MEDICINAL CHEMISTRY APPLICATIONS BOOK

Transcription

1 MEDICINAL CHEMISTRY APPLICATIONS BOOK

2 Introduction...3 The Role of LC and MS in Medicinal Chemistry...5 SCREENING System Management for a High Throughput Open Access UPLC/MS System Used During the Analysis of Thousands of Samples OpenLynx Open Access...15 CONFIRMATION New Tools for Improving Data Quality and Analysis for Chemical Library Integrity Assessment...23 PURIFICATION Scaling a Separation from UPLC to Purification Using Focused Gradients...29 Purification Workflow Management...33 Making a Purification System More Rugged And Reliable...39 Application of MS/MS Directed Purification to Identification of Drug Metabolites in Biological Fluids...45 Evaluating the Tools for Improving Purification Throughput...51 A Novel Approach for Reducing Fraction Drydown...57 PROFILING ProfileLynx Application Manager for MassLynx Software: Increasing the Throughput of Physicochemical Profiling...63 An Automated LC/MS/MS Protocol to Enhance Throughput of Physicochemical Property Profiling in Drug Discovery...65 OPTIMIZATION Synthetic Reaction Monitoring Using UPLC/MS...71 ACQUITY UPLC System: and Cost Savings in an Open Access Environment...73

3 The Role of Liquid Chromatography and Mass Spectrometry in Medicinal Chemistry Medicinal chemistry is a scientific discipline at the intersection of chemistry and pharmacy involved with designing, synthesizing, and developing pharmaceutical drugs. Medicinal chemistry involves the identification, synthesis and development of new chemical entities suitable for therapeutic use. Wikipedia.com The objective of medicinal chemistry is to design and discover compounds that offer the potential to become beneficial and profitable therapeutic drugs. Confidently confirming the identity and quality of these new chemical entities is a major challenge, particularly when labs are asked to maximize throughput and efficiency and to manage all the data generated by a variety of systems and users. Medicinal chemistry is also an iterative process that demands rapid turnaround times. High throughput liquid chromatography/ mass spectrometry (LC/MS), together with advanced data-handling software, has become the standard technique for drug discovery compound identification and purification, addressing needs for high throughput screening, optimization, and physicochemical property profiling. Waters UltraPerformance LC (UPLC ) technology is providing a sea change in capacity for medicinal chemistry labs. UPLC uses subtwo-micron column particle sizes to produce faster, more sensitive and high-resolution separations. Our UPLC systems are available with fast-scanning detectors, both optical and mass, and can be easily controlled by software that facilitates sample analysis in open-access laboratory environments. In this applications book, we look at a variety of system solutions that address the unique challenges of medicinal chemists in five key areas. n In Screening, we will demonstrate the use of high UPLC throughput and fast-scanning MS to obtain high quality and comprehensive data about compounds in the shortest possible time. n For Compound Confirmation, we will show how an open access interface, used with UPLC technology and advanced detection, enables chemists with minimal instrument training to determine the identities of known compounds, to rapidly identify unknowns, and to characterize complex sample components. n In Purification, we provide several examples on how chemists can use UPLC along with efficient time-saving techniques to dramatically increase throughput. n In Compound Profiling, we illustrate an automated UPLC/MS/ MS protocol that not only allows for automated MS method development and data acquisition, but also allows data generated from multiple assays to be automatically processed by a single processing method. n In Optimization, we will show how chemists were able to quickly and easily monitor their reactions, noting the relative amounts of starting materials and products by using a walkup UPLC/MS system. 3

4 THE ROLE OF LIQUID CHROMATOGRAPHY AND MASS SPECTROMETRY IN MEDICINAL CHEMISTRY Darcy Shave, Paul Lefebvre, and Marian Twohig Waters Corporation, Milford, MA, U.S. INTRODUCTION Confirming the identity and quality of new chemical entities is a major challenge facing the pharmaceutical industry. Maximum efficiency is essential for laboratories challenged by throughput requirements and the management of data from a variety of systems and users. Liquid chromatography with mass spectrometry has become the standard technique for confirming the identity and purity of drug discovery compounds to support high throughput screening (HTS), optimization, and physicochemical property profiling of these compounds. Medicinal chemistry is an iterative process and requires rapid turnaround times. High throughput solutions together with advanced data handling software must be employed. In this application note, we look at various solutions, including sub-2 µm column particle sizes, fast scanning mass spectrometers, and new software to assist the medicinal chemist in five key areas: n Screening n Confirmation n Purification n Compound profiling n Optimization METHODS AND DISCUSSION Screening It is important to verify the identity and purity of a compound before early activity studies. Chemists need to be sure they have synthesized the expected compound. Large numbers of compounds may be created, so it is necessary for this screening to be high throughput. Because only a small amount of material is synthesized, the screening must also consume as little material as possible, while generating a diverse amount of information. Figure 1. The ACQUITY SQD with the Sample Organizer plus PDA and ELS detectors. Samples were analyzed on a Waters ACQUITY UPLC System with a Sample Organizer. The column was an ACQUITY UPLC BEH C 18 (1.7 µm, 2.1 x 50 mm) run at 30 C. The injection volume was 5 µl. Compounds were separated using a generic water/acetonitrile gradient that was 1.1 min long. Detection was done with an ACQUITY UPLC Photodiode Array (PDA), ACQUITY UPLC Evaporative Light Scattering (ELS), and SQ Mass Detector with an ESCi source for ESI/APCI switching. Plates were logged into and processed with the OpenLynx Open Access Application Manager for MassLynx Software. By using an ACQUITY UPLC System with the Sample Organizer, we were able to analyze 3840 samples in under 7 working days on a single column. On a traditional HPLC system, this would take approximately 27 working days, assuming a 10-minute run time. The ESCi source on the mass spectrometer allowed the chemist to gather data in both electrospray and APCI (with positive/negative switching) modes during the same injection. In this way, the maximum amount of data was generated with a minimal amount of sample.

5 The open access interface allowed the user to log in the sample plates while providing a minimal amount of information. A series of methods, each including gradient conditions, MS conditions, and processing parameters, was designed by the system administrator. The user simply chose a method from this list, imported their sample lists, and placed their microtitre plates in the indicated positions. The samples were then analyzed and the data was processed. Once processing was finished, the data was automatically copied to a file storage PC. From here the users could do further processing, if desired. A report file was also generated from the processed file and converted to pdf. This facilitated storage of the results in a database. Confirmation Exact mass experiments permit elemental composition determinations of unknowns or confirmation of a suspected elemental composition. This allows the medicinal chemist to confirm identities of known compounds, to rapidly identify unknowns, and to characterize complex sample components. Samples were analyzed on an ACQUITY UPLC System. The column was an ACQUITY UPLC BEH C 18 (1.7 µm, 2.1 x 50 mm) run at 30 C. The injection volume was 5 µl. Compounds were separated using a generic water/acetonitrile gradient that was 1.1 min long. Detection was done with an ACQUITY UPLC PDA and an LCT Premier XE Mass Spectrometer with an ESCi source for ESI/APCI switching. Samples were logged into the system using OpenLynx Open Access and processed with MassLynx OpenLynx with i-fit exact mass processing. Figure 2. OpenLynx OALogin plate login wizard. A fast generic liquid chromatographic method was designed to provide excellent selectivity without compromising either chromatographic resolution or speed of analysis. To obtain such an analytical method, UPLC in conjunction with oa-tof MS detection was employed. With this analytical system, identification of the anticipated samples, isomers, and possible impurities with mass accuracy deviations less than 5 ppm from the actual were obtained using LockSpray. With such high accuracy data, the calculation of elemental compositions for each of the analytes was possible. Subsequent elemental composition results were produced using the i-fit algorithm, which takes into account the distribution of the spectral isotopes for the compounds of interest and employs novel data interpretation to simplify results lists returned. The Open Access interface allowed the medicinal chemist to log in the samples while providing a minimal amount of information. The results, including a pdf report showing the most probably elemental compositions, were then made available to the chemist. 6

6 Purification Having a pure building block is important for controlling the synthetic reactions and successfully making a pure target. A pure target is critical for understanding the results of screening and building quality structure/activity relationship (SAR) information. Reverse-phase HPLC has been successfully applied to the different aspects of the medicinal chemist s process. It is capable of purifying milligrams to multiple grams in a single system, and can be configured to automatically process hundreds of samples. The results can provide high purity and recovery of the desired compounds with minimal user intervention. Samples were analyzed on a Waters AutoPurification System, including a 2545 Binary Gradient Module, 2767 Injector, and Collector, and a System Fluidics Organizer (SFO). The compounds were purified on an XBridge Prep C 18 ODB column (5 µm, 19 x 50 mm) run at room temperature. Detection was done with 2996 PDA, ELS, and 3100 mass detectors. Fraction collection and processing was done with the FractionLynx Application Manager. Compounds were separated using 5-minute gradients that were chosen by the AutoPurify functionality of FractionLynx. A rapid LC/MS method was developed for the analysis of a medicinal chemistry library. The MS data confirmed the presence of the target compound and its retention time from a high resolution LC separation with a 1-minute cycle time. The retention time corresponded to a percent organic solvent at which the compound eluted. Based on this correspondence, a focused purification method for a 19 mm I.D. column with 5 micron particles was selected to maintain the analytical resolution. The isolated target was then separated by LC. The original analytical methodology was then used to determine the new purity for each compound collected. By logging in their samples just once, the medicinal chemists were able to get a purified product along with reports showing the initial and final purities. Compound profiling In an effort to avoid clinical failures, there is an emphasis across the pharmaceutical industry on examining pharmacokinetic and safety profiles earlier in the drug discovery process. Assays are developed in order to select compounds with the highest probability of becoming successful drugs based on preferred pharmacological properties. This step includes extensive testing for the absorption, distribution, metabolism, excretion, and toxicity (ADMET) and physicochemical properties of a compound. Samples were analyzed on an ACQUITY UPLC System with a Sample Organizer. The column was an ACQUITY UPLC BEH C 18 (1.7 µm, 2.1 x 50 mm) run at 30 C. The injection volume was 5 µl. Compounds were separated using a generic water/acetonitrile gradient that was 1.1 min long. Detection was done with an ACQUITY UPLC PDA, a ACQUITY UPLC ELS and a Quattro Premier XE Mass Spectrometer with an ESCi source for ESI/APCI switching. MS conditions were optimized using the QuanOptimize Application Manager. The samples were processed using the ProfileLynx Application Manager. Properties analyzed included solubility, logp, microsomal stability, and CHI. Figure 3. MS and UV chromatograms showing targeted mass and impurities. 7

7 Optimization Once a hit is generated through library screening, optimization of the compound of interest takes place. This step involves multiple repetitions of chemical modification of the hit to develop compounds with desired properties. Chemists need to know as soon as possible that these reactions are proceeding as desired. Samples were analyzed on an ACQUITY UPLC System with a Sample Organizer. The column was an ACQUITY UPLC BEH C 18 (1.7 µm, 2.1 x 50 mm) run at 30 C. The injection volume was 5 µl. Compounds were separated using a generic water/acetonitrile gradient that was 1.1 min long. Figure 4. ProfileLynx browser showing results of solubility experiment. Early screening of physicochemical properties (PP) is an integral process for modern drug discovery. Typical PP profiling practices include properties such as solubility, stability (ph and metabolic), permeability, integrity, etc. The critical factor to consider in PP profiling is throughput. The bottlenecks to throughput include MS method optimization for a large variety of compounds and data management for the large volume of data generated. Detection was done with an ACQUITY UPLC PDA, ACQUITY UPLC ELS and an SQ Mass Detector with an ESCi source for ESI/APCI switching. Single samples were logged into the system using OpenLynx Open Access and processed with the OpenLynx Application Manager. An automated UPLC/MS/MS protocol was developed that not only allowed for automated MS method development and data acquisition, but also allowed data generated from multiple tests to be processed by a single processing method, all in an automated fashion. As a result, the physicochemical profiling process was significantly simplified and throughput increased. The column manager bypass channel allowed users to easily switch to direct flow injection analysis for compound optimization without sacrificing one of the column positions. Chemists can choose the optimal conditions and chemistry for their compounds as the column manager is a thermostat-controlled oven with temperature regulation from 10 to 90 C and has automated switching for four columns. Figure 5. Chromatograms from various times during a 60-minute reaction. During the compound optimization stage of a discovery cycle, medicinal chemists are not only interested in determining the key structural features responsible for activity and selectivity, but also what structural changes need be made to improve these characteristics. Because the reactions necessary to bring about these changes may take a long time, chemists need to be sure they are progressing as expected. 8

8 By using a walk-up UPLC/MS system, chemists were able to quickly and easily monitor their reactions, noting the relative amounts of starting materials and products. They were also able to note the formation of any side products and make the necessary alterations to minimize these in their reaction protocol. CONCLUSION We were able to increase throughput and data quality by combining UPLC with a variety of detection techniques and software solutions. n Screening: By combining the speed of the ACQUITY UPLC System with the capacity of the Sample Organizer, we were able to nearly quadruple the screening throughput of the lab, without sacrificing data quality. n Confirmation: With the Open Access interface, medicinal chemists were able to confirm the elemental composition of their compounds, with minimal instrument training. The i-fit algorithm simplified the final exact mass determination by reducing the number of possible elemental formulas. n Purification: We were able to use analytical LC/MS data to tailor the purification method to maintain the analytical resolution. n Compounds profiling: The determination of physciochemical properties was simplified with the use of the ProfileLynx Application Manager, which automated the calculations of solubility, logp, metabolic stability, and CHI. The combination of the Column Manager and QuanOptimize facilitated the development of optimal MS/MS method. n Optimization: Chemists were able to quickly and easily log in their samples to determine the progress of the reaction. They were able to see the results of the analyses within minutes. Waters, ACQUITY UPLC, UPLC, and ESCi are registered trademarks of Waters Corporation. AutoPurification, AutoPurify, FractionLynx, i-fit, LCT Premier, LockSpray, MassLynx, ODB, OpenLynx, ProfileLynx, Quattro Premier, QuanOptimize, XBridge, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

9 SCREENING

10 SYSTEM MANAGEMENT TOOLS FOR A HIGH-THROUGHPUT OPEN ACCESS UPLC/MS SYSTEM USED DURING THE ANALYSIS OF THOUSANDS OF SAMPLES Darcy Shave, Warren Potts, Michael Jones, Paul Lefebvre, and Rob Plumb Waters Corporation, Milford, MA, U.S. INTRODUCTION Many compound libraries contain compounds that were synthesized several years prior or obtained from outside resources. It is important that the expected composition of each compound be confirmed. LC/ MS has become the standard technique for confirming the purity and identification of a compound that has demonstrated activity in a biological screen. If the library store is not routinely checked, false positives in an activity screen are highly possible. This will lead to wasted time, effort, and money on compounds that should not advance in the discovery process. Because these libraries may contain thousands, if not millions, of compounds, an Open Access UltraPerformance LC (UPLC )/MS system was investigated for high-throughput library quality control. Enhancements to HPLC and LC/MS technologies have provided useful tools to improve the throughput and accuracy of these assays. Throughput can be substantially increased with the use of UPLC/ MS, which makes use of small column particles (sub-2 μm) and high operating pressure (>10,000 psi). This can result in an up to 10-fold increase in throughput along with a three-fold increase in sensitivity. Due to the large number of samples analyzed and data generated during this testing, a new software package has been created that facilitates administration of this Open Access system. It created new project directories for the users and moved the resulting project data (such as raw data files) across the network as it was created. Data processing could then be done on a separate dedicated computer. The software also monitored the instrument PC, providing on-the-fly information about its status and the status of its sample queue from a centralized location. The ACQUITY UPLC System with the ZQ Mass Detector for open access laboratories. EXPERIMENTAL All experiments were conducted using the Waters ZQ Mass Detector, equipped with an ACQUITY UPLC System with a Sample Organizer, Photodiode Array (PDA) Detector, cooled Autosampler and Column Heater. The ZQ was equipped with an ESCi source, running in the ES+ ion mode. The instrumentation was controlled by MassLynx 4.1 Software with OpenLynx and OpenLynx Open Access Application Managers. Samples were run on a 1 min gradient from 5 to 95 organic at 0.8 ml/min. The column was a 1.7 µm, 2.1 x 50 mm ACQUITY UPLC BEH C 18 Column. The PDA was set to analyze a wavelength range from 210 to 400 nm. The mass detector analyzed a mass range from 100 to 500 amu with a dwell time of 100 ms and an interscan delay of 50 ms. Eight microtitre palates, each containing 96 pharmaceutical samples, were logged onto the system using OpenLynx Open Access. The first and last samples in each plate were used for quality control.

11 RESULTS AND DISCUSSION By using an ACQUITY UPLC System with the optional Sample Organizer, we were able to analyze 3840 samples in under seven working days on a single column. On a traditional HPLC system, this would take approximately 27 working days, assuming a 10-minute run time. The Open Access interface allowed users to log in the samples while providing a minimal amount of information. A series of methods, each including gradient conditions, MS conditions, and processing parameters, was designed by the system administrator. The users simply chose a method from this list, imported their sample lists, and placed their microtitre plates in the indicated positions. The samples were then analyzed and the data was processed. Once processing was finished, the data was copied to a file storage PC. From here the users could do further processing if desired. As well, a report file was generated from the processed file and converted to.xml format. This facilitated storage of the results in a database. Instrumentation Throughput was increased by using UPLC. This technique made use of 1.7 μm column particles and high operating pressure (12,000 psi). These properties resulted in a five-fold increase in throughput. Sensitivity was not investigated. Due to the large number of samples being run, an ACQUITY UPLC Sample Organizer was also used. This thermally-conditioned sample storage compartment extended the capacity of the system by adding space for seven deep-well microtitre plates (or 21 shallow-well plates). Total sample capacity was increased from 192 samples (two plates) to 768 samples (eight plates) when using 96-well plates. If using 384-well plates, maximum capacity would be 8064 samples. An added advantage of the Sample Organizer in an open access environment is the ability to add samples to the system without pausing the sample queue. When the door to the Sample Manager is opened, any movement whether of the sample plate or of the needle is paused for safety. This pause does not occur when loading the Sample Organizer. Software administration tools The Open Access software allowed chemists to walk up to a terminal and log in samples onto an instrument, inputting the minimum of information needed for the sample run. It also allowed the system administrator to maintain control over the Open Access systems and to track the performance of each system. It facilitated batch processing and reporting of results. The administrator selected the fields that appeared when remote users logged in samples. The administrator designated fields as mandatory so that login would not proceed unless the remote users entered values for these fields. They also defined upper and lower limits for the values of numeric fields. In addition, the administrator defined the format for text that remote users entered in the text fields. The Open Access Toolkit (OAToolkit) service ran on the Acquisition PC and copied open access users batch files and raw data to remote locations once their samples were run. The information about these users, and the locations to where their data was to be sent, is contained within the administration tool. This information is uploaded to the service on the Acquisition PC. The illustration in Figure 1 and following procedure describe the order of events during typical operation. 1. The administrator uses the Administration Tool to create a user. 2. The administrator uses the Administration Tool to add extra information about the OALogin user, for example, that the raw data of any of the user s samples should be moved to the File Storage PC whenever a user s sample is processed. 3. The administrator uploads the user information to the OALogin PC. This adds the user s name to the drop-down list in the login screen on the OALogin PC. 4. The administrator uploads the user information to the OAToolkit service on the Acquisition PC. The service now contains the instructions of how to proceed if the OALogin user logs in a batch. 5. The OALogin user logs in a sample using the OALogin terminal as normal. 6. oalogin logs the sample with MassLynx. 12

12 7. When MassLynx has finished running the sample, the OAToolkit service reads the batch file (.olb), and registers that it is from a recognized user. 8. The OAToolkit service moves the raw information to the specified location on the File Storage PC Acquisition PC File Storage PC Mass Spectrometer Figure 2. OpenLynx browser report. Figure 1. Data from the mass spectrometer is captured by the Acquisition PC, then is managed by the system administrator or accessed by the individual user via the OALogin tool. The raw data is also backed up to a File Storage PC. Reporting OAToolkit Administration PC OAToolkit Administration PC The Open Access software allowed the administrator to define how samples were processed. Once all the data for a sample set had been collected, the OpenLynx Application Manager automatically processed the data and created an OpenLynx Browser report (.rpt). The browser report (Figure 2) presented a summary of results as a color-coded map (found/not found/tentative) for easy visualization of analysis results. Users accessed and reviewed the data by simply pointing and clicking on the sample location of interest. Chromatograms, spectra, sample purity, peak height, peak area, retention time, and other information can easily be reviewed within the browser. The browser report was created in the report folder of the current project. A secondary report location could have been specified, but was not. The toolkit service also allowed for a copy of the report to be sent over the network to another location. That location was specific to each user a folder on their office PCs. The users no longer had to access the Acquisition PC to view their reports. In addition, the raw data folders were moved across the network to each user s PC and the users were able to reprocess it with a process-only version of MassLynx. Finally, the OAToolkit service was used to automatically convert the browser reports to.xml format. This was accomplished using the included.xml import and.xsl export schema. This data can then be easily incorporated into a database or shared with colleagues. System monitoring On the Administration PC, the Remote Status Monitor (RSM) monitored the status of the Open Access Acquisition PC, along with other Acquisition PCs on the network and wrote that monitoring information to an.xml file. The information could then be read and interrogated remotely in a browser (Figure 3). 13

13 CONCLUSION Figure 3. Status of the Open Access Acquisition PC. More detailed information about an instrument can be displayed by clicking anywhere in the instrument row (Figure 4). Waters Open Access systems give chemists the ability to analyze their own samples close to the point of production by simply walking up to the LC/MS system, logging in their samples, placing their samples in the system as instructed, and walking away. As soon as the analysis is completed, sample results are ed or printed as desired. System configuration and setup is enabled through a system administrator who determines login access, method selection, and report generation. OpenLynx OAToolkit enables administrators to manage open access users from a central point, assigning detailed configuration information and attributes for these users, and then exporting these details to multiple OALogin PCs and Acquisition PCs. OpenLynx OAToolkit also enables administrators and users to remotely monitor the status of Acquisition PCs. Figure 4. Detailed view of instrument status. Waters, ACQUITY UPLC, ESCi, UltraPerformance LC, and UPLC are registered trademarks of Waters Corporation. MassLynx, OpenLynx, ZQ, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

14 OPENLYNX OPEN ACCESS OVERVIEW Maximum efficiency is essential for LC/MS labs challenged by throughput requirements and the management of data from a variety of systems and users. Analyzing routine samples and returning the results to chemists can easily consume an analyst s entire day, leaving them with little time to focus on tasks that require their expert attention. Walk-up open access systems allow chemists to analyze their own samples, freeing up analysts time for more challenging analyses without compromising the quality of the final results. The Waters OpenLynx Open Access Application Manager for MassLynx Software offers the power of chromatography and mass spectrometry to chemists who are not analytical instrumentation specialists. To minimize the learning curve for instrument operation, OpenLynx Open Access leads chemists through sample submission, method selection, and reporting options. The system is maintained by a system administrator who predefines the system configuration, available experimental methods, processing criteria, and reporting options. By allowing chemists to submit their own samples, routine analyses can be performed more efficiently, leaving instrumentation experts more time to focus on advanced analyses. OpenLynx Open Access offers comprehensive capabilities: n Simplified sample submission process A single page login or a step-by-step, wizard-enabled process allows users to enter their name and sample information, and select pre-determined experimental methods and processing criteria n Exact mass measurement utilization For use with the appropriate mass spectrometers n Summary report generation Reports are automatically printed, ed, and viewed via the OpenLynx browser, containing sample found/not found information, purity, probable elemental composition (with exact mass MS), chromatograms, and spectra n Walk-up optimization of MS/MS methods and quantification of compounds of interest Combines OpenLynx Open Access with QuanOptimize and QuanLynx Application Managers n Advanced search Spectral library generation and searching n Automation of routine system administration tasks Through the use of OpenLynx Open Access Toolkit (OAToolkit) SOFTWARE SETUP Defining parameters INTRODUCTION Open access LC/UV, LC/MS, LC/MS/MS, and GC/MS The OpenLynx Open Access Application Manager is designed to allow chemists to walk up to a terminal and log in samples onto an instrument, while inputting the minimum of information needed for the sample run. OpenLynx Open Access allows the system administrator to maintain control over the open access systems and to track the performance of each system. It also facilitates batch processing and reporting of results. OpenLynx Open Access allows remote users to run samples on the acquisition computer. For OpenLynx Open Access users to be successful, the administrator defines (via the OpenLynx method) the sample information that users must provide when running samples. An intuitive OALogin setup wizard simplifies the system configuration and administration workspace to include only the analytical features the administrator uses.

15 Figure 1. OpenLynx method showing some of the OpenLynx Open Access input fields. Figure 2. Administrator-set OpenLynx Open Access options. The administrator selects the fields that appear when remote users log in samples using OpenLynx Open Access via the Walk-up tab of the OpenLynx method (Figure 1). They can designate fields as mandatory so that login will not proceed unless the remote users enter values for these fields. They can also define upper and lower limits for the value of numeric fields. In addition, the administrator can define the format for text that remote users enter in text fields. Setting options for users Using the administrator mode of OpenLynx Open Access, the administrator defines how users login samples via a number of options (Figure 2). Login setup ranges from changing the window appearance to allowing users to create their own user name. Notification of users via can be enabled, as can barcode support. OALogin can be configured for use with either OpenLynx (sample processing) or AutoPurify (fraction processing). Setting file options The administrator sets several file options. These include specifying the location where the OpenLynx methods, OpenLynx status file, and HPLC files are located. The administrator can set which methods are visible to users, along with the format needed for the text fields. Configuring quality control runs The administrator can configure OpenLynx to check that the LC and MS instrumentation are working correctly, thus ensuring the consistency of the data. The quality control feature (Figure 3) allows users to run a standard and have it compared to the results of the same standard that was run at an earlier time. Values that can be used to confirm system operational performance include peak retention time, peak area, the presence of specific masses or wavelengths, and spectral intensity.

16 Figure 4. OpenLynx Toolkit Administrator Tool. Figure 3. OpenLynx Open Access quality control options. Before a QC comparison can be run to check the system, there must be an OpenLynx method that contains the expected results from a standard. The QC run acquires data from a sample with a known retention time and peak intensity and then compares the results to the values defined in the OpenLynx method. Figure 5. Remote Status Monitor. OpenLynx Open Access Toolkit (OAToolkit) OpenLynx OAToolkit allows the creation and administration of OpenLynx Open Access users. It can push user information to OpenLynx Open Access PCs across the same network, as well as gather existing OpenLynx Open Access user information from OpenLynx Open Access PCs. It can create new project directories for the OpenLynx Open Access users and can move the resulting project data (such as raw data files) as it is created. The software can monitor numerous instrument PCs, providing on-the-fly information about their status as well as the status of their batch queues all from a central location. It ensures confidence in analytical results with password protection for open access users. The OpenLynx OAToolkit includes the following key features: n Administration Tool (Figure 4) Enables an administrator to create and manage all OpenLynx Open Access users from a single PC, and replicates that information to multiple OpenLynx Open Access PCs and Acquisition PCs n OAToolkit Service Runs in the background on one or more Acquisition PCs, monitors sample batches submitted by OpenLynx Open Access users that were uploaded from the Administration Tool n Remote Status Monitor (Figure 5) Enables any user to monitor the status of Acquisition PCs and their batch queues from a single PC

17 Additionally, the OpenLynx OAToolkit Service: n Relocates data produced during the processing of an OpenLynx Open Access user s batch of samples n Creates new project folders in which to store the processing data on a timed basis n Converts report files to different formats (XML, HTML, or text) LOGGING SAMPLES Login samples window Running samples using OpenLynx Open Access (Figure 6) involves entering sample information to correctly identify the samples and loading the samples into the autosampler. The methods available to the users depend on selections made by the administrator. Figure 6. OpenLynx Open Access window. If the administrator enables user passwords (using OpenLynx OAToolkit), the user must enter their designated password before they can login samples (Figure 7). If they enter an incorrect password, an error message appears and they cannot continue until the correct password has been entered. Single-page log-in vs. wizard OpenLynx Open Access displays the wizard for sample login by default. However, the administrator can allow OpenLynx Open Access users to use a single-page dialog box (Figure 8) for single shot samples. Users can enter multiple samples in this way. OpenLynx Open Access views the samples logged in as a single job. Figure 7. Entering user password.

18 PROCESSING SAMPLES Processing data automatically The administrator determines how OpenLynx processes the Open Access results. To configure OpenLynx Open Access to process data automatically, the administrator must create an OpenLynx method that defines the processing parameters. Figure 9. With the wizard, walk-up users enter their name, choose a method, enter sample information, and place the sample in the autosampler. The single-page login contains most of the selections on the wizard pages (Figure 9) necessary to schedule samples. The benefit of the single-page login is the speed of entering information for a single sample in a single dialog box, rather than through a wizard. This wizard is beneficial when logging in larger sample sets. The administrator must define the integration parameters for the type of data they want to process: n MS+ data For positive ions (total ion chromatogram (TIC), base peak intensity (BPI), and mass chromatograms) n MS data For negative ions (TIC, BPI, and mass chromatograms) n Analog data For up to four channels of analog chromatograms n DAD data For total absorbance chromatogram (TAC), BPI, and wavelength chromatograms Specifying how peak detection occurs involves selecting the integration algorithm and parameters that control peak detection, enabling smoothing (if desired), and setting the smoothing parameters and setting threshold values. Loading samples into the autosampler There are two ways to load samples into the autosampler. The system administrator designates each plate in the autosampler as either single shot or whole plate login. If a plate is designated for single shot login, the user enters data for their samples manually or imports data from a tab-delimited text file. OpenLynx assigns available positions for the samples on existing plates. If a plate is designated for whole plate login, the user prepares data in a spreadsheet or as a text file and imports it into OpenLynx Open Access. This is useful if the user needs to run a large number of samples in one run. OpenLynx reserves the entire plate for samples and the user selects the sample locations. Typically, a system with multiple plates will have both single shot and whole plate login available. Figure 10. Chromatogram integration window. When setting the integration and peak detection parameters (Figure 10), the administrator can specify which integration algorithm (standard or ApexTrack ) to use; how the baseline will be treated for valleys, peak tailing, and drift; and how peak separation for fused

19 peaks and shoulders will be handled. By enabling smoothing, noise will be decreased by filtering data points. Smoothing types include Savitzky-Golay and mean. The threshold values are set for one or more of the four threshold parameters: relative and absolute height and relative and absolute area. This option is used to remove peaks whose height or area is less than a specified percentage of the highest peak. In addition to acquiring and processing data, quantitation and optimization can be performed through OpenLynx Open Access. Performing quantitation Open Access quantitation is a way for the user to run quantitation analysis through OpenLynx Open Access (Figure 11). OpenLynx stores the conditions required for a particular quantitation analysis in an OpenLynx method. OpenLynx Open Access users select the OpenLynx method during login. Using Open Access quantitation, OpenLynx Open Access users can quantify the results as data are acquired. The processing steps available include: n Integrating samples n Quantitating samples n Calibrating standards Using QuanOptimize with OpenLynx Open Access The optional QuanOptimize optimizes the acquisition and quantitation parameters for a particular experiment. Open Access QuanOptimize (Figure 12) generates MS and MS/MS parameters by optimizing the cone voltage, parent ion, and collision energy parameters. QuanOptimize then takes these MS methods and performs automated acquisition and processing using processing methods developed on the fly. It can quantify these results using specified methods. This technique is useful for high throughput screening. Figure 11. Open Access quantitation parameters. Figure 12. Open Access QuanOptimize parameters.

20 REPORTING Results reporting Reporting in Open Access systems is facilitated by the OpenLynx Application Manager. OpenLynx can report results using a flexible array of printed reports or through a results browser. The standalone OpenLynx browser (Figure 13) is an interactive tool for viewing OpenLynx results and can be run on any windows PC without requiring a full MassLynx installation. Chemists can use the browser on their desktop PC to view the results (.rpt file format) that had been automatically ed to them at the end of OpenLynx processing. The OpenLynx browser presents a summary of results as a colorcoded (found/not found/tentative) map for easy visualization of analysis results. Chemists can access and review the data supporting any found/not found/tentative assignment by simply pointing and clicking on the sample location of interest. Chromatograms, spectra, sample purity, peak height, peak area, retention time, and other information can easily be reviewed within the browser. Figure 13. OpenLynx browser.

21 Printing and distributing reports OpenLynx creates an OpenLynx browser report file (.rpt) after it finishes a run and processes the data. This file resides in the OpenLynx Open Access\Reportdb folder. The file is named with the job number followed by the extension.rpt when the user logs in to OpenLynx. OpenLynx report files may be exported in.txt,.tab,.csv, and.xml formats. The administrator can configure OpenLynx Open Access so remote users can find the reports that OpenLynx generates after running samples. Information such as where to store reports and what print report format to use can be specified. CONCLUSION The OpenLynx Open Access Application Manager provides comprehensive, easy, and flexible open access walk-up LC/UV, LC/MS, LC/MS/MS, and GC/MS systems operation management for laboratories that have chemists with varying levels of instrumental analysis experience. With customizable batch processing and results review to support the large amounts of data resulting from high throughput analyses, a highly productive environment is ensured for high-volume laboratories. Waters is a registered trademark of Waters Corporation. MassLynx, QuanOptimize, QuanLynx, ApexTrack, AutoPurify, OpenLynx, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

22 confirmation

23 N E W T OO L S FO R IM P ROV ING DATA QUA L IT Y A N D A NA LYSIS T IM E FOR CHEMICAL LIBRARY INTEGRITY ASSESSMENT Marian Twohig, Paul Lefebvre, Darcy Shave, Warren Potts, and Rob Plumb Waters Corporation, Milford, MA, U.S. INTRODUCTION The identity and purity of a candidate pharmaceutical is critical to the effectiveness of the drug screening process. LC/MS is employed extensively in drug discovery in order to exclude false positives and maintain the high quality of the product. This process can be time consuming and can potentially delay the progression of a drug through the discovery process. Thus, sample throughput is a critical issue in moving compounds from the hit to lead status. UltraPerformance LC (UPLC ) leverages sub-2 µm LC particle technology to generate high efficiency faster separations. When a photodiode array/evaporative light scattering/mass spectrometry (PDA/ELS/MS) detection scheme is used in conjunction with multiple-mode ionization, the potential for peak detection is greatly improved. Pharmaceutical chemical libraries often contain a great diversity of small molecules to cover a broad range of biological targets. 1 In this environment, the ability to obtain information pertaining to multiple MS acquisition modes, in addition to PDA and ELS, in a single injection is invaluable. Open Access software offers the power of chromatography and mass spectrometry to chemists who are not analytical instrumentation specialists. It allows them to quickly and easily know what they ve made and allows the experts to work on the difficult analytical problems. An Open Access UPLC/MS system was investigated for high throughput library QC. In this application note, we describe some of the enhancements to LC and LC/MS technologies that have generated useful tools that improve the throughput and accuracy of these assays. Figure 1. The ACQUITY SQD for open access. EXPERIMENTAL LC conditions LC system: Waters ACQUITY UPLC System Column: acquity UPLC BEH C 18 Column 2.1 x 30 mm, 1.7 µm Column temp.: 50 C Sample temp.: 8 C Injection volume: 2 µl Flow rate: 800 µl/min Mobile phase A: 0.1 Formic acid in water Mobile phase B: 0.1 Formic acid in acetonitrile Gradient: 5 to 95 B/0.70 min

24 MS conditions MS system: Waters SQ Detector Ionization mode: esi positive/esi negative, multi-mode ionization Capillary voltage: 3.0 KV Cone voltage: 20 V Desolvation temp.: 450 C Desolvation gas: 800 L/Hr Source temp.: 150 C Acquisition range: 100 to 1300 m/z Scan speed: 2500, 5000, and 10,000 amu/sec Sample login OpenLynx Open Access Application Manager for MassLynx Software is designed to allow chemists to walk up to a terminal and log in samples while entering the minimum information required to run the samples. A series of methods, each including gradient and MS conditions as well as processing parameters, are initially set up by the system administrator. The users choose an appropriate method from the list, importing their sample lists and placing their samples in the position designated by the software. Desired sample analysis is then performed by the configured system. The single page login window can be seen in Figure 2. Note: A low volume micro-tee was used to split the flow to the ELS and SQ. ELS conditions Gain: 500 N2 gas pressure: 50 psi Drift tube temp.: 50 psi Sampling rate: 20 points/sec PDA conditions Range: Sampling rate: 210 to 400 nm 20 points/sec Figure 2. OpenLynx Open Access single page login. RESULTS AND DISCUSSION Maximum efficiency is essential for labs challenged by throughput requirements and the management of data from multiple systems and users. The Waters Open Access suite of software streamlines the integration of analysis with data acquisition, processing, and reporting. The system and software are initially configured by a system administrator who defines login access, method selection, and reporting schemes. This allows users to analyze their own samples with minimal intervention from analytical support. Open access system Chromatographic separations were carried out using the ACQUITY SQD System coupled to detectors specialized for UPLC separations: the single quadrupole SQ Mass Detector, and PDA and ELS detectors that provided simultaneous signal collection. For additional flexibility, the UPLC system was configured with a Sample Organizer and a Column Manager. The sample capacity of the system totals twenty two 384-well plates, for 8448 library samples in total. This extends the overall walk-away time for the system. The column manager allows four UPLC columns to be installed, heated, and switched into line 24

25 based on the method requirements. This allows the chemist to take advantage of the broad range of stationary phases that encompass compound types, ranging from very hydrophilic to very lipophilic. Sample analysis Samples were analyzed using gradients less than one minute in length with a flow rate of 800 µl/min. When analyzing the narrow peaks generated by the UPLC/MS system, the data collection rate can compromise the number of points across the LC peak, resulting in a poor definition of the eluting peak and hence inaccurate results. The ability of the MS system to collect data at a high scan speed, 10,000 amu/sec, greatly improves chromatographic peak definition. This in turn facilitates the acquisition of a large number of individual acquisition modes in one run while maintaining adequate peak characterization. As can be seen from the data displayed in Figure 3, the result of operating at lower data collection rates can compromise the chromatographic resolution. To maintain chromatographic integrity, it is therefore advantageous to be able to scan at elevated scan speeds. The total cycle time of the method was 1 minute 20 seconds, facilitating increased sample throughput while still allowing generous washing steps to prevent sample-to-sample memory effects. Using a flow rate of 800 µl/min and a 2.1 x 30 mm column clears 9 column volumes/min. The spectral data quality of scanning experiments carried out from 2500 to 10,000 amu/sec were found to be comparable, thus providing confidence that operating at these rapid data collection rates does not compromise the spectral data quality. Figure 4 shows a comparison of an acquired spectrum with a software generated isotopic model. Isotope ratios of data collected at 10,000 amu/sec were within 1 of the isotopic model, again ensuring data fidelity is not compromised. In addition to obtaining mass confirmation by multiple MS modes, it is possible to add PDA and ELS detectors to obtain auxiliary information. A single run can then provide UV spectral information and an estimation of compound purity at low wavelengths amu/sec C17H20N2CIS Acquired Spectrum 10,000 amu/sec amu/sec C17H20N2CIS Isotope Model 10,000 amu/sec m/z Figure 3. Chromatograms shown at 2500, 5000, and 10,000 amu/sec. Figure 4. Spectrum for isotope model and for acquired spectrum. 25

26 ELS detection is an alternative to UV detection, and does not depend on the presence of a chromaphore. ELS detection works by measuring the light scattered from the solid solute particles remaining after nebulization and evaporation of the mobile phase. Chromatograms illustrating the use of triple detection (PDA/ELS/MS) are shown in Figure 5. The signal from an ELS detector can give a tentative estimation on the relative quantities of the components present. It has been known to give rise to similar responses for related compounds. 2 The chromatographic peak widths of the MS and ELS increased by 25 to 30 when compared with the PDA trace. This can be attributed to the use of a low volume micro-tee after the PDA. Data processing As soon as the analysis is complete, data is automatically processed and a sample report is generated. Reporting in Open Access systems is facilitated by the OpenLynx Application Manager. OpenLynx can report results using printed reports or through the OpenLynx browser. The browser presents a summary of the results as a color coded (found/not found/tentative) map for clear interpretation of the results. Chromatograms, spectra, sample purity, peak height, peak area, retention time, and other information can easily be viewed by the browser. The OpenLynx browser, shown in Figure 6, displays the results for the entire 384-well plate. The report can automatically be ed, converted to pdf, or printed as desired. 3 AU LSU - 1.0e PDA ELS APcI+ APcI ESI- ESI Figure 5. UPLC/PDA/ELS/MS with multi-mode ionization. The OpenLynx OAToolkit facilitates an even easier administration of an open access system, automating many of the system management tasks carried out by a system administrator. The software also remotely monitors the status (via the Remote Status Monitor module) of one or more acquisition PCs and writes monitoring information to an XML file. The status summary page opens in the browser and contains a list of acquisition PCs, and the number of samples pending in the queue. 4 This allows the chemist to select the instrument with the shortest wait time, again increasing productivity. Figure 6. The OpenLynx browser. 26

27 CONCLUSION It is important to verify the identity and purity of a compound before early activity studies. Chemists need to be sure they have synthesized the expected compound. Because large numbers of compounds may be created, it is necessary for this screening to be high throughput. And because only a small amount of material is synthesized, the screening must also consume as little material as possible, while generating a diverse amount of information. The described system and software combination can autonomously evaluate large numbers of samples with a cycle time of 1 minute and 20 seconds. Data can then be automatically processed and a summary report can be generated. The scan speed capabilities of Waters ACQUITY SQD System make it possible to better characterize narrow chromatographic peaks. This has become a necessity since the advent of sub-2 µm particle technology where chromatographic peaks can be 1 second wide or less. Signals from auxiliary detectors such as PDA and ELS can be collected simultaneously. Together with the MS data, they provide important information relating to purity and an estimation of the relative quantities of the components present. Open Access gives the chemist a walk-up system that is flexible for analytical data acquisition. It runs as a complete system, from sample introduction to end results. The use of the fast-scanning MS along with the throughput of UPLC technology and remote status monitor software allows the chemist to obtain high quality comprehensive data about their compounds in the shortest possible time. This combined with intelligent open access software allows informed decisions to be made faster, thus supporting the needs of the modern drug discovery process. References 1. Mike S. Lee, LC/MS Applications In Drug Development, Wiley-Interscience Series on Mass Spectrometry. 2002; (Chapter 6) Kibbey, C.E. Mol. Diversity. 1995; I: Darcy Shave, OpenLynx Open Access, Waters Application Library. 2006: EN. 4. Darcy Shave, OpenLynx OAToolkit for Open Access Systems, Waters Application Library. 2006: EN. Waters, ACQUITY, ACQUITY UPLC, UltraPerformance LC, and UPLC are registered trademarks of Waters Corporation. MassLynx, OpenLynx, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

28 purification

29 SCALING A SEPARATION FROM UPLC TO PURIFICATION USING FOCUSED GRADIENTS Ronan Cleary, Paul Lefebvre, and Marian Twohig Waters Corporation, Milford, MA, U.S. INTRODUCTION Purification laboratories face many of the same challenges that their counterparts in analytical laboratories face: the need to increase throughput and efficiency without sacrificing quality and quantity. Successful performance of a purification lab is measured in the ability to produce pure fractions in sufficient quantities in a timely manner. UltraPerformance LC (UPLC ) has been widely accepted by chromatographers because of the improvements over HPLC in sensitivity, resolution, and speed of separations. Now scientists are beginning to explore the use of this technology in the sample screening process as a screening tool to evaluate samples prior to purification. A typical run time for analytical screening in a preparative lab is 10 minutes. By capitalizing on the efficiency of UPLC, a 10-minute run time can be shortened to as little as 1 minute. This offers substantial time savings enabling for greater capacity, but also fits into the fail fast and fail cheap motto adopted by many pharmaceutical companies. This application note will discuss the use of focused gradients to maintain selectivity and resolution and to allow UPLC screening to be applied to preparative samples. This will offer the substantial time savings associated with UPLC to customers in the preparative environment. EXPERIMENTAL A standard solution of pharmaceutical-like compounds was prepared to simulate the conditions under which many purification systems operate. Figure 1. The mass-directed AutoPurification System. UPLC conditions LC system: Waters ACQUITY UPLC System with ACQUITY UPLC Photodiode Array (PDA) Detector Column: acquity UPLC BEH C 18, 1.7 µm, 2.1 x 50 mm Injection volume: 2.0 µl Flow rate: 0.8 ml/min, 2.1 x 50 mm Mobile phase A: 0.05 Formic acid in acetonitrile Mobile phase B: 0.05 Formic acid in water Gradient: Generic 5 to 95 over 2 minutes Focused Gradient HPLC conditions LC system: Waters AutoPurification System Column: Waters XBridge Prep OBD C 18, 5 µm, 19 x 50 mm Waters XBridge C 18, 5 µm, 4.6 x 50 mm Injection volume: 200 µl Mobile phase A: 0.05 Formic acid in acetonitrile Mobile phase B: 0.05 Formic acid in water

30 Flow rate: Gradient: 22 ml/min 0 to 0.25 min, 2 B to initial B 0.25 to 1.61 min, initial B to end B 1.61 to 1.86 min, end B to 95 B 1.86 to 2.71 min, 95 B 2.71 to 2.72 min, 95 B to 2 B AU MS conditions MS system: Waters 3100 Mass Detector Ionization mode: positive Switching time: 0.05 sec Capillary voltage: 3 Kv Cone voltage: 60 V Desolvation temp.: 350 C Desolvation gas: 500 L/Hr Source temp.: 300 C Acquisition range: 150 to 700 amu Acquisition rate: 5000 amu/sec RESULTS AND DISCUSSION In order to maintain the selectivity and resolution achieved by analytical analysis, the overall cycle time of a preparative analysis must be increase almost nine fold. 1 This long cycle time is not practical for most separation scientists. Therefore, we look to focused gradients to maintain selectivity and resolution in UPLC screening. The UPLC separation of the sample shows the compound of interest eluting at 0.48 min, and is partially resolved from the peak at 0.51 min. The separation is first directly scaled to a 19 x 50 mm XBridge Prep OBD C 18 Column. The XBridge chemistry is built on the same secondgeneration bridged ethyl hybrid (BEH) particle as the ACQUITY UPLC BEH chemistry, in order to maintain the selectivity and resolution of the analytical analysis. To maintain the resolution and selectivity, the overall cycle time must be increased over nine fold Figure 2. ACQUITY UPLC analytical separation. AU 4.0e+1 2.0e Figure 3. Direct scale-up maintains resolution and selectivity, with a run time of 8 minutes. In a preparative environment, where the compound of interest is being isolated from the other components in the sample, retaining analytical resolution is not as important as isolating and collecting the compound of interest. 2 A set of focused gradients can be created based on the relationship between percent composition and retention time. The system dwell time is used to determine that relationship. 3 Here, in the analytical screen the mobile phase is 2 organic solvent at 0.17 minutes and 17.5 at minutes, and so a series of gradients can be created. The theory behind the focused gradients is the same for HPLC and for UPLC, but the time window for the UPLC gradient is much smaller. 30

31 Based on Table 1, method C is selected to isolate the compound that eluted at 0.48 min in the UPLC analysis. Using the focused gradient, the separation and isolation of the compound was carried out in 3 minutes. Method (min) (min) B start B end A B C D E F Table 1. UPLC retention time windows and corresponding focused preparative gradient composition. Figure 5. AutoPurify processing report showing the color coded purity and found/not found of a 348-well plate Focused library purification AU 1.0e AutoPurify automatically selects the samples requiring purification and the corresponding focused preparative method. 5.0e Figure 4. Separation of the compound of interest using a 3-minute focused gradient. UPLC library purity screening This same methodology can be applied to the purity screening and purification of a large sample library. The ACQUITY UPLC System s large capacity ( well plates) and the rapid analysis cycle time provide the ideal tool for high throughput library screening. Data is processed and handled using AutoPurify, part of the FractionLynx Application Manager. 4 Figure 6. AutoPurify processing of the UPLC screening library. 31

32 UPLC fraction analysis The substantial time savings associated with analytical screening can be magnified by incorporating UPLC into the analysis of the collected fractions. The collected fractions are analyzed to determine the new sample purity, and sample lists are automatically generated for each step of the process. By incorporating fraction analysis by UPLC into the workflow, the efficiency of the lab is further increased. CONCLUSION n Scale-up from UPLC to preparative HPLC in an efficient manner is possible with the use of focused gradients. n The efficiency of UPLC can be carried through to purification, offering a substantial increase in throughput and productivity. n The AutoPurify capabilities of FractionLynx allows for automation from the initial UPLC QC, through purification, to UPLC fraction analysis. n AutoPurify is also capable of automatically selecting a focused preparative gradient based on the analytical results, giving better quality purification and eliminating the need for expert manual invention. References 1. Xia F, Cavanaugh J, Diehl D, Wheat T. Seamless Method Transfer from UPLC Technology to Preparative LC, Waters Application Note. 2007; EN. 2. Cleary R, Lefebvre P. The Impact of Focused Gradients on the Purification Process, Waters Application Note. 2007; EN. 3. Jablonski J, Wheat T. Optimized Chromatography for Mass Directed Purification of Peptides, Waters Application Note. 2004; EN. 4. Cleary R, Lefebvre P. Purification Workflow Management, Waters Application Note. 2006; EN. Figure 7. AutoPurify processing of the UPLC analysis of the collect fractions. Waters, ACQUITY UPLC, UltraPerformance LC, and UPLC are registered trademarks of Waters Corporation. AutoPurification, OBD, XBridge, AutoPurify, FractionLynx, Application Manager, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

33 Purification Workflow Management Ronan Cleary and Paul Lefebvre Waters Corporation, Milford, MA, U.S. INTRODUCTION A standard requirement for drug discovery screening of synthetic libraries is that the test compounds must have a minimum purity. Purity is based on the area percent of an LC chromatogram from a detector such as UV, evaporative light scattering (ELS), MS with a total ion chromatogram (TIC), or a combination of multiple detectors. If the screening compounds do not meet this standard, purification is required. Managing the flow of samples, subsequent fractions, and all the associated data through this process can often be difficult and time consuming. This application note illustrates how a sample is efficiently taken through a three-step purification process utilizing the AutoPurify capabilities within the Waters FractionLynx Application Manager for MassLynx Software, and the AutoPurification System for MS-directed analysis. This comprehensive informatics solution enables automation from the initial evaluation, through the purification, to analysis of the collected fraction. The software will decide which shallow gradient should be used to perform the purification (Figure 2). Figure 2. TIC chromatogram after purification, with fraction collection indicated by the shaded area. Then, it automatically performs analysis of the collected fractions (Figure 3). DISCUSSION The AutoPurify functionality uses the results of the analytical analysis to determine the purification process. By performing an analytical evaluation of the sample, the presence of the target compound is confirmed and its purity measured (Figure 1). Figure 3. TIC chromatogram of the analysis of the collected fraction. Information determined from analysis of the fractions can be used to help with post-purification handling such as fraction pooling and transfer to an evaporator. A report can be exported in different file formats such as.xml,.csv, and.tab, to easily interface with other sample handling software packages. Figure 1. TIC chromatogram of the analytical-scale analysis of the crude sample.

34 Step 1: Analytical interpretation In the first of the three-step process, the purity of the target mass is identified by integrating the chromatogram. In the example shown in Figure 4, the area percent of the target determined from the TIC (22) is then used to calculate the sample purity. The area percent can also be determined by total absorbance current, wavelength, or analog signal. The purity of the target is then classified as pass, tentative, or fail, based on user-defined limits. In this example, less than 10 pure means purification will not occur, 10 to 80 purity requires purification, greater than 80 is pure enough, and does not require further purification. Step 2: The purification process In the second step of the process, purification occurs. The software will determine the purification method best suited to improving the separation by choosing one of six different shallow gradients. Using the analytical retention time of the target, the appropriate shallow gradient-based method will be chosen. Shallow gradients, also referred to as narrow gradients, allow for optimal target separation from closely eluting impurities, thus improving the purity of the resulting fraction. Each narrow gradient, whose time window is indicated by the colored lines (Figure 5), is created to cover a different timed section of the analytical gradient. Figure 5. Graphical representation of analytical and prep gradients. Figure 4. Analytical evaluation of mass is 22 of the TIC, and the target sample is co-eluting with peak 2. An overlay chromatogram of the two co-eluting peaks, with the spectrum, indicates the potential fraction contamination that could occur. In a manual process, the analyst would evaluate the separation, and adjust the gradient to achieve the best results. However, in an open access environment or where large numbers of samples are being handled, automation is necessary. The analytical gradient is indicated by the dotted black line, and shows the solvent change over the course of the gradient to be from 5 to 95 B. With the relationship between the analytical retention time and the elution organic composition known, the software can choose which of the narrow gradients will be used to automatically purify the samples during the purification stage of the process. When the software evaluates the analytical sample, it creates a browser report defining the recommended strategy. The user has the opportunity to change the strategy if necessary. The part of the report that refers to the strategy is the results pane (Figure 6). In this example, there are several other samples analyzed, but the one that is of interest is that last one on the list, A

35 Figure 6. Browser results pane with sample purity and prep strategy displayed. The sample in this case eluted at 4.04 min (Figure 7), so the narrow gradient chosen for the purification was Narrow Gradient C, the one that targeted the solvent change that occurred between 4 and 5 minutes. This gradient is denoted by the green line, which changes from 24 to 37 organic over 6.5 min, and is defined graphically as below. Figure 8. Overlay of the chromatograms of the two masses that were co-eluting earlier, showing the improved separation that was achieved. Spectra highlight the success also. Step 3: Fraction analysis With the first two steps of the process complete, the user can also decide to analyze the fractions (Figure 9). AutoPurify creates a sample list containing the fractions required for analysis and automatically runs them. MS ES+ :358.1 (3) e Figure 7. Representation of the narrow prep gradient chosen for the purification of the compound eluting at 4.06 min, with improved separation showing the isolated peak at 3.74 min collected MS ES+ :TIC (3) 1.6e The improved separation is more clearly displayed when the chromatograms of the two co-eluting compounds, as seen in Figure 4, are extracted and their chromatograms reviewed. Figure 8 shows the two chromatograms of masses 255 and 358, overlaid, and the improved separation achieved Figure 9. Fraction analysis post-collection, and post-fraction mixing by the injector/collector. TIC shows no other compounds present in the collection vessel. 35

36 To ensure that the portion of the sample taken for analytical analysis is representative of the entire collected fraction, it may be necessary to pre-mix fractions prior to injection (done with the injector/collector). Once homogenized, analysis can be performed on an analytical scale. Automating the process Automation of the three-step purification process is accomplished through AutoPurify. A FractionLynx browser is created after each of the three stages to display results of the analysis and to report the recommended strategy for the next stage in the process. The software can automatically create and run the list of samples that are to continue to the next step. The user has a choice whether to allow the three stages to run unattended, or to manually review the results of each stage and edit the software s decision. The determined strategy can be adjusted as necessary by the user through the interactive browsers that are produced. By automating the process, decisions can be made after regular work hours, allowing the work to continue unattended, saving time and resources. The root name of the data, the sample ID, sample list, and the FractionLynx browser, A123, as shown in Figure 10, are edited by the software and carried through the purification process to make sample and results tracking easier. Analytical interpretation FractionLynx browsers also include chromatograms and spectral information that are not shown in this application note. The portion of the browser file in Figure 10 shows sample purity and the prep strategy decision that was determined after the samples were analyzed on an analytical scale. The preparative sample list is automatically created and run after the analytical analysis. Once the purifications are complete, the results are processed and a new FractionLynx browser report is generated (Figure 11). Figure 11. Purification results, indicating where the fractions were collected, including fraction volume and spectral purity. Blue = collected fraction of the sample highlighted in the injector plate, green = passed spectral purity assessment, burgundy = review required, and red = failed purity assessment. Purification process Upon completion of the processing of the purification results, a sample list is generated and automatic analysis of the fractions generated is performed (Figure 12). Figure 10. Browser report created after the analytical evaluation. The resulting strategy is displayed using different colors for the injection plate. Green = mass is found, purity level between 20 and 80, and sample requires purification; and red = mass is either not found or sample is already pure enough, and purification will not be performed. Figure 12. Fraction analysis results, indicating the sample purity of the collected fractions. 36

37 Fraction analysis The final report shows the locations of the fractions, chromatograms, and spectra. The information in the reports can then be easily exported in different file formats such as.xml,.csv, and.tab, to easily interface with sample handling software packages such as liquid handlers or weighing devices. CONCLUSION This application note shows how a library of compounds can easily and efficiently be purified using the AutoPurify capabilities within the FractionLynx Application Manager. The software is capable of automating the entire purification process, from the original analytical purity assessment, to purification, and finally to the analysis of the fractions. AutoPurify allows the process to be performed intelligently. Analytical results are used to determine if the target is present and its purity. Based on these criteria, only samples that truly require purification continue on through the process. Samples that do not contain the target compound, not enough of the target, or are already pure enough can simply be excluded from purification. The benefits of using AutoPurify can be measured in time savings, reduced solvent consumption, and overall productivity gains. This is noticeable in several main areas: n Automated evaluation of samples before purification prevents unnecessary purification from being performed by removing samples that do not require purification. n Computerized evaluation of samples throughout the entire process saves analysts from having to manually review batches between stages of the process, and enables the subsequent analysis to be performed immediately without waiting for the analyst to be present. n Computerized determination of methods required during the process saves analysts from having to make or decide which gradients should be used to improve separations. n The use of narrow gradients allows for the use of shorter, more focused gradients, saving time and solvents. n Automation from stage to stage allows for unattended operation, combining all the savings of the process. Waters is a registered trademark of Waters Corporation. MassLynx, AutoPurification, AutoPurify, FractionLynx, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

38 MAKING A PURIFICATION SYSTEM MORE RUGGED AND RELIABLE Ronan Cleary, Paul Lefebvre, and Warren Potts Waters Corporation, Milford, MA, U.S. OVERVIEW The demand for the number of samples requiring purification continues to grow. This increase requires purifications systems to be able to run more efficiently and with less user intervention. However, there are a number of serious, corporate concerns with running unattended purification. These include losing samples due to system failure, solvent leaks, overflowing waste containers, and solvent reservoirs running dry. Another concern is the verification that the system is actually running properly and collecting fractions as expected. This application note highlights how the Waters AutoPurification System hardware and software can be utilized to alleviate theses concerns. Examples include software tools for monitoring solvent usage and that can monitor the number of injections without fraction collection. We also show how the system can be efficiently shut down in case of error to minimize the risk of sample loss. Finally, we demonstrate how a new splitter can increase recovery rates and how a post-fraction collector detector can be used as a quality control monitoring tool. DISCUSSION System configuration System configurations can vary depending on customer applications and requirements. Waters has developed a purification system based on input from our customers. The requirement for chemists to be able to make analytical injections to evaluate a sample before purification led to the development of the Waters 2767 Sample Manager, which has two separate flow paths one analytical, and one for preparative. A separate and additional flow path allows for fractions to be collected onto the instrument bed for further analysis. This injector/collector requires a solvent delivery system that is capable of delivering reproducible and accurate Figure 1. The mass-directed AutoPurification System consists of the 2545 Solvent Manager, 2767 Sample Manager, System Fluidics Organizer, and PDA detector. analytical and preparative flow rates. Additional pumps are regularly added to the system for other purposes, such as post-column splitter make-up, at-column dilution (US Patent #6,790,361), off-line column regeneration, and pre-column modifier solvent addition. Mass spectrometry was added to further increase the selectivity and efficiency of the systems. These components comprise the Waters AutoPurification System. Solvent monitoring The various pumps and vessels configured in a purification system can be defined in the monitoring software. The volume of solvent pumped from a solvent reservoir or into a waste container is monitored using the solvent monitor software. Graphical solvent level indicators allow for easy viewing of the system status. Each solvent reservoir has information specific to that container, maximum volume, and various warning levels. The status of the vessels is indicated by symbols, indicating that the system is either OK, or in Warning or an Acute Warning state. The response to the warning level is determined by the administrator.

39 A color-coded status page is also available, and can be accessed remotely through the remote status monitor component of the software. Once all the solvents are defined, monitoring occurs in the background without any user interaction. Any volume of solvent pumped, either during an acquisition or while idle, will be accounted for. Even the amount of solvent used to prime the pump is monitored. Figure 2. Solvent monitoring interface with both graphical and numerical reporting of system status. When the software monitoring the solvent vessels identifies a solvent level that has generated a warning condition, multiple notifications and responses can occur, such as: n Warning notification on the instrument page n Color-coded notification on the remote monitoring software n condition report sent to primary responsible party n Terminate the analysis or batch n Secondary s can be sent to different individuals, notifying them of the condition of the particular system Figure 3. Color-coded system status page, with icons that indicate the need to refill or empty the containers. Figure 4. configuration with primary and secondary contacts. Once the administrator has been notified, they can choose to manage the condition by emptying or refilling the containers as necessary, or allow the software to deal with the error condition and shut the system down safely. 40

40 Figures 5 and 6. The user can partially add or remove solvents as necessary. Shutdown software allows the user to configure a response produced when either the warning or acute level is reached: n Shut down immediately n Shut down after delay n Shut down after sample n Shut down after batch n Ignore the warning The shutdown procedure configured is linked to a particular shutdown method. This allows for an orderly shut down of the system to occur, allowing for columns to be flushed and returned to the correct conditions for storage, thus reducing the risk of damage. Tracking failures A critical component to ensure rugged and reliable unattended operation is to have the system be able to stop after a defined number of consecutive samples without fraction collection. There are various reasons why a system may not have collected fractions, and yet not be in an error state, such as a blocked splitter or MS sample cone that prevents detection, or a blocked injection port that keeps the sample from being loaded onto the column. User error can also be a contributing factor. Incorrect information such as mass or wavelength can also contribute to fractions not being collected. Figure 7. The user can define the number of injections that can occur without fraction collection before the run is ended. Additional collectors Frequently, analysts find that compounds other than the primary compound of interest are of importance, so it may be necessary to capture them in a separate collector. Examples include collection of a starting material or impurities along with the primary target. Another example is collecting all the other major peaks in addition to the primary target. This is shown in Figure 8 with a complex natural product separation. Figure 8. The top chromatogram shows collection of peaks detected by ELS detection. The lower chromatogram shows the peaks detected by the MS and collected by mass trigger. 41

41 There is no such thing as a universal detector, so it is possible that some compounds may not be detected. A waste collector can be added to the system, enabling all column eluent not diverted for collection earlier to be collected separately. In Figure 8, any of the sample not collected by either the primary or the secondary collectors was captured in a separate waste collector, thus minimizing the possibility of any sample loss. Splitter performance On any purification system where a destructive detector is being used, a splitter is necessary to isolate a portion of the primary flow for analysis, allowing the rest of the sample to be directed to the fraction collector. The flow to the collector must also go through a delay coil to prevent this much faster flow from reaching the collector before the triggering detector has identified the peaks to collect. Figure 10. The upper chromatogram shows the low-flow split to the fraction trigger detector. The middle chromatogram shows the high-flow split of the sample after using another commercially available splitter to the waste detector. The lower chromatogram shows the highflow split of the sample using the Waters splitter to the waste detector. Figure 11. Overlay of the trigger and collected fraction trace using a Waters splitter. The collected fraction is the purple trace, and shows little or no peak dispersion. Figure 9. The Waters splitter is matched to column dimensions for optimized performance. Figure 12. Overlay of the collections with the vertical axis linked. The green trace shows what would have been missed if a non-waters splitter had been used. The most important requirement of the splitter is that peak shape and resolution achieved from the column be retained in both the low- and high-flow solvent streams. The low-flow stream is sent to the detectors used to trigger fraction collection. If the peaks shapes differ between the triggering detector and the fraction collector, the collection of the fraction will be less than optimal. Laminar flow can cause the peaks on the high-flow side of the system to be larger than the peaks on the low-flow side of the system. This can contribute to decreased recoveries and impure fractions. We evaluated a new Waters splitter against another commercially available splitter to highlight the improvements that have been made with the splitter technology. Figure 13. AutoDelay results page with delay time and results export. 42

42 Collector delay time Delay time determination can be easily accomplished with the use of AutoDelay software, which will perform injections to determine the delay time and confirm injection for the determined delay time. Figure 14 shows the effect of delay time on the amount of missed fraction detected in the waste detector. The larger the detected peak corresponds to a lower recovery or increased sample loss. When the delay time is set optimally there is only a small peak, just above the noise. But as the delay time drifts from 1 to 3 seconds away from the optimal, the increase signal becomes more and more substantial. The measured recovery is greater than 99 at the optimal delay time. With the 3 seconds too early, the recovery is only 60. CONCLUSION Purification systems should include functionality that allows for unattended operation such as: n Solvent monitoring with tiered responses such as notification n Solvent monitoring with intelligent shut down n Remote system monitoring n Secondary fraction collection for use with other detectors n Waste collection to enhance user confidence Flow splitters should not increase band broadening and decrease fraction recovery rates. The new Waters flow splitters maintain equal peak shape for both the high and low flow for optimal fraction recovery and purity. The AutoPurification System, with technology that allows for rugged and reliable operation, is available from Waters. Figure 14. Different collection delay values have different responses in the waste detector. Waters is a registered trademark of Waters Corporation. AutoPurification 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

43 THE APPLICATION OF MS/MS DIRECTED PURIFICATION TO THE IDENTIFICATION OF DRUG METABOLITES IN BIOLOGICAL FLUIDS Paul Lefebvre, Robert Plumb, Warren Potts, and Ronan Cleary Waters Corporation, Milford, MA, U.S. INTRODUCTION The identification of drug metabolites following animal or human volunteer studies is essential to the drug discovery and development and regulatory submissions process. Traditionally, this has been achieved by the use of liquid or gas chromatography coupled to mass spectrometry. 1,2 More recently, the use of hyphenated techniques such as LC/NMR and LC/NMR/MS have become more commonplace in the drug metabolism laboratory, allowing a more precise identification of the site of metabolism. 3,4 While LC/NMR and LC/NMR/MS are extremely powerful tools, they are typically low throughput and limited in sensitivity. The capacity of analytical columns restricts the amount of material that can be loaded on to the column before the column exhibits either volume or mass overloading effects and the chromatographic resolution is lost. Thus LC/NMR is less attractive for the analysis of highly potent compounds dosed at low levels or those compounds that undergo extensive metabolism. In such cases, it is often necessary to perform a pre-concentration step, such as SPE or liquid/liquid extraction, both of which are time consuming and run the risk of losing of valuable information. The use of MS-directed purification, using semi-preparative scale columns (typically 19 mm i.d.), is now commonplace within the pharmaceutical industry, especially to support lead candidate purification. This approach has also been applied to the isolation of drug metabolites with some success. 5 The extra sensitivity and selectivity of MS/MS mass spectrometry allows for more precise selection of drug metabolites. Furthermore, the use of neutral loss and precursor ion scanning detection modes facilitates the collection of drug metabolites without the need for prior knowledge of compound metabolism. Figure 1. The Alliance HT System with the Quattro micro Mass Spectrometer. This application note shows how tandem quadrupole mass spectrometry has been employed for the isolation of the metabolites of common pharmaceuticals from urine. The application also demonstrates different modes of data acquisition, including scan, MRM, constant neutral loss, and precursor ion. We also demonstrate how the use of MS/MS-directed purification facilitates the combination of samples from several chromatographic runs. METHODS AND DISCUSSION A Waters Alliance HT System was used with a SunFire C 18 5 µm 4.6 x 100 mm column at 40 C. Eluent flow was split 1:20 with a Valco tee. 95 of the flow passed the 2996 Photodiode Array (PDA) Detector to the Fraction Collector III. The other 5 of the flow was routed directly to the Quattro micro Mass Spectrometer equipped with an ESCi multi-mode ionization source.

44 Caffeine metabolites methods Separation Water/acetonitrile in 0.1 formic acid, 1.25 ml/min total flow gradient. 0 to 5 min: 0; 5 to 35 min: 0 to 10 B; 35 to 35.5 min: 10 to 95 B; 35.5 to 39.5 min: 95 B; 39.5 to 40 min: 95 to 5 B; 45 minutes end. MS detection MS detection Electrospray negative, 3 kv capillary voltage, 30 V cone voltage, 20 V collision energy. Metabolites of interest Figure 3 shows the fragmentation patterns of the ibuprofen gluceronide metabolite. 7 Electrospray positive, 3 kv capillary voltage, 30 V cone voltage, 20 V collision energy (for MS/MS experiments). Metabolites of interest Figure 2 shows a portion of the caffeine metabolism pathway by demethylation. 6 Target metabolites maintain the methyl group in the 1 position. They also have a common fragment ion, m/z 57. Paraxanthine, m/z 181 1,7 Dimethylxanthine Caffeine, m/z 195 1,3,7 Trimethylxanthine 1Methylxanthine, m/z 167 Figure 3. Ibuprofen gluceronide metabolite with a common product ion of m/z 193. Common fragment of xanthine a methyl in position 1, m/z 57 Ibuprofen metabolites Separation Theophylline, m/z 181 1,3 Dimethylxanthine Figure 2. Metabolism of caffeine by demethylation: metabolites that maintain the methyl group in the 1 position have a common fragment ion, m/z 57. Water/acetonitrile/10 mm ammonium formate, 1.25 ml/min total flow gradient. 0 to 5 min: 5; 5 to 35 min: 5 to 60 B; 35 to 35.5 min: 60 to 95 B; 35.5 to 39.5 min: 95 B; 39.5 to 40 min: 95 to 5 B; 45 minutes end. Single quadrupole directed purification With single quadrupole directed purification, all ions generated in the source are passed through the quadrupole and detected. This is possible on the Quattro micro Mass Spectrometer by using the scan mode of acquisition. Only MS1 is scanned and there is no collision energy or scanning of Q3. Because all of the ions generated are detected in this mode, complex mixtures can contain numerous isobaric interferences. Consequently, multiple fractions can be generated from a single m/z value. Figure 4 shows the collection of the caffeine metabolites with m/z 167 and 181 detected using only the first quadrupole. There are eight fractions collected for m/z 167 and five fractions collected for m/z 181, with additional analysis required to determine the fraction of interest. 46

45 For a peak to be present in the MRM chromatogram, both the specific precursor and the specific product ion need to be detected. For each target, only one fraction was collected. m/z 181 OR Figure 4. Fractionation based only on scanning the first quadrupole. m/z 167 Tandem quadrupole directed purification: MRM collection With multiple reaction monitoring (MRM) data acquisition, MS1 is pre-selected on the precursor mass and MS2 is pre-selected on a specific product ion, as illustrated in Figure Figure 6. Fractionation based on MRM acquisition. Constant neutral loss collection A second possible mode of fraction triggering is from constant neutral loss acquisition. Here both MS1 and MS2 are scanned in synchronization, as illustrated in Figure 7. When MS1 transmits a specific precursor ion, MS2 looks for a product that is the precursor MS1 static Collision Cell RF only (all masses pass) MS2 static minus the neutral loss value. If the correct product is present, it registers at the detector. The constant neutral loss spectrum shows only the masses of all the precursors that lose the specific mass. Figure 5. MS/MS MRM data acquisition. By selectively detecting a product ion, the signal-to-noise ratio is optimized, thus reducing the isobaric interference and allowing only the target to be collected. This mode of acquisition requires previous knowledge of the exact precursor and the exact product ions before purification. Figure 6 shows the MRM acquisition and collection of the caffeine metabolites. The metabolites of interest for isolation have the transitions of 181 to 134, and 167 to 110. MS1 Collision Cell scanning RF only (all masses pass) Figure 7. MS/MS constant neutral loss data acquisition. MS2 scanning 47

46 m/z m/z m/z m/z Precursor ion collection A third mode of fraction triggering is from precursor ion acquisition, as illustrated in Figure 9. Here, MS1 is scanning and MS2 is fixed on a specific product ion. If the specific product ion is observed, it is registered at the detector. The spectrum only shows the masses that have that specific product. Neutral loss of 57 TIC Figure 8. Fractionation based on constant neutral loss acquisition. MS1 scanning Collision Cell RF only (all masses pass) MS2 static Figure 8 displays the constant neutral loss of 57 acquisition and collection of the caffeine metabolites with m/z 167 and 181. It shows that two fractions are collected, one for each mass. These fractions contain the target mass and have the specific neutral loss. Applications for fraction collection from constant neutral loss acquisition Mass triggered collection With constant neutral loss acquisition, the only peaks detected are the ones with the loss of the specific mass, in this case, 57. Depending on the specificity of the loss, numerous ions can be detected. This leads to complex total ion chromatograms. Therefore, when triggering by a specific mass, the collected target must contain the precursor of interest and have a specific neutral loss. Figure 9. MS/MS precursor ion data acquisition. Fraction collection from a precursor ion acquisition has to be from the TIC, since the precursor mass is unknown. This mode of fraction collection is valuable when the metabolites are unknown, but there is a common fragment of the core compound that can be detected. To illustrate the common fragment ion collection capability, Figure 10 shows the glucuronic acid conjugates collected from the ibuprofen urine samples using the precursor ion scan mode of m/z 193. There are three fractions that are collected, m/z 273 (not drugrelated), m/z 397 (hydroxyglucuronide conjugate), and m/z 381 (glucuronide conjugate). Collection triggered on TIC When using this mode of acquisition and collection, all the peaks with a specific neutral loss are collected. This functionality is valuable when the metabolites have a specific loss related to the drug s structure. It could also be used for isolating a class of metabolites with a generic loss (e.g., sulfates ( 80) or glucuronides ( 176)). The precursor mass for each fraction can then be extracted and used to aid in the identification of the metabolites m/z m/z m/z m/z m/z m/z In the constant neutral loss example shown, collection could also have been triggered from the total ion chromatogram (TIC). All peaks in the 57 TIC would be collected and then additional analysis or data review would be required to find the desired fractions. Parents of 193 TIC Figure 10. Fractionation based on the precursor ions of the m/z 193 TIC acquisition. 48

47 Additional collection options The ESCi multi-mode ionization source enables both ESI +/- and APCI +/- acquisition to occur within the same run. This allows for fraction collection to be triggered from any of the acquisition channels, thus proving useful if the metabolites require different ionization modes. Prior to this enabling technology, the only options for collection would be to split the sample and run in different modes, or rely upon time-based fractionation and then analyze all the fractions by both modes to determine the targets. The selectivity of the ESCi-enabled fraction collection process can be further enhanced by the use of mixed triggers. This approach uses Boolean logic strings to trigger collection from multiple data traces (e.g., collection can occur only when Mass A is present and Mass B is not, or a peak has to be present in two different traces at the same time for fractionation). n Constant neutral loss mode can be used for collecting a class of compounds with a target-specific loss or a generic group loss for a broader study, or can be used as a second filter where the target has to have a specific mass and the neutral loss. n Collection in precursor ion mode allows for all the precursors with a specific product ion to be collected, which is valuable when the metabolites are unknown, but there is a common fragment of the core compound that can be detected. Thus, these different modes of collection add value to a wide variety of applications previously accomplished with more laborious, time consuming, and less specific methodologies. References 1. Ismail IM and Dear GJ. Xenobiotica. 1999; 29(9): Dear GJ, Mallett DN, and Plumb RS. LCGC Europe. 2001; 14(10): Dear GJ, Plumb RS, Sweatman BC, Parry PS, Robert AD, Lindon JC, Nicholson JK, and Ismail IM. Journal of Chromatography B. 2000; 748: CONCLUSION Fraction collection with a tandem quadrupole mass spectrometer is now possible using four different modes of data acquisition: scan, MRM, constant neutral loss, and precursor ion, which enables improved versatility for triggering options. n Scan mode has the potential to increase the number of isobaric inferences detected and collected. n MRM mode is the most selective because it only monitors a specific precursor/product ion transition and greatly reduces the isobaric interferences, but requires previous knowledge of the transition. 4. Dear GJ, Plumb RS, Sweatman BC, Ayrton J, Lindon JC, and Nicholson JK. Journal of Chromatography B. 2000; 748: Plumb RS, Ayrton J, Dear GJ, Sweatman BC, and Ismail IM. Rapid Communications in Mass Spectrometry. 1999; 13(10): Bendriss E, Markoglou N, and Wainer IW. Journal of Chromatography B. 2000; 746: Kearney G, et al. Exact Mass MS/MS of Ibuprofen Metabolites using Hybrid Quadrupole-Orthogonal TOF MS Equipped with a LockSpray Source. Waters Application Note. 2003; EN. Waters, Alliance, and ESCi are registered trademarks of Waters Corporation. SunFire, Quattro micro, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

48 Evaluating the Tools for Improving Purification Throughput Paul Lefebvre, Warren Potts, Ronan Cleary, and Robert Plumb Waters Corporation, Milford, MA, U.S. INTRODUCTION Chemists are constantly looking for ways to improve the overall throughput of their purification system. is the limiting factor for throughput, and there are two areas where time savings can be achieved: the amount of time required to perform a separation, and the amount of time between injections. Making the purification system as efficient as possible requires optimizing and minimizing both of these times. The challenge, however, is minimize these times without impacting the purity and recovery of the fractions. In this application note, we examine tools available for increasing the overall throughput of a purification system. We will use information from the analytical separation to optimize the purification, and will examine the steps required between injections to then determine the most efficient way to minimize run time. Figure 1. The Waters mass-directed AutoPurification System. OVERVIEW In order to correctly compare time-saving techniques, we first established a baseline separation to define a standard analysis and collection time. We purified 10 drug-like compounds with a generic 10-minute preparative gradient. This baseline analysis time was then used as the comparison time for the analysis performed when the different time-saving chromatographic functionalities were applied. The major areas for improving throughput are: n Decreasing the time required for the analysis n Decreasing the time between injections One approach for decreasing the analysis time uses shallow or narrow gradients. Approaches for decreasing the time between injections include column regeneration techniques and automatically ending the purification run after the desired target has been collected. METHODS AND DISCUSSION Components The Waters AutoPurification System is comprised of: n 2545 Binary Gradient Module (BGM) n 2767 Sample Manager n System Fluidics Organizer (SFO) n 2996 Photodiode Array Detector n 3100 Mass Detector n 515 makeup pump n Passive flow splitter, 1:1000 n All components are controlled by MassLynx and FractionLynx software The 10-sample library consisted of various drug-like compounds at a sample concentration of about 20 mg/ml dissolved in DMSO.

49 The chromatographic methods used water with 0.1 formic acid as mobile phase A, and acetonitrile with 0.1 formic acid as mobile phase B. Methanol was used as the makeup solvent for the preparative analysis. Analytical gradient SunFire C x 50 mm, 5 µm, 1.5 ml/min total flow gradient and a 10-minute total run time. Gradient Name Analytical Retention B Start B End A 0.00 to B 1.67 to C 2.84 to D 4.00 to E 5.17 to F 6.34 to Table 2. The narrow gradients used relative to the analytical retention time. The time window in which the analytical sample eluted defines the conditions for the prep run. For example, if the compound eluted at 4.04 min, then the purification method would ramp up the organic percentage so that is was 50 at 0.5 min. Figure 2. Analytical gradient table. Generic preparative SunFire C x 50 mm, 5 µm, 25 ml/min total flow gradient. The same gradient table, as shown in Figure 2, was used. The only difference was the flow rate. Narrow or shallow preparative gradient SunFire C x 50 mm, 5 µm, 25 ml/min total gradient. The start and end percent B composition is variable and dependant on the sample retention time during its analytical analysis. (Minutes) Composition (B) 0.00 to to B start 0.50 to 1.67 B start to B end 1.67 to 2 B end to 95 2 to to 5 End Table 1. Narrow gradient table. See Table 2 for percent B start and end. Baseline throughput The generic gradient was used to perform the purification of 10 samples and the overall run time was measured. This time is used to compare the improvements. Sample Retention (min) Run (min) Between Injections (min) Total Run 120 minutes Table 3. The overall throughput with the generic gradient. The total run time was 120 minutes. 52

50 Narrow gradients Narrow gradients can be used to improve preparative chromatographic resolution. 1 However, if the resolution is adequate in the analytical separation, a shorter narrow or focused gradient can be used to increase throughput. The short method will focus its gradient on the same organic concentration, but in a shorter time frame. Figure 3. The different narrow gradients possible to focus on either improved resolution or throughput. Figure 4 shows an example of one of the 10 samples being purified by both a generic and a narrow gradient. The target was successfully isolated using narrow gradient D. The results show that the resolution is maintained over the focused section of the gradient (the blue bracket). Note that there is a loss in resolution, as expected, in the non-focused areas of the gradient. This would have to be considered when the compound elutes at the very beginning or end of the focused gradient Generic Gradient Narrow Gradient EIC = 270 EIC = 270 TIC TIC Figure 4. Comparison of the 10-minute generic and the 5-minute narrow purification. The blue bracket corresponds to the focused area of the gradient, where the resolution is maintained. Sample Generic Retention (min) Narrow Gradient Table 4. The overall throughput increases by 1.7 fold when incorporating narrow gradients, compared to using a generic gradient. Rinsing and equilibration Narrow Retention (min) Run (min) Between Injections (min) A E A D C B B D B B Total Run 70 minutes = 1.7 Fold Increased Throughput It is important for high-quality chromatography that the column is rinsed and re-equilibrated with the appropriate volume of solvent, typically defined in column volumes. Insufficient rinsing can cause carryover, and equilibration time also has a significant impact on the overall throughput, with inadequate equilibration leading to retention time variability, poor chromatographic peak shape, or even sample breakthrough. The quantity of rinsing solvent is dependant upon the sample matrix, the retentiveness of the column, and the elutropic strength of the rinsing solvent. Typically, two to three column volumes is required to rinse. For equilibration, various articles report anywhere from three to 20 column volumes can be used. 2-3 For example, a 19 x 50 mm column has a volume of about 12 ml. Two column volumes or 24 ml of 95 B were used to flush the column, and 60 ml of 5 B were used to re-equilibrate the column. With the gradient flow of 25 ml/min, the flush takes about 1 minute, and the equilibration takes about 2.5 minutes. 53

51 100 Early termination Chromatographic run time Area for potential time savings Flush and equilibration time Injection time To further reduce the time required for analysis, a software tool can be used to automatically end the run after the target has been collected. The throughput improvements of this feature will be illustrated for both generic and narrow gradients Figure 5. Illustration of an injection cycle with chromatographic analysis time, equilibration and flush time, and injection cycle for next injections time displayed. The area where time could potentially be saved is noted. For either gradient approach used, once the target has finished collecting, the gradient will stop and flush with 95 B to wash the remaining material off the column. After a defined time of rinsing, the column will then be re-equilibrated with the initial gradient solvent. (Note: 2 minutes of equilibration time is performed between injections.) However, the flow rate can be elevated above optimal chromatographic conditions (30 ml/min for 5 µm packing), so long as the system can withstand the overall pressure increase. We found that the flow could be increased to 40 ml/min, only generating an additional 1300 psi of backpressure, reducing the flush time to 0.6 min and the re-equilibration time to 1.5 min, for a 1.5-minute savings. Off-line regeneration To increase throughput, a regeneration pump can be used to flush and re-equilibrate the first column off-line, while the next sample is running on a second column. In this method, the run is terminated at 2.5 min for the narrow gradients, or 7 min for the generic and the next injection started. The first column is switched off-line and its flush started, while the second column is put in-line to receive the next sample. As mentioned earlier, the time required for the injection to be performed is 2 min. The run-time savings for a generic preparative saw a reduction of 3 min per sample, for a reduction in the total run time from 120 to 90 minutes, or a 1.2-fold savings. The run-time savings for a narrow gradient was more significant. Injection-to-injection time was reduced from 12 min with the generic method to 4.5 min using narrow gradients and off-line column regeneration. This reduced the total run time from 120 to 45 minutes, a 2.7-fold savings. Sample Generic Run Generic with Regeneration Narrow Run Narrow with Regeneration Total Run min = 2.0 Fold Increased Throughput min = 2.3 Fold Increased Throughput min = 2.6 Fold Increased Throughput min = 3.0 Fold Increased Throughput Table 5. The overall throughput improvement using the run termination function can range from a two- to three-fold increase, depending on what additional tools are used. Using the regeneration pumps saves 0.6 min per injection when compared to a single column method. This corresponds to the time required to rinse the column. The re-equilibration time is incorporated into the 2 min to make an injection. 54

52 Optimized injection routine Throughput can be further improved by reducing the time between injections. The injection cycle can be divided into three segments: n Aspiration of the sample into the needle n Dispensing the sample into the loop n Washing the assembly Optimizing the speed of the aspiration enables the sample to be quickly drawn into the needle and holding loop. Care must be taken to ensure the increased syringe speed does not create air bubbles in the system. Once the sample has been drawn into the holding loop, it is dispensed at an optimized flow rate. Care must again be taken to ensure that a high-pressure condition does not occur by operating the syringe too quickly. Two options are available for positioning the sample in the loop. The default setting is to center the sample in the loop, but the sample centering can be disabled to allow the sample to be more quickly loaded onto the front of the sample loop. When sample centering is removed, it is possible to operate with only one wash solvent and to be able to perform this wash at the beginning of the injection sequence, decreasing the injection time. Cumulative time-savings The time required to inject and rinse was reduced from 2 min with the standard partial loop injection to 0.4 min with the new settings. Table 6 shows the throughput possible by combining optimized injection settings with the various other tools. Tool Original Total Run Optimized Injection Total Run Default Injection Routine Overall Increase with Optimized Injection Routine Generic Generic + End Run Generic + End Run + Regeneration Fold Increased Throughput Narrow Narrow + End Run Narrow + End Run + Regeneration Fold Increased Throughput Table 6. Using optimized injection routines can improve the overall throughput. The improved injection routine has a greater impact when using regeneration because the 2 min for the normal injection is used to re-equilibrate with a single column. But with regeneration, the reequilibration is done off-line and the injection time is dead time. 55

53 CONCLUSION Throughput can be increased by about five-fold using a combination of narrow gradients, early run termination, off-line column regeneration, and an optimized injection routine. This correlates to an 80 percent decrease in run time. n Narrow gradients can be used to improve throughput, but require additional information about the target. n Off-line column regeneration has a greater impact on throughput as the run time is reduced. n Early run termination improves throughput and reduces the amount of consumed solvent saving both time and money. n Optimizing the wash sequence and adjusting when it is performed will save additional time between injections. n Various combinations of throughput-enhancing tools can be used based on the specific requirements. References 1. P Lefebvre, A Brailsford, D Brindle, C North, R Cleary, W Potts III, BW Smith, Waters Poster Presentation, PittCon A.P. Schellinger, P.W. Carr, Journal of Chromatography A. 2006; 1109: UD Neue, American Laboratory. 1997; March. Waters is a registered trademark of Waters Corporation. FractionLynx, MassLynx, SunFire, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

54 A NOVEL APPROACH FOR REDUCING FRACTION DRYDOWN TIME Paul Lefebvre, Ronan Cleary, Warren Potts, and Robert Plumb Waters Corporation, Milford, MA, U.S. INTRODUCTION Recent advances in purification technology have shifted the throughput bottleneck from purifying samples to fraction drying. Some of the technologies employed for sample drying include vacuum centrifugation, heated nitrogen blow-down, and lyophilization. However, each one has the same rate limiting factor the quantity of water present. This quantity is dependant on the separation technique used to generate the fractions. The most commonly used technique is reverse phase- (RP-) HPLC, which can generate fractions with the water content as great as 95. One approach experimented with is to collect fractions directly onto solid phase extraction (SPE) cartridges. In theory this method is perfect, but making it automated and rugged has continued to be a challenge. A drawback to this approach is that a very high flow dilution pump is required to trap the compound on the cartridge. This high flow rate requires a large quantity of sorbent with large volume cartridges, and generates large volume fractions. Another problem with collection onto SPE cartridges is the possible change in selectivity that could result in poor trapping or breakthrough of the analyte. This application note shows the development and optimization of a method that removes the water and reduces the overall volume of the collected fraction. This method works by injecting and trapping the previously collected fraction onto a preparative column. The fraction is trapped by diluting the loading flow with 100 aqueous mobile phase. After the trapping has been completed, 100 organic mobile phase is passed through the column to elute the sample. Collection of the target is triggered by the MS detector and the collected fraction is now in 100 organic mobile. Figure 1. Plumbing diagram for the concentration system. Both fraction collection and concentration was performed on the same mass-directed AutoPurification System. Fraction collection was triggered by MS. METHODS AND DISCUSSION The standard components of the Waters AutoPurification System were used to perform the fraction concentration. In the plumbing diagram shown in Figure 1, the aqueous flow out of the gradient pump is directed into the first tee (T1). This tee acts as a mixer, diluting the organic concentration of the injected fraction, so that it will not break through the trapping column. The organic flow out of the gradient pump is directed to a second tee (T2) and is used to elute sample from the column. Proof of principle To establish a baseline performance of the method parameters, 10 drug-like compounds were initially purified. These purified fractions were collected in different concentrations of organic solvent and then used as the samples to evaluate the concentration method. The samples were loaded onto a trapping column and eluted in 100 organic solvent. Once it was determined that the initial method was successful, the process was optimized for minimum fraction volume and maximum throughput. The examples shown have initial fraction volumes as great as 30 ml of aqueous/organic and are reduced to as little as 1.5 ml of organic solvent.

55 Purification method n 10 mg sample load n Generic 5 to 95 gradient with water/acn/formic acid n Fraction volume of 5 to 8 ml with recoveries of greater than 95 Method optimization Once the trapping method was determined to be successful, we looked into optimizing the conditions. The parameters evaluated included the column dimension and packing, the dilution ratio, and the elution flow rate. An initial fraction of 10 mg of diphenhydramine collected in 8 ml of 60 water was the concentration test sample. Column dimensions Figure 2. Generic 5 to 95 gradient. Concentration method The collected fractions were injected onto the same column as was used for purification. The samples were loaded onto the column with a loading pump at 6 ml/min 100 A, and 29 ml/min aqueous from a dilution pump. After 6.5 min, the loading and dilution flow is stopped. Now that the sample is retained on the column, the elution is started at 29 ml/min of 100 B. The column must be able to trap the target fraction and yet give a minimum elution volume for the concentrated fraction. The maximum flow rate and the minimum loading time were determined to establish a minimum run time. These factors are dependant upon the column I.D., particle size, and injection loop m/z= Purification Acid Concentration m/z=235.0 m/z= RESULTS Diphen _02a : Scan ES+ TIC 1.58e Base Concentration (50 µl NH 4 OH) m/z=235.0 m/z= Acid Concentration _01e m/z= #1,1:1 to 1#1,1: : Scan ES+ TIC 1.58e m/z= #1,1:72 Purification Figure 3. Seven of the 10 were successfully concentrated in the acidic mobile phase in which they were collected. All recoveries were greater than 85. Base Concentration (200 µl NH 4 OH) m/z= Figure 4. Two of the remaining three were successful after adding base to fraction. This indicates that these samples should have been purified at a basic ph to keep the target neutral. 19 x 50 mm trap column Although the remaining sample was purified using the SunFire Column, it was not retained on the column during the concentration process. However, because fraction collection was triggered by MS, no sample was lost. Additional work is required to determine why it was not retained. 5 and 10 µm packing gave the same fraction volume. The only difference was the system back pressure. 58

56 Diphen Conc _02a : Scan ES+ TIC 1.58e8 Diphen Conc 10x50 mm 5 µm column 4 ml/min load, 24 ml/min dilution _12b : Scan ES+ TIC 1.41e8 Elution Flow = 24 ml/min -2 m/z= #1,1:1 to 1#1,1: Volume = 5.8 ml Figure 5. Concentration of the test fraction on a 19 x 50 mm column. 10 x 50 5 µm trap column -2 m/z= #1,1: The overall flow rate was reduced when the method was transferred to the 10 mm column. By reducing the elution flow rate, from 24 to 12 ml/min, the concentrated fraction volume was reduced from 8 ml to 2.9 ml. By reducing the flow rate even further, to 8 ml/min, the original 8 ml of 60 water was reduced to 1.6 ml of 100 organic solvent. Diphen Conc 10 mm col 4 load / 22 dilution / 12 ml/min elute _14b Elution Flow = 12 ml/min 1: Scan ES+ TIC 1.31e8 There is minimal loss of the overall speed of the analysis with the reduced elution flow rate. The loading and dilution pump operate at 24 and 4 ml/min, respectively, until 6.5 min. The flow rate was then reduced to the lower elution flow, accounting for the smaller volume, concentrated fractions. Volume = 2.9 ml Improving throughput Sample loading rules n The injection volume must be less than half the volume of the sample loop. Because the injection volume was 8 ml, the minimum loop volume was found to be 15 ml. n 3 to 5 times the loop volume is required to clear the sample from the sample loop. The minimum volume found to clear all the sample was 45 ml. Dilution ratio The dilution ratio (dilution flow/loading flow) is a critical factor in this method. The dilution ratio is a measure of the amount of aqueous solvent used to dilute the fraction s organic content to -2 m/z= #1,1: Figure 6 and 7. Elution flow was reduced with minimal adjustment to peak width. allow it to be trapped onto the column. If the dilution ratio is too small, it will cause breakthrough. If it is too large, it will decrease the throughput because of the additional time required to load the sample. Figure 9 shows the effect of the concentration with varying dilution ratios. The results show that at a ratio of 4.5 there is a jagged breakthrough of the target compound that is not present at a ratio of 5 or higher. 59

57 Diphen Conc 10 mm col 4 load / 22 dilution / 8 ml/min elute _16a Elution Flow = 8 ml/min Volume = 1.6 ml 1: Scan ES+ TIC 7.57e7 Scaling the method Based on the minimum loading time and dilution ratio, it is possible to establish the relationship between the loading time and the total flow rate (Table 1). To reduce the loading time to less than 5 min, the table shows that a loading and dilution flow of 10 and 50 ml/min, respectively, are required. This gives a total flow of 60 ml/min across the column. -2 Diphen Conc 10 mm col 4 load/ 22 dilution / 8 ml/min elute _14a 98 Ratio = 5.5 m/z= #1,1: Figure 8. Concentration of the test fraction on a 10 x 50 mm 5 µm column at an elution flow rate of 8 ml/min : Scan ES+ TIC 1.11e8 Loading Flow (ml/min) Loading (minutes) Dilution Flow (ml/min) Total Flow (ml/min) Table 1. Relationship between loading time and total flow. -2 Diphen Conc 10 mm col 5 load/ 25 dilution / 8 ml/min elute _19a 98 m/z= #1,1: Ratio = : Scan ES+ TIC 1.12e8 Handling increased back pressure n Increase the particle size: a 2x increase equals a quarter of the back pressure n Waters SunFire column, 10 x 50 mm, 10 µm n 60 ml/min only generated 2,300 psi Diphen Conc 10 mm col 5 load/ 25 dilution / 8 ml/min el _09 1: Scan ES TIC e7-2 m/z= #1,1: Concentration of 10 mg test fraction to under 2 ml in 5.5 min with greater than 95 recovery Diphen Conc 10 mm col 5 load/ 20 dilution / 8 ml/min elute _10a : Scan ES+ TIC 1.39e8 Ratio = 4.5 m/z= #1,1: m/z= #1,1: Figure 10. Results from the optimized method. Figure 9 A-C. Concentration of test fraction with varying dilution ratios. 60

58 Mass load One concern with these optimized parameters is the mass load on the smaller trapping column. To evaluate this, the compounds were purified with increasing mass load on the preparative column until overload conditions were achieved. The collected fractions were concentrated using the optimized method. Two examples are shown. Example 1: 60 mg of Ketoprofen The initial purification generated a 10 ml fraction containing about 50 water. The concentration method successfully reduced the volume to 3.6 ml of organic solvent with a recovery greater than Purification: 10 ml fraction containing about 50 water Concentration: 3.6 ml of organic solvent with greater than 95 recovery Figure 11 A-B. The purification and concentration of 60 mg of ketoprofen. Example 2: 20 mg Phenyl-tetrazole The purification generated two fractions with a total volume of 18 ml containing about 60 water. The concentration successfully reduced the volume to 3.2 ml of organic solvent with the recovery greater than 95. When the chromatography begins to overload for the purification on a 19 x 50 mm, the fraction will not be completely trapped on the 10 x 50 mm column. 0 Purification: 10 ml fraction containing about 50 water Figure 12 A-B. The purification and concentration of 20 mg of phenyl-tetrazole. 0 Concentration: 3.6 ml of organic solvent with greater than 95 recovery Automatic pooling Fraction pooling on the trapping column can also increase throughput. In Figure 13, a 3 ml fraction was collected for each of the 10 injections. The fractions were then individually loaded onto the trap column and concentrated. A single 1.5 ml fraction was collected Purification: 10 injections of same sample 10 3 ml fractions collected 30 ml total volume Concentration: All 10 fractions loaded on the trap column eluted together in 1.5 ml or organic solvent Figure 13. An example of automatic pooling of 10 fraction tubes into a single concentrated fraction. 61

59 CONSIDERATIONS The pka of the target compound should be considered when performing purification. The target compound should be neutral during the purification. This means that a basic compound should be run in a basic mobile phase and, conversely, an acidic target should be run in an acidic mobile phase. This will result in better loading and chromatography 1 and will also ensure that the collected fraction is not ionized in solution. By being neutral, it is more likely to be successfully trapped during the concentration process. The amount of collected material, in both mass and volume, will dictate the required system configuration. The volume of the fraction will determine the size loop required. The mass of collected material will determine the column size. Both the loop and column size will determine the overall throughput of the system. In the examples shown, all of the concentrated fractions were triggered by MS. However, this was done only for method development purposes. It is possible to collect these fractions by UV or even just by time. When collecting by time, each tube has the same volume and organic concentration, so the time required for drying is constant. With typical fractionation, each tube can have a different volume and organic concentration, so the time required for drying is variable. This variability can lead to inefficiency, by either drying for too long, or by stopping too early then checking multiple tubes to find that you need to restart for only a few of the tubes. BENEFITS Dry-down time Composition Volume Dry Down Aqueous/Organic 5 to 30 ml 5 to 15 hours 100 organic 1 to 3 ml less than 30 min Concentrating the fraction to about 3 ml of organic solvent can be accomplished in 6 min. Process enhancements n Shorter drying times equals more efficient use of the driers. n Automatic pooling of multiple fraction tubes reduces the post-purification sample handling. Acknowledgements n Ian Sinclair, AstraZeneca n Giovanni Gallo, Waters, Manchester, UK n Paul Rainville, Waters, Milford, MA References 1. Neue UD. Wheat TE. Mazzeo JR. Mazza CB. Cavanaugh JY. Xia F. Diehl DM. J. Chrom. A. 2004; 1030: Waters is a registered trademark of Waters Corporation. AutoPurification, SunFire, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

60 profiling

61 PROFILELYNX APPLICATION MANAGER FOR MASSLYNX SOFTWARE Increasing the throughput of physicochemical profiling The client: Physical Chemistry group at a major pharmaceutical company BACKGROUND The Physical Chemistry group at a major pharmaceutical company was created to support Discovery projects in hit identification, lead identification, and lead optimization phases with early physicochemical data. The Discovery groups send test requests for selected compounds simultaneously to respective departments via the Chemical Support (CS) team within the Chemistry department. The Chemistry department is where all synthesized compounds are collected and stored. Compounds are sent out for testing according to the requests, as either standard stock solutions or solid samples. The Physical Chemistry group is made up of three analytical chemists running two LC/UV/MS systems. These systems each consist of a Waters Alliance HT System with a 2996 Photodiode Array (PDA) Detector and a ZQ Mass Detector, running on MassLynx Software. Testing is done in a 96-well plate format. Among the analyses performed by the team are identification, purity, stability, and solubility tests. ID and purity evaluations are always included in all solubility and stability tests and demand additional processing of data. THE CHALLENGE A screen solubility test of 48 samples took approximately 51 hours of analyst time, from when the samples were received to when the data was entered into the database. For a plate containing 48 duplicate samples, the variety of tasks involved: n 4 hours doing sample prep and running the samples n 18 hours in the office collecting compound and plate information codes, predicted properties, structures and creating appropriate sample lists n 8 hours evaluating purity n 19 hours doing the solubility calculations n 2 hours inputting the final data into the company s database. The analyst would get results well over a week later. The Physical Chemistry group recognized that tests were taking too long to deliver results. They needed to significantly reduce bottlenecks in data management and analysis, as well as instrument resources, to improve their ability to support discovery projects especially as incoming work volume was increasing.

62 THE SOLUTION Creating the proper tools for collecting sample information from the database, formatting sample lists, and analyzing the data generated consumed a great deal of analyst time. By implementing ProfileLynx a specialized Application Manager for MassLynx that automates processing of physicochemical property analyses into their existing LC/MS workflow, the chemists reduced the amount of time it takes to perform these tests from 51 to just 20 hours (Figure 1). The office time was reduced from 17 to 2.5 hours. Because of the improved reporting capabilities of ProfileLynx, the solubility evaluation now takes just 4 hours instead of Manual Process 2005 ProfileLynx Figure 1. Chemist s time distribution for a screen solubility test of 48 compounds using a manual process (top) vs. ProfileLynx implementation (bottom). BUSINESS BENEFIT (hours) Lab time Office time Evaluation ID/Purity Evaluation Solubility Database As a result of the overall reduction in time, the group is able to analyze more samples, as well as provide the critical information necessary to make decisions about possible lead candidates more quickly. Because of the success of ProfileLynx with this evaluation, the software will be implemented with other tests within the Physical Chemistry group, including solid solubility, stability, and ELogD. WATERS SOLUTIONS FOR LEAD OPTIMIZATION Waters System Solutions for lead optimization provide an automated, efficient selection process for determining compounds that have potential to become successful therapeutics. These solutions combine the strengths of Waters instruments, chemistries, software, and customer support to assist discovery labs in characterizing compounds faster, easier, and more cost-effectively. Waters MassLynx software and its ProfileLynx Application Manager streamline data management for physicochemical property profiling. MassLynx interfaces with upstream data systems to build Sample Lists used for data acquisition, while ProfileLynx automates the processing of chromatography-based data for physicochemical property analysis. While the LC/MS sample analyses was efficient for the screen solubility test, processing the data and interpreting the results required tedious and time-consuming data manipulation and calculation. By introducing ProfileLynx and other tools such as MassLynx templates into their workflow, the customer has saved about 30 hours on the solubility screen for each set of 48 compounds. The time is now used in the implementation of other tests. Waters and Alliance are registered trademarks of Waters Corporation. MassLynx, ProfileLynx, ZQ, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

63 AN AUTOMATED LC/MS/MS PROTOCOL TO ENHANCE THROUGHPUT OF PHYSICOCHEMICAL PROPERTY PROFILING IN DRUG DISCOVERY Peter Alden, Darcy Shave, Kate Yu, Rob Plumb, and Warren Potts Waters Corporation, Milford, MA, U.S. INTRODUCTION The synthesis of large, focused chemical libraries allows pharmaceutical companies to rapidly screen large numbers of compounds against disease targets. Active compounds, or hits, that result from these screens are traditionally ranked based on their activity, binding, and/ or specificity. Turning these hits into leads requires further analysis and optimization of the compounds based upon their physicochemical and ADME characteristics. The critical factor to consider in physicochemical profiling is throughput. The bottlenecks to throughput include MS method optimization for a large variety of compounds and data management for the large volume of data generated. Currently, experiments including solubility, chemical and biological stability, water/octanol partitioning, PAMPA, Caco-2, and protein binding are used to generate physicochemical profiles of compounds in drug discovery. The measurement of physicochemical properties from these studies is easily enabled using chromatographic separation and quantitation using LC/MS/MS/UV. While the sample analyses may be efficient, processing the data and interpreting the results often requires tedious and time-consuming manual manipulation and calculation. This application note describes an approach to solving these problems by using MassLynx Software s ProfileLynx Application Manager, a fully automated software package that allows for the design of experiments, data acquisition, and data processing as well as report generation. To demonstrate the use of this software package, we have developed an automated UPLC /MS/MS protocol for data generation. The data acquired from multiple assays was processed by a single processing method, all in an automated fashion. As a result, the physicochemical profiling process was significantly simplified and throughput increased. ACQUITY TQD with the TQ Detector. EXPERIMENTAL LC conditions Instrument: Waters ACQUITY UPLC System Column: ACQUITY UPLC BEH C 18 Column 2.1 x 50 mm, 1.7 µm Column temp.: 40 C Sample temp.: 20 C Injection volume: 5 µl Mobile phase A: 0.1 Formic acid in water Mobile phase A: 0.1 Formic acid in acetonitrile Gradient: A B Curve Flow ml/min ml/min ml/min ml/min

64 MS conditions MS system: Waters TQ Detector Software: MassLynx 4.1 with ProfileLynx ESI Capillary voltage: 3.20 kv Polarity: positive Source temp.: 150 c Inter-scan delay: 20 ms Desolvation temp.: 450 C Inter-channel delay: 5 ms Desolvation gas flow: 900 L/Hr Dwell: 200 ms Cone gas flow: 50 L/Hr Solubility 950ul µl ph 7.4 buffer Buffer 2 mm Samples in DMSO 50 µl 50 µl 50 µl 950ul µl Buffer/ buffer/acn ACN 950ul µl ACN Shake for for 24 hours 24 hours at 37 at C ºC 37 Property profiling assays n A set of 30 commercially available compounds were randomly chosen to demonstrate the ProfileLynx Application Manager. n QuanOptimize Application Manager allows for the automated optimization of the MS multiple reaction monitoring (MRM) conditions for each compound. n Each compound and a reference standard were analyzed by solubility, ph stability, LogP/LogD, and microsomal stability assays based on methods previously published. 1,2,3 n For quantitative experiments, single point or multipoint calibration curves were used. n To mimic the current practice in discovery labs, 96-well plate formats were used in this study. n ph stability assays were carried out at three different phs: stomach (ph 1.0), blood (ph 7.4), and colon (ph 9.4). n Solutions were shaken overnight and vacuum filtered through a Sirocco plate. n Fractions were quantified against single point 1 µm calibration standards. Centrifuge for 15 min at 3000 RPM Dilute supernatant 1 to 100 in DMSO Analyze and and quantitate Quantita against te standards ph stability 200 µm Samples in DMSO 50 µl 50 µl 50 µl 950 µl ul µl 950ul µl ph ph M HCl 7.4 ph 7.4 Buffer ammonium Ammonium buffer Formate formate Sample 50 µl at times 0, 5, 19, 15, 30, and 60 min Neutralize 50 µl samples with 450 µl, 0.02 M Ammonium ammonium Hydroxide hydroxide Neutralize 50 µl samples with samples 450 µl water with Neutralize µl samples with with 450 µl, 0.02 M HCl HCl Analyze and Q uantita te Analyze and quantitate against standards 66

65 LogP/LogD 50 µl sample µl ph 7.4 buffer* 475 µl ph 7.4 octanol** Shake overnight at 37 C Inject from octanol phase Set Alliance HT needle depth to 18 mm to sample top phase*** 20 µl samples in DMSO 50 µl sample µl water* 475 µl octanol** Shake overnight at 37 C *Octanol-saturated buffer (or water) **Water-saturated octanol Manually separate organic and octanol phases into separate vials and analyze or... Octanol phase Aqueous phase Inject from aqueous phase Set Alliance HT needle depth to 0 mm to sample bottom phase*** ***Using 2 ml 96-well plate Microsomal stability Solution A (4 C) Phosphate buffer + NADAPH A + NADAPH B Solution B (37 C) Phosphate buffer + rat liver microsomes Add 50 µl of 5 µm sample solution µl of solution A µl of acetonitrile µl of solution B 5 µm samples in phosphate buffer 50 µl of 5 µm samples in 1 ml 96-well plate Add 100 ml solution A Heat 37 C for 20 min Shake 37 C for 20 min Add 100 ml solution B Then add 500 µl acetonitrile T 0 Plate T 20 Plate 67

66 Data processing and report generation n The ProfileLynx results browser contains up to three sections: a results table, the chromatogram, and the calibration curve. n A pass/fail indicator column and user-selected highlight flags allow fast review of the data. n The chromatogram is interactive for manual integration if needed. Solubility browser LogP/LogD browser Metabolic stability browser ph stability browser 68

67 DISCUSSION n The 30 compounds were analyzed with the LC/MS/MS protocol including MS MRM parameter optimization, MS acquisition method creation, data acquisition, data processing, and report generation. n The data generated from the variety of assays were all processed with the same software automatically. n A single report was created for the 30 compounds that contained results from all property profiling assays, increasing throughput. n Results are displayed in an interactive, graphical summary format based on sample or experiment. n Additional improvements to throughput were achieved for the LogP/LogD assay by utilizing the needle height adjustment of the Alliance HT system to inject directly from the two phases of the octanol/water mixture without the need to manually separate the two phases. CONCLUSION Using the ProfileLynx and QuanOptimize Application Managers allows for: n Automated MS method development and data acquisition. n A single approach for data processing and report generation from multiple assays. n Complete and automated analysis, processing, and reporting. n Increased laboratory throughput. References 1. Kerns E. Journal of Pharmaceutical Sciences. 2001; 90 (11): US Pharmacopia. 2000; 24: Di L, Kerns E, Hong Y, Kleintop T, McConnell O. Journal of Biomolecular Screening. 2003; 8(X). Other assays supported: n Protein binding (plate or column) n Membrane permeability (PAMPA, Caco-2, etc.) n Chromatographic hydrophobicity index (CHI) n Immobilized artificial membrane Waters, Alliance, ACQUITY, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. MassLynx, ProfileLynx, QuanOptimize, Quattro micro, Sirocco, SunFire, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

68 OPTIMIZATION

69 Synthetic Reaction Monitoring Using UPLC/MS Marian Twohig, Darcy Shave, Paul Lefebvre, and Rob Plumb Waters Corporation, Milford, MA, U.S. INTRODUCTION Once a chemical hit is found through a library screening process and is verified, optimization of the compounds desired properties takes place. This step involves an iterative process of synthesis and reactivity measurement of the new compounds to further develop drug candidates into the lead phase. Because these reactions may take a long time, chemists need to know as soon as possible if their syntheses are proceeding as desired. This means utilizing measurement capabilities that require minimal sample preparation and provide a fast response giving low detection limits. Another advantageous property might be the ability to measure multiple parameters simultaneously. 1 High throughput approaches can provide important time savings in the optimization of process parameters. Open access LC/MS is replacing TLC as a reaction monitoring tool. 2 Sample preparation of reaction mixtures can be as minimal as filtering and dilution before injecting into the LC/MS system. This allows fast turnaround of results to allow the chemist to advance to the next step. The purpose of this application note is to demonstrate the advantages of speed and ease of use that self-service UPLC with photodiode array (PDA)/evaporative light scattering (ELS)/MS detection brings to reaction monitoring studies. Figure 1. The ACQUITY SQD for synthetic reaction monitoring. EXPERIMENTAL Chromatographic separations were carried out using an ACQUITY UPLC System coupled to an ACQUITY SQ Mass Detector. PDA and ELS signals were collected simultaneously. Samples were analyzed using gradients less than 1 minute. For chromatographic flexibility, a column selection module was added. LC conditions LC system: Waters ACQUITY UPLC System Column: acquity UPLC BEH C 8 Column 2.1 x 30 mm, 1.7 µm Column temp.: 45 C Flow rate: 800 µl/min Mobile phase A: 0.1 Formic acid in water Mobile phase B: 0.1 Formic acid in acetonitrile Gradient: 5 to 95 B/0.7 min

70 MS conditions MS system: Waters SQ Detector Ionization mode: esi positive/esi negative Capillary voltage: 3.0 KV Cone voltage: 20 V Source temp.: 150 C Desolvation temp.: 450 C Desolvation gas: L/Hr Cone gas: 50 L/Hr Acquisition range: 100 to 1300 m/z Scan speed: 10,000 amu/sec Note: A low volume micro-tee was used to split the flow to the ELS and SQ. ELS conditions Gain: 500 N2 gas pressure: 50 psi Drift tube temp.: 50 psi Sampling rate: 20 points/sec PDA conditions Range: 210 to 400 nm Sampling rate: 20 points/sec To illustrate the functionality of such a system, the synthesis of atenolol (Figure 2) was used as a reaction model. The increase in the formation of atenolol was monitored, as was the decrease in the intermediate 4-hydroxyacetamide 3 (Figure 3). A reaction by-product 4-hydroxyphenylacetic acid was also observed. O NH 2 OH OH 4-Hydroxyphenlyacetamide 4-Hydroxyphenlyacetic Acid C 8 H 9 NO 2 C 8 H 9 O 3 Atenolol, C 14 H 22 N 2 O 3 Figure 2. Structures of atenolol and 4-hydroxyacetamide. O OH Increase Decrease RESULTS AND DISCUSSION During the compound optimization stage of a discovery cycle, medicinal chemists are not only interested in determining the key structural features responsible for activity and selectivity, but also what structural changes need to be made to improve these characteristics. Because the reactions necessary to bring about these changes may take many steps, chemists need to be sure they are progressing as expected during the course of the reaction synthesis. t=5 min t=45 min t=50 min t=60 min Atenolol 4-Hydroxyacetamide Figure 3. UPLC/MS chromatograms. The reaction mixture was sampled at various time points. 72

71 The ACQUITY SQD is capable of scan speeds of up to 10,000 amu/sec. Consequently, it is possible to employ a large number of scan functions in a single run while still maintaining adequate peak characterization. The fast scan speed is essential for this functionality, as peak widths of 1 second or less are common with the use of UPLC. Scanning multiple functions allows confirmation of compound synthesis to be obtained on reaction components whether they ionize in positive ion mode or negative ion mode, ESI or APCI. The total cycle time of the method was 1 minute 20 seconds, facilitating increased sample throughput. A single run can also provide UV spectral information and an estimation of compound purity at low wavelengths. ELS detection is based upon the degree to which solute particles scatter light. It has been known to give rise to similar responses for related compounds. 4 The signal can give a tentative estimation on the relative quantities of the components present (Figure 4). It is also an alternative detector to UV, which depends on the presence of a chromaphore. As can be seen from Figure 4, atenolol ionizes in ESI positive ion mode (retention time 0.28 min). The reaction AU e PDA 2.5e intermediate 4-hydroxyphenylacetamide ionizes in both positive and negative ion mode (Rt 0.34 min) and 4-hydroxyphenylacetic acid (Rt 0.39 min) only ionizes in negative ion mode. The OpenLynx Open Access Application Manager, part of MassLynx Software, allows chemists to walk up to a terminal and log in samples while entering the minimum information required to run the samples. The OpenLynx OALogin screen shown in Figure 5 allows the administrator to set up the system such that the user only needs to input the information requested, and then upon completion, select the Login Samples button. This will either tell the user the designated autosampler position, or confirm the position that the user has chosen, and ask for confirmation of position before it will run the sample. In addition to a simplified sample submission process, the OpenLynx Application Manager can then process data automatically and produce a summary report that can be ed or printed as desired. The information contained in the summary report is viewed via the OpenLynx browser shown in Figure 6. It clearly defines what components are found or not found. Chromatograms and spectra are generated based on the processing parameters set up by the administrator in the OpenLynx method. LSU ELS ESI Positive ESI Negative Figure 4. UPLC/PDA/ELS/MS chromatograms. Figure 5. The OpenLynx single page login. 73

72 The described system and software combination can autonomously evaluate large numbers of samples, with a cycle time of 1 minute 20 seconds. Data can then be automatically processed and a summary report can be generated. The scan speed capabilities of the ACQUITY SQD make it possible to better characterize narrow chromatographic peaks. This has become a necessity since the advent of sub-2 µm particle technology, where chromatographic peaks can be 1 second wide or less. The fast scan speed allows the chemist to extract as much data as possible per injection by switching between APCI and ESI as well as positive and negative ion modes. Figure 6. The OpenLynx Application Manager browser. CONCLUSION During the compound optimization stage of a discovery cycle, medicinal chemists are not only interested in determining the key structural features responsible for activity and selectivity, but also what structural changes need to be made to improve these characteristics. Because the reactions necessary to bring about these changes may take a long time, chemists need to be sure they are progressing as expected. By using a walk-up UPLC/MS system, chemists were able to quickly and easily monitor their reactions, noting the relative amounts of starting materials and products. They were also able to note the formation of any side products and make necessary alterations to their reaction protocol to minimize these. Open Access gives the chemist a walk-up system that is flexible for analytical data acquisition. It runs as a complete system, from sample introduction to end results. The use of the fast-scanning MS along with the throughput of UPLC technology allows the chemist to obtain high quality and comprehensive data about their compounds in the shortest possible time. This combined with intelligent open access software allows informed decisions to be made faster, thus supporting the needs of the modern drug discovery process. References 1. Analysis and Purification Methods in Combinatorial Chemistry, Wiley-Interscience. (5): LC/MS Applications in Drug Development, Wiley-Interscience A Synthesis of Atenolol using a Nitrile Hydration Catalyst. Organic Process Research and Development. 1998; 2: Kibbey, C.E. Mol. Diversity. 1995; I: Waters, ACQUITY, ACQUITY UPLC, and UPLC are registered trademarks of Waters Corporation. MassLynx, OpenLynx, 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

73 THE WATERS ACQUITY UPLC SYSTEM and cost savings in an Open Access environment The client: A multi-national research-based pharmaceutical corporation BACKGROUND A laboratory supporting the medicinal chemistry department of a large global pharmaceutical firm relied on HPLC/MS systems in an open access environment to provide 150 synthetic chemists with critical information about the success of their reactions. The synthetic chemists wanted to ascertain quickly what compounds their reactions have made and whether any of the molecules are known to be toxic. To get the information they need, the medicinal chemists literally walk up to one of 21 open access systems configured for the purpose, add their sample to the cue, select one of three pre-set HPLC/MS scouting methods and walk away. Minutes later the results are ed back to them. At this facility, each open access system handles 600 to 700 samples per month; in 2004 the lab ran a total of 204,000 samples, with much higher numbers expected for subsequent years. The average run time for an HPLC/MS scouting method is 6.6 minutes. Turnaround time in this high-throughput environment is critical. As the lab manager has said, Anything I can do to save any amount of time, I do it. CHALLENGE Overall, the wait for results has been cut in half, while solvent consumption has been cut by 85 percent. The demands placed on the medicinal chemistry department for high-quality new drug candidates dictate that speed is of utmost importance. Despite this lab s best efforts to reduce turnaround times by pushing their HPLC methods to the limits, the wait for results was sometimes as long as an hour. Things needed to change in order to reduce drug development timelines and cost. Despite the larger workload, the lab manager had set as his goal a five-minute or less turnaround time for results. This ambitious goal clearly required a new approach. Another key concern for this laboratory manager: injection reproducibility. When chemists are tracking a reaction, any shift in retention times from one analysis to the other is a red flag and suggests that something unintentional might have been created in the reactor. THE SOLUTION In 2004, the laboratory acquired a Waters Open Access ACQUITY UltraPerformance LC (UPLC ) System, which they put on the front-end of a single quadrupole Waters ZQ Mass Spectrometer. The goal was to see by how much they could shorten the run time of their scouting methods without losing sensitivity or resolution.

74 By eliminating as little as one minute per analysis, the lab could save 3400 hours of total analysis time and increase the number of tests they perform by 34, percent savings by comparison. With no increase in lab space, and further savings captured in consumables and solvents, the lab now has a strengthened investment strategy for increasing capacity and productivity going forward. BUSINESS BENEFIT The support laboratory began to see their work pay off with UPLC in ways they hadn t imagined. In short order, they have reduced what was a 6.6-minute run to just 2.3 minutes, a three-fold improvement in overall run time. Now, sub-two-second peak widths are standard and the lab manager has reported, I can offer my clients the same peak capacity in one-half the time. Overall, the wait for results has been cut in half, while solvent consumption has been cut by 85 percent. Moreover, the lab manager has reported getting more than 2500 injections on a single column without any degradation in results. I am extremely impressed with the robustness of the column very happy, he has said. Perhaps the most important benefit of the Open Access ACQUITY UPLC System relates to the increase in the number of samples expected in the near future. The lab manager anticipated an increase of 15 to 20 percent in the next year, which would normally require the addition of up to four complete LC/MS systems, at a cost of over $600,000 in capital investment. Add to that increases in much-needed laboratory space, service, and maintenance and consumables. The lab manager has been able to develop an alternative plan to achieve the same increase in sample capacity by replacing the inlets on two of their existing systems with ACQUITY UPLC Systems. This could be achieved for $120,000 in capital investment, an WATERS AND UPLC The Waters ACQUITY UPLC System synergistically combines instrumentation, column chemistries, software for data acquisition and processing, and support services, creating a singular solution with superior sensitivity, resolution, efficiency, and sample throughput. When coupled with Waters MS Technologies, UPLC provides a level of separation, quantification, and characterization previously unattainable with traditional HPLC. UPLC today is employed by companies to bring their laboratories measurable improvements in analytical sensitivity, resolution, and speed. Ultimately, these firms are looking for meaningful ways to increase laboratory productivity, decrease operational costs, facilitate product development, and increase revenue generation. WATERS OPEN ACCESS SOLUTIONS Waters Open Access systems give chemists the ability to analyze their own samples close to the point of production by simply walking up to the LC/MS system, logging their samples, placing their samples in the system as instructed, and walking away. As soon as the analysis is completed, sample results are ed or printed as desired. System configuration and setup is enabled through a system administrator who determines login access, method selection, and report generation. Waters, ACQUITY UPLC, ACQUITY UltraPerformance LC, and UPLC are registered trademarks of Waters Corporation. ZQ 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. June EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

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76 Waters, ACQUITY, ACQUITY UPLC, ACQUITY UltraPerformance LC, Alliance, ESCi, UltraPerformance LC, and UPLC are registered trademarks of Waters Corporation. ApexTrack, AutoPurification, AutoPurify, FractionLynx, i-fit, LCT Premier, LockSpray, MassLynx, ODB, OpenLynx, ProfileLynx, Quattro micro, Quattro Premier, QuanLynx, QuanOptimize, Sirocco, SunFire, XBridge, ZQ 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. August EN LB-KP Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: