Bioequivalence and Bioavailability Forum

Hi Ladi!

❝ Currently, we do not have the strategy written in our SOP to handle this kind of situation.

IMHO, you can (and should) have an SOP for reanalysis or reinjection (if stability of extracts is validated).

❝ Since we think this is instrument issue, we have decided to reject the run and reanalysis the whole batch (please comment).

A remote diagnosis is difficult but I also think that it is a hardware-problem. IMHO, it will be almost impossible to write an SOP describing all potential steps to solve the problem. Have the system manual handy, if possible get a service/repair manual, make yourself familiar with the system, think about getting training by the vendor. Only as a last resort, call the service technician.

Reanalyse the batch only once you solved the problem – otherwise you are wasting your precious samples. If you are in a hurry and have another system with the same spec’s, run the batch on the other machine.

My guess:

• A faulty pump check valve could explain increased pressure and shoulders / peak splits because the flow temporarily stops.
  
• Since in a batch there are much more unknown samples than CCs and QCs, it is possible that by pure chance all CCs and QCs are OK.

What I would exclude:

• If you perform gradient elution a blocked solenoid valve before mixing.
  
• Blocked tubing between pump and column.
  
• Blocked inlet frit on the column.
  
• Contaminated column.

All of those will increase the pressure but not come back to normal.

I suggest to run a batch with analyte / IS in mobile phase (like in system suitability; not extracts). If there is a hardware problem I expect that you see “poor chromatography” in roughly the same percent of injections like in the sample batch. If yes, hopefully you don’t have one of these new fancy systems where you cannot do anything on your own.
General rule in troubleshooting: “Divide and conquer”, i.e., change one thing at a time (otherwise you will not discover the true cause).
Remove one pump value, disassemble, and inspect it with a magnifying glass. Deposit on the sapphire seats and/or the ruby ball? If yes, that’s the smoking gun. It seems that deposits occur more often with acetonitrile in the mobile phase than with methanol. Even if you don’t see anything, ultrasonic both (separately!) in a PP vial with a mild detergent. Rinse with deionized water and ultrasonic with methanol, let dry, assemble, and check whether the problem is resolved. If not, repeat with the other valve(s). I know, this is a time-consuming procedure.

BTW, a fantastic resource for any analyst (and nice reading matter while waiting for the result of the batch): John Dolan’s article series LC Troubleshooting in the LCGC magazine.

Good luck!