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.

Acknowledgements

Keith Worrall

Tim Jenkins

David Douce

Gary Harland