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Sciencefor a safer world
Case studies: Troubleshooting failed system suitability tests
Michael Wright
Bioanalytical Services
LGC
Fordham, UK
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13/10/2016 2
Case Study No.1
Expected for SST:
VMA
RT - 1.08 (0.98-1.18)
S:N - 80:1 (>20:1)
5HIAA
RT - 2.3 (2.15-2.45)
S:N - 100:1 (>20:1)
HVA
RT - 2.7 (2.6-2.9)
S:N – 30:1 (>15:1)
Pressure = 3300-3600 psi
NB: Uses different MRM periods
VMA
5HIAA
HVA
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1) Pressure = 2694 psi (lower
limit 3300)
2) New column….
Case Study No.1
“Where did everybody go?”
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Step 1 – start simple
• Correct method being run? Source?
• Is the LC connected to the MS?
YES
Want to see if the peaks have
moved outside of their MRM
periods
Moved the time periods back and…
Hello Peaks!
VMA
RT - 1.67 (0.98-1.18)
S:N - 40:1 (>20:1)
5HIAA
RT – 3.13 (2.15-2.45)
S:N - 50:1 (>20:1)
HVA
RT – 3.5 (2.6-2.9)
S:N – 11:1 (>15:1)
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13/10/2016 5
Step 2 – look for general causes of low back pressure
• Leak somewhere? Nothing detected
• Correct mobile phases? Made fresh and the same RT
• Lines not purged properly or leaking purge valve? No
• Fluctuating Back pressure? No
Step 1 cont….
Is to do with the new column? No
Struggling to find the cause
Pressure = 2406 psi (even lower)
Peaks eluting even later – VMA peak
now being cut off
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Same instrument – Clozapine run was submitted
Clozapine SST looked fine
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13/10/2016 7
Leak here
Leak worse when the pressure
was highest
Analyst had checked the front of
the valve for a leak (with a finger)
not the back
Learning points
• Now check the front and back
of the valve for leaks
• Now check for leaks using blue
tissue paper
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13/10/2016 8
Case Study No.2
SST – 25OH VitD
Retention Time Shift
Retention
time shift just
within the
criteria
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25OH Vitamin D batch submitted
Retention
time at start
of run
Retention
time at end
of run
The 2nd plate of samples had failed
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The following day the routine maintenance was performed
“Replace fluid in pump seal wash bottles (10% Ethanol)”
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Pump D seal
New Seal
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Learning points
• Our criteria for retention time wasn’t tight enough
to spot this
• We weren’t changing pump seals frequently
enough
Case Study No.2
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Case Study No.3…. 1 week later
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Unacceptable drop in
intensity & run failed
Started easy
Leak somewhere after the column? No
Gas OK? Yes
Vacuum OK? Yes
Correct ion probe? Yes APCI
Capillary extended in probe? Yes
Probe Blocked? No
APCI Corona needle positioned correctly? Yes
MS fouled or out of calibration? Ahhh
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A fibre in the
skimmer plate!
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Following removal
of the offending
fibre…
Learning points
• Our criteria for intensity wasn’t tight enough
• We needed to change the way in which we performed
a weekly source clean on the Mass Spec
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XIC of +MRM (4 pairs): 383.200/257.300 Da ID: 25OHD3 1 from Sample 224 (2016_03_30 TNHS) of SST.wiff (Heated Nebulizer) Max. 3850.0 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
Inte
ns
ity
, c
ps
4.74
4.98
5.29
Case Study No.4
XIC of +MRM (4 pairs): 383.200/257.300 Da ID: 25OHD3 1 from Sample 208 (2016_03_07 TN) of SST.wiff (Heated Nebulizer) Max. 4385.0 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
4385
Inte
ns
ity
, c
ps
4.95
5.20
XIC of +MRM (4 pairs): 383.200/257.300 Da ID: 25OHD3 1 from Sample 207 (2016_03_07 TN) of SST.wiff (Heated Nebulizer) Max. 1.0e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0Time, min
0.00
500.00
1000.00
1500.00
2000.00
2500.00
3000.00
3500.00
4000.00
4500.00
5000.00
5500.00
6000.00
6500.00
7000.00
7500.00
8000.00
8500.00
9000.00
9500.00
1.00e4
Inte
ns
ity
, c
ps
5.34
Supposed to
look like this
Now….
Purged mobile
phases…
XIC of +MRM (4 pairs): 383.200/257.300 Da ID: 25OHD3 1 from Sample 208 (2016_03_07 TN) of SST.wiff (Heated Nebulizer) Max. 4385.0 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
4385
Inte
ns
ity
, c
ps
4.95
5.20
Fresh mobile
phases….Checked the
obvious (e.g.
leaks)
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13/10/2016 19
Back pressure not
fluctuating and purge
valves not leaking
Back pressure looks
normal but tested flow
rate anyway – OK
Peaks are late but
drifting backward and
forward so unlikely to
be column at fault
XIC of +MRM (4 pairs): 383.200/257.300 Da ID: 25OHD3 1 from Sample 208 (2016_03_07 TN) of SST.wiff (Heated Nebulizer) Max. 4385.0 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
4385
Inte
ns
ity
, c
ps
4.95
5.20
XIC of +MRM (4 pairs): 383.200/257.300 Da ID: 25OHD3 1 from Sample 205 (2016_03_07 TN) of SST.wiff (Heated Nebulizer) Max. 1.3e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0Time, min
0.00
500.00
1000.00
1500.00
2000.00
2500.00
3000.00
3500.00
4000.00
4500.00
5000.00
5500.00
6000.00
6500.00
7000.00
7500.00
8000.00
8500.00
9000.00
9500.00
1.00e4
1.05e4
1.10e4
1.15e4
1.20e4
1.25e4
Inte
ns
ity
, c
ps
5.37
XIC of +MRM (4 pairs): 383.200/257.300 Da ID: 25OHD3 1 from Sample 206 (2016_03_07 TN) of SST.wiff (Heated Nebulizer) Max. 170.0 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0Time, min
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
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200
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220
230
240
250
260
Inte
ns
ity
, c
ps
XIC of +MRM (4 pairs): 383.200/257.300 Da ID: 25OHD3 1 from Sample 208 (2016_03_07 TN) of SST.wiff (Heated Nebulizer) Max. 4385.0 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
4385
Inte
ns
ity
, c
ps
4.95
5.20
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13/10/2016 20
Auto-sampler
Pump(s) 1 Pump(s) 2
Column
OvenMass
Spectrometer
2 position divert valve
On-line SPE cartridge
Analytical column
WASTE
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13/10/2016 21
Wanted to rule out issues with the
pumps and gradient mixers so
swapped pumps A/B with pumps
C/D
Wanted to rule out any issues with
the auto-sampler so bypassed it
with a manual injector
1st dimension of on-line SPE
seems to work fine in isolation….
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12
3
4 5
6
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12
3
4 5
6
12
3
4 5
6
A B
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Rotor seal in a switching valve
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13/10/2016 25
The scratched rotor seal was possibly causing liquid to leak to
varying degrees. It wasn’t leaking during the start of the run
(when BP was measured)
The instrument was recently brought into a higher throughput
environment but the maintenance schedule hadn’t been
changed
Learning points
• We introduced more regular servicing for the switching
valve.
• Highlighted the importance of having an system
capable of generating a pressure trace.
Case Study No.4
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Conclusions
Running and documenting SST can:
• Stream-line troubleshooting
• Guide to creating a more robust LC-MS service
• Help plan preventative maintenance
• Highlight areas where your systems could be
improved
13/10/2016 26
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Sciencefor a safer world
Questions?