1
The Waters® Xevo™ TQ Mass Spectrometer is capable of operating
at acquisition speeds up to 10,000 Da/sec, which aids in the adequate characterization of very sharp chromatographic peaks produced by the ACQUITY UltraPerformance LC® (UPLC®) System.
The Xevo TQ MS is equipped with a novel collision cell design that is continuously filled with collision gas, allowing rapid switching between MS and MS/MS modes in a single analytical run. This new collision cell is capable of enhanced high-sensitivity operation in MS/MS mode. In this Scanwave™ mode of operation, ions are constrained in the final third of the collision cell using both a DC and RF barrier. These ions are then ejected from the collision cell, in a controlled manner, from high to low m/z in synchronization with the scanning of the final resolving quadrupole. This increases the duty cycle of the instrument.
In this application note, we illustrate the ability of the Xevo TQ MS, in Survey Scan mode, to detect drug metabolites “on the fly” using a common diagnostic fragment ion.
EXPERIMENTAL
Rat plasma was spiked with ibuprofen and related major
metabolites. Samples were then precipitated using 2:1 acetonitrile to sample (v/v). The sample was evaporated to dryness and reconstituted in 9:1 water/methanol (v/v). The sample was then injected onto the UPLC/MS/MS system.
LC /MS conditions
LC system: Waters ACQUITY UPLC® System
Column: ACQUITY UPLC BEH C18 Column
2.1 x 50 mm, 1.7 µm Column temp.: 40.0 °C
Flow rate: 600 µL/min
Mobile phase A: 0.1 % NH4OH Mobile phase B: Acetonitrile
Gradient: 5% to 95% B/2 min
MS system: Waters Xevo TQ MS Ionization mode: ESI negative Capillary voltage: 2000 V
Cone voltage: 15 V
Collision energy: 7 eV
RESULTS
The superior efficiency of the ACQUITY UPLC System produces extremely narrow peaks, 2 seconds or less at the base. These narrow peaks require a fast data capture rate mass spectrometer to accurately define the peak. Figure 2 shows the MRM peak for ibuprofen using the transition m/z 205 to 161. The peak is 1.2 seconds wide at the base, and the high data capture rate of the Xevo TQ MS allows for more than 60 scans across the peak. This facilitates the accurate definition of the chromatographic peak, even if several MRM transitions are employed during analysis.
Figure 2. UPLC/MS/MS of ibuprofen using the MRM transition m/z 205 to 161.
Recent FDA guidelines have recommended that, during human clinical trials, the concentration and identity of any metabolites with an exposure of greater than 10% of the dosed compound must be determined. Mass spectrometry can detect and identify drug metabolites by various means. One method is to utilize Survey Scan mode. In this mode of operation, the MS is set to monitor a diagnostic fragment ion from the parent drug compound.
The use of a common fragment ion requires the mass spectrometer to scan the first quadrupole (Q1) while monitoring for a fixed m/z with the final resolving quadrupole (Q3). Ibuprofen gives rise to several distinctive product ions, m/z 113, 133, and 161.2 Figure 3
illustrates Xevo TQ MS operation in Survey Scan mode. In this example, the common fragment ion of m/z 113 was monitored by the resolving quadrupole. When a peak, containing a m/z of 113, was detected the MS switched to collect product ion data on the precursor ion containing the m/z 113. Peaks that exceed a user-defined detection threshold are used to trigger the acquisi-tion of product ion data.
Figure 4 illustrates the MS/MS spectra obtained for the peak detect-ed at 0.66 minutes. In this example, we can see that the precursor peak m/z value is 397. The m/z 397 produces major fragment ions at m/z 113, 175, 193, and 221.
Figure 3. Survey Scan: precursors of m/z 113 switching to product ion scan. Time 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 % 0 100 0.82 Scan 1820 1840 1860 1880 1900 1920 1940 1960 % 0 100 0.81
The m/z values and MS fragment pattern confirm the identity of this peak as the O-glucuronide metabolite of ibuprofen.2 The data
acquired for the peak eluting with a retention time of 0.88 minutes are shown below in Figure 5.
Figure 5. ScanWave DS of peak eluting at 0.88 minutes with a m/z value of 381.
This peak was determined to have a m/z value of 381. Resulting fragment ions produced from the product ion MS/MS were m/z 113, 161, 175, 193, and 205. This data confirmed that this peak was related to ibuprofen and, with the precursor ion m/z value of 381, was confirmed as the glucuronide conjugate of ibuprofen.2
Thus with one simple analytical experiment, along with the knowl-edge of the fragmentation pattern of the ibuprofen, the metabolites could be detected and the structure confirmed.
CONCLUSION
The quantification of pharmaceutical compounds in biological fluids is a regulatory requirement as part of any new drug submission, e.g., IND, CTX. More recently, these regulations have required that drug metabolites with an exposure greater than 10% of the active pharmaceutical be quantified and characterized. The Xevo TQ MS can perform data-directed MS/MS experiments, allowing metabolite structural confirmation using common fragment ions within a UPLC peak timeframe.
References
1. U.S.FDA. Guidance for Industry, “Safety Testing for Metabolites.” http://www.fda.gov/CDER/GUIDANCE/6897fnl.pdf
2. Plumb R., Rainville P, et al. Rapid Communications in Mass Spectrometry. 2007; 21: 4079-85. Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
Waters, ACQUITY UltraPerformance LC, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. Xevo, ScanWave, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. ©2008 Waters Corporation. Printed in the U.S.A.
IM P ROV INg QUA L ITAT IV E CON F I RMAT ION USINg X E VO T Q MS w IT H SU RV E y S C A NNINg
Marian Twohig, Andrew Aubin, Michael Jones, and Robert S. Plumb
Waters Corporation, Milford, MA, U.S.
INT RODUCTION
On a conventional tandem quadrupole mass spectrometer, the search for unknowns generally requires multiple injections: one injection in full-scan LC/MS mode followed by a second injection for targeted LC/MS/MS experiments. This results in increased time required to obtain the necessary data, in addition to the time the analyst needs to construct the MS/MS methods.
Real-time data-directed switching simplifies this experimental approach. In data-directed mode, a full spectrum LC/MS run is collected, with an LC/MS/MS experiment triggered if the signal in the LC/MS survey meets preset criteria.
Modern Linear Ion Trap (LIT) mass spectrometers allow the col-lection of MS, MRM, and MS/MS data in the same analytical run, enabling quantitative and qualitative data to be obtained simultane-ously. The duty cycle of these instruments when switching between MS and MS/MS modes is typically 2 to 3 seconds. With modern high-resolution, sub-2 µm particle chromatography, such as UPLC,®
peak widths of 2 to 3 seconds are now commonplace, thus with these LIT MS systems this results in just 1 to 2 points across the peak giving poorly defined peaks and possibly missed components. The Waters® Xevo™ TQ MS is capable of scan speeds up to 10,000
amu/sec. Consequently, it is possible to employ a number of scan functions in a single run while still maintaining good peak charac-terization with no loss in data quality.
EXPERIMENTAL
Survey Scans on Xevo TQ MS
Survey Scans on the Xevo TQ MS allow intelligent switching of LC/MS and LC/MS/MS data in one run, thus improving productivity. Conventional MS or ScanWave™ MS scanning experiments can be used to trigger MS/MS experiments in real time as the peaks are eluting from the LC column. A more targeted screen can also be performed using parent ion or neutral loss spectral acquisition, to screen for compounds that have common structural features. Conventional product ion or enhanced product ion spectra (ScanWave) data can be generated for all the components present in these complex samples. In ScanWave mode, duty cycle improve-ments result in signal enhancement in scanning acquisition modes, which facilitates the detection of low-level impurities.
An example of a survey scan for the active pharmaceutical ingredient (API) quetiapine, an antipsychotic medication, is shown in Figure 2, where the initial survey function is ScanWave MS switching to ScanWave DS.
Figure 2. Shown is an example of a Survey Scan of quetiapine (m/z 384) where the initial ScanWave MS Survey function switches to ScanWave DS mode.
In Figure 3, quetiapine (C21H25N3O2S) was analyzed in survey scan mode. The structure-characteristic fragments of quetiapine1,2
are m/z 253 (C15H13N2S) and m/z 279 (C17H15N2S). In the above example, a Precursor Ion Scan (m/z 253) was used to trigger the acquisition of a ScanWave product ion scan (ScanWave DS), generating a full product ion spectrum for the compounds potentially related to quetiapine.
More than 20 compounds were observed to have the fragments m/z 253 and m/z 279 as well as another signature fragment m/z 221 (C15H13N2). Shown in Figure 4 are spectra from the chromatographic peaks at retention times 7.86 min, 9.95 min, 10.13 min, 14.01 min, 15.94 min, and 17.52 min, respectively.
Figure 3. Survey precursor scan of m/z 253 (lower trace) switching to ScanWave DS.
Included are the API, quetiapine, and the previously-characterized1,2
quetiapine carboxylate and bis (dibenzo) piperazine, as well as three unknown compounds that have similar fragmentation patterns. This information was obtained from one Survey experiment without the need for extra confirmatory MS/MS analyses. This allows the analyst to acquire important structural information in a single run.
S N + N H S N N N O OH m/z 300 350 400 450 500 550 600 650 % 0 100 384 m/z 100 200 300 400 500 600 % 0 100 253 158 384 ScanWave MS ScanWave DS m/z 384 Quetiapine Diagnostic Fragment m/z 253 MS/MS of m/z 384 Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 % 0 100 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 % 0 100 8.41 7.86 6.11 2.68 7.18 9.26 17.15 15.94 14.01 11.04 12.42 18.27 9.79 9.27 7.87 8.38 17.51 14.00 Precursors of m/z 253 ScanWave DS
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
Figure 4. UPLC/MS/MS spectra in ScanWave DS mode from selected chromatographic peaks shown in Figure 3 (top).
CONCLUSION
The Waters Xevo TQ MS, with its unique collision cell design, where the collision gas is always on, facilitates the simultaneous acquisition of MS and MS/MS data in one LC/MS run. Its high scan speed allows for these experiments to be performed with sufficient points across the peak to accurately define the narrow peaks produced by UPLC. This capability facilitates data-directed experiments, where real-time switching between MS and MS/MS allows more information to be acquired from a single injection. This reduces the need for separate experiments and accelerates the process of structural identification and unknown compound determination.
References
1. Xu H, Wang D, Sun C, Pan Y, Zhou M. Identification of an unknown trace level impurity in bulk drug of Seroquel by high-performance liquid chromatography combined with mass spectrometry. J Pharm Biomed Anal. 2007 Jun 28; 44(2): 414-20. Epub 2007 Mar 7.
2. Bharathi Ch, Prabahar KJ, Prasad ChS, Srinivasa Rao M, Trinadhachary GN, Handa VK, Dandala R, Naidu A. Identification, isolation, synthesis and characterisation of impurities of quetiapine fumarate. Pharmazie. 2008 Jan; 63(1): 14-9. m/z 100 150 200 250 300 350 400 450 500 550 600 650 700 750 % 0 100 % 0 100 % 0 100 % 0 100 % 0 100 % 0 100
02_Sur_QT_3_10_001 2054 (7.856) 2: Auto ScanWave DS 482.46ES+ 8.93e7
253 221
143 279 482
02_Sur_QT_3_10_001 2636 (9.958) 2: Auto ScanWave DS 383.84ES+
8.93e7
253 221
158 210 279
02_Sur_QT_3_10_001 2683 (10.132) 2: Auto ScanWave DS 428.56ES+ 5.92e7 253
221
202 279
02_Sur_QT_3_10_001 3759 (14.007) 2: Auto ScanWave DS 456.48ES+
7.02e7
253
221 279 456
02_Sur_QT_3_10_001 4297 (15.946) 2: Auto ScanWave DS 510.38ES+
8.43e7
253
221 279 510
02_Sur_QT_3_10_001 4734 (17.524) 2: Auto ScanWave DS 505.53ES+ 3.88e7 253 221 279 505 Unknown Quetiapine Quetiapine Carboxylate Unknown Unknown Bis (dibenzo) Piperazine
Waters and UPLC are registered trademarks of Waters Corporation. Xevo, ScanWave, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2008 Waters Corporation. Printed in the U.S.A. October 2008 720002838EN LB-CP
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990
Waters, ACQUITY UPLC, UltraPerformance LC and UPLC are registered trademarks of Waters Corporation. XBridge and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
A NOV E L M E T HO d FO R MONIT O RINg MAT RIX INT E R F E R ENC E S IN BIO LOgIC A L SAM P L E S
USINg dUA L-S C A N M RM MO d E MA S S S P EC T ROM E T Ry
Paul D. Rainville, Joanne Mather, and Robert S. Plumb
Waters Corporation, Milford, MA, U.S.
INT RODUCTION
Development of a fast, sensitive, and robust bioanalytical LC/MS/MS assay is essential for cost-effective and compliant processing of samples in biological fluids.
However, any bioanalytical assay can be hampered by sample matrix effects. Components such as drug metabolites, proteins, and phospholipids within biological matrices frequently interfere with the robustness and sensitivity of the assay. Furthermore, regulatory authorities now require that matrix effects be deter-mined in these assays.
Therefore, it would be very advantageous to actively monitor and characterize the presence of matrix components coeluting with the compound of interest during LC/MS/MS assay method development. Significant time and effort could be saved by ensuring that compo-nents in the matrix are well resolved from the analyte of interest. However, conventional tandem quadrupole mass spectrometers cannot acquire multiple reaction monitoring (MRM) data while acquiring full-scan data at speeds fast enough for the narrow chromatographic peaks generated by modern separations techniques such as UPLC.®
In this application note, we describe the ability of a novel UPLC/MS/MS platform, the Waters® Xevo™ TQ Mass Spectrometer,
to monitor potential matrix interferences in plasma while monitoring a pharmaceutical compound of interest. Unique to the Xevo TQ MS platform is its ability to switch between MS and MS/MS modes in a UPLC run that typically generates peak widths of 2 to 3 seconds.
EXPERIMENTAL
Alprazolam was spiked into rat plasma at a concentration of 10 ng/mL and then precipitated with acetonitrile using a 2:1 acetonitrile/plasma ratio. The sample was then centrifuged at 13,000 RCF for 5 minutes. The supernatant was removed and injected onto the UPLC/MS/MS system.
LC conditions
LC system: Waters® ACQUITY UPLC® System
Column: ACQUITY UPLC BEH C18 Column
2.1 x 50 mm, 1.7 µm Column temp.: 40 °C
Flow rate: 600 µL/min
Mobile phase A: 0.1 % NH4OH Mobile phase B: MeOH
MS conditions
MS System: Waters Xevo TQ MS
Ionization mode: ESI positive Capillary voltage: 1000 V Cone voltage: 25 V Desolvation temp.: 500 °C Desolvation gas: 1000 L/Hr Source temp.: 150 °C Scan range: m/z 100 to 1000
Collision energies: High 20 V, low 3 V MRM transition: m/z 309 > 281
RESULTS
As previously stated, the large number of components found in matrices commonly employed in bioanalysis, such as plasma and urine, can pose a significant problem when developing and validat-ing a quantitative bioanalytical method. Techniques such as solid phase extraction (SPE) and high resolution chromatography are often employed to reduce their effects.1,2
Plasma, for instance, has many endogenous compounds that can interfere with the pharmaceutical compound undergoing quantifica-tion. Some of the major interferences in plasma samples that cause ion suppression or enhancement are phospholipids, specifically variants containing the choline head group. Consequently, scientists developing bioanalytical methods often will monitor phospholipids as they can be a major source of matrix effects.
Parent or precursor ion scanning of the indicative choline fragment ion (m/z 184, in positive ion mode) is commonly used to monitor phospholipids (Figure 2).
Figure 2. Fragmentation pattern for a phospholipid containing the choline head group. CH3 CH3 CH3 N+ O O O -P O O O O O R R' 184 CH3 CH3 CH3 N+ O O O -P O O O O O R R'
Figure 3 shows the MRM channel for alprazolam above a scan result for precursor m/z 184. In this example, we can see that UPLC meth-odology facilitates excellent resolution of the analyte of interest from the choline-containing phospholipids.
Figure 3 further illustrates the differences between the two scans of the two lots of plasma, indicating that each contains different choline-containing phospholipids. This variance in plasma lot A and lot B exemplifies the reason six different lots of matrix are required to be analyzed during the bioanalytical method validation process.3
Figure 3. Monitoring of the model drug alprazolam and matrix effects contributed by choline-containing phospholipids in a single analysis.
Recognizing that there may be other compounds in the matrix that could potentially interfere, and thus become a source of matrix effects, a full MS scan was acquired from the same injection as the MRM channel (Figure 4). Acquiring full-scan data gives the bioanalytical scientist the tools to observe more potential matrix interferences in the samples.
Figure 4. Simultaneous monitoring of the model drug alprazolam and matrix in the m/z 100 to 1000 range in a single analysis.
Figure 4 indicates the presence of other coeluting analytes from the matrix. The coeluting analytes have the potential to introduce matrix effects to the analysis of alprazolam, and may impact the quantification of the analyte of interest. The scientist is now in a position to adjust the conditions of the assay method, taking into account the interfering components.
The unique capability of the Xevo TQ MS to rapidly switch between MS and MS/MS modes facilitates simultaneous monitoring of coeluting components as well measuring the compound of interest, even when UPLC peaks are typically 2 to 3 seconds wide.
MRM alprazolam plasma lot A MRM alprazolam plasma lot B Parents of m/z 184 plasma lot A Parents of m/z 184 plasma lot B MRM alprazolam plasma lot A MRM alprazolam plasma lot B MS scan m/z 100 to 1000 plasma lot A MS scan m/z 100 to 1000 plasma lot B
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990
CONCLUSION
In this application note, we have shown the novel ability of the Waters Xevo TQ MS coupled with an ACQUITY UPLC System to acquire quality full-scan and MRM data in a single analysis. Using this technique, we monitored potential matrix interferences present in protein-precipitated plasma while monitoring the MRM transition for a model pharmaceutical.
Interferences due to coelution of matrix components can thus be detected “on the fly” in early method development, which reduces:
n Method development time n Method variability
n Problematic occurrences during validation
Thus utilizing the Xevo TQ MS enables researchers to increase the quality of the final MS/MS method while developing a fully-detailed scan record should reviewing the data be of interest.
References
1. Zhang S, Chen G. Journal of Chromatographic Science. 2008; 46: 220-224. 2. Chambers E, Diehl D, Lu Z, Mazzeo J. Journal of Chromatography B. 2007;
852 (1-2): 22-34.
3. Guidance for Industry, Bioanalytical Method Validation. U.S. FDA. 2001.
Waters, UPLC, and ACQUITY UPLC are registered trademarks of Waters Corporation. Xevo and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
R A P I d, SIM P L E IM P U RIT y C HA R AC T E RIz AT ION w IT H T H E X E VO T Q MA S S S P EC T ROM E T E R
Robert S. Plumb, Michael D. Jones, and Marian Twohig
Waters Corporation, Milford, MA, U.S.
INT RODUCTION
The detection and characterization of impurities and degradation products of an active pharmaceutical ingredient (API) are regulatory filing requirements. The detection and identification of impurities not only ensures medicine safety but can also be used as a finger-print for patent protection and counterfeit drug analysis. Impurity characterization and identification are normally carried out using information-rich analytical techniques such as NMR and LC/MS. Analysis by LC/MS provides parent ion mass from full-scan MS and structural information from the fragments generated in MS/MS experiments. With traditional tandem quadrupole instrumen-tation, the generation of this data requires multiple experiments to obtain MS and MS/MS information.
Modern Linear Ion Trap (LIT) mass spectrometers allow the collection of MS, multiple reaction monitoring (MRM), and MS/MS data in the same analytical run, allowing quantitative and qualitative data to be obtained simultaneously. However, the duty cycle of these instru-ments when switching between MS and MS/MS modes is typically 2 to 3 seconds. With modern high-resolution, sub-2 µm column par-ticle chromatography such as UPLC,® peak widths of 2 to 3 seconds
are now commonplace. With these LIT MS systems, this would result in just 1 to 2 points across the peak, with the peaks either poorly defined or missed completely; thus slower, lower-resolution LC systems must be used, resulting in reduced throughput and lower data quality. The Waters® Xevo™ TQ Mass Spectrometer is equipped with a novel
collision cell design that is continuously filled with collision gas, allowing rapid switching between MS and MS/MS modes. The Xevo TQ MS is capable of operating at up to 10,000 Da/sec and can correctly define the very sharp peaks produced by UPLC, with more than 10 points across a 2-second-wide peak, even on a multi-scan experiment. This new collision cell is capable of enhanced high-sensitivity operation in MS/MS mode. In this mode of operation, ions are constrained in the final third section of the collision cell using both DC and RF barriers. These ions are then ejected from the collision cell, in a controlled manner, from high to low m/z in synchronization
with the scanning of the final resolving quadrupole. This increases the duty cycle of the instrument, resulting in enhanced sensitivity that is ideal for the detection and characterization of low-concentra-tion impurities that may result in toxic effects.
EXPERIMENTAL
To evaluate the performance of this system, the impurities of the common pharmaceutical drug quetiapine, used to treat biopolar disorder, was investigated using UPLC/MS/MS.
LC /MS conditions
LC system: Waters ACQUITY UPLC® System
Column: ACQUITY UPLC BEH C18 Column
2.1 x 50 mm, 1.7 µm Column temp.: 65 °C
Flow rate: 800 µL/min
Mobile phase A: 20 mM Ammonium bicarbonate pH 10 Mobile phase B: Acetonitrile
MS system: Waters Xevo TQ MS Ionization mode: ESI positive Capillary voltage: 30 V Collision energy: 15 eV
RESULTS
The unique collision cell design allows the Xevo TQ MS to be oper-ated in several different modes of operation: full scan MS, MRM, as well as MS/MS mode. As the collision cell is continuously filled with collision gas, the instrument can rapidly switch between MS and MS/MS in the same analytical run. This allows MRM and MS scans to be performed in the same run. Combined with the high scan rate, this allows for rapid survey scans to be performed, such as MS neutral loss or parent ion, before switching to MS/MS.
This high data-capture rate allows for the accurate definition of the peak, even with the very narrow peaks produced by UPLC. Figure 2 shows the UPLC/MS chromatogram produced in the analysis of an API batch of quetiapine at a concentration of 1 µg/mL. Here we can see that impurity peaks are 2 to 4 seconds wide at the base. The data shown in Figure 3 illustrates the number of scans achieved in MS and MS/MS modes.
Figure 2. UPLC/MS analysis of quetiapine at 1 µg/mL.
Maximizing LC peak definition
In this example, the Xevo TQ MS was operated in ScanWave MS mode, switching to ScanWave DS (daughter ion scan) mode when a peak was detected above a user-defined threshold. In this mode of operation, the i
