©2012 Waters Corporation 1

Knowing your LC/MS system

- benchmark tests and

fundamental fault finding

Tore Caesar

3rd [Nordic MS Symposium], Jurmala 2012

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Agenda

Performance tracking

Automatic system checks

Basic trouble shooting

Remote service

Questions

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Performance tracking

Systems running routine analyses

– QC samples

– Internal standards

– Standards

Open Access Systems and systems with diverse projects

– Specific Test sample for a ”System Suitability Test”

– Not expensive, stable in solution, non-toxic and compatible with the

chemistry used.

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Test Mix MS

Component Empirical

Formula

Exact Mass

(as [M+H]+)

Exact Mass

(as [M-H]-)

UV Absorbance

Acetaminophen C8H9NO2 152.0712 150.0555 257 nm

Caffeine C8H10N4O2 195.0882 193.0726 273 nm

Sulfaguanidine C7H10N4O2S 215.0603 213.0446

Sulfadimethoxi

ne

C12H14N4O4S 311.0814 309.0658

Val-Tyr-Val C19H29N3O5 380.2185 378.2029

Verapamil C27H38N3O4 455.2910 453.2753

Terfenadine C32H41NO2 472.3216 470.3059

Leucine

Enkephalin

C28H37N5O7 556.2771 554.2615

Reserpine C33H40N2O9 609.2812 607.2656

Erythromycin C37H67NO13 734.4691 732.4534

©2012 Waters Corporation 5

Test Mix MS

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Test Compounds UV

Component UV Abs

2-Acetylfuran 254 nm

Caffeine 273 nm

Acetanilide 254 nm

Acetophenone 254 nm

Propiophenone 254 nm

Bytulparaben 254 nm

Benzophenone 254 nm

Valerophenone 254 nm

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Evaporative Light Scattering Fluorescence

Test Compounds

Caffeine

– λmax 273 nm

Anthracene

– λEx 246 nm

– λEm 402 nm

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What to look for

Retention time

– Pump, mobile phases, column temperature

Peak Area or Peak Height

– Injector, detector sensitivity

Signal to noise

– Background level, mobile phases, detector sensitivity

Blanks

– Carryover, contaminated mobile phase

Backpressure – absolute and ripple

– Clogged column / guard / prefilter / capillary, leak

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TargetLynx Trendplot

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Automatic system checks

”LC/MS system check” with IntelliStart

– Schedule a SST to run, process and report

– A report will tell the user if all specifications have been met

”QC Monitor” with TargetLynx

– Real time monitoring during batch

– Can take actions when specification is not met

o Reinject sample

o Pause sample list

o Skip to next batch

o Run shutdown

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LC/MS system check - IntelliStart

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LC/MS system check - IntelliStart

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TargetLynx QC Monitor

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TargetLynx QC Monitor

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TargetLynx QC Monitor

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TargetLynx QC Monitor

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Basic trouble shooting

Retention time

– Is back pressure ”normal” across the gradient?

– Reverse the gradient (run from B to A)

– For a binary (HP mixing) pump, e.g. Acquity Classic, I-class

o Check that both pumps generate the same backpressure at the

same flow with the same eluent.

– For a low pressure mixing pump (Alliance, H-Class, 600...)

o GPV test Alliance, put A and B line in 100 ml cylinders, pump at

50:50 @ 5 ml/min. Stop when A reaches 75 ml. B should be 72-78

ml.

– Column temp, column fitted correctly in compartment, pre-heaters.

– Mobile phase correctly prepared? Organic content and additives.

– Pressure decay test – use MeOH or 50:50 MeOH:H2O

©2012 Waters Corporation 18

Basic trouble shooting

Peak Area / Peak Height

– Injector, possible to test other injection mode?

– More than one detector in system?

– MS

o Check spray

o Readbacks ok?

o Clean cone or entire source in ultrasonic bath

– Is sample ok?

– Reproducibility

o Inject from same vial

o Method related? Try another compound.

o Make a simple MS method

©2012 Waters Corporation 19

Basic trouble shooting

Signal to noise

– Is the peak response low or is the baseline noisy or elevated?

– UV and PDA are sensitive to bad mixing when one eluent has higher

absorbance. Compare with a pre-mixed eluent.

– PDA

o Dirty flow cell – clean according to protocol

o Dirty mobile phase / no clean eluent in flow cell during from start

o Lamp intensity down – try new lamp

– MS

o Contaminated mobile phase – prepare new, don’t forget stock

solutions. Higher grade chemicals.

o Dirty source – clean in ultrasonic bath

o Check spray

o Can longer dwell time be used?

©2012 Waters Corporation 20

Basic trouble shooting

Blanks

– Carry over?

o Make several blanks. Usually response decreases from one injection to the next.

o Try other strong wash, e.g. 25:25:25:25 of IPA:MeOH:ACN:H2O + 0.1-2% FA or NH4OH. Use both.

o Increase wash volumes

o Try other needle and loop materials or different injection mode

– Contaminated mobile phase?

o Run gradient without injection or bypass injector?

o Deliver 100% A a longer time before gradient to see if response increase

o Document “Controlling Contamination in UltraPerformance LC®/MS and HPLC/MS Systems” on waters.com

©2012 Waters Corporation 21

Basic trouble shooting

Increased backpressure

– Column or pre-column clogged

– Correct column temperature

– Disconnect one thing at the time, starting at the end

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Connections Insight

– iAlert

– iHelp (iRequest on waters.com)

– iAssist

TeamViewer

– 3rd party software

– Remote desktop

Remote support

©2012 Waters Corporation 23

Thank You!