THE BASICS OF

LC-MSMS
TROUBLESHOOTING:
TOOLS, CASES,
STRATEGY
JUDY STONE, MT(ASCP), PH.D., DABCC
UNIVERSITY OF CALIFORNIA, SAN DIEGO
HEALTH SYSTEM, MASS
SPECTROMETRY/TOXICOLOGY LABORATORY
 THANKS TO:
• Larry McAndrew, AB Sciex
• Judy Chang, Camilla Otten,
Kathy Chen at TPMG Kaiser
Regional Laboratories
• Rob Fitzgerald, Heather
Hochrein, Josh Akin, Krista
Pratico at UC San Diego
 OUTLINE
• Troubleshooting tools
• Cases
• Strategy and ground-
work, the infrastructure
necessary for better
troubleshooting (Appendix)
 TROUBLESHOOTING
(DIAGNOSTIC) TOOLS
1. Chromatographic images
2. Pressure traces
3. System Suitability Test (SST)
results and Maintenance
Calendar annotation
4. Acquisition data file records
5. Divide and Conquer
 1. CHROMATOGRAPHIC
IMAGES
• Peak Review vs XIC Overlay

• Peak Review – single analyte

+ I.S. with internal standard
(integrated & presented using
a data analysis method for
quantitation)
1. PEAK REVIEW EXAMPLES
Quantifier
MRM

Qualifier
MRM

I.S. Quantifier
MRM

I.S. Qualifier
MRM
Courtesy of Grace vanderGugten (St. Paul’s Hospital, Vancouver, BC
1.TIC OR XIC OVERLAY
• Unprocessed data (no
integration, no assignment
of MRM to analyte or I.S.)

• Multi-analyte, qualitative
view

• Within run or between runs

1. XIC OVERLAY EXAMPLE
 1. ADVANTAGES OF
USING XIC OVERLAYS
• Relative change in
abundance between analytes,
within run, between run
(needs correct settings)
• Relative change in retention
times
• Peak shape – early vs late Rt
 2. LC PRESSURE TRACES
!
• Low pressure = leaks
• High pressure = obstruction
• Pattern changes = timing of
the problem during the LC
program, identity/location of
the problem LC component
2. PRESSURE TRACE
EXAMPLES
 3. SST
• SST – instrument check with
unextracted (neat) standards
• 3-6 injections to track X & %CV for:
• Peak areas
Establish the
• Peak Rts “reference
ranges” for your
• Peak width & S/N raw signals &
detect
• LC starting pressure abnormalities
 3. MAINTENANCE
CALENDAR
• Inline filter, guard column,
column # of injections &
changes Chart the “signs”, “symptoms”,
and “treatment” for the system
• Daily LC & vacuum pressures
• Tubing, connection, part, mobile
phase, needle wash changes
• Any event out of the routine
 POLLING QUESTION #1

Does your laboratory run a

System Suitability Test (SST)
each day you report patient
results?
 4.
ACQUISITION
DATA FILES

Electronic record –
what parameters were
actually used to
acquire a file?
5. DIVIDE & CONQUER
• SST: Sample Prep vs
Instrument
• Unextracted standard post-
column infusion: LC vs
MSMS
• Maintenance calendar: Silly
human error vs instrument
5. LC DIVIDE & CONQUER
• Disconnect components
sequentially & check
flow/pressure to find leaks
OR source of overpressure
• $64,000 question – what is
normal pressure for that
segment?
 CASES
• Peak shape
• Peak area/Peak Height
• Peak Retention Time (Rt)
PEAK SHAPE CASE 1
Quantifier
MRM

Qualifier
Normal
MRM

I.S.
MRM
PEAK SHAPE CASE 2
Morphine
Overlay
peakofafter
two Low QC injections
fix
for an opiates method
Hydromorphone
Oxymorphone

Morphine
peak
before fix
POLLING QUESTION #2
Would you change the guard
column or column to solve
this problem (without any
additional information)?
 PEAK SHAPE CASE 3

THC-COOH Normal
Quantifier
MRM

Qualifier
MRM

I.S.
MRM
 WHAT WOULD CAUSE
ABNORMAL PEAK SHAPES ?
(ALL SAMPLES)

1. Column, guard column or in-line filter frit aging

2. Column, guard column or in-line filter frit

changed (poor column connection created)

3. New tubing or fitting installed post-column

(poor post-column connection created)

4. Wrong injection solvent (too high % organic)

or too much volume injected (column
overload)

1. Wrong mobile phase, needle wash or column

 WHY - FRIT OR COLUMN AGING?

Uneven flow Head of

leading to peak column
shape distortion frit Residual
matrix in
extracted
samples
Altered stationary deposits on
phase – compounds the frits and
stationary
in the injection phase at the
Head of
volume can’t front of the
column
partition in to the column
stationary
stationary phase as
phase
a narrow band at the
head of the column
 WHY – POOR
CONNECTIONS?
WHY – POOR CONNECTIONS?
Courtesy of
Agilent
Technologies

Stainless steel
ferrule shape,
length, size and
compression
screw shape,
thread length,
size varies by LC
vendor
http://www.sepscience.com/Techniques/LC/Articles/265-/HPLC-
Solutions-55-PEEK-Fitting-Slippage
 MAKING A GOOD LC CONNECTION
REQUIRES TRAINING
1. Slide PEEK fitting quite far back from end of tubing
2. Bottom the tubing out in the mating port
3. Advance the fitting into the mating port while holding
the tubing firmly in the bottomed out position
4. Finger tighten, then + a ¼ turn, don’t over tighten
5. Tug to check that tubing doesn’t move
6. PEEK wears out – replace compression screws w
ferrule often
7. Cut PEEK tubing with perfectly flat ends, exact same
length as the old piece
of tubing (an ART)
 POLLING QUESTION #3
Have you ever received any
training about the importance
of extra-column dead volume
and how to minimize it by
making good LC connections
& optimizing LC plumbing?
 WHY - COLUMN OVERLOAD?
30μL
10 μLinjection
injectionof
or
low
high%%organic
organic
LC 2.1 mm
inner
column diameter
0.13 mm i.d. inlet
PEEK tubing

OR

3 sec wide, Wider, fronting Wider, tailing

gaussian peak peak w shifted Rt peak w shifted Rt
TROUBLESHOOTING TOOLS
1. Maintenance calendar:
• was anything changed from last good run?
• how many injections on the column, guard column,
inline filter?

2. Look at peak shape of the last run(s) – was

this a gradual or a sudden degradation in
peak shape?
3. Check volume injected, column and LC program
used in the acquisition data file record
4. Check SST or inject an extracted sample (if
stable) from last good run (✔ injection matrix)
DIFFERENTIAL DIAGNOSES
– CASES 1, 2, 3
Case 1 – 615 injections on the guard column
(should change at 500), gradual degradation of
peak shape during run No mobile phase, needle wash or LC
method changes (all cases)
Case 2 – 0 injections on the guard column
(changed today), last run had good peak shape,
abrupt degradation of peak shape – morphine
peaks in all injections of today’s run are bad

Case 3 – no guard, 850 injections on the column

(change at 2,500), correct volume injected (5 μL),
THC-COOH peak shape in SST is normal
CASE 1- AGED GUARD

Same vial with new guard

CASE 2–BAD LC CONNECTION

Same
vial
after
new
ferrule

Replaced the PEEK ferrule

 CASE 3 – WRONG INJECTION
MATRIX (COLUMN OVERLOAD)

100% 70:30 Quantifier

MRM
ACN ACN:H2O

THC-COOH Qualifier
After re-extraction MRM

I.S.
MRM
 POLLING QUESTION #4

Do you know the “rules” for

the correct composition of an
injection matrix (%s of solvent
& water in the mixture) that will
yield good chromatography
with isocratic versus gradient
LC analyses?
See the Appendix
 LOW S/N CASES

All low S/N cases – reported

problem was low QC or low
calibrator failure because
concentration or % accuracy
was out of range or S/N <10
 LOW S/N – CASE 1
Quantifier
MRM
,one two
three four
five

Abnormal
appearance of
appearance
today’s Qualifier of THC-
THC-COOH MRM COOH
LLOQ calibrator
LLOQ
calibrator

Internal
Standard
MRM
 LOW S/N – CASE 2
Today’s High
Benzodiazepine calibrator
Why does
the Fixed TIC w Fixed Y scale – 1.4 e8
Y scale 12%

matter?
100%

Yesterday’s High TIC w Fixed Y

Benzodiazepine calibrator scale – 1.4 e8
LOW S/N – CASE 3
 POLLING QUESTION #5

Without no additional
information – can we
conclude that these 3 low S/N
cases are all MSMS related
(MSMS is source of the
problem)?
 HOW TO PROCEED?
Pump – Sample Prep – Autosampler – Column – Divert Valve – MSMS
(Tubing & Connections) (Unauthorized method edits)

X
X
Column Oven
ALS
MSMS
X
X
Pumps
Q3 collision Q1
Divert
Valve
cell

Waste
WHAT WOULD CAUSE LOW PEAK AREAS, W
NORMAL RTS, NORMAL PEAK SHAPES?
1. Sample preparation error
2. Too little volume injected from autosampler
a. Programming error
b. Autosampler problem (leak, partially
plugged needle or needle seal, ran out of
needle wash)
3. Post-column leak (why only post column?)
4. MSMS needs cleaning or calibration or
source component replacement or detector
voltage too low or M.P. contaminant = ion
suppression Wrong method – unauthorized method edit
 HOW TO FIND THE SOURCE OF
LOW PEAK AREAS WITH NORMAL
CHROMATOGRAPHY?
1. Look at SST results, today & previous days
2. Inject stable extract from previous batch
6. Check for method edits
3. Autosampler problem 7. Check for solvent lot
change
a. Check vials & volume injected, check that
vial/plate caps were pierced
b. Inject extracts from other assays with similar
injection volume
4. Look for post-column leak, check LC pressure
5. Check MSMS signal, calibration, Rs - infusion
 LOW S/N CASE 1
1. No SST done for THC-COOH, but Vit D
SST passed (so is the LC-MSMS OK?)
2. Autosampler OK? Inject THC-COOH
LLOQ calibrator from previous batch –
normal (autosampler [LC] & MSMS OK)
3. No leak from column to MSMS found,
normal starting pressure & pressure
trace Instrument looks OK –
problem with Sample Prep?
4. Ask the analyst who extracted the batch
– anything abnormal happen?
 POLLING QUESTION #6

If I tell you that the extraction

method for this THC-COOH
case is solid phase extraction
(SPE) – can you guess what
error is most likely?
 “NOTHING WENT WRONG
WITH THE EXTRACTION !!!”
OK - Inject several vials from problem batch on
another instrument – same low peak areas.

“Are you absolutely sure there was nothing

abnormal during extraction?”

“Oh except - forgot to substitute vials in place of

waste bin from under SPE cartridges before
adding elution solvent -- but I stopped flow and
switched to vials immediately…….”
SOLUTION!
 LOW S/N CASE 1
Error in SPE workflow
CORRECT SPE workflow
The majority of the
Add Elution analyte is often
Solvent eluted in the first
10-20% of the
elution solvent
volume

If the first 100 µL of

the 1 mL of elution
solvent goes to
Waste Bin waste – expect
VERY low recovery
 Quantifier
MRM

Abnormal
appearance of
appearance
today’s Qualifier of THC-
THC-COOH MRM COOH
LLOQ calibrator
LLOQ
calibrator

Sample Prep Error Internal

(SPE eluate to waste) – Standard
re-extract the batch MRM
 LOW S/N CASE 2
1. SST passed
2. Autosampler OK? Inject Benzodiazepine
calibrator from previous batch – same
low peak areas
3. Obvious leak found at post-column
switching valve in column oven SOLUTION!
4. Why did SST pass with a leak present?
SST Threshold for acceptable peak area was set too low

5. Why is chromatography normal?

Separation occurred normally on the column, leak is post column,
post separation
 LOW S/N – CASE 2
Today’s High
Benzodiazepine calibrator

TIC w Fixed Y scale – 1.4 e8

12%
Post-
column
leak –
Fix the leak 100%
and re- Yesterday’s High TIC w Fixed Y
inject the Benzodiazepine calibrator scale – 1.4 e8
batch
 LOW S/N CASE 3
1. SST borderline, peak areas are low,
barely acceptable, Yesterday’s
calibrator injected today – also low.
2. No extraction reagent or solvent lot
changes, no sample setup error was
identified.
3. ALS - Inject opiate low calibrator =
normal peak area (autosampler OK)
4. No leaks found MSMS problem?
5. Review trend of SST results
 SST TRACKING
–
MEAN D2 I.S.
PEAK AREAS
FOR LC-MSMS
#1
LC-MSMS #2 (mirror image
of #1) has D2 I.S. mean
peak areas consistently
~90,000 cps
Today’s samples from #1
are injected on #2 -- D2 I.S.
peak areas are ~ 85,000 cps
on #2
What could be wrong with
the #1 MSMS?
 WHAT MSMS PROBLEMS WOULD
CAUSE LOW PEAK AREAS?
1. Aged ESI capillary (replace it)

2. Mass Calibration and/or Mass

Resolution drifted (check it)

3. Detector voltage too low (test it)

4. MSMS interface is dirty (clean

it)
 POLLING QUESTION #7
Is a key operator (or any/all users) in your
lab trained to perform any/all of the MSMS
maintenance described:
1. ESI capillary replacement/probe rebuild?
and/OR
2. Verify/Perform Mass Resolution & Mass
Calibration? and/OR
3. Check/Optimize detector voltage?
and/OR
4. Clean/Install MSMS interface (front-end)
 WHY ESI CAPILLARY?
Courtesy of ESI capillary is exposed
Waters
to high voltage &
temperature, acidic
mobile phases,
biological matrix residue
from extracted samples

Degradation

Decreased ionization
WHY MASS CALIBRATION
– MASS RESOLUTION?
Q1 Mass Resolution & Mass Calibration result table
Target Found Mass Pass? Peak Pass?
Mass at (Da) Shift Width
(Da) (+/- 0.1) (0.6-0.8)
59.05 59.04
58.03 -0.01
-1.02 YES
NO 0.72
0.54 YES
NO
175.13 175.15
174.32 +0.02
-0.81 YES
NO 0.68
0.58 YES
NO
500.38 500.37
499.76 -0.01
-0.62 YES
NO 0.76
0.57 YES
NO
616.46 616.46
616.03 +0.003
-0.43 YES
NO 0.69
0.60 YES
906.67 906.65
906.47 -0.02
-0.20 YES
NO 0.65
0.63 YES
1254.93 1254.95
1254.84 +0.02
-0.09 YES 0.68 YES
 POLLING QUESTION #8
With quadrupole mass
analyzers, higher mass
resolution (narrower MS peak
widths) = lower signal
abundance
True = YES
False or don’t know = NO
 WHY DETECTOR VOLTAGE?
• The detector counts ions & amplifies
signal *Not a substitute for cleaning the interface,
increases noise as well as signal!

• Higher voltage – shorter detector lifetime

• As the detector ages – voltage needs to
be increased to achieve the same
amplification
• Perform the detector voltage optimization
test to check and if necessary
increase the voltage*
WHY INTERFACE
CLEANING?
Region under vacuum
 LOW S/N – CASE 3
1. ESI capillary was changed last month
(less likely to be the problem)

2. MSMS calibration & resolution checked

with infusion - meets specifications

3. Detector voltage checked – no increase

is indicated

4. Vented the MSMS and replaced interface

components with clean, spare hardware
– pumped down for 24 hrs
LOW S/N – CASE 3

With clean
interface
components

With clean
interface
components
RT SHIFT CASE
 WHAT MIGHT CAUSE A
PROGRESSIVELY SHORTER RT?
• Typically think leak = late Rt
• Failing A pump or leak before
mixer might = early Rt
• Check the pressure trace
OVER-PRESSURE HISTORY
• Rarely a problem until last few
weeks, then…..
• Several Rt shifts per week on both
instruments for protein-crash 25-OH
Vitamin D method, sometimes
associated with system shutdown
for over-pressure
• What changed?
 INVESTIGATION
• Protein crash in ALS vials,
multi-mixer, centrifuge
• Checked multi-mixer &
centrifuge speeds – normal
• No extraction reagent
change or error found
• Checked ALS needle heights
• Raised needle 2 mm, no
further overpressure
 POLLING QUESTION #9
With the same column, flow rate and
column temperature – will a 50:50
(vol:vol) H2O:Methanol mobile phase
yield a higher column backpressure
than a 20:80 (vol:vol) H2O:Acetonitrile
mobile phase?
True = YES
False or don’t know = NO
 THE APPROACH TO
SUCCESSFUL
TROUBLESHOOTING
1. Know your instrument

2. Use troubleshooting tools

3. Set up a good production infrastructure

(including robust preventative
maintenance) – see Appendix

4. Divide and conquer (Sample Prep or LC

or MSMS)
LAST POLLING QUESTION

Do you want to see more

troubleshooting cases
presentations like these?
 EMAIL ADDRESS:
JASTONE@UCSD.EDU
APPENDIX
 1A. KNOW YOUR
INSTRUMENT - LC
1. Flow path details (between & within
components)

2. Cutting PEEK tubing, connecting PEEK

and stainless steel fittings correctly

3. Autosampler injection modes

4. Basic LC maintenance – changing

[plunger seals, check valves], [needle,
needle seal, rotor seal, stator, sample
loop]
 1B. KNOW YOUR
INSTRUMENT - MSMS
5. MSMS Source cleaning, normal appearance,
parts replacement (capillary, ESI & APCI
probes, corona discharge needle, heaters)

6. MSMS Interface cleaning

7. MSMS detector voltage optimization

8. Roughing pumps – oil, ballasting

9. Spare parts & Tools inventory (LC & MSMS)

 1C. KNOW YOUR
INSTRUMENT - IT
10.Project, Directory & File structure
(extensions)

11.How to find acquisition data file

information

12.How to edit LC & MSMS acquisition

and data analysis methods, divert
valve programs, apply MS Rs & Cal

13.Basics of PC maintenance & security

 2. KNOW TROUBLE-
SHOOTING TOOLS
1. Inspect LC baselines

2. Review LC pressure traces

3. Create XIC overlays (compare Peak

Shapes & Rt shifts between runs)

4. Carryover checks (L1-L2-H-H-L3-L4-L5)

5. Find leaks & source of over-pressure

(visual inspection, pressure testing LC
segments)
LC PEAK SHAPE– WHAT
CAN IT TELL YOU?
 BASELINE SIGNAL AND
PRESSURE TRACES – WHAT
CAN THEY TELL YOU?
 3A. SET UP A PRODUCTION
INFRASTRUCTURE
1. Good compliance with robust maintenance
charts – train everyone on how to make good
LC connections

2. Reliable tracking for & interpretation of SSTs

3. Set action limits for baseline, Rt, LC Pressure, &

MSMS response (peak area) changes,
acceptable peak areas & concentrations in
blanks, maximum batch sizes

4. Injection tracking for filters, guard columns,

columns – set maximums
 3B. SET UP A PRODUCTION
INFRASTRUCTURE
5. Use batch review sheets
6. Avoid mobile phase & autosampler
wash contaminants (LC-MS grade
solvents & water), maintain clean LC
reagent containers
7. Keep spare, clean MSMS interface
components
8. Lot to lot tracking of EVERYTHING
(including columns & solvents)
4. DIVIDE & CONQUER
 4. SAMPLE PREP OR
LC OR MSMS?
A. SST – Sample prep versus
Instrument
B. MSMS infusion – LC vs MSMS
C. False alarm – maintenance chart
review (silly human error suggests an
instrument problem when in fact - all is
well. Did someone touch the
instrument, change a fluid, make an
edit)?
LC PROBLEMS (MORE COMMON
THAN MSMS OR SAMPLE PREP)
A. Leaks & over-pressure can be detected using:
• Pressure traces, pressure monitoring, Rt changes
• Look at, touch every connection for leaks with flow on &
max. pressure normally seen for the method in question
• Isolate segments of the LC & check pressures (know what
flow rate, %A:B to use, expected segment pressures)

B. Failing column, guard, filter, plumbing connec-

tions (extra-column dead volume) will appear as:
• Rt changes, increased column, guard, filter pressure
• Peak shape, peak area (early > late eluters) changes - review
& compare with previous good batches
• Loss of resolution between peaks, carryover
• Usually related to use, residual matrix – track # of
injections!
 INJECTION MATRIX
“RULES” (REVERSE PHASE)
1. Isocratic separation –
injection matrix organic
solvent ≤ 90% of mobile
Russ Grant
phase organic solvent * rule – take
the MSACL
short course!
2. Gradient separation – determine
experimentally, more tolerant of higher %
organic & larger injection volume (affected by
column, analyte, mobile phase, starting gradient conditions,
injection matrix, injection volume)
 LC COMPONENT WEAR - 1
1. Pump normal wear & tear:
•Rt shifts, leaks, pressure trace changes,
pressure monitoring changes
1st - prime mobile phases thoroughly
(remove any air in the pump head)
2nd – Is correct/uncontaminated mobile
phase & needle wash in the reservoirs?
3rd - Check valve replacement, cleaning
4th - Plunger seal replacement
 LC COMPONENT WEAR - 2
2. Autosampler problems
• No peaks, decreased peak areas, peak shape distortion,
carryover, over-pressure from particulate in samples,
leaks, LC plumbing/connection errors
• Check for correct/uncontaminated needle wash
solutions
• Prime needle wash solutions – no air in the lines/valves
• Over-pressure, leak investigation
• Replace:
• Needle
• Needle seal
• Sample loop
• Rotor seal
 MSMS INVESTIGATION
1. Infusion – verify mass resolution and mass calibration
2. Infusion – can be misleading, signal is decreased but
infusion meets specifications (ionization differences at
infusion rates vs LC flow rates and with %B are VERY
important)
3. Rely on good SST tracking – then R/O the LC

1. With low signal – first -- what can be done w/o venting?

a. Silly human error (source not connected or flow not
directed to MSMS)
b. Rule out LC, sample prep
c. Rule out mobile phase contaminants
d. Detector voltage check, mass resolution & calibration
check
e. Source capillary change needed?
f. Interface cleaning needed?
SAMPLE PREP INVESTIGATION
SST – be sure review is detailed, action limits are robust

SST rules out the instrument - then:

1. Inject a sample from a previous, good run
2. Inject the suspect sample/batch on another
instrument
3. Look at the vial/plate caps, look at the contents
4. Was there a lot change – I.S., calibrator,
reagent?
5. Review protocol steps with the analyst
6. Missed I.S. addition, double I.S. addition
7. Mixing, labeling, pipetting variance – transfer
errors
COMMON SAMPLE
PREP ERRORS