Uncompromised Simplicity in a Workflow-Oriented Approach for Routine Gas Chromatography-Mass Spectrometry Data Processing

Editor's Notes

• #3: What are the Challenges of a modern GC-MS workflow? The top-level challenge is that of Methodology Selection: if in business of water analysis, should the lab choose LC-MS or GC-MS or GC-MS/MS? Single quad or triple quad technology? Typically, the Methodology is specific to the Industry and is determined by industry standards and prevailing practices. In that regard, we can talk of the framework, mindset, notion space, and terminology specific to the Environmental Analysis, Contract Research or Clinical Research Organizations, or Methodology of the Forensic Toxicology field. In most cases, it is a Given.
• #4: Within a given Methodology, Analytical Method Development and Validation becomes the first challenge the Analyst faces. Developing a broad and universal or compound-class-specific method, it is always a challenge if it is your First method. Even if it is not, it still takes time and money. Thankfully, in many cases the analyst does not have to start from scratch.
• #5: As soon as we “Escape from Freedom” and step into the realm of the regulated analytical methods, we get the blessing and the curse of them.We get very well-documented guidance, sometimes stipulation in the methods the lab has to follow (to stay in business).Which is good – no need to “invent the wheel”.The other side is that there is a lot of requirements to be met, a lot to adhere to, and a lot to document in the lab. The analyst gets an “Express Delivery” of the Method – depicted here by name-sake painting of Jacek Yerka “Express Delivery”. Those methods may often be dated and odd-looking from a modern instrumentation prospective – just like the depicted delivery method would look odd to a FedEx air jet pilot. For one, how about taking an approved and validated EPA GC-MS Method and running it with Hydrogen as a carrier gas in the modern world of Helium scarsity?
• #6: Once the methods are developed, validated, and are set off to go – the analytical sequence run is usually the least problematic of them all. Provided you have a change of shoes in form of consumables and an autosampler, and your instruments are under Service Plan: so that when they do not run, you do not have to.
• #7: After the run is over, the game only begins: data review and reporting takes majority of the analyst’s time. For many analytical chemists in the routine laboratories data review may even seem like an endless Moebius-like Knot depicted here by Maurits Escher “Knot”.
• #8: And after all of those challenges, there is one of GC-MS hardware maintenance and cleaning. These are the top three challenges in the modern GC-MS workflow: Method Development Data Review and ReportingHardware Maintenance and CleaningLet’s see how the Unity of the modern GC-MS Hardware and Software can help in tackling those challenges. Let’s start with the software as the most important piece with the most “face time”.Jacek Yerka “Afternoon with Brothers Grimm”
• #9: Welcome to TraceFinder. The GC-MS software that was designed and built with all those workflow challenges (and solutions) in mind.TraceFinder is the latest software package from Thermo Scientific designed to increase your lab’s efficiency using a high throughput, workflow oriented approach.TraceFinder is available in four different methodology mindsets - General Quan, EFS for Environmental and Food Safety, Forensic Toxicology and Clinical Research.
• #10: TraceFinder software is hardware-agnostic. Develop your methods, run, review the data and make your reports with any of Thermo Scientific instruments including the – ISQ single quadrupole, ITQ ion trap, TSQ 8000 and the TSQ Quantum XLS Ultra triple quadrupole.
• #11: as well as Thermo Scientific LC-MS systems including TSQ Quantum Series, Exactive Series and Ion Traps . What about analog, FID, PDA and UV detectors for stand alone LC and GC? TraceFinder is compatible with these as well. Acquire FID and MS data and TraceFinder will deal with them all.
• #12: Welcome to TraceFinder. The GC-MS software that was designed and built with all those workflow challenges (and solutions) in mind.TraceFinder is the latest software package from Thermo Scientific designed to increase your lab’s efficiency using a high throughput, workflow oriented approach.TraceFinder is available in four different methodology mindsets - General Quan, EFS for Environmental and Food Safety, Forensic Toxicology and Clinical Research.
• #13: TraceFinder software is hardware-agnostic. Develop your methods, run, review the data and make your reports with any of Thermo Scientific instruments including the – ISQ single quadrupole, ITQ ion trap, TSQ 8000 and the TSQ Quantum XLS Ultra triple quadrupole.
• #14: as well as Thermo Scientific LC-MS systems including TSQ Quantum Series, Exactive Series and Ion Traps . What about analog, FID, PDA and UV detectors for stand alone LC and GC? TraceFinder is compatible with these as well. Acquire FID and MS data and TraceFinder will deal with them all.
• #15: This is the dashboard display that appears when TraceFinder is opened.Three modes of operation are available from the dashboard. The Configuration mode, Method Development mode and Analysis modeLet’s take a quick peak at each mode of operation
• #16: TraceFinder uses a workflow oriented approach for quick method development, batch creation and acquisition followed by user friendly data review and culminating in the creation reports even offering custom report options<click>This is a screen shot of the Configuration mode. You will notice the blue button in the bottom left hand of the screen indicates the active mode. Within this mode, we are looking at the Application Configuration View. Here you have access to the application wide settings shown in the workspace such as reports and detection options.<>The method development mode offers a streamlined approach to method development. Here we see the master method where the processing parameters are quickly and easily optimized.<>This is the Data Review area where analysts typically will spend the majority of their time. We’ll illustrate later how this view enables efficient review of data while ensuring the utmost data quality.
• #18: One of the challenges faced in all analytical laboratories is creating a new method. The TraceFinder Method Forge feature can ease this burden by using a previously acquired data file or by acquiring one during the method forge process. Method forge reviews the raw data file and identifies compounds that are present in the sample according to the processing parameters selected thus minimizing the tedious tasks often encountered during method development. This process only takes a few minutes to complete and can be a real time saver as you will see shortly.
• #19: Method Forge is super cool. After making a few selections on the screen shown here, such as which method template to use, what to name the method, and what type of file to use- a newly acquired one that can be acquired on the spot from this screen or a previously acquired data file and you will be on your way to creating a new method. Just click ok and the method forge process begins…
• #20: Here we have an EPA Method 8270 standard acquired to develop a new method using Hydrogen carrier gas. Anyone familiar with this method knows the compound list is quite long and just adding all of the compounds names can be pretty labor intensive.Method Forge can save tons of method development time. In an automated manner, Method Forgewill perform peak detection and integration across the run; perform any peak list winnowing (i.e. removing small peaks); perform a library search of each detected peak; and add each of those now-identified peaks to the master method. Along with the peak name and retention time, the base peak ion in the mass spectrum will be set as the primary quantitation ion; confirming ions, if selected, will also be added, in decreasing relative abundance order (i.e. second largest ion after the base peak will be first confirming ion). A reference mass spectrum will also be stored, and the basic calibration model will be defined and loaded into the method.No automated method development tool is perfect, but Method Forge gets you a long way down the road to a production-ready method. In the next few slides, let’s take a look at how the method is fine-tuned for optimum performance.
• #21: When the method forge process is finished, the remainder of the method development process will need to be completed and/or fine tuned. This is the resulting Detection tab of the master method defined during the method forge process.Target compound definition, covering the scan filters, mass ranges, peak detection algorithm and settings, etc. can be customized at the per-ion level – each ion, whether used for quantitation or for confirmation or qualifying purposes, can have unique settings. This is sometimes necessary due to complex matrix background being different on different ion channels, or different scan modes used for different ion measurements. Multiple quantitation ions can be used for each compound, and compound classes, where perhaps isomers or related compounds elute at different retention times.Additional compounds, from any RAW file, can also be added automatically to the master method using chromatogram and mass spectrum plots at the bottom of the pane – simply click on a peak you want to add, and a mini-method-forge operation will begin. The compound will be identified along with retention time, quantitation and confirming ions, and reference mass spectrum andwill be added to the master method. It’s that easy.
• #23: There are more and more application areas incorporating GC-MS/MS triple quadrupole technology to deal more effectively with complex matrices.We’ve seen how Method Forge is a super tool for developing full scan methods for the ISQ, but what tools are available GC-MS/MS?Method development in which transitions from a precursor to a product ion are monitored will be a bit more involved than straight forward full scan methods, but just because it appears complicated, doesn’t mean it has to be complicated.Let’s take a look at how a seemingly complex method development can be conducted very easily using the TSQ 8000 and the specialized Auto SRM software.If you’ve created a GC-MS/MS method in the past you know the process can take a week or two to complete. What if there was a way to complete this process in a day or two instead? Creating a new method on the TSQ 8000 is a three part process. The software tool AutoSRM provides the means to automatically provide the selected reaction monitoring method, or MRM, for a new method. You can start with a sample in the autosampler or you can start with a single quadrupole GC-MS method. AutoSRM will select a precursor based on the most intense peak. You can make manual adjustments if you would like.AutoSRM will then select the product ion. Again this is based on the most intense peak, you can however make adjustments.Finally, AutoSRM will check several different collision energies looking for the most intense product ion peaks.This will require a couple of different injections, however, the software will automatically handle the control of this and all the work can be done over night without attendence.
• #24: The first step is to pick the precursor ion for each compound from a full scan analaysis.When the FS sample has been analyzed, a list of ions is produced in decreasing intensity. For the selected compound,we see the list of the ions and their intensities along with a visual of the spectrum. It is generally best practice to pick the most intense peak, 112 in this case. However, in some cases there will be reasons to select other peaks such as when peaks coelute or when heavy matrix interferes.Precursor ions are selected for each compound in this manner.Once all of the precursor ions are selected, they are pushed to the next stage of the process.
• #25: The software arranges for the injection of the next sample or samples depending upon the nature of the target analytes. Auto SRM is intelligent enough to determine if one or more injections is necessary for this stage taking into account coeluting compounds.Not only does the software schedule the injections for you, but it also produces data at three separate collision energies, 10, 20 and 30 eV. As you can see in this screen, at 10 eV, the precursor ion at 112 is still the dominant ion, we would therefore expect the optimal collision energy to be somewhere in the 20 to 30 eV range.
• #26: And, if we look closely in the list of product ions, the m/z ion at 63.4 is selected as the most intense ion using a collsion energy of 20 eV. The spectrum at the bottom of the screen is for the 10 eV collision energy. The collision energies for the ions vary within this view because the software automatically alters the collision energies to determine the most intense product ions.
• #27: In step three, the software evaluates collision energy vs intensity by applying multiple energies in Q2, the collision cell. In this case we are monitoring the transition from 112 to 63 and as we can see from the energy vs intensity profile, 20 eV is in fact the optimal collision energy.This step is optional, and for this compound we found the original 20 eV determination to be the best choice, but this isn’t always the case. The highest intensity /collision energy pair is listed at the top of the list for each SRM transition monitored. All you need to do is make a selection for each compound and SRM transition to be prepared to export to the instrument method.
• #28: Here is a screen shot of the instrument method containing all of the information collected using AutoSRM, once again, with minimal user input.There is a seamless transition into TraceFinder from the TSQ 8000 instrument method by just the click of a button which ultimately creates a file for importing into TraceFinder
• #29: Now we are back in TraceFinder where a new Compound Data Store is created using the import/export functionality. AutoSRM is just one of many ways the Compound Data Store can be built, appended and modified. Naturally, any and all entries in a given Compound Data Store can be shared between the analysts and instruments, and adapted in simple but uncompromised way to the changing analytical conditions.
• #30: Now that we have Target-Analytes-Compound-Specific method entries, instead of using the method forge functionality that we looked at first, we will simply use the Compound Data Store to create the complex MRM method created using AutoSRM. It is that easy!
• #31: You click and check the compounds you want to be included– or all of them – in your method, and the resulting master method is created with a click of a mouse.In a regulated analytical environment, the corresponding version of TraceFinder (for example, EFS for Environmental and Food Safety industries) will know the industry-specific terminology and will guide you through additional set up of the criteria pertaining to the calibration and QA/QC objectives that you choose (or not) to use. Once all of the desired parameters are defined for the method, samples can be acquired.
• #32: An example case of a method created for 80 compounds with a total of 250 transitions was developed in a 24 hour period with minimal user interaction. This is quite impressive.
• #33: From the TraceFinder’s Home Page, select “Analysis” …
• #34: Create a Batch (that is, a sequence of certain length) of samples. Batch creation in TraceFinder is as simple as a click of a mouse in the automated Batch Wizard functionality. The batches are quickly created without overlooking any of the QA/QC sampleswhich are automatically inserted according to the template for routine analyses in the Master Method.
• #35: Finally, some play for the Hardware. The data acquisition from Thermo Scientific GC-MS instruments is interactive. The real time status view at the bottom of the page allows you to view samples as they are being acquired while the status buttons at the top of the page quickly indicate which samples have been acquired, are being acquired and have not yet been acquired, just by using a simple color code. Just-acquired data from the batch (sequence) in progress can be reviewed while the acquisition is still in progress. That brings us to the next challenge: Data Review.
• #36: Once the data is acquired, we move into the Data Review stage.Data isn’t always going to be perfect. There will be times when certain compounds or samples will require some additional attention. TraceFinder has incorporated an array of flags to easily identify areas that require some further review. We see in the sample list at the top of the screen that some of the samples have yellow caution signs in the flags column while others do not. The yellow caution signs quickly attract the attention of the data reviewer indicating further review is necessary.When the user clicks on the yellow caution sign the details are displayed as shown on our next slide.
• #37: The sample flag details are promptly listed so the necessary action can be taken. There is no need to dig and search through the batch to see what the issue may be. This really makes the data review process flow much better and helps to eliminate overlooking problem areas.
• #38: The individual compounds will also display colored flags when additional review is necessary. Just hoovering over the colored compound flag will display the associated details..
• #39: While speaking of flags to alert reviewers to data issues, I’d like to illustrate what happens when an ion ratio failure is detected. The confirming ion peak was integrated poorly for illustration purposes only. Whenever the ion ratio does not fall within the defined range, the corresponding ion pane is highlighted with a red perimeter.The expansive flagging features built into TraceFinder will help ensure that data quality issues are identified immediately making the analysts job easier as well as the QA/QC coordinators job easier.
• #40: Data review is very thorough. The sample list contains all of the information you need regarding your samples. As the horizontal scroll bar is moved, the Status, Filename, Sample Type, and Level columns stay fixed while the other columns scroll right and left for easy review of the data.
• #41: The Peak display at the bottom of the page is very flexible and can be customized to meet the analysts needs. Here we have the Quan Peak on the left and the Confirming ions view on the right while an additional parameter, the calibration curve, will be displayed in the middle.
• #42: Now we have the calibration curve displayed.The flexibility of data review is terrific and it is easy to use at the same time. There is no need to reprocess data, this is done automatically and there is no need to move about into other windows allowing quick, efficient review of data- an analyst’s dream!
• #43: This is an example of the Quantitation Report. Once again, flexibility has been incorporated in to the report generation process as well. Reports can be viewed within the application and then printed or saved as .pdf or .xml files or they can be created on the fly as the samples are acquired and processed. The choice is up to the analyst.The list of report options is extensive and there is even a custom reporting feature available to create special reports as needed.
• #45: Let’s review some features of the new TRACE 1300 Series Gas Chromatograph, which has been designed with mass spectrometry and mass spectrometer it can be coupled to, in mind. The injector of the TRACE 1300 Series GC has been designed in such a way as to minimize as much as possible the septum bleed. When combined with immesurable air diffusion into the GC components, it allows your mass spectrometer to stay cleaner – longer. In addition, within the GC, there are no gas lines to be plumbed (or re-plumbed). How’s that possible?
• #47: Another design feature of the TRACE 1300 Series GC, aiming at maximizing time between maintenance: the speed of heating and speed of cooling. With typical cooling time of less than 4 minutes (from 450 °C to ambient) and maximum heating rate of up to 125 °C/min, you can fit more samples between the maintenance events and shut downs … that is, if you ever need to shut down your GC-MS at all.
• #48: For the mass spectrometer cleaning and maintenance, one usually needs to disrupt the vacuum – and consequentially the flow of data, for at least couple of hours at best. Not so with Thermo Scientific ISQ and TSQ 8000 mass spectrometers. For one, ISQ and TSQ 8000 are designed for robustness and naturally, by the design, provide longer time periods between any interventions needed. Let’s look at those design features.
• #49: There are two filaments. When one burns or no longer produces enough emission current, switch to another one without any interruption. Simple? Yes. But uncompromising, too – the second filament is positioned in the exactly same geometry relative to the ion source – that is, with exactly the same distance, using the same focusing lenses and magnetic field, - as the first one, minimizing the variability of the switch.
• #50: The key design feature of both ISQ and TSQ 8000 mass spectrometers that is as simple as uncompromising in attaining longer time periods between any interventions are necessary – is the curved, S-shaped ion guide acting as a pre-filter, strategically positioned right after the ion source and filtering out the excited neutrals but letting the ions through and on to the analyzing quadrupole. Majority of the ions that do not get through the analyzing quadrupole, end up on the very beginning of the rods. Hence, by design, the very beginning of the quadrupole has been made removable, together with the entire ion source, without disrupting the vacuum.
• #51: And if you are one of those very few laboratories that end up with a need to clean your analyzing quadrupole rods? Well, you can! Made of solid molybdenum, the quadrupole rods can be cleaned.
• #52: When it comes to detecting your ions, the ion beam is already practically free of the excited neutrals – for we got rid of them right after they were formed in the ion source, and the Linear-Logarithmic detector is ready for a wider range of the electronic current from the conversion dynode, thus providing to you-the-analyst a wider dynamic range of concentrations that can be detected and quantified. Less re-runs because you sample did not fit into your detector’s range means, again, longer times between maintenance..