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Instrumentation issues for reliable Mass Spectrometry

Data

Dr Jackie Mosely

Durham University

6th June 2006RSC/AAMG

Choosing the right instrumentation

Aim of experiment• Identification/elucidation of an

unknown compound

• Analysis of a complex mixture

• Low volume or low concentration

• High throughput

• Quantitation

Instrumental considerationsmass accuracy, resolution, MSMS, pos/neg switching, …

chromatography, resolution, dynamic range, mass range, ..

Sensitivity, duty cycle,..

Duty cycle, stability, …

Duty cycle, sensitivity, stability, reproducibility, dynamic range, SIM, …

Having faith in data comes from the assurance in instrument performance at any time

Consider…

API (ESI, nanospray, APCI etc)• Need a stable spray/signal so balance gas flow, temperature,

voltages and distance/angle from sampling orifice. The optimum distance/angle from sampling orifice will depend on the components of the mobile phase and flow rate; note this can change during RPLC runs

Factors affecting performance – Ion sources

Sheath gas

spray

nebuliser

Mass spectrometer entrance

API sourcesS O L V E N T # 2 4 9 R T : 0 .7 1 A V : 1 N L : 9 .9 5 E 5T : IT M S + c E S I F u ll m s [ 1 5 0 .0 0 -2 0 0 0 .0 0 ]

2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0m /z

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5

1 0

1 5

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1 9 1 .0

2 0 8 .9

2 4 9 .6

2 5 1 .7

2 5 3 .8

3 6 5 .03 8 2 .92 6 7 .7

6 1 0 .0

2 6 9 .8 3 9 1 .0 6 1 1 .03 9 2 .92 7 1 .8 6 1 1 .9 6 8 5 .05 3 5 .9 5 7 5 .54 3 3 .3 4 7 6 .6 7 5 8 .97 1 4 .9 9 0 0 .6 9 3 2 .38 7 6 .98 1 4 .2 9 7 8 .8

Dirty acetonitrile!

•Clean volatile polar solvents. Mobile phase additives can help ionisation.•Blank analyses will identify and validate background and contaminants can suppress ions of interest

Consider…

MALDI • Choice of matrix and ratio of sample to matrix • Homogeneous sample preparation with small equally sized crystals

• A very flat target surface is particularly important for MALDI-ToF

• Operating laser at the power that is the threshold for ion generation

Factors affecting performance – Ion sources

Ideal world More usual real world

acceleration region

field free flight path

Instrumentation Issues for Reliable Mass Spectrometry Data

Jackie Mosely - Durham University, Durham

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Factors affecting performance - Analysers

Time-of-flight (ToF)…

Vacuum• Poor vacuum will cause a loss in resolution and ultimately

inaccurate mass measurement.

Temperature m/z = 2eVacc(t/d)2

• Fluctuations in room temperature can cause very slight changes in length of flight tube (d), thus affecting measured time of flight (t).

• Fluctuations in temperature of the electronics can mean fluctuations in voltage output (Vacc) thus affecting measured time of flight (t).

Factors affecting performance - AnalysersQuadrupoles and Paul Ion traps and FTICR traps

Vacuum• A poor vacuum will affect the ability to trap ions in an ion trap and will affect the stability of an ion

passing through a quadrupole, resulting in loss of resolution, sensitivity and signal stability.

Ion Energy• Too much kinetic energy can mean ions are hard to trap efficiently. This is the reason for the helium

bath gas in a Paul ion trap. Collisions between neutral inert gas molecules and ions will help ‘cool’the ions and keep them in the centre of the trap. If this gas becomes contaminated by a non-inert gas the trapping conditions can become very unstable.

• Cannot use a bath gas with FTICR cells so need to reduce ion energy before they are trapped

Ion population• Too many ions in a confined area will interact with each other. The resulting space charge effect can

affect the resonant frequencies of the ions, affecting their ejection from the Paul trap and or the cyclotron frequency in the FTICR. Ultimately resolution and m/z value measured will be compromised.

Voltage (RF)• Must be stable

EndcapRing

ElectrodeEndcap

entranceexit

Helium bath gas

Factors affecting performance - Recording and processing data

Consider…

• Digitiser and detector

• Signal averaging

• Centroid/smoothing/peak threshold

• Database searching criteria

CONSISTANCY IS KEY

What to look for

Consider…

• Resolution and peak shape

• Mass accuracy and precision – previous talks

• S/N and absolute intensities and isotopic profile

• Reproducibility

Peak shape

T:

278.2 278.4 278.6 278.8 279.0 279.2 279.4 279.6 279.8 280.0m/z

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Centroid m/z value is wrong

Resolution

T:

278.2 278.4 278.6 278.8 279.0 279.2 279.4 279.6 279.8 280.0m/z

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279.09094

279.15901

279.???

RP = 1,500 FWHH

RP = 150,000 FWHH

Instrumentation Issues for Reliable Mass Spectrometry Data

Jackie Mosely - Durham University, Durham

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279.0 279.5 280.0 280.5 281.0 281.5 282.0 282.5m/z

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279.09094

280.09439281.08676

281.70224280.35889279.09102

280.09429

281.08691282.09029

NL:5.03E4ESI_pos_Sulphamethazine_FT_FSb#25 RT: 0.83 AV: 1 T: FTMS + p ESI Full ms [ 100.00-750.00]

NL:1.92E4C 12 H15 S 1 O2 N4: C 12 H15 S 1 O2 N4p (gss, s /p:40) Chrg 1R: 80000 Res .Pwr . @FWHM

Mass spectrum of Sulphamethazine

Theoretical profile of C12H15N4O2S

S/N, absolute intensity and Isotopic profile Qualify the type of MS for your application

• Performance– Read manufacturers specs or other literature, i.e. is the

accuracy/precision or resolution sufficient…

• Functionality– Can you set up the experiment to answer you question

i.e. msms, quantitation…

Verify general performance

A few easy steps…

• Set up hardware to fit the experimental aim• Tune and optimise mass spectrometer• Calibrate mass spectrometer• Verify performance by running a range of test samples,

aiming to match manufacturers specifications

Verify general performance

A few easy steps…

• Set up hardware to fit the experimental aim• Tune and optimise mass spectrometer• Calibrate mass spectrometer• Verify performance by running a range of test samples,

aiming to match manufacturers specifications

Need to know all-round instrument performance is acceptable

Validating experimental set-up

• Run something very similar to your application and check results – this now includes software!

• Record and archive validation data, it gives a great evaluation of long term instrument status

• Repeat at regular intervals or if there is reason to believe performance has dropped.

DON’T CHANGE ANYTHING

Summary

Qualify instrumentation for your application

Verify instrument can meet an acceptable level of specification

Validate performance regularly, recording data

Change application or requirements

Run analysisClean instrument or regular maintenance

NO

YES

YES

YES

NO

Instrumentation Issues for Reliable Mass Spectrometry Data

Jackie Mosely - Durham University, Durham

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Take home message

Spend a little time to get to know your instrument, it will save you time in the long run

Resolution

Peak Shape Isotopic profile

Mass accuracy

Precision

S/N

Reproducibility

Instrumentation Issues for Reliable Mass Spectrometry Data

Jackie Mosely - Durham University, Durham