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Case studies: Troubleshooting failed system suitability tests

Michael Wright

Bioanalytical Services

LGC

Fordham, UK

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

1) Pressure = 2694 psi (lower

limit 3300)

2) New column….

Case Study No.1

“Where did everybody go?”

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)

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

Same instrument – Clozapine run was submitted

Clozapine SST looked fine

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

13/10/2016 8

Case Study No.2

SST – 25OH VitD

Retention Time Shift

Retention

time shift just

within the

criteria

25OH Vitamin D batch submitted

Retention

time at start

of run

Retention

time at end

of run

The 2nd plate of samples had failed

The following day the routine maintenance was performed

“Replace fluid in pump seal wash bottles (10% Ethanol)”

Pump D seal

New Seal

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

Case Study No.3…. 1 week later

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

A fibre in the

skimmer plate!

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

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

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800

1000

1200

1400

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2000

2200

2400

2600

2800

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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)

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

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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

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

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….

12

3

4 5

6

12

3

4 5

6

12

3

4 5

6

A B

Rotor seal in a switching valve

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

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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Questions?