SCIEX PA 800 Plus Development Manual
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SCIEX PA 800 Plus Development Manual

Pharmaceutical analysis system methods development guide
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PA 800 Plus Pharmaceutical Analysis
System
Methods Development Guide
RUO-IDV-05-5330-A
January 2018

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Summary of Contents for SCIEX PA 800 Plus

  • Page 1 PA 800 Plus Pharmaceutical Analysis System Methods Development Guide RUO-IDV-05-5330-A January 2018...

  • Page 2: Example

    This document is provided to customers who have purchased SCIEX equipment to use in the operation of such SCIEX equipment. This document is copyright protected and any reproduction of this document or any part of this document is strictly prohibited, except as SCIEX may authorize in writing.

  • Page 3: Table Of Contents

    Interlocks, 24 UV Detector Optics, 25 The Photodiode Array (PDA) Detector, 26 Laser-Induced Fluorescence (LIF) Detector, 27 Laser Module, 29 488 nm Laser Module, 29 Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 3 / 158...
  • Page 4 Data, 54 Filter, 54 Relay 1 and Relay 2, 54 Reference Channel, 55 Absorbance Signal, 55 Electropherogram Channel Data, 55 LIF Detector Initial Conditions, 56 PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 4 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 5 Components, 93 Qualitative Analysis, 93 Quantitative Analysis, 93 Creating a Calibration, 94 Developing a Calibration Method, 94 Generating the Calibration Curve, 97 Analyzing Unknowns, 102 Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 5 / 158...
  • Page 6 Law, 125 Corrected Migration Time, 125 Corrected Area, 126 Mobility, 126 APPENDIX C: Understanding PDA Data, 129 Overview, 129 Mixed View, 131 Contour Plot, 132 PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 6 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 7 Example 2, 142 Summary, 145 APPENDIX F: Sequence Vial Report, 147 Overview, 147 Sequence Vial Increment Preview, 147 Sequence Vial Report-Confirmed, 149 Revision History, 157 Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 7 / 158...
  • Page 8 Contents PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 8 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 9: About This Guide

    Guide for detailed information on the safe use and operation of the system. Overview The PA 800 Plus Methods Development Guide is a self-paced introduction to the operation of the SCIEX PA 800 Plus Pharmaceutical Analysis System, which includes the 32 Karat software. It contains a brief introduction to the technique of capillary electrophoresis (CE), an overview of the PA 800 Plus System hardware, tutorials for the most frequently used software features, and appendices of useful information.

  • Page 10: Instructions For Using The Sample

    • multi calibration level 5.dat • multi calibration level 6.dat The methods folder contains the following files: • CE calibrate.met • multilevelcalibration.met • pda estd.met PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 10 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 11: Purpose Of These Files

    Save all methods and sequences you create as part of the tutorial in this new folder. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 11 / 158...
  • Page 12 About This Guide PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 12 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 13: Introduction To Capillary

    All of these modes are generally referred to as capillary electrophoresis (CE). In the PA 800 Plus system, the sample is injected into the capillary using either pressure, vacuum, or voltage. Under the influence of an electric field, sample components migrate differentially through the capillary.

  • Page 14: Common Modes Of Electrophoresis

    At the end of the process, the various analytes are located in discrete zones in the capillary. In a separate mobilization step, the bands are moved past the detector for analysis. This technique is most often used for protein and peptide analysis. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 14 / 158...

  • Page 15: Capillary Electrochromatography

    Under some conditions, electrophoresis can also participate in the separation. CEC is primarily employed to separate small molecules such as drugs. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 15 / 158...
  • Page 16 Introduction to Capillary Electrophoresis PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 16 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 17: System Overview

    System Overview Instrument The main components of the PA 800 Plus Pharmaceutical Analysis System include trays that hold vials of sample, buffer, and other solutions, an interface block, a high-voltage power supply and electrodes, a source optics module and detector, temperature control hardware, and a sample injection mechanism (Figure 3.1,...
  • Page 18 System Overview Figure 3.1 PA 800 Plus System 1. Outer Door (Open Position) 4. Clamp Bar and Cable Connection 2. UV Detector 5. Capillary Cartridge 3. Two-ended Fiber Optic Cable 6. Interface Block PA 800 Plus Pharmaceutical Analysis System Methods Development Guide...

  • Page 19: The Sample Handling System

    System Overview Figure 3.2 PA 800 Plus System Side View 1. Outer Door or Sample Cover (open) 5. Sample Trays 2. Inner Door or Cartridge Cover (open) 6. Buffer Trays 3. Indicators 7. Fluid Bubble Indicator 4. Fluid Fill Port 8.
  • Page 20 System Overview Figure 3.3 PA 800 Plus System Trays 1. Inlet Sample Tray (48) or 96-Well Plate 3. Outlet Sample Tray (48) or 96-Well Plate 2. Inlet Buffer Tray (36 Vials) 4. Outlet Buffer Tray (36 Vials) Each buffer tray has slots for 36 universal vials. Sample trays hold either 48 vials or a 96-well plate. Each tray is...
  • Page 21 Figure 3.4 Sample and Buffer Trays 1. Buffer Tray 2. 48-Vial Sample Tray for Universal Vials or Universal Vials Holding Microvials 3. 96-Position Sample Tray Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 21 / 158...

  • Page 22: Capillary Cartridge

    The universal vials are pressurized during rinse and separation-with-pressure events. To reduce the risk of breakage and expelled particles, use only SCIEX vials (PN A62251), and inspect every vial for damage before use. Do not use any vial that appears cracked or damaged in any way.
  • Page 23 One type of plug is used for UV and PDA detectors, and a second type is used for LIF detectors. Refer to the PA 800 Plus Pharmaceutical Analysis System Maintenance Guide or procedures to install a capillary in a cartridge.

  • Page 24: Fluid Delivery, Power Supply And Syringe Pump

    Cartridge and Sample Cover Interlocks The hinged doors of the PA 800 Plus instrument have interlock sensors that prevent unsafe access to the inside of the instrument. The outer door is called the sample cover; the second inner door is called the cartridge cover (Figure 3.2).

  • Page 25: Uv Detector Optics

    214 nm) 3. Deuterium Lamp 7. Motor 10. Fiber Optic Cable 4. Lamp Power Supply 8. Position Filter Wheel 11. Fiber Optic Connector 12. Capillary Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 25 / 158...

  • Page 26: The Photodiode Array (Pda) Detector

    The PDA detector is calibrated using discrete emission wavelength bands generated by a mercury lamp. The mercury lamp is an important part of the detector system. When requested by the user, the calibration is performed automatically. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 26 / 158...

  • Page 27: Laser-Induced Fluorescence (Lif)

    7. Motor Laser-Induced Fluorescence (LIF) Detector The LIF detector consists of the LIF Detector Module and a Laser Module. A PA 800 Plus capillary cartridge with an LIF detector plug installed is required for use with this system. Methods Development Guide...
  • Page 28 For more information on the LIF system, refer to the System Maintenance Guide. The initial installation of the LIF detector is performed by a SCIEX Field Service Employee. The PA 800 Plus system can easily be changed between LIF and UV/PDA modes because the detector components are modular. Refer to Figure 3.8...

  • Page 29: Laser Module

    System Overview Laser Module The following section describes the 488 nm laser module and how it interfaces with the PA 800 Plus instrument and the LIF detector. WARNING During normal operation of the LIF detector, laser light is not accessible to the user. To prevent...
  • Page 30 System Overview PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 30 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 31: 32 Karat Software

    The 32 Karat software opens to the current location / group window that lists the configured instruments. Figure 4.1 32 Karat Software Enterprise Window (Main Menu) Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 31 / 158...

  • Page 32: Tool Bar

    IMPORTANT This section assumes that all hardware and interface boards have been installed. If they have not been installed, refer to the System Maintenance Guide for instructions. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 32 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 33: Creating A New Instrument

    Configuring the instrument is a process in which the software is prepared to control the hardware and process the data. It consists of first identifying the PA 800 Plus instrument as a unique instrument and then identifying the hardware components that are present. Each physical instrument should be represented by at least two icons.
  • Page 34 32 Karat Software Figure 4.3 Instrument Configuration Dialog Click . The PA 800 Plus Configuration dialog is launched with the modules available for Configure configuration listed in the left pane. Figure 4.4 PA 800 Plus Configuration Dialog PA 800 Plus Pharmaceutical Analysis System...

  • Page 35: Auto Configuration

    32 Karat Software Auto Configuration The 32 Karat software can automatically configure the PA 800 Plus module in the Instrument. The instrument must be connected to the PC and turned Click at the bottom of the dialog and listen for tray movement in the PA 800 Plus Auto Configuration instrument.
  • Page 36 . Verify that the instrument was properly detected and configured. Open Figure 4.7 PA 800 Plus Instrument Configuration for UV Detector Each PA 800 Plus instrument connected to the computer must have a unique device ID. The default setting is After the device address is selected, click to send this information to the instrument.

  • Page 37: Configuration Options

    Configuration Option Analysis Options Analysis options define what software functions will be available when data is reprocessed. Figure 4.8 PA 800 Plus Instrument Configuration Options Dialog (Analysis Options) Allows the analysis of multichannel data from the photodiode array detector. System Suitability Enables automatic review of results.

  • Page 38: Qualitative Analysis

    Use the Instrument Options function to select (default) or Standard CE CEC/LC Figure 4.9 PA 800 Plus Instrument Configuration Options Dialog (Instrument Options) Standard CE The time for a peak to reach the window is called Migration Time. CEC/LC The time for a peak to reach the window is called Retention Time.

  • Page 39: Manual Configuration

    The detector icon is added and must now be configured. Figure 4.10 UV Detector as Configured Module Click the new icon and right-click . The PA 800 Plus System Instrument Configuration dialog opens. Open Methods Development Guide PA 800 Plus Pharmaceutical Analysis System...

  • Page 40: Manual Configuration Of The Pda And Lif Detectors

    32 Karat Software Figure 4.11 Instrument Configuration Dialog For information on setting the PA 800 Plus system instrument configuration parameters, refer to the 32 Karat Software Online Help. Click to exit and save the module configuration. Manual Configuration of the PDA and LIF Detectors Manual configuration of the PDA and LIF detectors is performed by selecting the appropriate icons in place of the UV detector.

  • Page 41: Chapter 5: Direct Control

    Additional information regarding Direct Control features can be found in the 32 Karat Software Online Help. Direct Control Window To open the Direct Control window, go to the and click Control Menu Direct Control > View Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 41 / 158...
  • Page 42 9. Trays to Load Position The UV detector is shown above. The PDA and LIF detectors are similar. The functions are defined in Table 5.1. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 42 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 43 Indicates present, target, and maximum voltage Current Text/Graphic/Button Indicates present, target, and maximum current Power Text/Graphic/Button Indicates present, target, and maximum power Pressure Text/Graphic/Button Indicates present, target, and maximum pressure Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 43 / 158...

  • Page 44: Exercise: Conditioning The Capillary

    • 75 µm i.d. capillary, 60 cm total length (50 cm to the detector) installed in cartridge appropriate for detector type configured. Refer to the PA 800 Plus Pharmaceutical Analysis System Maintenance Guide for instructions on building a new cartridge or installing a capillary.
  • Page 45 Designates whether the flow of fluid is Forward (from inlet to outlet or left to right) or Reverse • Direction: (from outlet to inlet or right to left). Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 45 / 158...
  • Page 46 Duration Pressure direction should be Forward and Pressure Type should be Pressure. The dialog should now look like the one shown above in Figure 5.2. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 46 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 47 The amount of time the power supply takes to reach the voltage (0.10 minutes to 999.9 • Ramp Time: minutes). Ramp time cannot exceed the Duration time. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 47 / 158...
  • Page 48 Status returns to idle. This completes the Direct Control exercise. It may be helpful to experiment with the other Direct Control functions before proceeding to the next section. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 48 / 158...

  • Page 49: Chapter 6: Creating And Editing A Method

    It is possible to open the method editing dialog by clicking from this dialog. Create or Modify a Method Click to close the instrument wizard. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 49 / 158...

  • Page 50: Initial Conditions Tab

    A window opens that contains three or four tabs, depending on the detector configuration. Click Initial Conditions The dialog in Figure 6.1 opens. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 50 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 51 Peak integration is discussed later in this guide. Trigger Settings The PA 800 Plus system can be forced to wait until certain conditions are met before beginning a run. These are selected by clicking the appropriate check box. If...

  • Page 52: Detector Initial Conditions

    UV Detector Initial Conditions Click the tab. UV Detector Initial Conditions Figure 6.2 Instrument Setup Dialog - UV Detector Initial Conditions Tab PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 52 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 53: Electropherogram Channel

    Relay 1 and Relay 2 The two relays incorporated into the PA 800 Plus system can be used to control or signal other devices. This dialog allows you to set the state of the relays at the start of the method to (open) or (closed).

  • Page 54: Pda Detector Initial Conditions

    UV Detector Initial Conditions for information on this topic. Relay 1 and Relay 2 Refer to UV Detector Initial Conditions for information on this topic. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 54 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 55: Reference Channel

    APPENDIX A, Additional Resources for information on this feature. Peak detect: For this exercise, set up the PDA Detector Initial Conditions to match Figure 6.3. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 55 / 158...

  • Page 56: Lif Detector Initial Conditions

    The LIF detector is available in one and two channel versions. The programming of these channels is identical. In this exercise use only one channel. If your system is configured with non-SCIEX lasers, the test mixture used in this exercise might not perform as described.

  • Page 57: Signal

    Inlet and Outlet Vial: a system-generated description of the event. Summary: a user-generated annotation of the event. Comments: Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 57 / 158...

  • Page 58: Separate Dialog

    The Separate dialog is used to control the conditions under which the separation process takes place. Every method must have at least one separation step. The step at time = 0.00 is usually a separation. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide...
  • Page 59 The inlet position, outlet position, or both can be incremented automatically by selecting the appropriate check boxes and typing the desired number of cycles between changes. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 59 / 158...
  • Page 60 Changes the way the instrument manages the power supply. Select this check box only if the External Adapter accessory is in use. Refer to the System Maintenance Guide for more information. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide...

  • Page 61: Rinse Dialog

    The inject event is used to deliver a precisely measured amount of sample into the capillary. This step is always untimed, and usually precedes the first separation step. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 61 / 158...
  • Page 62 The capillary fill option is used for cIEF separation, which requires the capillary to be completely filled with the sample mixture. For more information on cIEF separations, visit www.CELeader.com or refer to the PA 800 Plus Pharmaceutical Analysis System cIEF Application Guide. Tray Positions Functions exactly as described in the Separation event. Sequence Table When a method is used in a sequence table, certain parameters in the inject event can be overridden by values input for the Sequence.

  • Page 63: Autozero

    The next part of this exercise creates a time program to go with the initial conditions previously entered. The example method does the following: • Performs a mini-regeneration on the capillary. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 63 / 158...
  • Page 64 Time Program spreadsheet. The key parameters have been entered into the table automatically. This rinse is an untimed step, so there is no value in the Time window. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 64 / 158...
  • Page 65 Select a new field, and choose as the event type. Event Separate Use the button to select positions Trays BI:A2 BO:A1 Click Select a separation. Voltage Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 65 / 158...
  • Page 66 . When this item is entered into the Time Program spreadsheet, it At Time 1.00 automatically moves to the correct time sequence. At this point the Time Program tab should look like that in Figure 6.14. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 66 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 67: Saving The Method

    In the next chapter, you run the method just created. The resources in APPENDIX A, Additional Resources and the 32 Karat Software Online Help describe events and parameters not covered here. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 67 / 158...
  • Page 68 Creating and Editing a Method PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 68 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 69: Running The System

    • For UV or PDA Detection: Test Mix B (PN 501333) • For LIF detection using a 488 nm argon-ion laser: LIF Detector Test Mix (PN 477615) Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 69 / 158...

  • Page 70: Single Run

    You must provide at least a Sample ID, Method, and Data File. The Data File is stored in the directory identified in the Data Path. Sample ID can be any text string that identifies your sample. This ID appears on all subsequent PA 800 Plus Pharmaceutical Analysis System Methods Development Guide...
  • Page 71 . The system examines the method to verify that it is appropriate for the Start instrument configuration. The method is then downloaded to the PA 800 Plus system. The instrument first performs a brief start-up check and then executes the method.

  • Page 72: Programming A Sequence

    Open Under click . Amount Values are not used in this exercise. Data File Type For Acquisition Click Next PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 72 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 73 Open Data Path Type a text string into the Data File field. The blue arrow to the right of the text field opens this menu: Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 73 / 158...
  • Page 74 Refer to the resources in APPENDIX A, Additional for more information on calibrating through the sequence table. Resources PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 74 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 75 Figure 7.8 Sequence Wizard - Calibration Page This example is not calibration. Calibration is covered in CHAPTER 9, Calibration. For Number of calibration levels, type , and click to continue. Next Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 75 / 158...
  • Page 76 . Line 2 will now execute twice, giving a Reps total of four runs in this three-line sequence. Scroll to the right until the window resembles the one shown here. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 76 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 77: Running The Sequence

    You must be online before proceeding. Click the icon from the Instrument window toolbar. This icon is not available if you are in Offline mode. The dialog shown here opens: Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 77 / 158...

  • Page 78: Sequence Validation

    If the validation is successful, the method in the first line is downloaded to the PA 800 Plus system and the run will begin. Observe the operation of the instrument as a guide to future operations and troubleshooting. You can...
  • Page 79 If your method allows override, this can be done with a single method by editing the Sample Inject Duration column in the sequence table. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 79 / 158...
  • Page 80 Running the System PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 80 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 81: Integration

    (resolved), it is also necessary to estimate where to divide the area of the two peaks. A very simple data set is shown in the following figure. Figure 8.1 Typical Data Set Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 81 / 158...

  • Page 82: Integrating Data

    CHANNEL A WINDOW Ctrl-Z data is used. Auxiliary data (current, voltage, power, pressure) cannot be integrated. Right-click the electropherogram and select Annotations The following dialog opens: PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 82 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 83 Width = 0.2 and Threshold = 50. Every Integration Table must contain at least one Width and one Threshold value. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 83 / 158...

  • Page 84: Optimizing Integration

    Increasing the value of Threshold removes background artifacts. If taken to extremes, it can also remove real data. Set the Threshold value to and analyze the data again. 1000 PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 84 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 85: Integration Parameters

    Not all 150 components are of interest. Some parameters need to be changed to reduce the number that are identified. In this part of the exercise, graphical programming allows you to build the integration table. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 85 / 158...

  • Page 86: Width

    Add to Table Analyze Now but it also causes the integration to be redone with the new parameter in place. Click Add to Table PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 86 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 87: Threshold

    As with the Width parameter, change the start and stop times of the new Threshold event to , and deselect the old Threshold parameter. Again for consistency, adjust the new Threshold value to exactly Analyze the sample. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 87 / 158...

  • Page 88: Integrating A Peak Cluster

    In this case, we are using the graphical function to define Start and Stop times rather than to define Width. In the dialog that opens, change the Value to Click . The cluster should appear similar to the one shown here: Analyze Now PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 88 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 89: Integration Off

    To turn off integration, right-click Graphical Programming > Integration Off Click at about 35.8 minutes, at the end of the electropherogram, and then click Analyze Now Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 89 / 158...
  • Page 90 Instrument Setup dialog of this method, and this omission has been detected by the Auto-Validation function of the software. In actual practice, Integration Events would be added to the method used to acquire the data. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 90 / 158...

  • Page 91: Integration Results And Reports

    . A report opens showing the integration results in tabular form. If you want a hard copy of this report (and Area% if a printer connected to your system), right-click the report and click Print Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 91 / 158...
  • Page 92 Integration PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 92 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 93: Calibration

    It uses an external standard calibration where data from standards and unknowns are acquired in separate runs. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 93 / 158...

  • Page 94: Creating A Calibration

    Click to integrate the data set. (The integration parameters have already been optimized.) Analyze The two large peaks are well integrated with the default parameters. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 94 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 95 A table opens listing the two peaks just added. As these peaks have not yet been named, the system creates a name based on the migration time. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 95 / 158...
  • Page 96 You may get a warning message indicating the data collection time of the method is 0.0. Click Yes to proceed with the save. Right-click the electropherogram and click Annotations Select parameters so that only are in the Show box. Name ESTD concentration Click PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 96 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 97: Generating The Calibration Curve

    Scroll right to the column. The five files should be listed in order. Filename Scroll back left to the column. Change the level in line 1 from Level Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 97 / 158...
  • Page 98 Selecting this type causes any previous calibrations of this data set to be cleared before the new calibration is performed. Figure 9.3 Sample Run Types Dialog PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 98 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 99 Start The processing requires a few seconds to complete. As each data file is analyzed, the status column in the sequence table changes to Complete Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 99 / 158...
  • Page 100 Zero Do not click Zero in line 2. Save the method and look at the calibration results again. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 100 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 101 Saving the method saves the calibration. It is not necessary to reprocess the sequence. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 101 / 158...

  • Page 102: Analyzing Unknowns

    This exercise has covered only the basics of quantitative analysis. For more detail and more options, refer to the resources in APPENDIX A, Additional Resources. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 102 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 103: Chapter 10: Qualitative Analysis

    The mobility of peaks of known standard sizes is used to create a standard curve that can be used to estimate the size of an unknown fragment. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 103 / 158...
  • Page 104 From the menu, click to open the window shown below. Type the molecular Method Qualitative analysis weight (Y) and the mobility (X) into the method. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 104 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 105 Make sure the scale for the X-axis and the Y-axis are 1/X and Logarithmic respectively. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 105 / 158...
  • Page 106 This method can now be used to estimate the molecular weight of an unknown protein. To do this, analyze the unknown using the same method used to analyze the standards. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 106 / 158...

  • Page 107: Chapter 11: Mobility

    The software is also capable of displaying electropherograms with the X-axis scaled in terms of mobility rather than time. In this exercise, you review these features of the software. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 107 / 158...

  • Page 108: Mobility Markers

    Standards for other peaks expected in the unknowns should be included in this run. Figure 11.1 Advanced Method Options Window PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 108 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 109 If the results are acceptable, the method can now be edited to allow the generation of an electropherogram with mobility as the X-axis in place of time. This option is selected in the tab of the Initial Conditions dialog: Instrument Setup Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 109 / 158...

  • Page 110: Mobility Plot

    After selecting the appropriate mobility channel options, save the method. Run the sample again. At the end of the run, the mobility trace is generated. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 110 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 111: Demonstration: Using The Mobility Features

    The following is not intended to be a hands-on exercise, but rather a step-by-step example of the process described above. This example uses a UV detector and SCIEX Test Mix B. You previously determined the optimum conditions for the separation of a mixture of two components, ALPHA and BETA.
  • Page 112 The mobility of BETA has now been calculated relative to the mobility of ALPHA. You can now set up to collect data for a mobility plot. Open the dialog and click the tab. Instrument Setup Initial Conditions PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 112 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 113: Peak Identification By Mobility

    The option to use mobility for peak identification must be selected from the tab of the Method Options Properties dialog (under the Method menu). Click and click Mobility based Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 113 / 158...
  • Page 114 BETA to some other value like –0.00025227 and again. Analyze Because the mobility of the peak is now outside the window, it is integrated, but not associated with the ID BETA. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 114 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 115: Chapter 12: Creating Reports

    You must create a Method Custom Report, which becomes part of the method. Save the report as a standard template that is added to the reports list. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 115 / 158...

  • Page 116: Creating A Method Custom Report

    This moves the Print Time field to the same line as the Delete Acquired field. Press the key twice to place space between the two fields. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 116 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 117 Select the Current Data (the file currently Trace Setup open in the Instrument window) or a data file (or files) stored on the computer. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 117 / 158...
  • Page 118 The new graph can be formatted as described above. Below the graph is a table with a variety of peak parameters. Some of these must be changed for the new report. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 118 / 158...

  • Page 119: Saving A Report As A Template

    Method menu (if not already open). Method Custom Report The report is blank, because a new method has no defined Custom Report. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 119 / 158...

  • Page 120: Saving A Report As A Default Report

    Other run types requiring templates include Suitability, QC Check Standards, Duplicates, and Sequence Vial Report. Refer to APPENDIX F, Sequence Vial Report. Sequence Custom Report templates are created and edited using the Sequence Custom Report editor. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 120 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 121: Standard Report Templates

    • Calibration • Summary • Duplicate • QCCheckStd • Spike • SysSuit They are located in the 32 Karat Software/Sequence directory and have the .brp extension. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 121 / 158...
  • Page 122 Creating Reports PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 122 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 123: Appendix A: Additional Resources

    SCIEX Service If you have determined that a problem exists with the instrument that cannot be fixed by following the procedures in the System Maintenance Guide, contact your local SCIEX Field Service Employee. There may be a charge for this service.
  • Page 124 Additional Resources PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 124 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 125: Appendix B: Pa 800 Plus System Calculations

    = migration time of peak of interest tref = actual migration time of reference peak t'ref = expected migration time of reference peak (from peak id table) Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 125 / 158...

  • Page 126: Corrected Area

    Lt = capillary total length V = average voltage up to migration time of peak t = migration time of the peak v = velocity PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 126 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 127 Mobility calculations depend on an accurate measurement of the average applied voltage up to the point that the component passes the detector. The output from the PA 800 Plus voltage monitor is used to calculate average applied voltage as follows:...
  • Page 128 PA 800 Plus System Calculations PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 128 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 129: Appendix C: Understanding Pda Data

    CE PDA Data Sample.dat. The multiple components of the PDA data set are available by choosing from the menu bar. View > PDA View Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 129 / 158...
  • Page 130 Figure C.1 View > PDA View Each item in the menu represents a different way of presenting the data in the window. Only one of these selections can be made at a time. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 130 / 158...

  • Page 131: Mixed View

    The views available here are: Table C.1 PDA Data Set Views Peak Name Left Right Upper Contour Plot Spectrum Lower Electropherogram 3D Plot Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 131 / 158...

  • Page 132: Contour Plot

    To adjust the display, right-click the 3D plot and click to open the 3D Data Graph Properties window. properties Figure C.3 3D Data Graph Properties Window PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 132 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 133 Peaks \ Groups table and Integration Events table, that are available from the Method menu only when the data channel has been selected. More information about using PDA data is available in the resources listed in APPENDIX A, Additional Resources. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 133 / 158...
  • Page 134 Understanding PDA Data PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 134 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 135: Appendix D: System Administration

    When all the names are entered (or at least the name of the administrator), user privileges must be assigned. New users do not have any privileges until privileges are assigned. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 135 / 158...
  • Page 136 It should not be used for methods and data that are not intended to be shared. For more information on System Administration, refer to the System Administration Guide. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 136 / 158...

  • Page 137: Appendix E: Vial Incrementing

    The Inject event, unlike Rinse, Separate, and Wait, can be programmed through both the method or sequence table. Inject event has the option of allowing you to introduce samples from the vial positions stated in the Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 137 / 158...
  • Page 138 5 for a single method means the instrument executes 5 cycles of the same method. A set of repetitions makes up a run. A set of runs makes up a sequence. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide...

  • Page 139: Examples Of Use

    4. Wait: Increment inlet and outlet vials after every cycle of the method. 5. Separate: Increment inlet and outlet vials after every cycle of the method. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 139 / 158...
  • Page 140 Incrementing is also done from the vial position of the highest row of one column to the lowest row of the next column. For example, A1, A2, A3, A4, A5, A6, B1, B2, etc. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 140 / 158...
  • Page 141 You can then generate another sequence table (Sequence Table B2) with 4 separate runs, each run consisting of only one repetition of the same method B: Figure E.7 Sequence Table Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 141 / 158...

  • Page 142: Example 2

    Vial incrementing also works when there are different methods interspersed with one another in the sequence table. Consider 2 methods, methods A and B, that alternate in the sequence table. Figure E.8 Method Time Program A PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 142 / 158...
  • Page 143 The Run Range dialog of the Sequence Run command gives you the choice of running only some sequence table run lines: Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 143 / 158...
  • Page 144 (Table E.4, Range of Sequence Table AB from Example 2). PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 144 / 158 RUO-IDV-05-5330-A | A51965AF...

  • Page 145: Summary

    Vial incrementing is not carried over from the first single run to the second and third single runs. In the second example of run queue that includes sequence runs of Sequence Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 145 / 158...
  • Page 146 Table B1, the first, second, and third sequence runs are independent of each other. Vial incrementing is not carried over from the first sequence run to the second and third sequence runs. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide...

  • Page 147: Appendix F: Sequence Vial Report

    To view the preview report before starting a sequence, click Sequence > Sequence Vials Preview Figure F.1 Sequence > Sequence Vials Preview Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 147 / 158...
  • Page 148 This is the case for users who choose to inject from the sequence table. However, the sequence does not run since no values are entered in the Sample Inject Inlet/Outlet and Sample Inject Duration columns of the sequence table. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 148 / 158...

  • Page 149: Sequence Vial Report-Confirmed

    • Filenames for the sequence run are now included in the filename column. • Only the vial combinations that have been successfully used in the course of the sequence run by the system are shown. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 149 / 158...
  • Page 150 . The report template name should Sequence Vial Summary CE.brp Open be in the Report Template field after opening. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 150 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 151 Summary Run as the run type. There is no need to open a report template. Click to return to the sequence table. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 151 / 158...
  • Page 152 Sequence Vial Report Figure F.7 Return to Sequence Table Figure F.8 Sample Run Types PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 152 / 158 RUO-IDV-05-5330-A | A51965AF...
  • Page 153 For users who want to generate the Sequence Vial Report - Confirmed, for any single sequence table run line that consists of one or more repetitions, the process is similar: Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 153 / 158...
  • Page 154 Sequence Vial Report - Confirmed. A Complete message in the Status column of the sequence table confirms the end of the sequence run. PA 800 Plus Pharmaceutical Analysis System Methods Development Guide...
  • Page 155 Report - Confirmed for all three repetitions. Figure F.14 View Sequence Reports Dialog You can highlight any of the reports in the list and click View to open it. Methods Development Guide PA 800 Plus Pharmaceutical Analysis System RUO-IDV-05-5330-A | A51965AF 155 / 158...
  • Page 156 The Print Sequence Report dialog opens, and it looks exactly the same as the View Sequence Report dialog. Select the desired Sequence Vial Report - Confirmed and click Print PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 156 / 158...

  • Page 157: Revision History

    This guide applies to the latest software and firmware listed above, and any higher subsequent versions. When a subsequent software or firmware version affects the information in this guide, a www.sciex.com new issue will be released to the SCIEX website. For updates, go to and download the latest version of the guide.
  • Page 158 Revision History PA 800 Plus Pharmaceutical Analysis System Methods Development Guide 158 / 158 RUO-IDV-05-5330-A | A51965AF...

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