6 applications that will make a difference in your bioanalytical lab

Transcription

1 [ 6 applications that will make ] a difference in your bioanalytical lab Streamline method development Increase sensitivity in negative ion mode Enhance full-scan sensitivity in metabolite detection Quantify therapeutic peptides with high sensitivity Automate calculations in matrix evaluation Maximize flexibility and efficiency

2 [ INTRODUCING XEVO TQ-S ] [ ] You re going to need a bigger graph. The Xevo TQ-S from Waters. Featuring high sensitivity with StepWave, our revolutionary off-axis ion-source technology lets you quantify compounds at lower levels than you ever thought possible. And its sensitivity, speed and selectivity will take your results to an entirely new level. Learn more at waters.com/xevotqs 2010 Waters Corporation. Waters, Xevo, StepWave and The Science of What's Possible are trademarks of Waters Corporation.

3 Step up to the challenges of bioanalysis with a step change in MS sensitivity. Table of Contents At Waters, we understand the unique challenges faced by the bioanalysis community requirements to inc rease sensitivity, improve throughput, and effectively explore new analytical techniques. We partner with leading laboratories to understand the best way to develop and apply UPLC /MS/MS technologies to meet your everyday assay sensitivity and quality needs, but also drive new innovations for up-and-coming applications such as 2D separations, microfluidics-ms/ms, dried blood spot samples for PK and TK analysis, and novel sample cleanup and concentration strategies. As a result, Waters solutions for quantitative bioanalysis are built around a logical workflow that combines robust sample cleanup and fast separations with innovative and highly-sensitive tandem quadrupole mass spectrometry. Our intuitive system allows any user to quickly develop sensitive and robust bioanalytical assays whatever their level of expertise. And the ultimate flexibility and functionality available with Waters technologies means that the right solution for your application can be configured to solve any bioanalytical challenges, whether in regulated or discovery bioanalysis. ACQUITY UPLC : Fast, high-resolution separations from the innovators of sub- 2-µm chromatographic technology that no one else can match. Xevo TQ-S: Now you can quantify compounds at concentrations lower than ever thought possible. Delivering a step change in sensitivity, the Xevo TQ-S provides highest quality, most comprehensive information for bioanalysis so you can accurately, robustly, and reproducibly measure compounds at much lower concentrations than you ever thought possible. Oasis SPE: Achieve robust, selective, and sensitive sample preparation and clean-up for excellent recovery and cleanliness with no compromises. MassLynx MS Software: Flexible and comprehensive software to match your data analysis and reporting goals, and improve the productivity of your laboratory. Our no-compromise technology is ready for you: Rapid and streamlined bioanalytical method development High sensitivity for the most demanding assays Minimize and monitor matrix effects Easier and faster identification of peaks One-step detection and confirmation of metabolites Maximize analytical productivity Learn more at 5 RADAR, a Xevo TQ-S function, combined with Low-High ph UPLC Screening for Fast, Simple Bioanalysis Methods Development 9 High Sensitivity Analysis of Prostaglandin D2 in Plasma using UPLC and Xevo TQ-S 13 Increasing Bioanalytical Assay Sensitivity for Low Exposure Compounds with Xevo TQ-S 17 Enhanced Full Scan Sensitivity For Metabolite Detection using UPLC and Xevo TQ-S 21 High Sensitivity Quantitative Analysis of a Therapeutic Peptide in Plasma using UPLC and Xevo TQ-S 2 TargetLynx Matrix Calculator: A Tool for Robust Analytical Methods Development 33 Maximizing Flexibility and Efficiency for a Pharmaceutical CRO with ACQUITY UPLC and Xevo TQ MS 3

4 Dried Blood Spot Bioanalysis: Addressing the Sensitivity Challenge Watch this informative webinar, hosted by spectroscopynow.com Dr. Christopher Evans GlaxoSmithKline Dr. Christopher Evans is a Section Manager of the Bioanalytical Sciences and Development group at GlaxoSmithKline Pharmaceuticals R&D. He co-leads the worldwide effort responsible for the investigation, evaluation, and implementation of novel technologies and processes to enhance the way that the quantitative bioanalysis of drugs is performed, including the investigation into Dried Blood Spot (DBS) technology and fundamentals. Dr. Robert S. Plumb Waters Corporation Dr. Robert Plumb is a Senior Manager in the Pharmaceutical Business Operations Division at Waters Corporation. Dr. Plumb has published over 80 papers on bioanalysis and metabolite identification. He is a recognized expert in the use of liquid chromatography with mass spectrometry, capillary-scale LC, purification-scale LC, and metabonomics, giving many invited papers at international meetings around the world. Dried blood spots (DBS) have been used for years in neonatal testing, using a heel or finger prick onto a piece of paper which is then dried and shipped for analysis. This approach has now been applied to the field of bioanalysis in preclinical, toxicokinetic, and clinical studies. One major difference between the neonatal studies and bioanalytical studies is the rigorous quantitative nature of the bioanalytical studies. Although there are many benefits of the DBS approach for the bioanalyst, both ethical and financial, the use of DBS involves a reconsideration of current workflows. Notably, the small volumes typical of the DBS sample require highly-sensitive LC/MS/MS assays. Thus, the area of dried blood spot analysis is attracting a significant amount of research into best practices. In this webinar, hear from experts in the field about the challenges and solutions associated with DBS analysis and promising analytical solutions to address them. What the advantages and disadvantages of DBS are in toxicokinetics How this affects the DMPK laboratory Strategies to increase assay sensitivity for low-exposure compounds How to choose the right card, matrix, and background Strategies for validating and running DBS-derived studies

5 RADAR, a Xevo TQ-S function, combined with Low-High ph UPLC Screening for Fast, Simple Bioanalysis Methods Development Paul D. Rainville and Robert S. Plumb Waters Corporation, Milford, MA, U.S. APPLICATION BENEFITS We demonstrate the productivity gains possible with the Xevo TQ-S tandem quadrupole mass spectrometer for use in bioanalytical method development. Where traditionally this activity requires several analyses to collect the necessary full scan, MRM, and precursor ion data to capture information on the analyte peak phospholipids and other endogenous compounds, Xevo TQ-S employs unique RADAR functionality to simultaneously collect these data in the time scale of a narrow 1- to 2-second UPLC peak. INTRODUCTION For a bioanalysis method to be effective it must be transferable, reproducible, and robust to variations in matrix caused by diet, phenotype, age, and gender that can cause assay variability. The development of a reliable LC/MS/MS bioanalysis assay involves the optimization of sample preparation, MS detection, and chromatography conditions. This can be a time-consuming process requiring the analyte peaks(s) to be resolved from resolved endogenous matrix components that cause ion suppression and assay irreproducibility. This often requires multiple analytical runs to acquire the necessary multiple reaction monitoring (MRM), product ion, and full-scan data. Ranitidine Plasma Waters solutions Xevo TQ-S ACQUITY UPLC key words RADAR, T-Wave, Bioanalysis, method development, ph, ranitidine hydrochloride Time Figure 1. In this application note we describe the use of of Xevo TQ-S with RADAR, a novel dual-scan data collection capability to simplify bioanalysis method development. The Xevo TQ-S employs a novel T-Wave collision cell design 1 that allows the simultaneous collection of full scan MS and MRM data. 5

6 EXPERIMENTAL Chromatography LC system: ACQUITY UPLC Column: ACQUITY UPLC BEH C x 50 mm Mobile phase: Flow rate: A: 0.1 Formic acid or 0.1 aqueous ammonium hydroxide B: Methanol or acetonitrile Gradient 5 to 95 organic over 0 to 2 min 600 µl/min Mass spectrometry MS system: Xevo TQ-S Positive ion electrospray MRM: 315 => 129 Full scan MS: 50 to 500 mz at 5000 amu/sec Product ion scan: Precursors of m/z = 184 from 200 to 600 RESULTS AND DISCUSSION Endogenous compounds in serum/plasma, in particular phospholipids, can cause ion suppression, leading to reduced sensitivity and reduced robustness. The key to a successful bioanalytical method is to adjust the chromatographic conditions such that the analyte peak is positioned away from the endogenous materials in the sample such as phospholipids, amino acids, and nucleosides. As the concentration and nature of these endogenous materials can vary according to subject age, diet, state of health, and phenotype it is critical that as much robustness is built into the method at an early stage to support the methods use in the later stages of drug development and clinical trials. Traditional method development activity usually requires several analyses to collect the necessary full scan, MRM, and precursor ion data to capture information on the analyte peak phospholipids and other endogenous compounds; this can significantly reduce productivity. This problem is resolved with the Xevo TQ-S by using its unique RADAR functionality, which enables the simultaneous collection of full scan, MRM, and precursor ion scanning data, all in the time scale of a narrow 1- to 2-second UPLC peak. Previously we have described the use of low and high ph mobile phases to simplify methods development. 2 To illustrate the use of the RADAR technology, a common H2 receptor antagonist ranitidine hydrochloride was spiked into rat plasma, precipitated with acetonitrile (2:1) and analyzed using an ACQUITY UPLC System coupled Xevo TQ-S. The data shown in Figure 2 illustrates the chromatogram obtained when a conventional acidic aqueous buffer and acetonitrile organic modifier gradient is employed, and when a basic aqueous buffer is used. The RADAR MRM data shows that, with an acidic aqueous modifier, ranitidine is not retained, eluting with the void of chromatography system, whereas when a basic aqueous modifier is employed, the compound is retained, eluting at 0.9 min and thus making it the best option. The full-scan and parents of m/z 184 data show that with both the acidic and basic separation the analyte is resolved from phospholipid and other endogenous materials in the matrix. 6 RADAR, a Xevo TQ-S function, combined with Low-High ph UPLC Screening for Fast, Simple Bioanalysis Methods Development

7 Ranitidine Plasma MRM Signal ACIDIC SEPARATION Acidic Separation 1: MRM of 1 Channel ES+ TIC (Ranitidine) 3.25e4 Ranitidine Plasma BASIC SEPARATION Basic Separation MRM Signal 1: MRM of 1 Channel ES+ TIC (Ranitidine) 2.24e : Parents of 184ES TIC Lipid Signal e Lipid Signal : Parents of 184ES+ TIC 1.17e Full-Scan Signal : MS2 ES+ TIC 1.31e9 Time Full-Scan Signal : MS2 ES+ TIC 1.84e9 Time Figure 2. Differences in analyte retention using acidic and basic modifiers as detected by the Xevo TQ-S. Ranitidine Plasma A 1: MRM of 1 C hannel ES+ TIC (Ranitidine) 1.66e MAR2010_PR_114 3: Parents of 184ES+ 100 TIC 1.18e MAR2010_PR_114 2: MS2 ES+ TIC e8 4 Time Ranitidine Plasma 100 3B 2: MS2 ES+ TIC 2.37e8 Ranitidine Time Figure 3. LC/MS/MS analysis of ranitidine hydrochloride in rat plasma using aqueous basic methanol gradients. The use of a methanol organic modifier, instead of acetonitrile, with a basic aqueous buffer increased the analyte retention to 1.30 minutes, as shown in Figure 3A. The analyte signal response was also increased by a factor of 4. However, with a simple 5 to 95 basic/ methanol gradient, the full-scan data revealed that the analyte peak coeluted with the endogenous material in the sample. This coelution could cause ion suppression and assay irreproducibility. To resolve the analyte peak from the endogenous material, the gradient steepness was adjusted. The RADAR functionality was used to quickly select the best LC conditions with the best throughput. The data displayed in Figure 3B shows the final chromatography conditions. A gradient of 20 to 65 methanol over 1.5 minutes was employed with a 95 organic wash at 1.5 to 2 minutes. As can be seen from the full-scan trace (green), in Figure 3B the analyte peak is well resolved from the endogenous material in the sample. This approach, of simultaneous full-scan MS and MRM data collection, can also be used during sample analysis to check for drug-related metabolites, co-administered therapies, and variations in matrix that could affect the veracity of the results. RADAR, a Xevo TQ-S function, combined with Low-High ph UPLC Screening for Fast, Simple Bioanalysis Methods Development 7

8 CONCLUSIONS Bioanalytical method development is significantly simplified using the Xevo TQ-S with RADAR technology. The ability to simultaneously acquire full-scan data as well as MS/MS and MRM data allows the endogenous sample matrix to be monitored at the same time as the analyte peak. Thus the Xevo TQ-S facilitates: Faster method development Removes the need for repeat injections to obtain background signal Matrix monitoring during sample analysis Easy troubleshooting Detection of in vivo metabolites during preclinical and clinical development References The Traveling wave device described here is similar to that described by Kirchner in U.S. Patent ; Mather J, Rainville PD, Potts WB, Smith NW, Plumb RS. Development of a high sensitivity bioanalytical method for alprazolam using ultra-performance liquid chromatography/tandem mass spectrometry. Drug Testing and Analysis. 2010; Waters, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. Xevo, RADAR, T-Wave and The Science of What s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Printed in the U.S.A. May EN LB-CP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

9 High Sensitivity Analysis of Prostaglandin D2 in Plasma using UPLC and Xevo TQ-S Robert S. Plumb, Gareth Booth, and Paul D. Rainville Waters Corporation, Milford, MA, U.S. APPLICATION BENEFITS This new tandem quadrupole MS system provides significant increase in sensitivity for the analysis of prostaglandins in negative ion mode. INTRODUCTION Prostaglandins are lipid compounds, containing 20 carbon atoms, including a five-carbon ring. They are derived enzymatic action from fatty acids and have many important functions in the animal body including causing dilation in vascular smooth muscle cells, aggregation of platelets, regulation of inflammatory mediation, control of hormone regulation, control of cell growth, and acts on the thermoregulatory center of the hypothalamus to produce fever. Conventional transfer optics StepWave transfer optics Figure 1. Comparison of the peak response for prostaglandin D2 with convention transfer optics (left) and StepWave transfer optics (right) when analyzed by UPLC /MS/MS in MRM mode. Waters solutions Xevo TQ-S MassLynx with TargetLynx ACQUITY UPLC ACQUITY UPLC BEH column chemistry key words StepWave, T-Wave, bioanalysis, prostaglandin These prostaglandins have several clinical uses such as to induce childbirth, prevent closure of patent ductus arteriosus in newborns with cyanotic heart defects, and treat peptic ulcers. During treatment, the systemic concentrations of these prostaglandins is very low, thus the accurate quantification of prostaglandins in biological fluids requires a very high-sensitivity assay. In this application note we describe the use of a new high-sensitivity tandem quadrupole mass spectrometer for the analysis of prostaglandin D2 in plasma. 9

10 EXPERIMENTAL Chromatography LC system: ACQUITY UPLC (binary solvent manager, sample manager, HT column oven) LC column: ACQUITY UPLC BEH C 18, 1.7 µm, 2.1 x 50 mm Gradient: 0.1 NH 4 OH (Aq), Methanol 5-95 over 1.5 min Flow rate: 600 µl/min Injection vol.: 10 µl Mass Spectrometry MS system: Xevo TQ-S and Xevo TQ Negative ion electrospray mode MRM data acquisition 351 =>189 prostaglandin D2 Voltages: Capillary, cone, and collision voltage where optimized for each mass spectrometer as well as cone gas flow Source temp.: 140 C Desolvation temp.: 625 C Nebuliser gas flow: 1200 L/Hr Data Management MassLynx 4.1 Quantification using TargetLynx Application Manager RESULTS AND DISCUSSION The Xevo TQ-S is a new ultra-high-sensitivity tandem quadrupole mass spectrometer. It is equipped with new StepWave technology, which is a revolutionary off-axis ion source. The design of this source significantly increases the efficiency of ion transfer from the source to the quadrupole analyzer while the off-axis ion path eliminates neutral contaminants, Figure 2. These two factors combine to dramatically increase the sensitivity of the LC/MS/MS system. The use of stacked ring electrode, T-Wave,* ion optics allows the use of very fast multiple reaction monitoring (MRM) acquisition rates, less than 10 ms dwell time, with no loss in sensitivity. This makes the Xevo TQ-S the ideal tandem quadrupole MS to be used with narrow peaks developed by ACQUITY UPLC. Ion sampling maximized Neutral compounds eliminated Figure 2. Schematic of the StepWave transfer optics, showing the path of the charged analyte ions of interest (left) and the neutral compounds being exhausted to waste (right). 10 High Sensitivity Analysis of Prostaglandin D2 in Plasma using UPLC and Xevo TQ-S

11 Prostaglandin D2 is found in the brain and mast cells in mammalian systems. It binds to the receptor PTGDR, as well as CRTH2, and is critical to development of allergic diseases such as asthma. To evaluate the performance benefits of the new tandem quadrupole mass spectrometer, prostaglandin D2 was prepared and an aliquot injected onto both the Xevo TQ and the new Xevo TQ-S utilizing an ACQUITY UPLC System. The compound was eluted using a linear gradient from 5 to 95 aqueous ammonium hydroxide/methanol over 1.5 minutes at a flow rate of 600 µl/min. The peak of interest eluted with a retention time of 1.14 minutes. The data displayed in Figure 1 shows the sensitivity benefits of the new StepWave ion source versus a conventional ion optics design. Here we can see that the new design gives a 400-fold increase in peak area and 34-fold increase in signal-to-noise (RMS). The data displayed in Figure 3 show a comparison of the signal response of the two systems with the baseline expanded. Here we can see that with the conventional quadrupole based transfer optics, the prostaglandin peak is not detectable, whereas with the new StepWave design the peaks are clearly visible above the base line. Conventional transfer optics StepWave transfer optics Figure 3. UPLC/MS/MS analysis of prostaglandin D2 with the baseline expanded to show the limit of detection. Conventional transfer optics on left, StepWave optics on right. High Sensitivity Analysis of Prostaglandin D2 in Plasma using UPLC and Xevo TQ-S 11

12 CONCLUSIONS The new Xevo TQ-S combined with ACQUITY UPLC offers significant increases in both peak area and signal-to-noise performance for the analysis of prostaglandins in negative ion mode, by 30-fold. The novel off-axis geometry design prevents unwanted, neutral compounds from entering the analyzer stage of the instrument. Meanwhile, the use of T-Wave ion optics and collision cell design allowed more than 12 points to be acquired over a very narrow UPLC peak with no loss in sensitivity. For bioanalysis, these factors translate into: Lower levels of detection that can be achieved easier Methods that are developed faster Peak detection is simplified Assays are more robust * The traveling wave device described here is similar to that described by Kirchner in U.S. Patent ; Waters, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. Xevo, RADAR, StepWave, T-Wave, MassLynx, TargetLynx, and The Science of What s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Printed in the U.S.A. May EN LB-CP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

13 Increasing Bioanalytical Assay Sensitivity for Low Exposure Compounds with Xevo TQ-S Paul D. Rainville and Robert S. Plumb Waters Corporation, Milford, MA, U.S. APPLICATION BENEFITS The new Xevo TQ-S tandem quadrupole MS system provides a significant increase in sensitivity for the analysis of low-systemic-concentration pharmaceutical compounds. INTRODUCTION Accurate quantification of pharmaceuticals in biological fluids facilitates the correct determination of the pharmacokinetics of a medicine. Low-systemicexposure compounds such as inhaled products or those undergoing extensive metabolism require very high sensitivity assays to accurately define the elimina - tion phase of the pharmacokinetics curves. This need challenges the sensitivity of modern LC/MS/MS instrumentation. The Xevo TQ-S is an ultra-high-sensitivity tandem quadrupole mass spectrometer. It is equipped with StepWave optics featuring a revolutionary off-axis ion source design. This design significantly increases the efficiency of ion transfer from the source to the quadrupole analyzer while the off-axis ion path eliminates neutral contaminants. These two factors combine to dramatically increase the sensitivity of the LC/MS/MS system. StepWave Transfer Optics Fluticasone propionate Waters solutions Xevo TQ-S MassLynx with TargetLynx ACQUITY UPLC ACQUITY UPLC BEH column chemistry Conventional Transfer Optics key words StepWave, T-Wave, bioanalysis, pharmaceutical compounds, fluticasone propionate, salmeterol succinate Figure 1. Comparison of the peak response for fluticasone propionate analysis by UPLC /MS/MS in MRM mode using StepWave transfer optics (top) and conventional quadrupole transfer optics (bottom). In this application note, we describe the increased sensitivity obtained for the analysis of two pharmaceuticals compounds, fluticasone propionate and salmeterol succinate, which are inhaled medicines and are present at very low systemic levels in the circulatory system. The high data capture rate of Xevo TQ-S allows the rapid collection of numerous multiple reaction monitoring (MRM) transitions as well as simultaneous full-scan/mrm data acquisition. 13

14 EXPERIMENTAL Chromatography LC system: ACQUITY UPLC (binary solvent manager, sample manager, HT column oven) LC column: ACQUITY UPLC BEH C 18, 1.7 µm, 2.1 x 50 mm Gradient: A: 0.1 NH 4 OH (Aq) B: Methanol 5 to 95 B over 1.5 min Flow rate: 600 µl/min Injection vol.: 10 µl Mass Spectrometry MS systems: Xevo TQ-S and Xevo TQ operated in electrospray positive mode MRM data acquisition: 501 => fluticasone => 380 salmeterol Voltages: Capillary, cone, and collision voltage where optimized for each mass spectrometer as well as cone gas flow Source temp.: 140 C Desolvastion temp.: 625 C Nebuliser gas flow: 1200L/Hr Data Management MassLynx 4.1 Quantification using TargetLynx Application Manager RESULTS AND DISCUSSION The accurate quantification of pharmaceutical compounds in biological fluids is dependent upon the discrimination of the peaks of interest from the background noise. The StepWave source in the Xevo TQ-S is specifically designed to optimize this task. The source consists of two ion transfer stages which are differentially pumped; both stages are T-Wave-enabled 1, stacked-ring RF devices. As the ion beam passes through the source sampling orifice it undergoes expansion. The entrance of the StepWave is designed to be large enough to efficiently capture all of the ions in this expanded ion cloud. The design of the first stage ensures that all the ions are efficiently focused and directed up into the second stage. The off-axis design ensures that any neutral materials entering the source sampling orifice are actively extracted from the system, as shown in Figure 2. Ion Sampling Maximized Neutral Compounds Eliminated Figure 2. Schematic of the StepWave transfer optics, showing the path of the charged analyte ions of interest (left) and the neutral compounds being exhausted to waste (right). In Figure 2, we can see how the ion cloud expands as it enters the MS transfer region and then is refocused into a narrow beam as it moves onto the quadrupole analyzer region. The neutral compounds, not being charged, are unable to traverse this offaxis path and are exhausted to waste. This results in a significant improvement in the limit of detection, allowing bioanalysts to quantify analytes in plasma, serum, and urine at levels previously unobtainable. With this StepWave design, we observed gains in sensitivity in the order of 10- to 25-fold, depending on the compound being analyzed. For example, modern medicines to treat asthma and rhinitis, such as beta 2 agonists and steroids, are designed not to enter the circulatory system and are often dosed via the inhaled route. These pharmaceutical compounds are, by design, present at extremely low levels in the systemic system, and thus are ideal for analysis with the Xevo TQ-S System. 14 Increasing Bioanalytical Assay Sensitivity for Low Exposure Compounds with Xevo TQ-S

15 Fluticasone propionate and salmeterol succinate were dissolved in methanol and then spiked at various physiologically-relevant levels into plasma. The plasma samples were then precipitated with acetonitrile 2:1, centrifuged, and the supernatant solution injected onto the chromatography system. The data displayed in Figure 1 show the sensitivity increase obtained using the Xevo TQ-S for fluticasone propionate, and the data in Figure 3 show the increased response obtained for salmeterol succinate. For fluticasone propionate, the peak height was increased by a factor of 12, and for salmeterol succinate the increase in peak response was 15-fold. StepWave Transfer Optics Salmeterol succinate Conventional Transfer Optics Figure 3. Comparison of the peak response for salmeterol succinate analysis by UPLC/MS/MS in MRM mode using StepWave transfer optics (top) and conventional quadrupole transfer optics (bottom). The overall increase in peak response for these two pharmaceutical compounds and other model pharmaceuticals is given in Table 1. Here we can see that the increase in peak response ranged from 12- to 25-fold. Compound Compound Class Increase in Response Fluticasone propionate Steroid 12 Salmeterol succinate b2-agonist 15 Alprazolam Benzodiazepine 13 Formoterol b2-agonist 20 Desmopressin Peptide 25 Nefazodone Antidepressant 16 Table 1. Comparison of increased peak response for several pharmaceutical compounds when analysed using UPLC/MS/MS with the Xevo TQ-S s StepWave transfer optics. Increasing Bioanalytical Assay Sensitivity for Low Exposure Compounds with Xevo TQ-S 15

16 The increased peak sensitivity has other benefits beyond lower levels of sensitivity. The greater peak response will allow faster method development, as the desired detection limits will be obtainable without the need for complex method development. The increased peak height will also make peak integration simpler, and assays more robust in general use. CONCLUSIONS The StepWave transfer optics in the Xevo TQ-S significantly increase the efficiency of ion sampling in the source. Its novel off-axis geometry design prevents unwanted, neutral compounds entering the analyzer stage of the instrument. This new design resulted in a significant increase in sensitivity, from 10- to 25-fold. This increase in assay sensitivity will allow: Lower levels of detection for low exposure compounds Faster method development Simpler peak integration More robust assays 1 The traveling wave device described here is similar to that described by Kirchner in U.S. Patent ; Waters, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. Xevo, StepWave, T-Wave, MassLynx, TargetLynx, and The Science of What s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Printed in the U.S.A. May EN LB-CP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

17 Enhanced Full Scan Sensitivity For Metabolite Detection using UPLC and Xevo TQ-S Robert S. Plumb, Gareth Booth, and Paul D. Rainville Waters Corporation, Milford, MA, U.S. APPLICATION BENEFITS The new Xevo TQ-S tandem quadrupole MS system provides significant increase in full-scan sensitivity, allowing useful metabolite structural information to be obtained at much lower levels than previously achievable on a tandem quadrupole MS instrument. INTRODUCTION The rapid detection of metabolites in drug discovery DMPK studies allows the project team to quickly evaluate and compare compounds with a similar core structure produced from a parallel synthesis experiment. The ability to obtain both qualitative and quantitative data in one simple analytical run improves laboratory productivity. This is of particular importance in an area where tens to hundreds of new candidate discovery compounds are analyzed per week. To address this productivity issue requires an instrument that is capable of acquiring both high sensitivity quantitative and qualitative data. StepWave Transfer Optics Glucuronide Metabolite Hydroxyl Glucuronide Metabolite Keytone Metabolite Ibuprofen MRM Waters solutions Xevo TQ-S ACQUITY UPLC ACQUITY UPLC BEH column chemistry MassLynx with TargetLynx Conventional Transfer Optics Glucuronide Metabolite Hydroxyl Glucuronide Metabolite key words StepWave, T-Wave, metabolite detection, ibuprofen Keytone Metabolite Ibuprofen MRM Figure 1. Comparison of the extracted ion LC/MS chromatograms for the glucuronide (m/z 381), hydroxyl glucuronide (m/z 397), and ketone glucuronide (m/z 411) of ibuprofen using StepWave transfer optics and conventional transfer optics. 17

18 EXPERIMENTAL Chromatography LC system: ACQUITY UPLC (binary solvent manager, sample manager, HT column oven) LC column: ACQUITY UPLC BEH C µm, 2.1 x 50 mm Column temp.: 40.0 C Gradient: A: 0.1 Ammonium hydroxide (Aq) B: Acetonitrile, 5 to 95 B in 1.5 min Flow rate: 600 µl/min Injection vol.: 10 µl Mass spectrometry MS system: Xevo TQ-S and Xevo TQ operated in electrospray negative mode Full-scan data acquisition: Scan Range 200 to 600 m/z MRM data acquisition 205 => 161 Voltages: Capillary, cone, and collision voltage where optimized for each mass spectrometer as well as cone gas flow Collision energy: 7 ev MRM acquisition, 3 ev full-scan acquisition Source temp.: 140 C Desolvation temp.: 625 C Nebuliser gas flow: 1200 L/hr Data management MassLynx 4.1 LC/MS/MS using tandem quadrupole instruments is the technique of choice for quantitative analysis in drug metabolism studies, however it is typically less sensitive in full-scan than ion trap or time-of-flight instruments. The Xevo TQ-S tandem quadrupole mass spectrometer is equipped with novel StepWave transfer optics and large sampling orifice which dramatically increases both quantitative and qualitative assay sensitivity. Here we demonstrate the increase in full-scan sensitivity obtained with this new StepWave design using the metabolism of the common non-steroidal anti-inflammatory (NSAID) drug ibuprofen. RESULTS AND DISCUSSION The detection of low-concentration drug metabolites in biological fluids such as plasma and urine allows informative data to be obtained about a candidate drug discovery molecule from a very low dose to a rat or mouse. However, this requires an analytical system capable of acquiring high sensitivity full-scan data. While tandem quadrupole instruments are the most sensitive instruments for acquiring quantitative data, via multiple reaction monitoring (MRM) mode, they are usually significantly less sensitive in full-scan mode. This means that the acquisition of the necessary qualitative and quantitative data normally requires two analytical runs on two separate instruments. This reduces productivity. StepWave Transfer Optics Conventional Transfer Optics Figure 2. Comparison of full scan spectra for the glucuronide (m/z 381), hydroxyl glucuronide (m/z 397) and ketone glucuronide (m/z 411) using StepWave transfer optics and conventional transfer optics. 18 Enhanced Full Scan Sensitivity For Metabolite Detection using UPLC and Xevo TQ-S

19 The Xevo TQ-S is a new tandem quadrupole MS equipped with the latest StepWave ion optics which dramatically increases the efficiency of ion transfer from the ion source to the MS analyzer. The use of a two off-axis, stacked ring electrodes design in the transfer region ensures that only the ions of interest are directed to the analyzer. Complementing this increased ion sampling is the ScanWave technology employed in the collision cell. Here, ions within the collision cell accumulate and are then separated according to their mass-to-charge (m/z) ratio. By synchronizing the release of these ions with the scanning of the second quadrupole mass analyzer significantly improves the overall duty cycle of the instrument and hence product ion spectral sensitivity. The data in Figure 1 shows the comparative full-scan negative ion sensitivity obtained from a tandem quadrupole instrument using the conventional transfer optics and that obtained with the new StepWave ion optics. In this example, urine was collected from a human volunteer two hours after a 100 mg oral dose of ibuprofen. The urine was then diluted 1:1000 with water and injected onto the chromatography system. In this data set we can see the extracted ion chromatograms for the three major classes of ibuprofen metabolites. These are clearly visible in the new StepWave-enabled mass spectrometer whereas they remain undetected in the conventional instrument. A comparison of the full-scan spectra obtained from the new instrument design and that achieved with an instrument equipped with conventional ion optics is shown in Figure 2. This example demonstrates that the new instrument is capable of acquiring high-quality, full-scan MS spectra for the three types of metabolites whereas this was not possible with the instrument equipped with conventional ion optics. The spectrum with the m/z 411 peak corresponds to the ketone glucuronide, the m/z 397 peak is from the hydroxyl glucuronide, and the m/z 381 peak is from one of the glucuronide metabolites of ibuprofen. This enhanced sensitivity of the new Xevo TQ-S not only facilitated the detection of the metabolites and collection of the full-scan MS data but also was sufficiently sensitive to allow MS/MS data to be acquired, as shown in Figure 3. The glucuronide metabolite m/z 381 gave rise to three fragment ions, m/z 205, 192, and 113, whereas the hydroxyl glucuronide, m/z 397, gave rise to the product ions, m/z 221, 192, and 113. The keytone glucuronide, m/z 411, gave rise to two major fragment ions, m/z 174 and 113. Figure 3. Full-scan MS/MS spectra obtained from the human urinary metabolites of ibuprofen. Keytone glucuronide m/z 411 (top), glucuronide m/z 381 (middle), and hydroxyl glucuronide m/z 397 (bottom). Enhanced Full Scan Sensitivity For Metabolite Detection using UPLC and Xevo TQ-S 19

20 CONCLUSIONS The rapid detection and identification of drug metabolites in biological fluids plays a critical role in drug discovery. The ability to detect and characterize these metabolites from low-dose rodent studies can allow studies to be performed with less compound earlier in the discovery process and hence reduce costs. The Xevo TQ-S is a new tandem quadrupole mass spectrometer equipped with the latest ion transfer optics enabling metabolites to be detected at extremely low levels. The Xevo TQ-S is also equipped with RADAR, which allows the simultaneous acquisition of quantitative MRM and qualitative full-scan data in one analytical run. These novel features of the Xevo TQ-S allow: Detection of metabolites at extremely low levels Simultaneous acquisition of MRM and full-scan MS data Elimination of unnecessary duplicate analysis Acquisition of MS/MS data at low levels Waters and ACQUITY UPLC are registered trademarks of Waters Corporation. Xevo, MassLynx, TargetLynx, StepWave, T-Wave, and The Science of What s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Printed in the U.S.A. May EN LB-CP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

21 High Sensitivity Quantitative Analysis of a Therapeutic Peptide in Plasma using UPLC and Xevo TQ-S Robert S. Plumb, Gareth Booth, and Paul D. Rainville Waters Corporation, Milford, MA, U.S. APPLICATION BENEFITS The Xevo TQ-S, a new tandem quadrupole MS system, provides the highest possible sensitivity for peptide bioanalysis. Its fast data capture rate and dual scan MRM capability allows for the accurate quantification of the narrow chromatographic peaks produced by UPLC as well as confirmation of the peak identity. INTRODUCTION The use of peptides as therapeutic agents is increasing due to their high tolerability, target receptor selectivity, and high potency. The ability to accurately quantify these therapeutic peptides in biological fluid requires a selective isolation process, a high-resolution chromatography system, and a high-sensitivity detector. A highresolution chromatography system is required to separate the target analyte from the endogenous peptides in plasma and blood, some of which may be isobaric or form identical fragment ions. As these therapeutic peptides imitate or replace the activity of endogenous peptides, the detection process must be able to differentiate between these endogenous and exogenous compounds. StepWave Transfer Optics Conventional Transfer Optics Waters solutions Xevo TQ-S ACQUITY UPLC ACQUITY UPLC BEH column chemistry MassLynx with TargetLynx Figure 1. UPLC/MS/MS quantitative analysis of desmopressin in rat plasma extract using MRM mode. The top chromatogram shows the peak response with the new StepWave optics of the Xevo TQ-S; the lower chromatogram shows the peak response with conventional transfer optics. Chemical structure of desmopressin. key words StepWave, T-Wave, peptide bioanalysis, desmopressin Despite the fact that they are observed as multiply-charged compounds, the high molecular weight of peptides, 1000 to 4000 amu, the detection of these peptides requires a mass spectrometer with an upper mass range in the region of 2000 m/z for successful analysis. In this application note, we describe the use of the ACQUITY UPLC System and a new high-sensitivity tandem quadrupole MS, Xevo TQ-S, for high-sensitivity peptide bioanalysis. 21

22 EXPERIMENTAL Chromatography LC system: ACQUITY UPLC (binary solvent manager, sample manager, HT column oven) Column: ACQUITY UPLC BEH C 18, 1.7 µm, 2.1 x 50 mm Column temp.: 40 C Gradient: Acetonitrile 0.1, Formic acid (Aq) 5 to 45 over 1.5 min Flow rate: 450 µl/min Injection vol.: 10 µl Mass spectrometry MS system: Xevo TQ-S and Xevo TQ operated in electrospray positive mode MRM data acquisition 535 => 328 Voltages: Capillary, cone, and collision voltage were optimized for each MS as well as cone gas flow Source temp.: 140 C Desolvation temp.: 625 C Nebuliser gas flow: 1200 L/hr RESULTS AND DISCUSSION Peptide therapeutics are extremely potent but are also quickly eliminated from the body either via metabolism or as unchanged drug. In order to accurately characterize the pharmacokinetics (PK) of these medicines, it is necessary to have a high-sensitivity LC/MS/MS system to define the later time points and hence the elimination phase of the PK curve. The Xevo TQ-S is a new tandem quadrupole instrument equipped with a novel source design. This new design significantly improves the efficiency of the ion sampling process allowing more analyte ions to be transferred to the analyzer. This is achieved by the use of a larger sampling orifice and differentially pumped region, and off-axis stacked ring transfer optics to prevent neutral compounds entering analyzer region of the instrument. To evaluate sensitivity increase for the analysis of peptides in biological fluids, desmopressin was spiked into plasma and extracted via protein precipitation with acetonitrile (2:1). The data obtained for conventional optics and new StepWave optics are shown in Figure 1. Here we can see that the desmopressin peak is barely visible with the conventional transfer optics instrumentation, whereas there is a significant peak with the Xevo TQ-S. The data in Figure 2 compares the data with the baseline expanded for the conventional source. We can clearly see that there is a peak at 0.73 minutes for the desmopressin in the conventional instrumentation chromatogram. This increase in sensitivity was determined to be 25-fold. Data management MassLynx 4.1 TargetLynx Application Manager 22 High Sensitivity Quantitative Analysis of a Therapeutic Peptide in Plasma using UPLC and Xevo TQ-S

23 StepWave Transfer Optics Conventional Transfer Optics Figure 2. UPLC/MS/MS quantitative analysis of desmopressin in rat plasma extract using MRM mode. The top chromatogram shows the peak response with the new StepWave optics; the lower chromatogram shows the peak response with the conventional transfer optics with the baseline expanded to show the desmopressin peak at 0.74 minutes. CONCLUSIONS Peptide therapeutics offer the opportunity to treat new diseases with low risk of side effects, drug-drug interactions, or toxicity. The Xevo TQ-S combined with ACQUITY UPLC provides the ideal platform for the analysis of peptides in biological fluids. This combination offers: Highest possible levels of sensitivity Fast analysis times Resolution from endogenous peptides Sufficient mass range in the analyzer to quantify large molecular weight peptides High Sensitivity Quantitative Analysis of a Therapeutic Peptide in Plasma using UPLC and Xevo TQ-S 23

24 Waters, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. Xevo, MassLynx, TargetLynx, StepWave, T-Wave and The Science of What s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Printed in the U.S.A. May EN LB-CP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

25 TargetLynx Matrix Calculator: A Tool for Robust Analytical Methods Development Marian Twohig, Joanne Mather, and Alex Hooper Waters Corporation, Milford, MA, U.S. APPLICATION BENEFITS The TargetLynx Matrix Calculator provides a convenient way to evaluate the matrix factor during the development of bioanalytical methods. This is a helpful technique to identify methods that may be susceptible to matrix effects that could potentially affect long-term method robustness. INTRODUCTION In quantitative bioanalysis, the analytical technique of choice is LC/MS/MS due to the high sensitivity and selectivity that it affords. Quantitative bioanalytical methods development by LC/MS/MS is complicated by the interference of the matrix, as the analyte response can differ significantly within a matrix. 1 It is necessary to develop robust analytical methods to ensure the long-term integrity of the results. Matrix effects, resulting from coeluting matrix components that compete for charge in the ionization process, manifest themselves as suppression or enhancement of the analyte signal. Matrix effects are caused by numerous factors: Phospholipids n Coeluting metabolites Subject differences n Degradation products Impurities All of the above can cause significant errors in the accuracy and precision of bioanalytical assays. 2 WATERS SOLUTIONS ACQUITY UPLC Xevo TQ MS MassLynx Software TargetLynx Application Manager KEY WORDS RADAR Scanning Technology, matrix effects As part of the method validation process, it is necessary to measure the ion suppression due to the matrix and calculate a matrix factor. This is often a timeconsuming process often requiring the transfer of the MS data to external software programs. This application note describes the use of a simple algorithm to estimate the matrix factor automatically. How does the TargetLynx Matrix Calculator work? Using the TargetLynx Application Manager, three sample types are defined in the sample list: Analyte One compound of interest (and its internal standard if present) is injected onto the column to determine retention time Solvent A solvent blank is injected through the column with a post-column infusion of the analyte and the internal standard Matrix blank An aliquot of the blank-extracted matrix is injected through the column with a post-column infusion of the analyte and its internal standard 25

26 The solvent and matrix blank experiments (Figure 1) are run multiple times and the coefficient of variation (CV) is calculated when the data is processed. LC Column MRM Signal 1) Determine Analyte Profile AN and IS are injected through the column 2) Inject Solvent Blank 3) Inject Extracted Matrix Blank Solvent blank injected with post-column infusion of AN and IS Extracted Blank injected with post-column infusion of AN and IS AN and IS are infused post-column AN = Analyte IS = Internal standard MRM = Multiple reaction monitoring Figure 1. Illustration of the three matrix factor experiments. Figure 2 illustrates the workflow for calculating the matrix factor. A sample matrix factor calculation is shown in Figure 4. (MF= Matrix Factor, A = Area). This formula is incorporated into the matrix calculator, and the MF and IS normalized MF are calculated automatically when the data from the three LC/MRM experiments are processed. STEP 1 (Figure 1) Run LC/MS/MS experiments for analyte, solvent, and matrix blank. STEP 2 (Figure 3) A. Select Multiply Traces in the TargetLynx method editor B. Process data from sample list as usual. STEP 3 The chromatograms for the traces labeled Solvent and Matrix are multiplied on a point-per-point basis with the chromatographic trace labeled Analyte. STEP 4: VIEW REPORT (Figure 5) The matrix factor and (if present) the internal standard-normalized matrix factor will be displayed. Figure 2. Matrix factor calculation workflow. 26 TargetLynx Matrix Calculator: A Tool for Robust Analytical Methods Development

27 Figure 3. TargetLynx method editor. Figure 4. Sample matrix factor report. Figure 5. Sample matrix factor calculation. TargetLynx Matrix Calculator: A Tool for Robust Analytical Methods Development 27

28 EXPERIMENTAL LC conditions Solvent delivery: Sample delivery: Waters ACQUITY UPLC Binary Solvent Manager Waters ACQUITY UPLC Sample Manager Column: ACQUITY UPLC BEH C µm, 2.1 x 50 mm Column temp.: 45 C Sample temp.: 4 C Injection vol.: 5 µl Flow rate: 600 µl/min Mobile phase A: 0.1 ammonium hydroxide in water Mobile phase B: Methanol Gradient 1: 5 to 95 B in 0.70 min Gradient 2: 5 to 95 B in 2.00 min DISCUSSION Using the Matrix Calculator Fluticasone proprionate was analyzed in plasma. Acetonitrile (2:1) was used to precipitate plasma proteins. Two sets of experimental conditions were used, a short gradient of 0.70 min and a longer gradient of 2.00 min. The elution profile of fluticasone was determined for each chromatographic method. Fluticasone and its D3 internal standard were infused into the LC stream post-column. A solvent blank was injected (n=5) followed by blank-extracted plasma (n=5). TargetLynx was used to process the data and to automatically calculate the matrix factor for both sets of experimental conditions. F O S O CH 3 O HO F H 3 C CH 3 CH 3 MS conditions MS system: Waters Xevo TQ MS Ionization mode: ESI positive Capillary voltage (ESI): 1.0 kv Compound: Fluticasone proprionate 501 > 293 O F H H Figure 6. Structure of Fluticasone proprionate. Cone voltage: 18 V Collision energy: 20 ev Compound: Fluticasone proprionate D3 504 > 293 Cone voltage: 18 V Collision energy: 20 ev Compound: Phospholipids 184 > 184 Cone voltage: 75 V Collision: 4 ev RADAR: Qualitative Scan100 to 900 amu Scan speed: 10,000 amu/s Source temp.: 150 C Desolvation temp.: 450 C Desolvation gas: 1000 L/hr Sample extraction Plasma proteins were precipitated using acetonitrile (2:1 ratio). 28 TargetLynx Matrix Calculator: A Tool for Robust Analytical Methods Development

29 Gradient method 1: 5 to 95 MeOH in 0.70 min It can be seen in Figure 7 from the LC/MS/MS chromatogram that the fluticasone has a retention time of 0.97 min. The analyte chromatogram is superimposed on the plasma-blank injection where there is a post-column infusion of the analyte and the internal standard. The fluticasone elutes in a region of the chromatogram where there appears to be a suppression event occurring. The calculated matrix factor of 0.34 shown in Figure 8 reflects this significant matrix effect. The deuterated internal standard ensures that the results are normalized to This matrix factor is not acceptable for a bioanalytical assay. We can see from the infusion chromatogram that the peak of interest elutes at the same retention time as significant matrix material. Fluticasone Fast gradient Plasma blank with post-column infusion Time Figure 7. LC/MS/MS chromatograms from a fast gradient. Analyte peak is superimposed on the plasma blank chromatogram where there is a post-column infusion of fluticasone and its D3 internal standard. Figure 8. Matrix factor report for gradient method 1. TargetLynx Matrix Calculator: A Tool for Robust Analytical Methods Development 29

30 Gradient method 2: 5 to 95 MeOH in 2.00 min When a 2.00-min gradient was used, the retention time of the fluticasone peak was 1.95 min (Figure 9). The matrix factor report shown in Figure 10 was calculated to be 0.91, which normalizes to 1.01, due to the presence of the internal standard. It can be noted that the proximity of the analyte peak to the region of suppression has changed to a region of less coelution. The Xevo TQ MS employs RADAR Scanning Technology to acquire both MS and MS/MS data simultaneously (formerly referred to as dual scan-mrm). This technology provides a convenient way to monitor the background while collecting MRM data for quantitation. 3 Fluticasone 2.00-min gradient Plasma blank with post-column infusion Time Figure 9. LC/MS/MS chromatograms from a 2.00-min gradient for the analyte peak superimposed on the plasma-blank chromatogram, where there is a post-column infusion of fluticasone and its D3 internal standard. Figure 10. Matrix factor report for gradient method TargetLynx Matrix Calculator: A Tool for Robust Analytical Methods Development

31 Depending on the chromatographic conditions, analytes can coelute with endogenous matrix components and/ or metabolites, leading to potential matrix effects and possible reduced-assay robustness. When MRM is used exclusively, only the specified precursor > product ions are seen. Qualitative RADAR scans show all masses that are defined in the MS method scan. In Figure 11, the proximity of the analyte peak to the phospholipids can readily be visualized. It has been reported that the concentration of the phospholipids at the retention time of the analyte can greatly influence the existence of matrix effects. It can be seen from the RADAR qualitative scan of the plasma blank in Figure 12 that there is more chromatographic resolution between the matrix peaks and the analyte when it elutes at 1.95 min. This is supported by the data from the matrix calculator min gradient 2.00-min gradient Fluticasone 501> Fluticasone 501> RADAR Solvent blank RADAR Plasma blank Phospholipids 184> RADAR Solvent blank RADAR Plasma blank Phospholipids 184> Time Time Figure 11. LC/MS/MS chromatograms from a 0.70-min gradient for (a) fluticasone and (d) lipid fraction; RADAR scan of the solvent and plasma matrices. Figure 12. LC/MS/MS chromatograms from a 2.00-min gradient for (a) fluticasone and (d) lipid fraction; RADAR scan of the solvent and plasma matrices. TargetLynx Matrix Calculator: A Tool for Robust Analytical Methods Development 31

32 CONCLUSIONS The TargetLynx Matrix Calculator is a convenient way of evaluating the matrix factor during the method-development process. This can help to identify methods that might be susceptible to matrix effects that could potentially affect long-term method robustness. The Xevo TQ MS allows RADAR scanning to be used to monitor the background and detect non-targeted compounds while also collecting targeted MRM data for quantitation. 3 RADAR qualitative scanning used in conjunction with the TargetLynx Matrix Calculator is a useful tool for the rapid development of robust bioanalytical methods. Different plasma lots can easily be screened to calculate the matrix factor in the method validation stage. References Tang P, Tang L. Anal. Chem. 1993; 65: 972A 986A. Chambers E, Diehl, DM, Lu Z, Mazzeo JR. Journal of Chromatography B. 2007; 852: Dual Scan MRM Mode: A Powerful Tool for Bioanalytical LC/MS/MS Method Development. Waters Application Note. 2009; en. Waters and ACQUITY UPLC are registered trademarks of Waters Corporation. Xevo, RADAR, and The Science of What s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. July EN AG-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

33 Maximizing Flexibility and Efficiency for a Pharmaceutical CRO with ACQUITY UPLC and Xevo TQ MS Background BioFocus DPI aims to expand its partners drug pipelines by accelerating the gene-tocandidate discovery process. This is achieved through a comprehensive discovery platform, which includes target discovery in human primary cells, focused as well on diverse compound libraries, in vitro and cell-based screening, structural biology, medicinal chemistry, ADME/PK services, supported by unique chemogenomic and informatics tools, and compound library acquisition, storage and distribution services. Challenge Within its ADME/PK laboratory, BioFocus DPI provides in vitro and in vivo services to support drug discovery projects. This typically involves performing high-throughput screening analyses on drug like molecules together with more in-depth studies frequently involving challenging analytes such as peptides and peptidomimetics. Such studies often involve limited sample numbers from a wide range of biological fluids and tissues. On occasion, with their existing analytical instrumentation, BioFocus DPI had to spend considerable time developing robust, cost-effective analytical methods that achieved the required levels of sensitivity. Operating within an increasingly competitive environment, and with a mission to provide the very best service to customers, the challenge for BioFocus DPI was to maximize flexibility and maintain the efficient analytical workflows that would be required to sustain both current capacity and future development, customer satisfaction, and a competitive edge. The Solution To deliver on its promise of providing the very best customer service, BioFocus DPI turned to Waters for a powerful analytical solution that would enable them to deliver the high quality, rapid results its customers demand. The Xevo TQ MS combined with Waters UltraPerformance LC Technology provide a higher throughput and greater sensitivity. The enhanced compatibility of Xevo TQ with UPLC Technology ensures that the system operates optimally at the highest data acquisition rates, maximizing the number of compounds that can reliably be assayed in a single method. The Xevo TQ increases our ADME/PK laboratory throughput and allows more accurate measurements to both quantify new chemical entities and confirm the identity of metabolites in a wide range of biological fluids and tissues. For molecules that would previously have a poor response using standard technologies, the Xevo TQ increases the likelihood of detection significantly. Chris Newton, SVP, BioFocus DPI BioFocus DPI is experiencing workflow benefits gained from enhanced sensitivity, selectivity, and linearity, delivered through the unique ScanWave collision cell and ion source technology. The company can now more easily quantify and confirm trace components in highly complex matrices, with much greater speed and accuracy.

34 Business benefits BioFocus DPI is achieving clear business efficiencies and can provide more cost-effective studies since adopting the Xevo TQ MS System solution with (the) ACQUITY UPLC (System). Its increased throughput and faster turnaround times have enabled the company to expand its current capacity. With an order of magnitude improvement in sensitivity, the company has been able to broaden the range of studies it offers. We ve been able to accept PK studies with much lower dose levels, and have been able to successfully quantify the compounds in plasma and tissues from those studies. That really would have been very challenging without the Xevo TQ, said Dawn Yates, ADME/PK s Laboratory Manager. Further efficiencies are being experienced in sample extraction. In some cases, the increased sensitivity negates the need for sample concentration in the preparation stage. We are building ADME/PK Laboratory s advanced bioanalysis service to provide the best ADME data to support drug discovery programs for our UK, US, and European clients. This newly installed instrument provides greater sensitivity, accuracy, and speed, thus enabling us to further enhance the service that we deliver whether this be for a small-scale study, or as part of an integrated drug discovery program, concluded Chris Newton, SVP, BioFocus DPI. Waters, UltraPerformance LC, UPLC, and ACQUITY UPLC are registered trademarks of Waters Corporation. The Science of What s Possible, Xevo, and ScanWave are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. August EN LB-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

35 [QUALITY] Minimize Assay Variability and Increase Productivity Waters sample preparation products and chromatographic columns are proven to improve data quality for bioanalysis. The combination of the Oasis family of Solid-Phase Extraction products (SPE) and ACQUITY UPLC columns routinely enables bioanalytical scientists to achieve the most robust, accurate and reproducible methods. These complementary technologies enhance the productivity of bioanalytical laboratories by significantly reducing matrix interference, improving the data quality and sensitivity, while simultaneously reducing the number of repeated assays or failed validation runs. SPE/UPLC Method Development Simplified Waters Corporation. Waters, Oasis, ACQUITY UPLC and The Science of What s Possible are trademarks of Waters Corporation.

36 ULTRA HIGH-SPEED LC FAST LC EXPRESSLC HIGH SPEED LC RRLC [ ] CALL IT WHAT YOU WANT. NOTHING DELIVERS PROVEN PERFORMANCE LIKE ACQUITY UPLC. SEE THE PROOF AT WATERS.COM/ONLYUPLC 2009 Waters Corporation. Waters, ACQUITY UPLC, and The Science of What s Possible are trademarks of Waters Corporation EN LL