Thermo Scientific GC and GC-MS Systems Analysis of chemical contamination in nutraceuticals and other matrices.

Gas Chromatography Products Trace 1300/1310 Gas Chromatograph ISQ Single Quadrupole GC-MS These are the GC and GC-MS systems that we will be talking about today. TSQ 8000 Triple Quadrupole GC-MS/MS ITQ Ion Trap GC-MS/MS

TRACE 1300 Series Gas Chromatograph Launched at PittCon March 12

TRACE 1300 Series – Two Systems Available The TRACE 1300 Series GC comes in two standard systems, the 1300 and the 1310. The 1300 has a limited user interface, with two user buttons and four indicator lights. The 1310 provides a touch screen interface and local control of the system. The modules that are currently available for these two systems are: Inlets: split/splitless, PTV and backflush capabilities. Detectors: FID, ECD, TCD, NPD and mass specs. Other options available on these systems is oven cryogenics and extra auxilliary functionality. You also have a choice of software options with this GC, Chromeleon, Xcalibur, ChromQuest and ChromCard. Modules available: INLETS: SSL - SSL backflush - PTV - PTV backflush DETECTORS: FID - TCD - ECD - NPD - MS* OTHER OPTIONS: Oven Cryo - Aux carrier Software: Chromeleon, Xcalibur, ChromQuest, ChromCard

Expand throughput with flexible automation TRACE 1300 series GC is part of the Thermo Scientific GC/MS solutions TriPlus RSH: Liquid, Headspace and SPME injections in a single sequence, with unattended syringe switching Sample preparation capabilities, without increasing the bench space The functionality of the GC can be expanded through the use of autosamplers. The AI 1310 on the left provides auto injector capabilities of 8 samples. On the right you see the TriPlus RSH. It is able to perform sample preparation steps, liquid injections, large volume injections, SPME, and Headspace injections. It is even able to change tools in the middle of a sequence so that it can do more than one in a sequence.

TRACE 1300 Series: Tailor instrument configuration In the case of the Trace 1300 series GC you are able to replace and add injectors and detectors, simply and easily. It really does only take a few minutes to make the change with the instant connect concept, and you are ready to go. Modules are user installable in only two minutes just removing three screws you’ll put the new module in place No special training, dedicated tools or on-site service engineers required

USP 467 Analysis using the TRACE 1310 Triplus RSH Dilute Headspace Liquid injection TRACE 1310 GC Split/splitless injector TR-V1 column FID detector Let’s take a quick look at one of the Trace 1310 applications. Here we have the system that was set up to analyze residual solvents.

USP 467 Analysis using the TRACE 1310 1_ methanol 2_ acetonitrile 3_dichloromethane 4_trans-1,2-dichloroethane 5_cis-1,2-dichloroethane 6_tetrahydrofuran 7_cyclohexane 8- methylcyclohexane 9_ 1,4-dioxane 10_toluene 11_chlorobenzene 12_ethyl benzene 13_ m-xylene/p-xylene 14_ o-xylene Here is a partial list of the solvents that were analyzed. This is the Class 2 A list. The B list and Class 1 were also analyzed. Example calibration curves are also shown.

ISQ Single Quadrupole GC-MS Now let’s move to the mass spectrometers. First off is the single quadrupole.

ISQ: Features Dual Filament assembly Ion Source ISQ Ion Path On the left is the linear dual filament assembly On the right is the Removable extractabrite ion source Contains the ion volume, repellor, lenses, RF prefilter Solid, highly inert material ensures reliable performance in all ionization modes, including EI and CI Below is a side view of the ion path though the ISQ Single quad The S-Shaped Ion Guide reduces neutral noise created by excited helium neutrals striking the detector, resulting in lower detection limits and better selectivity

Full Scan/SIM Methodology for Drinking Water To demonstrate the benefits of the ISQ and one of the functionalities using full scan/SIM analysis, we’ll look at this volatiles analysis of drinking water. Here we see the first part of the full scan method run from 47- 300 amu and the second part from 35 to 300 amu range to avoid problems as stated in the method requirements. Later in the analysis we see the alternating full scan/SIM data. The collection of SIM data enables the analysis down to lower concentrations for these three compounds. All of this is completed within the same analytical run and eliminates the need to run the sample twice, once in full scan and again in SIM for the compounds with very low detection limits.

Low Level Drinking Water Analysis FS/SIM 0.020 ppb SIM Full Scan 1,2,3-trichloropropane 0.100 ppb Full Scan 0.020 ppb SIM 1,2-dibromo-3-chloropropane 0.020 ppb Full Scan SIM Dibromomethane The difference that can be seen in the SIM portion of the analysis can be dramatic. For these three compounds you can see the difference in the noise at the same levels between the full scan and SIM. The third compound can not even be seen in full scan at the 0.02 ppb level. The method detection limits for these compounds are 2 ppt for the first 2 and 4 ppt for the third using SIM analysis. The full scan precision at the lower levels varies much more than the SIM data does, resulting in much higher detection limits. In order to meet the QC requirements and the detection limits for places like California, SIM is necessary. Regulatory agencies generally push limits lower and lower, so the flexibility afforded by having a system capable of alternating Full Scan/SIM will likely increase your productivity as it allows for you to get the work done in one injection rather than two.

Source Maintenance without Breaking Vacuum Step 2. Remove source Step 1. Insert removal tool Flexibility and ease of maintenance is very important for the long term use of a GC/MS single quad. This slide demonstrates how to remove the source without breaking vacuum. This feature is used for three main reasons Changing from dedicated EI source to a dedicated CI source for maximum sensitivity in either one. Putting a sample directly into the source, without chromatography. Quickly determine what a sample is. Removing the source for cleaning and replace it with a clean one without venting, saving hours of down time. Step 4. Push source out of tool Step 3. Hot source is held in tool

ITQ Ion Trap GC-MS/MS Next we move to ion trap technology. On the surface it may seem like a quadrupole. It is a little different. The flexibility that is part of the ion trap comes in very handy when dealing with very difficult samples.

Why an ion trap and MS/MS? Gamma BHC Methiocarb Mevinphos Single Quadrupole – SIM Mode at 5 ppb Gamma BHC Methiocarb Mevinphos Why an ITQ ion trap instead of the single quad for this analysis? Here are three compounds at 5 ppb in lettuce run on a single quadrupole in SIM mode. The pesticides can be found but in the case of mevinphos it is difficult to impossible to prove that is the peak. Integration is difficult at best. [click] The same sample was also run on an ITQ ion trap in MSMS mode. Notice the increased S/N due to the elimination of the matrix interferences in the ion trap data. This is the advantage of using the MSMS mode on an ion trap for analyses in complex matrices. The peaks are easily found and integrated by the software. There would be no need to do any manual integrations. Ion Trap – MS/MS Mode at 5 ppb 15

Multi-Residue Pesticide Analysis in Tea ITQ Ion Trap GC-MS/MS Split/splitless injector, 250 °C, 2 µL inj. Surge pressure 250 kPa, 30 s Low Endrin/DDT breakdown TRACE™ TR-Pesticide 35% diphenyl/65% dimethyl polysiloxane 30 m x 0.25 mm x 0.25 μm w/ 5 m guard column 1 mL/min, const. Flow For example we developed a method for the analysis of pesticides in tea. Tea is a matrix that has a lot of components in it, as you might imagine. Most of these get in the way of analyzing for the pesticide contaminants. This is the GC method and the column that we used. We used DDT and Endrin breakdown calculations as part of the QC of the method.

ITQ Chromatograms in MS/MS and Full Scan GC-MS/MS GC-Full Scan-MS Looking at the results. The left side has a region of that full scan chromatogram that has a lot of matrix in the way of the analysis. The right side shows the MS/MS scan function for that time frame. All of the pesticides that were spiked into the tea, eluting at that time, can be easily found and quantitated by the software in the MS/MS portion of the data file, no need for the manual integrations in the full scan portions of the data file (if you could even find them there). Interference free quantitation by MS/MS Unknown peak investigation by Full Scan

Analysis of Phthalates in Serum The Problem: EI GC-MS analysis of phthalates generates a non-specific full-scan spectrum with prominent m/z 149, and not many other identifying ions The Solution: PCI with ammonia generates a prominent [M+1] at m/z 391, perfect for MSn analysis for improved specificity and confident identification & quantitation Next we move to an application that addresses needs showing up in the news recently. DEHP has been found in drinks from specific regions. The problem isn’t that they are in the drinks, the real problem is DEHP and other phthalates get into the body. Analyzing serum can be difficult and phthalates generate a very strong signal at mass to charge 149, this is not very specific. [click] The answer is to use another tool, ammonia PCI with MS/MS. Here we see the result with PCI using ammonia. This ion is then isolated and fragmented in the ITQ ion trap giving the specificity that is needed for the analysis.

Results – All Work Done in Serum Calibration curve 1 to 10 µM DEHP in serum Results from sample 5.3 µM DEHP in serum

TSQ 8000 Triple Quadrupole GC-MS/MS Launched at ASMS May 20 Now on to the triple quad technology. Not just 1quadrupole, there really are three quadrupole fields. This adds a level of specificity that is unmatched by the single quad or even the ion trap.

Achieve Low Detection Limits With Confidence Malathion extract at 5 ppb Cut through chemical background High selectivity in matrix compared with single stage MS Selected Reaction Monitoring (SRM) High precision at low concentrations Femtogram level detection Confirming ions SIM SRM Selectivity the TSQ 8000 delivers through Selected Reaction Monitoring (or SRM) is the key to productive analysis The challenge in trace level analysis in single stage MS systems (using full scan or Selected Ion Monitoring (SIM) ) is the limitation in detection due to chemical background matrix (example malathion) The TSQ 8000 through a combination of high sensitivity and selectivity allows low level contaminants to be comfortably detected in very complex matrices well within the regulatory performance requirements (eg EU pesticide regs).

Not Just MS/MS Performance High full scan spectral quality Searchable with NIST etc. High sensitivity SIM & Full Scan Virtually indistinguishable from a single quad instrument Dual acquisition modes Quan & qual in the same run Full Scan SIM When adding MS/MS to your laboratory, you should not compromise capability especially if you need still to performance single quad like analysis The TSQ 8000 was designed to be a powerful instrument for Full scan and SIM experiments for when you need them. Mass spectra is preserved and library searchable with no unnecessary gas collisions taking place on the way to or through the mass analyzer In addition, analyzer speed means that simultaneous targeted and untargeted acquisition modes are possible. That’s in both MS and MS/MS mode. Lindane isomers 40 ppb in cucumber in simultaneous full scan/SIM

Designed For More Uptime, More Results Reported Design based on strong reputation Thermo Scientific ISQ Single Quadrupole GC-MS Routine-grade robustness ExtractaBrite ion source-no-vent maintenance Heated sample path zones for reduced ion burn Innovative GC modularity Thermo Scientific TRACE 1300 Series GC Optimized maintenance %RSD pesticides (10 ppb) before and after 1,400 vegetable matrix injections EU limit for LOQ %RSD Before After The TSQ 8000 shares the DNA of the highly reputable ISQ™ Single Quadrupole GC-MS instrument. The TSQ 8000 inherits the routine grade robustness delivered from the ExtractaBrite ion source and associated heated zones in the sample path. The innovative modularity of the TRACE™ 1300 Series Gas Chromatograph is a perfect fit for GC-MS/MS which often handles dirty samples. A very fast swap of injectors or injector parts means you needn’t be down during maintenance peroids. The real test of robustness is the long term analysis of real samples. The EU QC criteria for LOQ is within 10% RSD. The detection requirement is 10 ppb. The chart shown here represents the calculated precision of the TSQ 800 before and after 1400 injections of vegetable matrix over 35 batches. The mean RSD does not rise significantly or anywhere close to the limit. The robustness to matrix is outstanding. If, after further matrix exposure, we hit the limit. We can be back up and running very quickly with no vent ion source removal.

SRM Enables Optimized Sample Preparation 1,3 –dichlorobenzene 1,4-dichlorobenzene and 1,2-dichlorobenzene (left to right) in ASE soil extract at 5 ppb. Explore generic sample approaches Method consolidation Eg. QuEChERS Reduce number of steps Pre-concentration Clean-up Bring sample preparation onboard TSQ 8000 TriPlus RSH autosampler The selectivity and sensitivity of the TSQ 800 means that sample preparation can relax on pre-concentration and clean-up. Sample prep selectivity becomes less important. This brings the opportunity to optimize sample preparation step to speed up the analysis. It also brings the possibility of more generic sample preparation approaches that can capture more compounds, helping consolidate efficient analytical methods. The example shown is the low level SRM analysis of dichlorbenzenes in a direct ASE (accelerated solvent extraction) soil extract with no clean-up. With robotic autosamplers, some steps can even be handled onboard the GC-MS/MS system.

Consolidated, Faster Methods With More Compounds Real example of method consolidation on the TSQ 8000. Five SIM methods combined to form a powerful MRM method for 350 pesticides (700 transitions) Method consolidation Combine separate MS analyses Driven by selectivity of SRM Faster run times Compress chromatography Using MS/MS resolution Clean peak detection Less manual integration changes Fast data processing We already talked about method consolidation in sample preparation, here it is on the MS/MS side. The busy chromatogram on the right is a multiple reaction monitoring (MRM) method analyzing for 350 pesticides with 700 transitions. This is a real example of method consolidation from 5 SIM monitoring methods on a single quadrupole instrument. The selectivity of the TSQ 8000 makes this possible. TSQ 8000 selectivity also allows in a lot of cases the compression of the chromatography into a faster GC run to allow a higher sample through-put. As discussed before, the ability to have clean peak detection in this situation really helps to automate and speed up data processing.

A Fast, Simple Route to High Performance MS/MS AutoSRM Unknown SRM Transitions The very beginning or a SIM Method TSQ 8000 Inst. Method Known SRM Transitions From an existing method or SRM Transition list Thermo Scientific TraceFinder SW Optimized SRM Transitions From Compound Data Store or existing TSQ 8000 method SRM management tools SRM creation and method management independent of your starting point SRM Optimization Method Sync One of the challenges of MS/MS is the very nature of how the selectivity is delivered. With two mass analyzers comes more masses to manage and more method development. Especially if consolidated methods are to be used the development, and continuous maintenance of these methods can be really complex and time consuming. The TSQ 8000 has been designed to take the pain away in the creation, optimization and continues management of MS/MS methods. Integrated software tools allow easy access to high performance MS/MS routinely, regardless of where you are starting from...

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