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LC-MS/MS Practical Mass Spectrometry Training Track: Understanding the Importance of System
Suitability Testing for Monitoring Instrument Health
P H I L I P S OBOLESKY, P H . D.
U C SAN D I EGO H EALT H SYSTEM
Center for Advanced Laboratory Medicine
MSACL Website Resources
What is a System Suitability Test (SST)?
• Tool for monitoring LC and MS health status– General
– Assay Specific
• Sample injected onto LC-MS instrument before submitting patient sample batch
Acceptable SST Provides Assurance
SST Does Not Assure Everything
Ordering Errors
Collection Errors
Labeling Errors
Transport Issues
Processing & Handling
Storage
Reporting ErrorsPre- and Post-Analytical
Errors Still Apply
What Should be Monitored During a SST Run?
Peak Symmetry
Background Noise
Peak HeightSignal
Retention Time
Signal
Noise
1. 2.Retention
TimetR (± 0.2 min)
3.Peak Area
4.Peak Symmetry
tR
± 0.2 min
Example of SST Tracking Data
Why Develop and Run a SST
• Increased Efficiency by Avoiding• Re-extractions
• Re-injections
• QNS reports
• Identify Problems sooner
• Tracking records help to identify• Proper maintenance (column or guard replacement, MS
cleaning etc.)
• Convincing vendor to perform PM when MSMS infusion is “within spec”
SST Troubleshooting Cases
PROBLEM ANALYSIS SOLUTION
Case 1: Importance of Tracking Assay Specific SST Data
• 25-OH Vitamin D SST passes after annual PM (service rep leaves)
• Benzodiazepine SST ran next day and FAILS.
Normal Benzo SST
Benzo SST after PM25-OH VitDSST after PM
?
Case 1: Important Differences Between the Assays
25-OH Vitamin D Benzodiazepine
1. Column 2.1x75 mm, 2.7 μm particle size, C18, HSS T3, 35°C
2. Isocratic LC method
3. Average Column Pressure ~5000 psi
1. Column 2.1x50 mm, 1.7 μm particle size, C18, BEH, 40°C
2. Gradient LC method
3. Average Column Pressure ~7500 psi
Mobile Phases Identical
Case 1: Resolution & Lessons Learned
• Problem: Leaky Check valve at pressures >7000 psi
• Solution: Replace check valve
• Lessons Learned:1. A single SST on a “within spec” instrument does not mean its okay
for all assays.
2. Run all assay SSTs on an instrument after a PM before letting engineer leave.
Case 2: Spotting Column Degradation using SST Data
Normal Conditions >50 Injections
Case 2: Developing a SST to Assure Acceptable Chromatographic Resolution
1. Make Neat SST solution containing more Oxazepam than Desalkylflurazepam
2. If unacceptable – Change the guard and/or column
Unacceptable Resolution
Overlay 289>140 MRM QC 10ng/mL Desalkylflurazepam detecting the Desalkylflurazepam peak
Acceptable Resolution
289>140 MRM Desalkylflurazepam detecting Oxazepam
289>140 MRM Desalkylflurazepam detecting Desalkylflurazepam
Case 2: Resolution & Lessons Learned
• Problem: Poor chromatographic resolution due to sample buildup
• Solution: Run SST that assures peak resolution acceptable
• Lessons Learned:1. Some post-validation issues can be addressed with SST preventing
revalidation
Case 3: Troubleshooting Blank SST TIC• Blank SST Chromatogram Empty vial?
Was the vial pierced?
Run errors?
Key Points to Remember about SST
1. A single SST on a “within spec” instrument does not mean its okay for all assays.
2. Run all assay SSTs on an instrument after a PM before letting engineer leave.
3. Some post-validation issues can be addressed with SST preventing revalidation
4. Start by checking the obvious when troubleshooting
AcknowledgementsThanks to the staff of the UCSD Toxicology/Mass Spectrometry Laboratory who solved and documented this troubleshooting case:• Judy Stone, MT(ASCP), Ph.D., DABCC
• Josh Akin
•Heather Hochrein
•Dawn Francisco
•Robert Fitzgerald, Ph.D., DABCC
•UCSD Clinical Chemistry Fellows• Imir Metushi, Ph.D., ABCC
• Jacqueline Hubbard, Ph.D.
https://www.surveymonkey.com/r/7WQPWJC
Please let us know what training resources you need
[email protected] [email protected]
Speaker and Presentation Evaluations for Sobolesky/Akin/ Metushi Survey Monkey
Zen and the Art of LC-MS/MS Maintenance Assuring Optimal Instrument Performance
Joshua AkinUC San Diego Health
DisclaimerThis talk should in no way be associated with that great body of factual information relating to orthodox Zen Buddhist practice,
though it is very factual in chromatography and mass spectrometry.
Overview and ObjectivesGoal: Assure optimal and reliable performance of your LC-MS/MS instrument
Understanding the importance of regular maintenance
Develop a checklist to aide in the tasks and intervals required of instrument maintenance
Why is Maintenance so ImportantIrv Gordon’s 1966 Volvo 1800S w/ 3,039,122 miles
"Just follow the owner's manual; it's a piece of machinery and can't take care of itself. Have its scheduled maintenance
completed… and when it makes a funny noise, listen to it."
Monitor gauges
Change oil
Check pressure
Clean Intake
Top-off fluids
Why is Maintenance so Important
LC-MS/MS instruments are no exception
Monitor gauges
Change oil
Top-off fluids
Check pressure
Clean Intake
Maintenance Checklists –A Roadmap to Success
LC-MS/MS instruments are no exception
Where to Begin?CLSI Document C50-A “Mass Spectrometry in the Clinical Laboratory: General Principles and Guidance - Approved Guidelines”
CLSI Document C62-A – “Liquid Chromatography-Mass Spectrometry Methods - Approved Guidelines” No Guidance on
Maintenance!
Where to Begin?CLSI Document GP31-A – “Laboratory Instrument Implementation, Verification, and Maintenance; Approved Guidelines”
◦Provides guidelines on:
◦Establishing procedures
◦Proper documentation
◦Responsibilities
Not Specific to LC-MS/MS Instrumentation!
Where to Begin?Manufacturer Documentation
◦Maintenance schedules
◦Procedures
◦Troubleshooting
For use only as GUIDANCE to prepare a procedure
SPECIFIC to your laboratory
READ
THE
MANUAL!
Before Moving ForwardAdequate source of power/backup (UPS)
Data repository/server
Gases (i.e., nitrogen, argon) are properly supplied
LC-MS/MS grade reagents and ultrapure H20
Service contract – more extensive service
Start with the Basics
Start with the Basics
Start with the Basics
1) Month and Year2) Day of the Month3) Frequency4) Tech Initials
1
2
3
4
Maintenance intervalsDaily
Weekly
Monthly
Yearly
As needed
Daily Maintenance
Is there enough mobile phase for start-up and analysis?
Assure adequate volume for duration of analysis
Can be approximated:
◦ flow rate (ml/min) X length of run (min)= volume needed
• “Drying up” fluidics system can damage pumps
Mobile Phase Reagents
Daily Maintenance
Are the instrument vacuum readings within acceptable range?
Are the turbo pumps operating as expected?
Vacuum Monitoring
0.00E+00
5.00E-05
1.00E-04
1.50E-04
2.00E-04
2.50E-04
1 4 7 1013161922252831 3 6 9 12151821242730
Vac
uu
m (
torr
)
Collision Cell Vacuum
0.00E+00
5.00E-04
1.00E-03
1.50E-03
2.00E-03
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61
Vac
uu
m (
torr
)
Ion Source Vacuum
Tracking longitudinally can give insight into problems
Leak coming from source enclosure
Interruption in the argon supply
Date
Date
Daily Maintenance
Was system suitability performed on this instrument?What column/method was used?
See Phil’s Lecture!
Weekly Maintenance
Were the interface cones properly cleaned?
Cleaning Sample ConesDepends on the degree of contamination
• Regular use:
1:1 MeOH/H20
• Grossly contaminated: 45:45:10 MeOH/H20/ Formic Acid
• 10 min sonication• Blow dry
Weekly Maintenance
Was the computer rebooted and disk cleanup performed?
Reboot removes “Operating Footprint”
◦ Background processes◦ Hoards valuable system resources (usually
RAM)
• Disk Cleanup frees disk space on HD
– Consult with IT for specific files
Reboot and Disk Cleanup
Weekly Maintenance• Was the oil level
checked on the roughing pumps
• Were the pumps ballasted?
Pump Ballast and OilBallast Valve: purging pump oil of condensate (water/solvent vapors)
◦ Recommended: 15-30 minutes at operating temperature
Ballast Valve
Pump Ballast and OilCheck oil level and note color
◦ Change every 6 months or sooner
Weekly Maintenance
Were LC guard columns replaced?
Guard columnsChange at regular time or injection intervals
Inspect ferrule and replace if needed
Minimize dead-volume by assuring tight connections
Column Guard column
Flow Path
Monthly Maintenance• Is data backed-up
properly?• Are security
updates current?
Data Back-upReal-time Server-based (LIS Intranet, LAN)
PeriodicScope? (full vs. incremental)
Frequency?
Backup media (tape, floppy, disk, etc.)
Windows Security Updates
• Security or Critical updates: malware and security exploits.
• Other updates: typically unrelated to security or enhance functionality.
Friday May 12th, 2017
Key PointsRoutine maintenance is the cornerstone of a happy, healthy LC-MS/MS
Craft a checklist that is suited for your particular lab
Implement a maintenance procedure that assures the reliability and longevity of your instrument.
AcknowledgementsUCSD Toxicology/Mass Spectrometry Laboratory
◦ Rob Fitzgerald, Ph. D., DABCC◦ Judy Stone, MT (ASCP), Ph. D., DABCC◦ Heather Hochrein, MT (ASCP)◦ Dawn Francisco, MT (ASCP)
Questions? Need help?
Thank you!
Choosing the right internal standard for LC-MS/MS
Imir G. Metushi (PhD, DABCC, FAACC)
Director, LabCorp (Endocrine Sciences)
Calabasas CA
2
Disclosures
None
3
Acknowledgments
The following slides include troubleshooting cases documented by LabCorp, Burlington and RTP
Slides are courtesy of Russ Grant and Brian Rappold (LabCorp, Burlington and RTP).
4
Outline
What is the value of an internal standard in LC-MS/MS
How do we use it
What can go wrong
External Calibration
“known” amounts of analyte
1 5
10 50
? ?
?
External Calibration Samples
Unknown Test Samples ?
1 5 10 50
External Calibration curve
Unknown Samples
? ? ? ?
Total Efficiency
Total Efficiency
Analyte must have identical matrix effects and extraction efficiency (i.e., total efficiency) between calibrators and samples for accuracy
External Calibration can demonstrate recovery differences
Hydrolysis (100% Efficiency)
Hydrolysis (50% Efficiency)
CA
LIB
RA
TO
RS
S
AM
PL
ES
-50% Bias
What is an Internal Standard
An internal standard in analytical chemistry is a chemical substance that is added in a constant amount to samples, the blank and calibration standards in a chemical analysis. This is done to correct for the loss of analyte during sample preparation, injection and ionization.
Source: wikipedia
S L I D E
8
Internal Standards
UTILITY:
Normalization of recovery differences
Injection variance
Identification of analyte retention time shift
• And peak shape
Normalization of ionization effects between calibrators and samples
One of the most valuable components in LC-MS/MS analytical quality
Retinol D4 Retinol
0.05 0.15 0.25 Time, min
5.0e4
1.0e5
1.5e5
2.0e5
Inten
sity, cps
0.35
1.0e5
2.0e5
3.0e5
Inten
sity, cps
0.05 0.15 0.25 Time, min
0.35
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
Inten
sity, cps
0.05 0.15 0.25 Time, min
0.35
5.0e4
1.0e5
1.5e5
2.0e5
Inten
sity, cps
0.05 0.15 0.25 Time, min
0.35
Retinol D4 Retinol
Good IS informs what the analyte retention time and peak shape should be Allows diagnosis of problems
9
What Does the IS Tell You Qualitatively
Qc1 (injection #12) Qc1 (injection #94)
10
IS Peak Area Trend: Outliers and Drift
Tecan Liquid handler IS addition Using same 8 tips with aqueous D4-Cortisol
Drift across run observed
IS peak area precision enables outlier detection
With 0.1% BSA (aq) D4-Cortisol solution and Pre-wetted tips (x3) prior to dispensing
IS peak area CV = 8.6%
External Calibration with Internal Standardization “known” amounts
of analyte
1 5
10 50
? ?
?
External Calibration Samples
Unknown Test Samples ?
External Calibration Samples
Unknown Samples
Total Efficiency
Total Efficiency
1 5 10 50
? ? ? ?
D[A/IS] = 0
Difference in the matrix effects and extraction efficiency (total efficiency) incurred by the Analyte between calibrators should be identical to the difference incurred by the Internal Standard
DA=DIS
DA=DIS
External Calibration with Poor Internal Standardization
Hydrolysis (100% Efficiency)
Hydrolysis (50% Efficiency)
CA
LIB
RA
TOR
S SA
MP
LES
+Labeled-IS
50% Bias
External Calibration with Good Internal Standardization
Hydrolysis (100% Efficiency)
Hydrolysis (50% Efficiency)
CA
LIB
RA
TO
RS
S
AM
PL
ES
0% Bias
+Labeled-IS
Internal Standards: Needs and issues
NATURE:
Structurally unique (exogenous) – Not observed in samples
Resolved (separated) from analyte(s) by MS, but Co-elutes
Stable labeled isotope (2H, 13C, 15N, 18O) - +3 amu*, pure
Structurally similar (analog) – No recovery or matrix effect correction
Structurally dissimilar - injection check at best?
XIC of +MRM (77 pairs): 150.1/87.1 Da ID: Lysine D3 from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Turbo Spra... Max. 9.4e5 cps.
2 4 6 8 10 12 14 16 18 20 22 24 26Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.0e5
8.5e5
9.0e5
9.4e5
Inte
ns
ity
, c
ps
12.57
19.03
D3-Lysine (m/z): 150-87 Tr = 18.97
XIC of +MRM (77 pairs): 150.1/104.1 Da ID: Methionine from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Turbo Sp... Max. 3.8e5 cps.
2 4 6 8 10 12 14 16 18 20 22 24 26Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
Inte
ns
ity
, c
ps
12.57
15.95
15.43
Methionine (m/z): 150 – 104 Tr = 12.57
XIC of +MRM (77 pairs): 153.1/107.1 Da ID: Methionine D3 from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Turb... Max. 1.6e5 cps.
2 4 6 8 10 12 14 16 18 20 22 24 26Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
Inte
ns
ity
, c
ps
12.57
13.68
D3-Methionine (m/z): 153 – 107 Tr = 12.57
XIC of +MRM (77 pairs): 153.1/88.1 Da ID: Glutamic Acid D5 from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Tur... Max. 1.9e5 cps.
2 4 6 8 10 12 14 16 18 20 22 24 26Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
Inte
ns
ity
, c
ps
13.68
13.54
D5-Glutamic Acid (m/z): 153 – 88 Tr = 13.68
XIC of +MRM (77 pairs): 119.1/73.1 Da ID: Proline D3 from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Turbo Spr... Max. 1.1e6 cps.
2 4 6 8 10 12 14 16 18 20 22 24 26Time, min
0.00
5.00e4
1.00e5
1.50e5
2.00e5
2.50e5
3.00e5
3.50e5
4.00e5
4.50e5
5.00e5
5.50e5
6.00e5
6.50e5
7.00e5
7.50e5
8.00e5
8.50e5
9.00e5
9.50e5
1.00e6
1.05e6
1.10e6
1.15e6
Inte
ns
ity
, c
ps
12.89
13.87 D3-Proline (m/z): 119-73 Tr = 13.84
XIC of +MRM (77 pairs): 118.1/72.1 Da ID: Valine from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Turbo Spray) Max. 1.3e6 cps.
2 4 6 8 10 12 14 16 18 20 22 24 26Time, min
0.00
5.00e4
1.00e5
1.50e5
2.00e5
2.50e5
3.00e5
3.50e5
4.00e5
4.50e5
5.00e5
5.50e5
6.00e5
6.50e5
7.00e5
7.50e5
8.00e5
8.50e5
9.00e5
9.50e5
1.00e6
1.05e6
1.10e6
1.15e6
1.20e6
1.25e6
Inte
ns
ity
, c
ps
12.89
14.15
Structurally Unique in specimens
Valine (m/z):118-72 Tr = 12.89
Internal Standards
NATURE:
Structurally unique (exogenous) – Not observed in samples
Resolved (separated) from analyte(s) by MS, but Co-elutes
Stable labeled isotope (2H, 13C, 15N, 18O) - +3 amu*, pure
Structurally similar (analog) – Limited recovery or ionization effect correction
Structurally dissimilar - injection check at best?
Too many Deuterons can hurt you
D10-Gabapentin 182.2→147.1
Gabapentin 172.2→137.1
XIC of +MRM (4 pairs): 172.200/137.100 Da ID: Gabapentin 1 from Sample 9 (Blank) of ASMS_Gaba_res5.wiff (Turbo Spray) Max. 4.4e4 cps.
0.69 0.70 0.71 0.72 0.73 0.74 0.75 0.76 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84 0.85 0.86 0.87 0.88Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
4.0e4
4.2e4
4.4e4
Inte
ns
ity
, c
ps
0.78
Fast LC: Co-elution
XIC of +MRM (4 pairs): 172.200/137.100 Da ID: Gabapentin 1 from Sample 9 (Blank) of ASMS_Gaba_res4.wiff (Turbo Spray) Max. 2.6e4 cps.
1.33 1.34 1.35 1.36 1.37 1.38 1.39 1.40 1.41 1.42 1.43 1.44 1.45 1.46 1.47 1.48 1.49 1.50 1.51 1.52Time, min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e4
1.9e4
2.0e4
2.1e4
2.2e4
2.3e4
2.4e4
2.5e4
2.6e4
Inte
ns
ity
, c
ps
1.43
Slow LC: Separation
1:1 Gabapentin:D10 IS in Clean Urine
D10-Gabapentin IS not Co-eluting
Under-labelled IS
11-Desoxycortisol Concentration (ng/dL) 400 800 1200 1600 2000
0
5
10
15 An
alyte Area / IS
Area
Calibration curve non-linear 3-log range for 11-Desoxycortisol
IS only 13C2 labelled (*)
Analyte isotopically contributes to IS -
Solutions:
Truncate to linear range assay neat and pre-dilute samples (two analysis!),
Repeat on dilution > mid point
Increase IS concentration or alternate IS
Clinically acceptable as is
* *
No
rmal
Abnormal (>156ng/dL)
S L I D E
19
Internal Standard
ADDITION:
FIRST step after mixing/pipetting sample (see Glucoronide IS example)
Solution ideally miscible with sample – to correct analyte recovery
Mixed well prior to extraction – Equilibrated identically to analyte
Internal Standard stability to be checked – In solution and H/D exchange in ion source
Reproducibly added (precise!) to samples, calibrators, QC’s except double blanks
Protein Precipitation – Serotonin Blood vs Serum
Spike & Recovery
Mix and Pipet 100uL Sample
Add 300uL D4-Serotonin IS in Acetonitrile
Mix and centrifuge 10 min each
Transfer to 96-well plate and inject
Internal Standard Equilibration in Samples Reverse Timing Study
S L I D E
21
Aliquots of sample pool plus IS equilibrated with mixing Decreasing amounts of time prior to extraction in parallel (i.e., reverse timing)
IS 120 min
IS 60 min
IS 30 min
IS 15 min
IS 5
Equilibration w/ Mixing Extraction
Plateau in A:IS ratio indicates IS has reached binding equilibrium Use double the minimum time!
Serotonin: IS Binding and RBC’s
S L I D E
22
Equilibration/Mixing Time (min)
An
alyt
e:IS
Rat
io
S L I D E
23
Internal Standard
ADDITION:
FIRST step after mixing/pipetting sample (see Glucoronide IS example)
Solution ideally miscible with sample – to correct analyte recovery
Mixed well prior to extraction – Equilibrated identically to analyte
Internal Standard stability to be checked – In solution and H/D exchange in ion source
Reproducibly added (precise!) samples, calibrators QC’s except double blanks – Hard to know with manual pipetting
Analyte observed in IS solution: H/D Exchange
Neat Dopamine D4 solution appeared to contain mostly Dopamine
Labelling is in an active region of the molecule
Deuteriums exchange with Protons in the APCI source
Solution: Used ESI
XIC of +MRM (6 pairs): 154.1/91.1 Da from Sample 12 (IS APCI) of H-D exchange.wiff (Heated Nebulizer) Max. 1.0e5 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70Time, min
0.00
5000.00
1.00e4
1.50e4
2.00e4
2.50e4
3.00e4
3.50e4
4.00e4
4.50e4
5.00e4
5.50e4
6.00e4
6.50e4
7.00e4
7.50e4
8.00e4
8.50e4
9.00e4
9.50e4
1.00e5
Inte
ns
ity
, c
ps
0.56
XIC of +MRM (6 pairs): 158.2/139.0 Da from Sample 10 (IS ESI) of H-D exchange.wiff (Turbo Spray) Max. 3.7e5 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.7e5
Inte
ns
ity
, c
ps
0.55
XIC of +MRM (6 pairs): 154.1/91.1 Da from Sample 10 (IS ESI) of H-D exchange.wiff (Turbo Spray) Max. 1133.3 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70Time, min
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1133
Inte
ns
ity
, c
ps
0.00
0.02
0.69
0.29
0.190.09
0.10
0.14 0.21 0.30
0.110.05 0.08 0.17 0.56
0.12 0.34 0.58
0.31 0.590.51
0.670.36 0.52 0.71
0.61 0.63 0.660.430.400.38 0.72
XIC of +MRM (6 pairs): 158.2/139.0 Da from Sample 12 (IS APCI) of H-D exchange.wiff (Heated Nebulizer) Max. 2.5e6 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70Time, min
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
1.1e6
1.2e6
1.3e6
1.4e6
1.5e6
1.6e6
1.7e6
1.8e6
1.9e6
2.0e6
2.1e6
2.2e6
2.3e6
2.4e6
2.5e6
Inte
ns
ity
, c
ps
0.55
D4-Dopamine IS
1e5 cps
Dopamine
2e6 cps
D4-Dopamine IS
4e5 cps
Dopamine, 1100 cps
IS in Water APCI Source IS in Water ESI source
It can get more complicated: just ask Russ
IS Peak Area vs Index (004102 DHEA)- "Mean" Regression ("No" weighting): mean = 8.11e+004 x (std. dev. = 1.44e+004)
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95Index
0.00
5000.00
1.00e4
1.50e4
2.00e4
2.50e4
3.00e4
3.50e4
4.00e4
4.50e4
5.00e4
5.50e4
6.00e4
6.50e4
7.00e4
7.50e4
8.00e4
8.50e4
9.00e4
9.50e4
1.00e5IS
Pe
ak
Are
a,
co
un
ts
D5-Phenylalanine Peak Area Response in a run
Final thoughts
S L I D E
26
Rule 1: Internal standards are NOT a universal accuracy fix.
Rule 2: Ensure adequate binding/equilibration for analyte and IS prior to extraction (time, pH, temperature, mixing)
Rule 3: IS stability during extraction relative to analyte will impact accuracy (e.g., H/D exchange).
Rule 4: IS recovery (and matrix effect for D-labeling) should be identical between calibrators and samples
Rule 5: Don’t abuse the IS – it is literally your best friend
27
Acknowledgments
Philip Sobolesky at UC San Diego
MSACL Committee
Russ Grant and Brian Rappold (LabCorp Burlington and RTP) for the slides
28