Exactive &

Q Exactive MS
Operational Qualification 1.1
Procedure & Worksheets

Revision E
© 2012 Thermo Fisher Scientific Inc. All rights reserved.

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protected and any reproduction of the whole or any part of this document is strictly
prohibited, except with the written authorization of Thermo Fisher Scientific Inc.

The contents of this document are subject to change without notice. All technical
information in this document is for reference purposes only. System configurations
and specifications in this document supersede all previous information received by
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Thermo Fisher Scientific Inc. makes no representations that this document

is complete, accurate or error-free and assumes no responsibility and will
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conflicting information between the two documents.

For Research Use Only. Not for use in diagnostic procedures.

Exactive & Q Exactive MS Operational Qualification 1.1
Procedure
 
Document Version History
Version Date Revision Author Summary of Changes

John
15 May 2009 A New document.
Jameson

John Updated legal disclaimer. Updated

1 August 2010 B
Jameson Introduction and OQ Scope.

Included Q Exactive support.

22 July 2011 C Brian Heft Added additional testing to support
Orbitrap capability.

20 September 2011 D Brian Heft Clarified the OQ test settings.

Re-clarified the OQ test settings to

16 January 2012 E Brian Heft be consistent with the IQ Acceptance
document.

Date Authored By:

Brian Heft
16 January 2012
Senior Regulatory Product Support Engineer

Justification Author

Date Reviewed By:

Bryan Shade
16 January 2012
Senior Regulatory Product Support Engineer

Justification Technical Review

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Date Approved By:

Dan Furlano
16 January 2012
Regulatory Support Manager

Justification Supervisory Approval

 
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MS Operational Qualification
Introduction
Documented evidence must be provided in order to demonstrate the
integrity of data collected and to validate the results obtained on
laboratory instrumentation.

Many laboratories achieve this by formal quality systems, which are

generally implemented in accordance with one or more of the following
recognized quality standards and other quality guidelines:

• Good Laboratory Practice (GLP)

• Current Good Manufacturing Practice (cGMP)
• ISO Guidelines
• USP

These standards are written in broad terms, to make them as widely

applicable as possible. All stipulate general requirements specifying
instruments must be fit for purpose, properly maintained, and
calibrated to national or international standards. The procedure used
for the Thermo Fisher Scientific qualification methodology is adapted
to these standards.

This procedure is intended to guide a trained and qualified person

through the Operational Qualification of Thermo Scientific Exactive
Series Hybrid Mass Spectrometer systems using the Xcalibur software
platform to include the following:

• Exactive
• Q Exactive

Recommended Documents
Use this document along with the Exactive or Q Exactive Operations
Manual (P/N: 1249360 or 1288120) and individual component
operators/service manuals in order to complete the OQ.

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Recommended Materials
• 1 liter MS grade water
• 1 liter MS grade acetonitrile
• MS grade methanol (MeOH)
• Formic acid
• Qualification Test Loop (25001-60001)
• 5 µL Injection Loop (00109-99-00023)
• Disposable transfer pipettes
• Two clean 1 L solvent bottles
• 1 mg/mL Buspirone specification solution
• Dilution vials
• Positive Ion Mode Calmix Solution (88323)*
• Negative Ion Mode Calmix Solution (88324)*

*Refer to the MS operating manual for detailed information.

Operational Qualification Scope

Hardware OQ is the process of establishing confidence through
documented evidence that a system operates within established limits
and tolerances according to written and pre-approved specifications.

Qualification testing is recommended:

• At initial installation or upon relocation of the instrument

• When a new module is added to an existing instrument
• After a firmware update
• After replacing a non-consumable hardware component that has a
serial number

OQ documents the following items:

• Qualification Engineer Information: Document the Qualification

Engineer information and signature.
• Customer and System Information: Document the customer
information. Record the specific system hardware type and serial
number.
• Specifications: Record a list of manufacturer recommended
specifications for ensuring that the system is operating as
expected.
• Instrument Operational Tests and Results: Test important
instrument functions to verify that the instrument operates as
intended by the manufacturer and required by the user. This is a
group of selected important instrument parameters for testing
according to the instrument's intended use.

 
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Overview of Tests
Instrument Parameter Description OQ Limits
Mass Calibration Passes:

(+)RMS error of the

calibration < 2.5 ppm
An automated mass with lock mass (524) and
calibration check is 5 ppm external mass
Mass Calibration (+/-) performed for accuracy.
positive/negative ions using
respective CalMix solutions. (-) RMS error of the
calibration < 2.5 ppm
with lock mass (514) and
5 ppm external mass
accuracy.

>62,000
Resolution of MRFA is Exactive
Mass Resolution (+/-) observed at m/z 524 at
Ultra High resolution. >87,000
Q Exactive

Signal-to-noise ratio of >8,000:1

caffeine at m/z 195 is Exactive
Dynamic Range (+) observed in Ultra High
resolution and at High >15,000:1
Mass Spec Q Exactive
Dynamic Range.

Checks the Stability of the

External Mass Calibration of
External Mass Calibration
the MS, in Positive Ion < 5.0 ppm
Stability (+)
Mode, over a sustained
period of time.

(+) Relative Peak Height

The performance of the HCD at m/z 138 > 15%
is checked by comparing the
HCD Performance intensity of caffeine in (+) Sum of Relative Peak
(+/-) positive ion mode Heights at m/z 138 and
if installed (Ultramark in negative 195 > 30%
mode) with and without
HCD fragmentation. (-) Relative Peak Height
at m/z 1305 > 50%

Three injections of
≥10:1
5 µL of 100 fg/µL Buspirone
Exactive
solution are injected into the
Signal-to-Noise
mass spec. The data is ≥100:1
collected and evaluated for Q Exactive
the signal-to-noise ratio.

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Mass Spec Signal-to-Noise (Sensitivity) Specification

A standard of Buspirone is injected three times, using an injection

volume of 5 μL for each injection and full MS scan mode. The relative
signal-to-noise ratio the target peak is averaged over the three
injections. The average signal-to-noise ratio is calculated and
compared to the set specifications.

Mass Calibration

A calmix solution is infused and automated mass calibration checks are

performed on positive/negative ions.

Mass calibration checks are performed in positive ion mode at m/z 74,
138, 195, 524, 1222, 1422 and 1622.

Mass calibration checks are performed in negative ion mode at m/z

265, 514, 1280, 1380, 1480, 1580, 1680 and 1780.

Mass calibrations are performed at m/z 200 at resolutions of 25,000

for the Exactive and 35,000 for the Q Exactive.

Mass Resolution

A MRFA peak is observed at m/z 524 at Ultra High resolution (100,000

for the Exactive and 140,000 for the Q Exactive).

Dynamic Range

Signal-to-Noise of caffeine at m/z 195 is observed in Ultra High

resolution (100,000 for the Exactive and 140,000 for the Q Exactive)
and at High Dynamic Range (3e6).

External Mass Calibration Stability

After a period of two hours, the RMS error of the external mass
accuracy is checked to verify stability.

HCD Performance (if installed)

The performance of the HCD is checked by comparing the intensity of

caffeine at m/z 138 with and without HCD fragmentation. In positive
mode the combined intensity of the fragment ion and its parent ion
(m/z 195) is also observed.

In negative mode the performance of the HCD is checked by

comparing the intensity of Ultramark 1621 at m/z 1305.

 
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Performing the Operational

Qualification
Preparing the system for Operational Qualification
Prior to performing the Operational Qualification, completion of a
preventative maintenance routine is highly recommended. It is also
recommended that any Factory Installation Acceptance steps (not
covered in the qualification procedures) be performed.

Loading the OQ Templates

The appropriate OQ templates will be automatically installed into the
C:\Xcalibur\Operational Qualification\Hardware folder when the
Qualification Installer program is run.

Please refer to document 25001-97305 Qualification Installer

Installation and Instructions for information on running the
Qualification Installer program.

A template file named Exactive Log.doc is located in the Tools

directory of the Qualification Installer CD, which can be used to collect
all report output into a single file prior to printing.

Preparing the OQ Worksheets

The worksheets for the Exactive or Q Exactive MS system Operational
Qualification are built into a single MS Excel workbook. The OQ
Worksheets are arranged on different MS Excel worksheets in the MS
Excel workbook to collect information on the various tests performed
during the Operational Qualification. The System Information tab
summarizes the tests specifications and results. The Qualification
Engineer will need to input data into the worksheets manually or
through predefined macro programs designed to quickly and
accurately import the data acquired during the Operational
Qualification.

The Exactive or Q Exactive MS System Operational Qualification

Worksheet will be referred to as “OQ Worksheet” from this point
forward.

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Note: If using Office 2007 or 2010, ensure that the file is saved in the
.xls format and not the default .xlsx format. To save in .xls format,
choose, Save As, and choose the file type, Excel 97-2003
Workbook (*.xls).

Qualification Engineer Information

Open the MS Excel file located in C:\Xcalibur\Operational
Qualification\Hardware\Report Template folder. This file is the
OQ Worksheet. Click on the tab labeled Executor Information.
Complete the Qualification Engineer information.

Customer Information
Click on the tab labeled Customer Information. Complete this
information.
System Information
Click on the tab labeled System Information. Click on the cell
marked Choose Mass Spec. Using the drop down list for this cell,
choose the mass spec that is to be qualified.
The Thermo recommended specifications for each Operational
Qualification test are listed in the column labeled Suggested OQ
Limits. If different OQ limits are desired, the column labeled Tested
OQ Limits can be updated to reflect desired limits.

Note: If a specific MS probe is not present or is not being tested,

enter N/A in the cell for the Tested OQ Limits for that particular
probe. N/A should also be entered into the appropriate cells on the
MS Signal-to-Noise tab.

Note: Thermo will not be able to support limits more restrictive than
Suggested OQ Limits. If Tested OQ Limits do not match the
Suggested OQ Limits this will be denoted by a highlighted field in the
Tested OQ Limits section.

Standards and Test Equipment

Click on the tab labeled Test Equipment and Standards. Enter the
information required for each standard, solvent, or test equipment to
be used in the OQ. Click on the Save button to save the changes to
the OQ Worksheets.

 
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Preparing Standards and Mobile Phase
Mobile Phase

1. Fill the bottle with HPLC grade water and label the bottle HPLC
Water.
2. Fill a bottle with a mix of 70:30 acetonitrile:water with 0.1% formic
acid. Label this bottle 70:30 acetonitrile:water with 0.1%
formic acid.

Standard Diluent

1. Fill a 10 mL scintillation vial with a mix of 75:25 MeOH:water with

0.1% formic acid. Label this vial Standard Diluent.

Preparation of Buspirone Sensitivity Test Solution

The procedures for preparing buspirone sensitivity test solution consist

of dissolving buspirone in methanol solution containing 0.1% formic
acid (Standard Diluent) and subsequent dilutions with 75:25
MeOH:water with 0.1% formic acid. While the stock solutions can be
stored in refrigerator for up to 6 months, the final test solution expires
in 24 hours. Alternative dilution procedures that result in a 100 fg/µL
buspirone in 0.1% formic acid MeOH solution with a reasonable level
of accuracy are acceptable.

1. Prepare 100 mL of a 1 mg/mL Buspirone Stock Solution, as follows:

a. Carefully weigh 100 mg of buspirone.
b. Transfer the 100 mg of buspirone into a 100 mL volumetric
flask.
c. Add methanol with 1% acetic acid until the 100 mL mark is
reached.
d. Mix the solution thoroughly.
e. Transfer the 1 mg/mL Buspirone Stock Solution to a clean
dry vial.
f. Label the vial 1 mg/mL Buspirone Stock Solution.
2. Prepare 1 mL of 10 ng/µL Buspirone Stock Solution as follows:
a. Pipette 10 µL of the 1 mg/mL buspirone stock solution into a
clean dry vial.
b. Pipette 990 µL of the standard diluent (75:25 MeOH:water
with 0.10% formic acid) into the vial.
c. Mix the solution thoroughly and label the vial 10 ng/µL
Buspirone.

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3. Prepare 1 mL of 100 pg/µL Buspirone Stock Solution as follows:
a. Pipette 10 µL of the 10 ng/µL buspirone stock solution into a
clean dry vial.
b. Pipette 990 µL of the standard diluent into the vial.
c. Mix the solution thoroughly and label the vial 100 pg/µL
Buspirone.

4. Prepare 1 mL of 1 pg/µL Buspirone Stock Solution as follows:

a. Pipette 10 µL of the 100 pg/µL buspirone stock solution into
a clean dry vial.
b. Pipette 990 µL of the standard diluent into the vial.
c. Mix the solution thoroughly and label the vial 1 pg/µL
Buspirone.

5. Prepare 1 mL of 100 fg/µL Buspirone Stock Solution as follows:

a. Pipette 100 µL of the 1 pg/µL buspirone stock solution into a
clean dry vial.
b. Pipette 900 µL of the standard diluent into the vial.
c. Mix the solution thoroughly and label the vial 100 fg/µL
Buspirone. This solution will be used for the signal-to-noise
test.

Calibration Solutions

For detailed positive/negative calibration test solution preparation

instructions (aka CalMix), refer to the Exactive and Q Exactive
operating manuals.

 
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Performing the Exactive or Q

Exactive Mass Spec Tests
If any of the following tests will not be performed (i.e., the probe is not
available), open the System Information tab of the OQ Worksheets
and enter N/A into the appropriate Tested OQ Limits cells for the
tests.
A template file named Exactive Log.doc is located in the Tools
directory of the Qualification Installer CD; it can be used to collect all
report output into a single file prior to printing.

Mass Calibration: Positive Ion Mode (+)

Prior to running calibrations or calibration checks, it is necessary to
ensure that the H/ESI ion source is stable and the correct Tube Lens
(Exactive) or S-Lens (Q Exactive) voltage is set by optimizing the
CalMix peak height distribution.
A complete calibration (positive ion mode) must be performed prior to
running the calibration checks to prevent the incorrect masses from
being utilized.
Make sure that the covers have been put onto the instrument and the
General Instrument State has been set to Standby for at least 90
minutes.
1. Through the Tune application open the Calibration dialog window.
2. With the CalMix infusing, run the Positive Mass Calibration Check
(this is called Evaluation in newer Tune versions).
3. When the test has completed, print the results of the test or copy
and paste into the Exactive Log.doc.
4. Record the results on the FTMS tab of the OQ Worksheets.

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Mass Resolution: Positive Ion Mode (+)
Resolution is inversely proportional to the square root of the m/z. The
resolution and peak shape are checked at m/z 524.
1. In the Instrument Control/Scan Parameters window set the
following:
• Exactive:
i. First Mass (m/z): 300
ii. Last Mass (m/z): 2000
iii. Resolution: Ultra High (100,000 @ 1Hz)
iv. AGC Target: Balanced (1e6)
• Q Exactive
i. Scan type: Full MS-SIM
ii. First Mass (m/z): 200
iii. Last Mass (m/z): 1000
iv. Resolution: Ultra High (140,000 @ 1.5Hz)
v. AGC Target: Balanced (1e6)
Note: the above settings may need to be adjusted to see the base
peak.
2. Zoom into the MRFA peak m/z = 524.
3. To display the resolution, right-click the Spectrum window and
choose Display options. Enable Display option, Show
Resolution.
4. Print the spectra or copy and paste into the Exactive Log.doc.
5. Record the results on the FTMS tab of the OQ Worksheets.
6. Expected results are as follows:
• 100,000 (Exactive):
i. Resolution at m/z 524 > 62,000
• 140,000 (Q Exactive):
i. Resolution at m/z 524 > 87,000
• Intensity of Shoulders (vs. main signal) < 4%

 
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HCD Performance: Positive Ion Mode (+), if installed
To check the HCD performance, compare the intensity of caffeine m/z
138 with and without HCD fragmentation.
1. In the Instrument Control/Scan Parameters set the following:
• Exactive:
i. First Mass (m/z): 70
ii. Last Mass (m/z): 300
iii. Max Inject Time (ms): 50
iv. Resolution: Ultra High (100,000 @ 1Hz)
v. AGC Target: Ultimate mass accuracy (5e5)
vi. Fragmentation: HCD: 20-30 eV
• Q Exactive
i. Scan type: MS/MS
ii. Precursor Mass: 195.1 Width = 2
iii. Scan range (m/z): 70-1000
iv. Max Inject Time (ms): 30
v. Resolution: Ultra High (140,000 @ 1.5Hz)
vi. AGC Target: 5e4
vii. Fragmentation: HCD: NCE = 10
2. For the Exactive:
• In the Instrument Status Tree, choose
Control/Settings/HCD Event. If needed, optimize HCD by
maximizing the peak height of the caffeine fragment at
m/z = 138.
• Fix the vertical scale in the Spectrum window and print or
copy both spectra (with and without fragmentation) into the
Exactive Log.doc.
3. For the Q Exactive:
• Fix the vertical scale in the Spectrum window and print or
copy the spectra with low fragmentation into the Exactive
Log.doc.
• Increase the NCE setting to increase the peak height of the
caffeine fragment at m/z= 138. Optimize the normalized
collision energy to increase the 138 fragment to a relative
peak height above 15%. The expected result is a NCE
between 30 and 40.
• Print or copy the spectra with high fragmentation into the
Exactive Log.doc.

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4. Record the results on the FTMS tab of the OQ Worksheets.
5. Expected results are as follows:
• Relative Peak Height at m/z 138 > 15%
• Sum of Relative Peak Heights at m/z 138 and 195 > 30%

Dynamic Range (+)

1. For Exactive (Optimize on caffeine m/z 195):
• In the Instrument Control/Scan Parameters set the
following:
i. First Mass (m/z): 90
ii. Last Mass (m/z): 1800
iii. Max Inject Time (ms): 30
iv. Resolution: Ultra High (100,000 @ 1Hz)
v. AGC Target: High dynamic range (3 e6)
• In the Instrument Control/ESI Source window set the
following:
i. Tube Lens Voltage: 90v
2. For Q Exactive (Use the SIM mode to isolate the m/z 195):
• In the Instrument Control/Scan Parameters set the
following:
i. Scan type: SIM
ii. Center Mass (m/z): 195
iii. Isolation width (m/z): 5
iv. Max Inject Time (ms): 1000
v. Resolution: Ultra High (140,000 @ 1.5Hz)
vi. AGC Target: High dynamic range (3 e6)
3. Vertically zoom into the noise band and print or copy a screenshot
into the Exactive Log.doc.
4. Determine the dynamic range (signal-to-noise) by dividing 100
(peak height of m/z = 195) by the peak height of the noise band.
5. Record the results on the FTMS tab of the OQ Worksheets.
6. Expected results are as follows:
• Exactive: Signal-to-noise at m/z 195 > 8, 000:1
• Q Exactive: Signal-to-noise at m/z 195 > 15, 000:1

 
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External Mass Calibration Stability (+)
Prior to running the external mass calibration stability check:
• Assure the Mass Calibration was performed at least 12 hours
after the system was baked out.
• Assure that the instrument electronics were on for at least 2
hours.
• Assure that the H/ESI ion source is stable.
1. In Instrument Control/Scan Parameters set the following:
• First Mass (m/z): 120
• Last Mass (m/z) : 2,000
• Max Inject Time (ms): 30 (20 for Q Exactive)
• Fragmentation: in-source CID: 20 eV
• Resolution: High
• AGC Target: Balanced (1e6)
2. The stability check runs for 2 hours. Be sure to fill the 500 μL
syringe pump and set the pump flow to 4 μL/min (240 μL/hour)
or less so that sufficient sample is available for a 2-hour run. It is
necessary to have stable H/ESI conditions.
3. Choose Control/Procedures/Evaluate/Extra Evaluation and
run Long-Term Mass Accuracy Test for 2 hours.
Note: these menu options may differ slightly in older versions of the
software.
4. When the test has completed, print the results of the test or copy
and paste into the Exactive Log.doc.
5. Record the results on the FTMS tab of the OQ Worksheets.
6. Expected results are as follows:
• RMS Error After 2 Hours < 5 ppm

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Mass Calibration: Negative Ion Mode (-)
1. Switch the instrument to negative polarity.
A complete calibration (negative ion mode) must be performed prior
to running the calibration checks to prevent the incorrect masses
from being utilized.
2. Through the Tune application open the Calibration dialog window.
3. With the CalMix infusing, run the Negative Mass Calibration
Check (this is called Evaluation in newer Tune versions).
4. When the test has completed, print the results of the test or copy
and paste into the Exactive Log.doc.
5. Record the results on the FTMS tab of the OQ Worksheets.

Mass Resolution: Negative Ion Mode (-)

Resolution is inversely proportional to the square root of the m/z. The
resolution and peak shape are checked at m/z 514.
1. In the Instrument Control/Scan Parameters window set the
following:
• Exactive:
i. First Mass (m/z): 300
ii. Last Mass (m/z): 1000
iii. Resolution: Ultra High (100,000 @ 1Hz)
iv. AGC Target: Balanced (1e6)
• Q Exactive
i. Scan type: Full MS-SIM
ii. First Mass (m/z): 300
iii. Last Mass (m/z): 1000
iv. Resolution: Ultra High (140,000 @ 1.5Hz)
v. AGC Target: Balanced (1e6)
Note: the above settings may need to be adjusted to see the base
peak.
2. Zoom into the MRFA peak m/z = 514.
3. To display the resolution, right-click into the Spectrum window and
go to Display options. Enable Display option, Show Resolution.
4. Print the spectra or copy and paste into the Exactive Log.doc.
5. Record the results on the FTMS tab of the OQ Worksheets.
6. Expected results are as follows:
 
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• 100,000 (Exactive):
i. Resolution at m/z 514 > 62,000
• 140,000 (Q Exactive):
i. Resolution at m/z 514 > 87,000
• Intensity of Shoulders (vs. main signal) < 4%

HCD Performance: Negative Ion Mode (-), if installed

To check the HCD performance, compare the intensity of Ultramark m/z
1305 with and without HCD fragmentation.
1. For Exactive:
• In the Instrument Control/Scan Parameters set the
following:
i. First Mass (m/z): 700
ii. Last Mass (m/z): 2000
iii. Max Inject Time (ms): 50
iv. Resolution: Ultra High (100,000 @ 1Hz)
v. AGC Target: Ultimate mass accuracy (5e5)
vi. Fragmentation: HCD: 40-50 eV
• In the Instrument Status Tree, choose
Control/Settings/HCD Event. If needed, optimize HCD by
maximizing the peak height of the Ultramark fragment at
m/z = 1305.
• Fix the vertical scale in the Spectrum window and print or
copy both spectra (with and without fragmentation) into the
Exactive Log.doc.
2. For Q Exactive:
• In the Instrument Control/Scan Parameters set the
following:
i. Scan type: Full-SIM
ii. Scan range (m/z): Center=1480 Width =8
iii. Max Inject Time (ms): 30
iv. Resolution: Ultra High (140,000 @ 1.5Hz)
• Fix the vertical scale in the Spectrum window and print or
copy the spectra with low fragmentation into the Exactive
Log.doc.

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• Change the scan parameters to the following:
i. Scan type: MS/MS
ii. Precursor Mass: 1480 Width = 8
iii. Scan range (m/z): 600-1484
iv. Fragmentation: HCD: NCE ~ 25
• Print or copy the spectra with high fragmentation into the
Exactive Log.doc.
3. Record the results on the FTMS tab of the OQ Worksheets.
4. Expected results are as follows:
• Relative Peak Height at m/z 1305 > 50%

System Setup for ESI Sensitivity Specification

The ESI Sensitivity Specification was not designed for use with nano LC
pumps and autosamplers. If nano LC components must be used,
adjustments may be needed for parameters such as flowrate, injection
volume, standard concentration and the signal-to-noise specifications.
1. Attach the Rheodyne 5 μL sample loop between ports 1 and 4 of the
Divert/Inject valve (See Figure 1) on the Exactive or Q Exactive.
2. Connect the tubing from the outlet of the pump to port 2 on the
Divert/Inject valve.
3. Attach the 0.005 inch ID red PEEK sample transfer line tubing from
port 3 of the Divert/Inject valve to the ESI grounded union for the
inlet of the MS (See Figure 2).
4. Attach the syringe needle liner/fitting/ferrule combination to port 5
of the Divert/Inject valve.
5. Connect a waste line from port 6 (port 6 is labeled with the
Rheodyne® logo) of the Divert/Inject to a waste container.
6. Using a 250 µL syringe, draw up at least 100 µL of the 100 fg/µL
buspirone solution for the Exactive or the Q Exactive.

 
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Figure 1. Injector Valve Plumbing for Sensitivity Specification

Note: If an external Divert/Inject valve is utilized, the flow connections
may instead be:
• 3-6 Loop
• 5 from LC pump
• 4 to ion source
• 1 injection port
• 2 to waste

Figure 2. Sample Transfer Tube Setup

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7. In the Tune software, put the system in the On mode.
Note: Utilize positive mode.
8. Set up the source parameters as follows for the Exactive
(or the Q Exactive):
• H/ESI – Source (MaxIon, metal needle kit installed)
• Spray Voltage: 3.5 – 4.0 kV
• Sheath Gas Pressure: 20-30 constant spray conditions
• Aux Gas Pressure: 6-12 constant spray conditions
• Capillary Temperature: 275 (320 for the Q Exactive)
• Define Scan: Full MS-SIM
• Scan range: 300 to 1000
• RES: Ultra High 100,000 (70,000 for the Q Exactive)
• AGC target: balanced 1e6 (3e6 for the Q Exactive)
• Max Inj Time: 250 ms or higher, if MaxInjTime is reached
permanently
• microscans: 1
• Polarity: pos
9. Additionally, for the Q Exactive:
• S-Lens: 55%
• Heater Temperature: 310
• Lock Masses: On; 445.12003
10.Set the Divert/Inject valve to the Inject position.
11.Set up the LC Pump to run 70:30 Acetonitrile:water at a flow rate of
200 µL/min.
12.Put the LC Pump in the Run mode.

Acquisition of ESI Sensitivity Data (manual injection)

1. Open the Acquire Data dialog box and set up the acquisition
parameters as:
• Folder:
C:\Xcalibur\Operational Qualification\Hardware\Data
• File Name: ESI_Buspirone_ddMonthyy
• Sample name: Buspirone
• Comment: ESI Sensitivity Test with Buspirone
• Acquisition Time: Continuously
2. Click on the Start button to begin the acquisition.
3. Overfill the 5 µL sample loop with the appropriate Buspirone
solution.
4. From the Divert/Inject Valve dialog, click on the Inject radio
button to make an injection. After the injection, click on the Load
button to move the injector back to the load position.
 
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5. Make three injections to show repeatability. Perform the first
injection at approximately the one minute mark. Wait at least one
minute between injections to facilitate the signal-to-noise
calculation.
6. Stop the LC pump.

Acquisition of ESI Sensitivity Data (optional for use with a

syringe pump)
1. Set up the following method in the instrument method editor:

MS Detector
• Acquisition time: 5.50 min
• Segment 1: 5.50 min duration at 0.00
• Tune file: C:\Xcalibur\Operational Qualification\Hardware\
Exactive_ Buspirone_Test_ESI.mstune or
QE_Buspirone_Test_ESI.mstune
• Lock masses: Off
• Event 1:
o Positive Mode
o Scans: 1
o R=100,000 (140,000 for the Q Exactive)
o AGC=1,000,000
o Inject=250.00 ms [300.0 - 1,000.0] (120 – 1800 for the Q
Exactive)
o Source CID: disabled
o HCD: disabled
• Syringe Pump
o In use
o 1: 5.50 min duration at 0.00 in On
o End of run: Pump will be stopped
o Syringe type: Unimetrics 250 µL
o Syringe diameter: 2.300 mm
o Flow rate: 10.000 µL/min
• Divert valves
Divert valve 1: in use
o 1: 0.99 min duration at 0.00 in Pos2
o 2: 0.51 min duration at 0.99 in Pos1
o 3: 0.99 min duration at 1.50 in Pos2
o 4: 0.51 min duration at 2.49 in Pos1
o 5: 1.01 min duration at 3.00 in Pos2
o 6: 0.50 min duration at 4.01 in Pos1
o 7: 0.98 min duration at 4.51 in Pos2
Divert valve 2: not driven

2. Set the LC pump program to pump 70:30 acetonitrile:water with

0.10% formic acid at 200 µL/minute.
3. Save the method as OQ_Exactive in the
C:\Xcalibur\Operational Qualification\Hardware folder.
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4. Create a sequence to run the OQ_Exactive method.
5. Save the sequence as OQ_Exactive to the
C:\Xcalibur\Operational Qualification\Hardware folder.

6. Start the sequence by clicking on Actions/Run Sequence.

7. Click on Change Instruments and remove the AS from the
instruments in use. The pump and the Exactive or Q Exactive should
all be marked in use as YES. The mass spectrometer should be the
Start instrument (YES).
8. Choose OK.

Determining Signal-To-Noise for the Sensitivity Data

1. From the Qual Browser, open the Sensitivity data file
C:\Xcalibur\Operational Qualification\Hardware\Data\
ESI_Buspirone_ddMonthyy.
2. Activate the chromatogram by clicking on the pushpin icon.
3. From the File menu, select Layout, then Apply, and browse to the
C:\Xcalibur\Operational Qualification\Hardware folder to
select the file:
• busp_sens.lyt for the Exactive
• Qex_senstest_woLC_layout.lyt for the Q Exactive
4. Record the signal-to-noise ratio for each injection on the OQ
Worksheets tab labeled MS Signal-to-Noise.
5. Print the data results or copy and paste into the Exactive Log.doc.
(landscape will display better).

 
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Sensitivity Specification Example

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Exceptions and Comments
1. If an exception is made during the qualification, the exception must
be recorded on Exceptions/Comments tab.
2. Open the OQ Worksheet and click on the tab labeled
Exceptions/Comments.
3. Record any exceptions, changes, or comments.
4. Click the Save button to save the changes to the OQ Worksheets.

Printing and Saving Data

1. Open the OQ Worksheets.
2. Holding down the Ctrl key, select only the tabs that are going to be
printed (the tabs will turn white when they are selected).
Note: In Office 2007 and 2010 it is difficult to see the tabs that are
selected, thus it is recommended that the Print Preview be viewed prior
to printing the selected sheets.
3. Select File, Print, Active Sheets (or use the print macro in the
Add-Ins menu).
4. Collect the workbook print outs and ensure that all pages have
printed:
a. Executor Information
b. Customer information
c. System Information
d. Test Equipment and Standards
e. MS Signal-to-Noise
f. FTMS
5. Ensure that all data chromatograms have been printed including:
a. MS Signal-to-Noise
6. Using a blank CD, save the contents of the director
C:\Xcalibur\Operational Qualification to the CD ROM. If an
Installation Qualification was performed at the same time, also save
the contents of the directory C:\Xcalibur\Installation
Qualification to the same CD ROM.

 
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Building Operational Qualification Report Binder

1. Place all documentation (paper and electronic) in the appropriate
section of the Qualification binder.
2. Documentation required is as follows:
a. Software Installation Qualification Procedure & Worksheets (if
conducted)
b. Software Installation Qualification documentation (if conducted)
c. Software Operational Qualification Procedure & Worksheets (if
conducted)
d. Software Operational Qualification documentation (if conducted)
e. Hardware Installation Qualification Procedure & Worksheets (if
conducted)
f. Hardware Installation Qualification documentation (if conducted)
g. Hardware Operational Qualification Procedure
h. Hardware Operational Qualification Worksheet pages
i. Hardware Operation Qualification data Chromatograms
3. Complete the information on the Qualification stickers, seal, and
place a sticker on each of the following:
a. Qualification binder (For Module serial number write Data)
b. Qualification data archival CD (For Module serial number write
Data)
c. Each module that was qualified

Returning the System to Original Configuration

1. Return the customer system to the configuration and state it was in
prior to the Qualification.

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OQ Completion
Required Documents
The following documents must be part of the OQ packet submitted for
customer review:

• This document with the completed OQ Worksheets

• Qualification Engineer’s Training Certificate(s)

Customer Review
• The OQ results should be reviewed by the Customer. If the
qualification is accepted, both the Customer and the Qualification
Engineer should sign the OQ Results, below.

• The OQ documentation becomes the property of the Customer.

OQ Results

Based upon the actual results obtained, this OQ Passed Failed the acceptance criteria described
in Operational Qualification Scope, above.

Qualification Engineer
I have completed all aspects of the operational qualification and have concluded that the system has been
successfully verified to be operating as required.

Printed Name Signature Date

Customer
I agree that all aspects of the operational qualification have been completed and that the system has been
successfully verified to be operating as required.

Printed Name Signature Date

 
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