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The Emergence of Benchtop Tandem Quadrupole Mass Spectrometers as a
Routine Tool in Quantitative GC/MS/MS Applications
Doug Stevens
Waters Corp.
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.
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
Introduction
Mass Spectrometers for GC
Quadrupoles—Single
—Tandem
Time of Flight—Low Resolution
—Elevated Resolution
Quadrupole Ion Trap
Magnetic Sector— EBE and BE configurations common
Single Quadrupole:Advantages/Disadvantages
Advantages
+ Ease of use
+ Cost
+ Dynamic range
O Disadvantages
ー No MS/MS ー Mass accuracyー Resolving powerー Scanning instrument
Advantages
+ Mass accuracy
+ Excellent resolving power
+ Excellent dynamic range
+ MS/MS, Linked Scans
O Disadvantages
ー Complexー Costー Sizeー Scanning instrument
Magnetic Sector:Advantages/Disadvantages
ZAB c.1975
None of the disadvantages are based on technical capabilities of
the instrument type which is why magnetic sectors were the gold standard for quantitative
MS assays for years.
However, the complexity, cost and size are definitely relevant
considerations in most labs decision to use or not use
magnetic sectors.
Mass Spectrometers for GC
Quadrupoles—Single
—Tandem
Time of Flight—Low Resolution
—Elevated Resolution
Quadrupole Ion Trap
Magnetic Sector— EBE and BE configurations common
In time, new types of GC/MS instruments were introduced.
Let’s consider ion traps briefly and then Tandem Quadrupole
instruments in detail later on.
Ion Trap:Advantages/Disadvantages
Advantages
+ Ease of use
+ Cost
+ MS/MS
+ Full scan sensitivity
O Disadvantages
ー Mass accuracyー Low resolutionー Dynamic rangeー Scanning instrument
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.
!
Constant sampling across the peak.
Irregular sampling rate dictated by ion
current.
Constant sampling rate
ensures precision and accuracy.
Irregular sampling rate leads to
higher variation.
Ion TrapTriple Quadrupole
Ion Trap Disadvantages in Quantitative Assays
LC PolarBasicAcidicLabileLarge Molecules
ElectrosprayEasy coupling to MSEfficient, soft ionization ofpolar, acidic, and basic compoundsSuppression possible
MS/MS Eliminates high backgroundRemoves matrix interference
LC/MS/MS 1990’s
Impact of Standing Still in the Face of Advancing Technology
Doesn’t look like a protein to me but it does to an CHNOS analyzer!
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
Update
…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
Tandem Quadrupole
+ Ease of use
+ MS/MS Capability
+ Excellent Dynamic range
ー No Accurate Massー Low Resolutionー Scanning instrument
MS/MS is used as an alternative to high resolution, accurate mass data.Quantitative assays no longer
require the use of full scan data for high selectivity. Consider using high resolution TOF for applications in which a full
spectrum is desired/required.
That doesn’t look like much left in the advantages list so let’s
take a look at what MS/MS adds to quantitative assays.
Endosulfan-ß EI Spectrum
The following slides will demonstrate how MS/MS is used to reduce the
complexity while increasing selectivity and sensitivity.
Endosulfan-ßProduct/Daughter Ion Spectrum
237 167 143 119119143167
Collision induced dissociation is used to fragment the 237Da peak into these m/z values.
Any one of them or all of them can then be allowed to pass through the second analyzer
for detection.
Daughter/Product Ion Spectrum
MRM vs SIM:Effect on Selectivity
MRM
SIR
Butylate 5.75mins
MRM vs SIM:Effect on Selectivity
MRM SIR
Ethion 13.14mins
MRM vs SIM:Effect on Selectivity
MRM SIR
Fluvalinates
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
Implications:MS v. MS/MS
SIR MRM
MRM vs SIM:Effect on LOD and Dynamic Range
Vinclozolin MRM 285>212
Vinclozolin SIM 285 ?
2.5ppb Vinclozolin in Plum Extract
EI MRM:Linearity Example
Com pound nam e: VinclozolinCorrelation coefficient: r = 0.999512, r^2 = 0.999023Calibration curve: 6.69366 * x + -1.3721Res pons e type: External Std, AreaCurve type: L inear, Orig in: Include, Weighting: 1/x, Axis trans : None
0 50 100 150 200 250 300 350 400 450 500pg/ul-1
3423
Res pons e
CI- Linear Dynamic Range Example: 5 Orders
Com pound nam e: OctafluoronaphthaleneCorrelation coefficient: r = 0.998950, r^2 = 0.997902Calibration curve: 11.9082 * x + 4.45084Res ponse type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans : None
0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000pg/ul0
595414
Res pons e
Dynamic Range Comparison to TOF
MS/MS and Speed
- 5.36 and 5.39mins
Retention times for these analytes are commonly 3 to 4 times longer when using single stage MS.
- 5.44 and 5.47mins
Repeatability
Repeatability of Hexachlorobenzene response on the Quattro micro GC.
1000
6000
11000
16000
210001 5 9 13 17 21 25 29 33 37 41 45 49 53 57
Sample number
Res
pons
e +/- 5%
Quantitation Reporting:TargetLynx Results
Overview table
Calibration curve
Interactive peak integration
Summary table
Sequenceo Is the blank response OK (81 checks)
o Have RT shifted (81 checks/81 data inputs)
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)
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).
Over 5200 checks required!Automatically performed by
TargetLynx
Quantitation Reporting:TargetLynx Results
Integrating GC/MS/MSinto 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.
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.
Full Scan
SIR: Selected Ion Recording
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.
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