XEVO TQ MS PHARMACEUTICAL APPLICATION NOTEBOOK

Transcription

1 XEV TQ MS PHARMACEUTICAL APPLICATIN NTEBK

2 Xevo TQ MS: Pharmaceutical Applications 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 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

3 The Changing Face of LC/MS: From experts to users Robert S. Plumb Senior Applications Manager, Pharmaceutical Business perations, 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 demonstrated 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 ionization (API) source interfacing was just beginning. Samples 3

4 requiring analysis were passed from the requesting scientist 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 spectrometers 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 operate 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 ed 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 25 units. This large number of units sold each year is also reflected in the increased number of users. In 198, the number of scientists attending ASMS was around 125; by 22 this had risen to greater than 4 with a growth rate of 1 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 appropriate experiments to be performed as well as to interpret the data automatically and recommend a course of action (pass/fail, pure/impure). Advances in chromatography Interfacing Liquid Chromatography with Mass Spectrometry (LC/MS) allows analytical chemists access to about 8 percent of the chemical universe unreachable by Gas Chromatography (GC); it is also responsible for the phenomenal 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 197s, LC/MS as we know it today reached maturation in the early 199s. 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 highperformance liquid chromatography (HPLC). In 24, further advances in instrumentation and column technology 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, psi (1, 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 197s. 4

5 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 nevertheless 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 ( 12 C). nce 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 proportional electrical current that a data system then reads. The data system converts the current to digital information, 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 ionization (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 represented in the spectrum. The general term atmospheric pressure ionization (API) includes the most notable technique, electrospray ionization (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 5 and 4 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 controversy. 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 reduction 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 3 Da while ion evaporation dominant 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. 5

6 superior signal-to-noise ratio. This dramatically improves assay sensitivity and specificity. The vast majority of quantitative experiments are performed on quadrupolebased instruments; whereas ion traps and accurate mass instruments are preferred for structural elucidation experiments. Figure 1. The Xevo TQ Mass Spectrometer. 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 MS n ) to produce a wealth of information about the molecule s structure. ther instruments such as magnetic sector instruments, hybrid quadrupole time-offlight (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 reaction 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 the detector, it all but eliminates the noise, resulting in 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. ne 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-trap instru- 6

7 ment 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 experiment (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 chromatographic 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 flexibility 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 technology 1, holds the potential to resolve many of these issues. The Waters Xevo TQ Mass Spectrometer employs traveling-wave technology that improves MS capabilities by performing simultaneous, 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 Figure 2. A tool-free source ensures easy access for any user to perform rapid maintenance. Figure 3. MS software advancements such as QuanPedia allow users to choose pre-defined tasks for ease of operation. 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 2-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. 7

8 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 is achieved by maximizing the duty cycle of the instrument ScanWave Daughter Ion Scan 371 ScanWave DS 725 Daughter Ion Scan DS H H H N CH 3 H 2 N H H 3C Cl H N N H C H H H H N H NH 2 H H Cl H N H NH H 3C CH 3 NH CH 3 In conventional mass spectrometers, the selected ions enter from the first quadrupole (Q1) 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 allowing 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 instrumentation 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 1 m/z per second over a mass range of 1 Da and a 2-second-wide peak, the instrument will only spend 2 x 1/1 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 collision 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 increasing the number of ions reaching the detector and hence increasing sensitivity. 8 Fragmentation m/z Figure 4. Daughter ion scans obtained with ScanWave, top, and without, bottom. 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 functionality can be employed to confirm identity 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 impurity analysis, MS experiments such as constant neutral loss or common fragment ion analysis are often employed to detect ions that are related to the parent API molecule, or to look for particular metabolites that may be toxic. nce 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 second analytical run, and, hence, improves productivity.

9 Figure 5. Monitoring of a model drug, alprazolam, and matrix effects of phospholipids, in a single analysis. Recent regulatory guidelines on bioanalysis have placed greater emphasis on the measurement of ion suppression and control of the matrix, and measurement of drugrelated 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 development. 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 phenotype, 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 simultaneously, they have also become easier to use thus improving instrument up-time and laboratory productivity. 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 highsensitivity 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 adoption even wider into the general analytical community. Acknowledgements The authors would like to thank Paul Rainville and Marian Twohig for their scientific contributions. Reference 1. The traveling wave device described here is similar to that described by Kirchner in U.S. Patent 5,26,56 (1993). 9

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11 Improving MS/MS Sensitivity using Xevo TQ MS with ScanWave Marian Twohig, Peter Alden, Gordon Fujimoto, Daniel Kenny, and Robert S. Plumb Waters Corporation, Milford, MA, U.S. INTRDUCTIN 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 structural 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 1, 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 independently controlled, which allows fragmentation and accumulation of ions to occur in the front of the gas cell while previouslyaccumulated ions are simultaneously ejected from the back of the gas cell. This provides 1 percent sampling efficiency. Ejection of ions from the gas cell is mass dependent, although low resolution. This low-resolution behavior allows for high spacecharge capacity without degradation of performance. The second concept behind ScanWave is that it links the lowresolution 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 resolution are all identical to that for operation in traditional scanning acquisition modes. Figure 1. Unique T-Wave and ScanWave-enabled collision cell technology for the very best MS/MS data. 11

12 Potential DC Barrier Low m/z Ion Intermediate m/z Ion RF Barrier To Scanning Quadrupole (MS2) 1 A 5 1 B ScanWave DS of 726ES+ TIC 1.25e8 High m/z Ion 2.88 Storage Region Ejection Region 3.73 Scan Traveling Wave 4 Traveling Wave Traveling Wave 2, 3 8 Traveling Wave Time Figure 2. Schematic depicting a ScanWave experiment, where ions are accumulated before being sequentially ejected. 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. Significant increases in sensitivity using ScanWave The data shown in Figure 3A are chromatograms for the conventional 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. 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 conventional product ion scan experiments was 5 amu/sec. This allowed more than 1 data points for the mass range 9 to 1455 amu to be collected across chromatographic peaks which were 3 seconds wide. 12

13 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 1, m/z 144, and m/z 136. The data illustrates that the Xevo TQ MS is capable of acquiring highquality spectral data while operating at the high scan speeds required to characterize narrow UPLC peaks. 1 ScanWave DS of 725ES+ 3.64e6 136 CNCLUSIN 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. 144 Reference 1. The traveling wave device described here is similar to that described by Kirchner in U.S. Patent 5,26,56 (1993) m/z Figure 4. ScanWave DS spectrum for vancomycin, [M+2H] 2+ m/z 725. 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. 28 Waters Corporation. Printed in the U.S.A. ctober EN LB-CP Waters Corporation 34 Maple Street Milford, MA 1757 U.S.A. T: F:

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15 Sim u lta n eous Confirmat io n a n d Qua n t i f ic at io n using X e v o T Q MS: Product Ion Confirmation (PIC) Marian Twohig, Gordon Fujimoto, Joanne Mather, and Robert S. Plumb Waters Corporation, Milford, MA, U.S. INTRDUCTIN 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-tonoise 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 confirmation (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. 15

16 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 qualitative data are acquired simultaneously. Flu_1_9_15d 1 A H F H 3C F S CH 3 H H CH 3 CH 3 MRM for Fluticasone 1: MRM of1 Channel ES > (Fluticasone) 4.9e7 F 1 B Time MRM Trace Switches here and acquires PIC Scan Switches back to MRM data acquisition m/z PIC spectrum from MRM peak at Rt = 1.8 min Figure 2. Schematic showing Product Ion Confirmation (PIC) switching after the peak top. Figure 3. Chromatogram from the analysis of fluticasone, with MRM 51 > 293, and an example of the ScanWave DS PIC spectrum. Figure 3 shows an example of an MRM chromatogram (3A) obtained from the quantification of the corticosteroid fluticasone, m/z 51. 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 4 to 511. 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 (2 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. 16

17 Flu_1_9_15d 2 (1.813) 1 Flu_1_9_14d 2 (1.814) 1 PIC spectrum ScanWave DS PIC spectrum DS : Product Ions of 51 ES > (Fluticasone) 5.26e A 1: Product Ions of 51 ES > (Fluticasone) 5.26e7 B CNCLUSIN The Xevo TQ MS can be used to perform quantification of fluticasone 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. Flu_1_9_17d 961 (1.796) 1 Spectrum ScanWave DS ScanWave DS of 51ES+ 5.26e m/z C Figure 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 51 (Vertical axis linked). 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. 28 Waters Corporation. Printed in the U.S.A. ctober EN LB-CP Waters Corporation 34 Maple Street Milford, MA 1757 U.S.A. T: F:

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19 Novel Dual-Scan MRM Mode Mass Spectrometry for the Detection of Metabolites during Drug Quantification Paul D. Rainville, Jose Castro-Perez, Joanne Mather, and Robert S. Plumb Waters Corporation, Milford, MA, U.S. INTRDUCTIN The measurement of the levels of circulating drugs and their metabolites 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, interfere 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 fullscan 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 4 mg of ibuprofen. The samples were stored frozen prior to analysis. Samples were prepared by centrifugation at 13, 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 C 18 Column 2.1 x 5 mm, 1.7 µm Column temp.: 4 C Flow rate: 6 µl/min Mobile phase A:.1 NH 4 H Mobile phase B: ACN Gradient: 5 to 95 B/2 min 19

20 MS conditions MS system: Waters Xevo TQ MS Ionization mode: ESI negative Capillary voltage: 2 V Cone voltage: 15 V Desolvation temp.: 55 C Desolvation gas: 1 L/Hr Source temp.: 15 C Scan range: m/z 1 to 5 Collision energies: MRM data 7 V, full-scan data 3 V MRM transition: m/z 25 > 161 H 3 C CH 3 H CH 3 1-Hydroxy Ibuprofen Glucuronide H 3C CH 3 CH 3 - Gluc H 3 C - Gluc Ibuprofen Glucuronide CH 3 CH 2 CH 3 H H H 3C CH 3 CH 3 CH 3 - Gluc 3-Hydroxy Ibuprofen Glucuronide CH 3 H 2-Hydroxy Ibuprofen Glucuronide - Gluc Figure 2. Ibuprofen and some of its associated metabolites. 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 continuously 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 1, 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). Ibuprofen MRM Diluted urine patient 1 Full scan Diluted urine patient 2 Full scan Figure 3. MRM of ibuprofen and full-ms scan data acquired from subject urine. 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. 2

21 Ketone XIC m/z 411 Ketone Glucuronides XIC m/z 381 Glucuronides Hydroxy glucuronides XIC m/z 397 Hydroxy glucuronides Full scan Figure 5. Product ion spectra of ibuprofen metabolites. 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 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. 21

22 CNCLUSIN 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. Reference 1. Plumb R, Rainville P., et al. Rapid Communications in Mass Spectrometry. 27; 21: 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 decisions as they direct their drug discovery and development studies. 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. 28 Waters Corporation. Printed in the U.S.A. ctober EN LB-CP Waters Corporation 34 Maple Street Milford, MA 1757 U.S.A. T: F:

23 Data-Directed Detection and Confirmation of Drug Metabolites in Bioanalytical Studies Robert S. Plumb and Paul D. Rainville Waters Corporation, Milford, MA, U.S. INTRDUCTIN 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 nonclinical 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 1, 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. Figure 1. Xevo TQ Mass Spectrometer. 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 C 18 Column 2.1 x 5 mm, 1.7 µm Column temp.: 4. C Flow rate: 6 µl/min Mobile phase A:.1 NH 4 H Mobile phase B: Acetonitrile Gradient: 5 to 95 B/2 min 23

24 MS system: Waters Xevo TQ MS Ionization mode: ESI negative Capillary voltage: 2 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 25 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 6 scans across the peak. This facilitates the accurate definition of the chromatographic peak, even if several MRM transitions are employed during analysis. 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 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 acquisition of product ion data. Figure 4 illustrates the MS/MS spectra obtained for the peak detected at.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 Scan Figure 3. Survey Scan: precursors of m/z 113 switching to product ion scan. Time Figure 2. UPLC/MS/MS of ibuprofen using the MRM transition m/z 25 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 1 of the dosed compound must be determined. Mass spectrometry can detect and identify drug metabolites by various means. ne 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. Figure 4. Survey Scan ScanWave DS spectrum of peak eluting at.66 minutes. 24

25 The m/z values and MS fragment pattern confirm the identity of this peak as the -glucuronide metabolite of ibuprofen. 2 The data acquired for the peak eluting with a retention time of.88 minutes are shown below in Figure 5. CNCLUSIN 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 1 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. Figure 5. ScanWave DS of peak eluting at.88 minutes with a m/z value of Plumb R., Rainville P, et al. Rapid Communications in Mass Spectrometry. 27; 21: 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 25. 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 knowledge of the fragmentation pattern of the ibuprofen, the metabolites could be detected and the structure confirmed. 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. 28 Waters Corporation. Printed in the U.S.A. ctober EN LB-CP Waters Corporation 34 Maple Street Milford, MA 1757 U.S.A. T: F: