MH QQQ Users workshop rev3 - Chemical Analysis, … 316.1 unit 270.1 unit 158 25 2.923 0.2 Positive Flunitrazepam 314.1 unit 268.1 unit 158 25 2.951 0.2 Positive Flurazepam 388.2 unit

Agilent 6400 Series Triple Quadrupole

LC/MS/MS Users Workshop

North America

QQQ Method Development and Optimization

MassHunter Quant:Method setupPeak detection optimizationQuant troubleshooting

Author: John Hughes Contributors: Anabel Fandino, Doug McIntyre, David Presser, Peter Stone, André Szczesnieski, Shane Tichy, David Weil, Laura Phillips

MH QQQ Users workshop 1 1/25/2012

Agenda

•QQQ method development and optimization– Source optimization for standard and Agilent JetStream sources– Compound optimization using flow injection or Optimizer software– Dynamic MRM

• MassHunter Quant software– Current revision B.05.00: What's New?– Review of Quant methods: creation from acquired data, existing batch– Global and Advanced parameters– Optimizing peak identification and rejection– Quant method troubleshooting: identification and integration


Agilent ESI and APCI sources:for polar to non-polar compounds

MH QQQ Users workshop 3

Vcap

Fragmentor V

Nebulizer Pressure

Drying Gas Temperature and Flow

Vcap

Corona current

NebulizerPressure

Fragmentor V

Drying gasTemperature

and Flow

Vaporizer

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Agilent Multimode source for flexibilityESI or APCI or Mixed mode (simultaneous ESI/APCI)


Fragmentor V

HPLC inlet

Nebulizer

Drying gas

Corona needle

ESI Zone: Charging V

APCI ZoneIR emitters:"Vaporizer"

Capillary V

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Agilent Jetstream Technology (AJT)The super-heated sheath gas collimates the nebulizer spray and presents more ions to the MS inlet.

*Nozzle voltage

Resistive samplingcapillary exit: FragV

Nebulizing gas: pressure

*Sheath gas: flow and temperature

Drying gas:flow and temperature

Enhanced efficiency nebulizer

Resistive samplingcapillary entrance: Capillary V

5MH QQQ Users workshop

*New parameters unique to AJT source

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Source optimization for Agilent LC/MS systemsESI, APCI, MM and Agilent JetStream Technology (AJT)

Flow dependent parameters:•Nebulizer pressure

•Drying gas temperature and flow

•Vaporizer temperature (MM)

•Sheath gas flow (AJT)

Compound dependent parameters:

•Capillary voltage

•Fragmentor voltage

•Collision Energy (QQQ, QTOF)

•Vaporizer temp (APCI, MM, AJT)

•Sheath gas temperature (AJT)

•Nozzle voltage (AJT)


Starting parameters for Agilent sources(small molecules)


Parameter ESI source APCI sourceNebulizer pressure 25 psi 0.2 mL/min

50 psi 1 mL/min60 psi

Drying gas flow 10 Lpm < 0.2 mL/min12 Lpm > 0.4 mL/min

5 Lpm

Drying gas temp 325-350°C 350°CCapillary voltage 3500V 3500VFragmentor voltagecompound dependent

70-100 V Single quads120-140V QQQ, TOF140-170V 6230, 6530


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Starting parameters for Agilent sources(small molecules)


Parameter Multimode source Agilent JetstreamNebulizer pressure 60 psi ESI

20 psi APCI40 psi Mixed mode

45 psi

Drying gas flow 5 Lpm 7 LpmDrying gas temp 250°C ESI, Mixed

300°C APCI300°C

Capillary voltage 2000V 3500VCharging voltage (MM) 2000VNozzle voltage (AJT) 500VFragmentor voltageCompound dependent



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Tuning and Calibration for Agilent QQQ systems•Agilent QQQ systems are very stable and require infrequent Autotuning

•Current software uses "ESI-Low" tune mix for all QQQs.

•Routine use only requires resolution and mass axis verification for polarity and resolution modes to be used.

• Run performance check sample (lab SOP)• Do Checktune (checks all three resolution modes)• In Manual Tune, turn on calibrant and observe MS1 and MS2 profiles. Use Adjust

Gain and Offset button if width or mass axis requires adjustment.•If using negative ion, Autotuning should be done monthly or less to minimize source exposure to TFA.

• Flush source extensively with LC flow after 30-minute Autotune before running samples in negative ion.

• Autotune at least quarterly to optimize ion transmission and update EM voltage.

MH QQQ Users workshop

9 1/25/2012

Optimizing the Agilent JetStream Technologybeginning with recommended starting values ( )

Order of effect on sensitivity

Sheath gas temperature (250°C)

Sheath gas flow (8 Lpm)

Nebulizer pressure (45 psi)

Capillary voltage (3500V)

Nozzle voltage (0V)

Drying gas temperature (250°C)

Drying gas flow (5 Lpm)

Things to note

Requires time to stabilize

Generally 10-12 Lpm for 0.2-0.7mL

LC flow- and mobile phase- dependent

Somewhat MW dependent

Very compound- and polarity-dependent

Interaction with flow and sheath parms

Generally 5-7 Lpm, higher when in doubt


Optimizing the Agilent JetStream Technology Typical values ( ) and Increments

Order of effect on sensitivity

Sheath gas temperature (325°C)

Sheath gas flow (10 Lpm)

Nebulizer pressure (40 psi)

Capillary voltage (3500V)

Nozzle voltage (0V)

Drying gas temperature (300°C)

Drying gas flow (7 Lpm)

Increments and ranges to test

50°C, 250-400°C

2 Lpm, 8 -12 Lpm

5 psi, 25 – 50 psi

500V, 2500-4500V

500V, 0 - 2000V

50°C, 250-350°C

2 Lpm, 5-11 Lpm


Optimization techniques

•Fragmentor V and Collision Energy (for MS/MS systems) should be optimized first using recommended starting parameters for source.

•Voltages, gas flows change quickly and can be optimized with series of flow injections using method with injector program and time segments.

•Temperatures (sheath gas, drying gas, vaporizers) require equilibration time between injections; perhaps best done with final chromatography conditions.


Injector program for series of flow injections


Check to use injector program

Injector program steps:Remote StartpulseRepeat n times [n = number of time segments]Draw default amount from sampleValve to Mainpass [injects sample without start signal]Wait 0.20 min [adjust acq time segments to sync with actual spacing of injections]End repeat

Example Injector Program with 11 repeats and needle wash step added (to prevent carryover buildup)

Note: Remote Startpulse and Inject commands work differently on QQQ and TOF/QTOF.

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MS acquisition for series of flow injections: Synchronize MS time segments with actual peak spacing


Example: MS time segments increment FragV in 10V steps

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Fragmentor and Collision Energy optimizationusing flow injection with injector programMethylmalonic acid, dibutyl ester


Fragmentor 60-160V 20V steps

Collision Energy 5-30V 5V steps

Maximize MH+ ion transmission, minimize CID with Fragmentor voltageMaximize product ion signal(s) with Collision EnergyCan fine tune each with smaller steps after initial experiments

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Fragmentor and Collision Energy optimizationusing flow injection with injector programAndrostenedione


Fragmentor 100-260V 20V steps

Collision Energy 5-45V 5V steps

Maximize MH+ ion transmission, minimize CID with Fragmentor voltageMaximize product ion signal(s) with Collision EnergyCan fine tune each with smaller steps after initial experiments

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Fragmentor and Collision Energy Determination with Optimizer software•Now included with MassHunter QQQ B.04 acquisition software

•Useful for frequent method development [new compound each week] or for multi-compound methods [e.g. toxicology, environmental applications]

•Many toxicology and pesticide transitions are available in new Agilent application kits and from Agilent application chemists.

•Can use Flow Injection before LC separation is developed

•Can use LC method to optimize multiple compounds in a few injections


MassHunter Optimizer – method type, parameter step selection


MassHunter Optimizer – precursor ion selectionUser-selectable adducts (H, Na, K, OAc- , HCOO-, etc.)Can run both positive and negative ion methods in same Project


MassHunter Optimizer – product ion selection and rejection(set mass range, minimum abundance)


MassHunter Optimizer user interface: Compound list - can import from Excel file or DB


Optimizer resultsAutomatically added to MRM database


MassHunter Optimizer – interactive parameter selection


MassHunter OptimizerResults for the project can be viewed immediately after run:precursor and product ions, Fragmentor and Collision Energies


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MassHunter OptimizerBrowse compounds in Acquisition for importCompounds can be

filtered by project, group, polarity or date


Compounds can be searched for by name or formula

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MassHunter OptimizerBuild LC/MS/MS method from compound database

• Select compounds in database after filtering• Select transitions for each compound (all or individual)• Easily import into template LC/MS method


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MassHunter Optimizer for Peptides


MassHunter Optimizer for Peptides – add and select multiple charge states


MassHunter Optimizer for Peptides


Peptide sequence used instead of compound name/formula

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Optimization of MRM Acquisition

•Good quantitation requires adequate number of data points across each peak (ideally 10-20)

•High confidence or regulated identification requires > 1 MRM per compound.

•Monitoring many compounds simultaneously lowers dwell time per MRM.

•Therefore for best sensitivity, only monitor compounds in retention time window where they elute.

•Traditional approach of time segments has limitations and is tedious to setup up and maintain.


Peaks widths with UHPLC can be 2 seconds or less> ten uniform measurements across peak REQUIRED for good quantitation

Page 31

W = 2.1 sec

Cycle time for several MRMs < 115 ms

Avg. W1/2 = 0.72 sec

MH QQQ Users workshop 1/25/2012

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Peak Capacities are very high with UHPLC 40 MRM Transitions in 1 minute time window


The solution: MassHunter Dynamic MRMIncluded in QQQ Acquisition B.02.01and later

For applications requiring quantitation of 100 – 1000 compounds in one run; some examples:• Food and environmental analysis (e.g. pesticides)• Targeted quantitation of proteins via peptides (proteomics)

WITHOUT Dynamic MRM:• Need to manually set up multiple time segments to maximize dwell times• Tedious to set up; problematic if changes in retention times

WITH Dynamic MRM:• Automatic setup of overlapping time segments without user intervention• Fewer MRMs per unit time results in longer dwell time => incr sensitivity• Unaffected by minor chromatographic time shifts


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Dynamic MRM: wide time windows for each transition

"Delta Ret Time" = 0.2 min (12 sec)

0.03min


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Dynamic MRM: smaller time windows for first 3 peaks


0.03min


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Dynamic MRM for 6400 Series Triple Quads monitors transitions only when compounds elute

1. # Concurrent MRMs fewer than with time segments more data points across each peak2. Allows longer dwell times better sensitivity, S/N

Cycle Time:Concurrent MRMsDwell Time:


Acquisition setup with Dynamic MRM:No time segments, instead

Retention Time and "Delta Ret Time" [MRM time range]


Dynamic MRM transitions – 100-compound Drug Screen Partial list (200 DMRMs in total, 5-min run time)

CompoundName Precursor Q1-Res Product Q2-Res Frag CE RT RT Delta IonPolarity7-aminoclonazepam 286.0 unit 222.0 unit 159 25 1.581 0.2 Positive7-aminoflunitrazepam 284.1 unit 135.1 unit 159 25 1.500 0.2 PositiveClobazam 301.1 unit 259.0 unit 138 17 3.011 0.2 PositiveClonazepam 316.1 unit 270.1 unit 158 25 2.923 0.2 PositiveFlunitrazepam 314.1 unit 268.1 unit 158 25 2.951 0.2 PositiveFlurazepam 388.2 unit 315.0 unit 158 21 2.755 0.2 PositiveLorazepam 321.0 unit 275.0 unit 113 21 3.033 0.2 PositiveLormetazepam 335.0 unit 289.0 unit 143 21 3.122 0.2 PositiveMedazepam 271.1 unit 91.1 unit 107 41 3.324 0.2 PositiveMidazolam 326.1 unit 209.0 unit 154 33 3.406 0.2 PositivenorDiazepam 271.1 unit 165.0 unit 117 21 3.325 0.2 PositiveOxazepam 287.1 unit 241.0 unit 143 21 3.032 0.2 PositiveOxazolam 329.1 unit 271.0 unit 123 21 3.316 0.2 PositivePrazepam 325.1 unit 271.0 unit 123 17 3.408 0.2 PositiveTemazepam 301.1 unit 255.0 unit 102 21 3.095 0.2 Positive7-aminoclonazepam 286.0 unit 121.1 unit 159 25 1.581 0.2 Positive7-aminoflunitrazepam 284.1 unit 256.3 unit 159 25 1.500 0.2 PositiveClobazam 301.1 unit 224.1 unit 138 17 3.011 0.2 PositiveClonazepam 316.1 unit 214.0 unit 158 25 2.923 0.2 PositiveFlunitrazepam 314.1 unit 239.1 unit 158 25 2.951 0.2 PositiveFlurazepam 388.2 unit 317.1 unit 158 21 2.755 0.2 PositiveLorazepam 321.0 unit 303.0 unit 113 21 3.033 0.2 PositiveLormetazepam 335.0 unit 317.0 unit 143 21 3.122 0.2 PositiveMedazepam 271.1 unit 207.1 unit 107 41 3.324 0.2 PositiveMidazolam 326.1 unit 223.1 unit 154 33 3.406 0.2 PositivenorDiazepam 271.1 unit 140.0 unit 117 21 3.325 0.2 PositiveOxazepam 287.1 unit 269.0 unit 143 21 3.032 0.2 PositiveOxazolam 329.1 unit 140.0 unit 123 21 3.316 0.2 PositivePrazepam 325.1 unit 140.0 unit 123 17 3.408 0.2 PositiveTemazepam 301.1 unit 283.0 unit 102 21 3.095 0.2 Positive


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Dynamic MRM – Update Method in Acquisition


Update RTs, RT windows, add new compounds from acquired QQQ datafile.


Some typical narrow peaks with Agilent UHPLC Dynamic MRMs with 6400 Series QQQ

Avg W1/2 = 1.3 sec 35 points across RSD [%] = 1.8




Verapamil, 0.75 min

Buspirone, 0.40 min

Dextromethorphan, 0.53 min

Diclofenac, 1.04 min


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Overlaid EICs of 10 Benzodiazepines in Oral Fluid

10 pg on-column


0.03min


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8 Minute Dynamic MRM Analysis with 6460 QQQ- 250-compound Pesticide Screen.

2x10

0.4

0.6

0.8

1

1.21.4

1.6

1.8

2

2.22.4

2.6

2.8

3

3.23.4

3.6

3.8

4

4.24.4

4.6

4.8

5

5.25.4

5.6

5.8

6

6.26.4

6.6

6.8

7

7.27.4

7.6

7.8

8

8.28.4

8.6

8.8

9

9.29.4

9.6

3.193427

1 1

Counts vs. Acquisition Time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4

500ppt, dynamic MRMRetention Time Window = 12 secPeak widths ~ 1 second

Zorbax Eclipse Plus C182.1 x 100mm (1.8μm)


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Summary – Optimization of QQQ Acquisition

•Source optimization: flow and compound dependent parameters

•Agilent Jetstream Technology source: increased sensitivity vs regular ESI source if parameters optimized correctly:

– Sheath gas temperature and flow are most important– Other parameters have less effect on response– Need less drying gas flow and temperature, but keep capillary clean

•Dynamic MRM is available for all 6400 models– More data points across peak– Longer dwell times for better sensitivity, S/N– Easier to set up and maintain than time segment methods


MassHunter Quantitative SoftwareReview and Quant Method OptimizationTopics • Method setup from acquired data• Method re-use and updating• Peak detection optimization• Peak identification troubleshooting


Important MassHunter Quant concepts and rules:Batches•A Batch is a file which contains all the Quant results from a set of data files AND the Quant method used. Very convenient for backup and moving data around.

•All the data files in a Batch must reside in a single directory, so put them all together before creating New Batch.

•Select the data directory BEFORE naming the new Batch

•Using the Browse to Copy Samples button when creating the Batch can be dangerous: you will have two copies of the same files (one with Quant results and one without)!


Important MassHunter Quant concepts and rules:Quant methods•THE most common method problem: Calibration/QC level names in Batch and Quant method do not match, e.g.

– Batch: levels are 1,2,…5, QC-Lo, QC-Hi– Method: levels are L1, L2…L5, QC-Low, QC-High

•2nd most common problem: a hidden column in the Method with a key parameter, e.g. Criteria in MRM Compound Setup for peak selection, or Ion Polarity if method created manually.•Many Quant parameters can be copied between compounds with Apply to All button.

•When in doubt, Right-Click to look for convenient features and shortcuts, like Fill Down or Fill Column.


MassHunter Quant prior to B.04.00 optimizing target compound identificationMake the Criteria column visible in MRM Compound Setup:


Using the default of Greatest Response may result in the wrong peak being chosen as the target compound:

Endogenous interference withwrong retention time incorrectly

identified as tramadol(no qualifier)

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MassHunter Quant – B.04.00 and lateroptimizing target compound identificationThe Criteria column is now visible in Compound Setup:


The default is now “Close RT”

Endogenous interference withwrong retention time incorrectly

identified as tramadol(no qualifier)

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MassHunter Quant –optimizing target compound identificationChange the Criteria to be more specific:


This might still not be selective enough, so specify how close the retention time must match the calibrated RT with Non-reference Window parameter in Globals:

Now the offending peak with the wrong RT is ignored:

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MassHunter Quant –optimizing target compound identification


Qualifying the Qualifier:For qualifiers, you can ensure that the qualifier and the quant ion have the same retention time (i.e. line up properly as they should if coming from the same chromatographic peak), by setting the Correlation Window in Globals correctly. The default of 2 minutes isWAY too large.

The Ignore Peaks Not Found checkbox will prevent reporting "Amount=0.00" for target compounds not present with your criteria.

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MassHunter Quant –Setting a reporting threshold:


You can also make Quant ignore peaks that meet these criteria but are below your desired detection or reporting threshold, or that really are background (if you can’t use Qualifiers). Set a Peak Filter (area reject) value in Advanced Tasks …Integration Parameters.

You can have the same Peak Filter for all compounds with Apply to All, or specific values for each compound. You can also limit integration to the N largest peak(s), like only one in this example.

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MassHunter Quantitation Integrators:


Agile – available since Quant B.04.00• No parameters• for MS and MSMS data• requires only 12 data points• default integrator

MS/MS series– No parameters, requires 64 data points in window

General – set parameters

Universal – set parameters

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Features added in MassHunter Quant

Software Enhancements (some are instrument-specific)

•Compound Math •Metrics plot (e.g. for IS areas)•Support for QQQ dynamic MRM data•More integrator choices•Fixed graphics to lowest or highest calibration level•Signal to noise setup•Continuing calibration•Dynamic background subtraction•Standard addition calibration•Compound library setup and searching•Peak purity and deconvolution•More choices in the Generate Reports dialog (e.g. no graphics)

•Direct Link to Getting Started Videos and Familiarization Guide


What's new in MassHunter Quant B.05.00(just type 'What's new' in the Online Help!)


Important features of Quant B.04 and B.05•B.05.00 compounds can have different number of calibration levels

•B.05.00 Reference library comparison for triggered MRM data

•B.05.00 Can display overlaid extracted ion chromatograms for sample chromatogram

•Agile Parameterless Integrator

•Compounds-at-a-Glance results reviewer

•Custom property settings for all graphic windows

•New outliers

•Faster report generation

•Single Sample/Compound reports

•“Fast” report templates

•Scripts – Food Safety ID Point System, and Calculate MDL, LOQ, and LOD





Compounds-at-a-Glance


Compounds-at-a-Glance allows the user to view many chromatograms for samples/compounds at the same time:

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An out-of-limit outlier is highlighted in red:

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Pop up manual integration tool:

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New Report Generation Dialogue Box


•Remembers the last template selected – just click “Ok”•Can move immediately to Advanced Reporting options•Only one template may be selected•Option to report by :

•Batch (all samples reported in a single file)•Single sample reports for selected samples (each sample selected in BAG reports in a single sample report file)

•Starts Queue Viewer unless unchecked

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Final recommendation for learning MassHunter Quant:


Uses data files included with the software

1/25/2012

Documents

MH QQQ Users workshop rev3 - Chemical Analysis, … 316.1 unit 270.1 unit 158 25 2.923 0.2 Positive Flunitrazepam 314.1 unit 268.1 unit 158 25 2.951 0.2 Positive Flurazepam 388.2 unit