The Emergence of Benchtop Tandem Quadrupole Mass Spectrometers as a Routine Tool in Quantitative GC/MS/MS Applications
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1 The Emergence of Benchtop Tandem Quadrupole Mass Spectrometers as a Routine Tool in Quantitative GC/MS/MS Applications Doug Stevens Waters Corp. Introduction The range of GC/MS instrument types has expanded in order to meet the limitations of earlier instruments designs or of increasingly stringent legislation in applications areas like environmental analysis, food safety screening, metabonomics, and clinical applications like forensics, toxicology and drug screening.
2 Introduction In the past GC/MS analyses were dominated by two different types of instrument. Magnetic sector High resolution, accurate mass analyses requiring the ultimate in sensitivity Single quadrupole Routine analysis of target compounds Mass Spectrometers for GC Quadrupoles Single Tandem Time of Flight Low Resolution Elevated Resolution Quadrupole Ion Trap Magnetic Sector EBE and BE configurations common
3 Single Quadrupole: Advantages/Disadvantages Advantages O Disadvantages + Ease of use + Cost + Dynamic range ー No MS/MS ー Mass accuracy ー Resolving power ー Scanning instrument Magnetic Sector: Advantages/Disadvantages Advantages + Mass accuracy + Excellent resolving power + Excellent dynamic range + MS/MS, Linked Scans O Disadvantages None of the disadvantages are However, the complexity, cost based on technical capabilities of and size ー Complex are definitely relevant the instrument type which is considerations why ー magnetic Cost in most labs sectors were the decision to use or not use gold ー standard Size for quantitative magnetic sectors. ー MS Scanning assays for instrument years. ZAB c.1975
4 Mass Spectrometers for GC Quadrupoles Single Tandem Let s In time, consider new ion types traps of GC/MS briefly and Time of Flight instruments then Tandem were Quadrupole introduced. Low Resolution instruments in detail later on. Elevated Resolution Quadrupole Ion Trap Magnetic Sector EBE and BE configurations common Ion Trap: Advantages/Disadvantages While the trap can perform MS/MS it has serious limitations that are problematic in quantitative applications as will be discussed here and on the following slide. Advantages O Disadvantages + Ease of use + Cost + MS/MS + Full scan sensitivity ー Mass accuracy ー Low resolution! ー Dynamic range ー Scanning instrument
5 Ion Trap Disadvantages in Quantitative Assays Triple Quadrupole Constant sampling across the peak. Ion Trap Irregular sampling rate dictated by ion current. Constant sampling rate ensures precision and accuracy. Irregular sampling rate leads to higher variation. LC/MS/MS 1990 s LC Polar Basic Acidic Labile Large Molecules Electrospray Easy coupling to MS Efficient, soft ionization of polar, acidic, and basic compounds Suppression possible MS/MS Eliminates high background Removes matrix interference
6 Impact of Standing Still in the Face of Advancing Technology Packed column GC method Non-specific detector, TCD Indirect determination of protein content Assumes all N is from protein Meanwhile. Direct measurement of protein content became possible Continued use of outdated technique Doesn t look like a protein to me but it does to an CHNOS analyzer! Update
7 Now: Quattro micro GC Quattro micro GC Diagram Dynamic Range 5 orders of linear dynamic range MS/MS Full Range of Experiments o MRM o Product Ion o Precursor Ion* o Neutral Loss/Gain* Dedicated EI and CI sources switchover in < 1 min Overlapping acquisition windows Dedicated to GC/MS Improves ease of use and uptime = Productivity
8 Tandem Quadrupole MS/MS is used as an alternative to high resolution, accurate mass data. That Quantitative doesn t look assays like no much longer left require in the advantages the use of full list scan so let s data + Ease of use take for a ー high look No selectivity. Accurate what MS/MS Consider Massadds using to quantitative high resolution assays. TOF for + MS/MS Capability applications ー Low Resolution in which a full + Excellent Dynamic range spectrum ー Scanning is desired/required. instrument Endosulfan-ß EI Spectrum The following slides will demonstrate how MS/MS is used to reduce the complexity while increasing selectivity and sensitivity.
9 Endosulfan-ß Product/Daughter Ion Spectrum Collision induced dissociation is used to fragment the 237Da peak into these m/z values Daughter/Product Ion Spectrum Any one of them or all of them can then be allowed to pass through the second analyzer for detection. MRM vs SIM: Effect on Selectivity MRM SIR Butylate 5.75mins
10 MRM vs SIM: Effect on Selectivity MRM SIR Ethion 13.14mins MRM vs SIM: Effect on Selectivity MRM SIR Fluvalinates
11 Implications: MS v. MS/MS Higher confidence ー Decrease/eliminate false positives/negatives Ease of integration ー Easier to automate ー Less manual reprocessing Time Savings ー Allows faster run times ー Reduce/eliminate sample clean up steps Reduced/eliminated chemical noise ー Increases accuracy ー Improves precision ー Improves dynamic range SIR MRM MRM vs SIM: Effect on LOD and Dynamic Range Vinclozolin SIM 285? Vinclozolin MRM 285> ppb Vinclozolin in Plum Extract
12 EI MRM: Linearity Example Compound name: Vinclozolin Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: External Std, Area Curve type: Linear, Origin: Include, Weighting: 1/x, Axis trans: None 3423 Response -1 pg/ul CI- Linear Dynamic Range Example: 5 Orders Compound name: Octafluoronaphthalene Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Response 0 pg/ul
13 Dynamic Range Comparison to TOF MS/MS and Speed Retention times for these analytes are commonly 3 to 4 times longer when using single stage MS and 5.39mins and 5.47mins
14 Repeatability Repeatability of Hexachlorobenzene response on the Quattro micro GC Response /- 5% Sample number Quantitation Reporting: TargetLynx Results Summary table Overview table Interactive peak integration Calibration curve
15 Quantitation Reporting: TargetLynx Results Sequence o Is the blank response OK (81 checks) o Have RT shifted (81 checks/81 data inputs) Over 5200 checks required! o Have ion ratios changed (81 checks/81data inputs) o Are all the standards within RT tolerance. (972 checks) o Are standard ion ratios OK (972 checks) Automatically performed by TargetLynx o Do RL standards satisfy S/N (322 checks) o Are calibrations OK (323 checks) o Are recoveries OK (162 checks) o Are any analytes above RL/MRL (2268 checks) o Do positives have correct RT and ion ratios (More checks). Integrating GC/MS/MS into the Lab The Quattro micro GC can perform all the experiment types available on a single quad - validation, capacity Minimal training is required for a quadrupole GC/MS operator to become proficient in quadrupole based GC/MS/MS. Full Scan SIR: Selected Ion Recording Databases of optimized MRM conditions exist for a wide variety of compounds including pesticides, dioxins, PCBS, steroids and other illicit drugs as well as environmental contaminants like PAHs.
16 GC/MS/MS Applications Toxicology, Forensics, Drug Screening Flavors/Fragrance Monitoring Impurity Monitoring Food/Beverage Metabonomics/Biomarker Monitoring Environmental Dioxins/PCB Screening, POPs Petrochemicals and growing!!! Summary Tandem quadrupoles have firmly established themselves as the gold standard for quantitative assays. The selectivity of MS/MS allows higher confidence data to be acquired in less time with better sensitivity and dynamic range. Today s benchtop tandem quadrupole instruments are small enough and easy to use enough for routine use in many application areas.
17 Acknowledgements Keith Worrall Tim Jenkins David Douce Gary Harland
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