Help needed on UPLC carryover issues

[biggirl20]

Hi people, I am currently using Waters Acquity UPLC to study Plasmalogen (both phosphatidylcholine and phosphatidylethanolamine) and I am experiencing carryover issues when doing target profiling (MRM), such problem did not exist in global run. I've changed to a new column and the very first run had appearances of the carryover from previous plasmalogen samples, so i doubt the problem is with the column.

Intensity decreased over blank injections, and peaks still appear when performed zero-volume injection.

100% ACN is used as strong wash and 95% H2O + 5% ACN is used as weak wash. Strong needle wash consists of 100% IPA and Weak needle wash consists of 90%H2O + 10% ACN. Mobile phase A is 40% H2O + 40% Methanol + 10% ACN while Mobile Phase B is 100% IPA. Other lipid compound studies uses same solvent composition and did not have carryover issue. So I think the problem is that the solvents are not that effective in getting rid of plasmalogen?

The column I am using now is Waters Acquity CSH C18 Microflow column, flow rate is set at 0.1mL/min and temp at 45 dC.

Not sure the problem lies with sample loop or unsuitable wash solvent? Hope some experts here can give me some suggestions.

Thanks in advance!

[Gerhard Kratz]

What injection system you are using? If it is a valve please check the rotor seal. That is mostly the source for carry over.

[biggirl20]

Hi Gerhard Kratz, thank you very much for your reply.

Would like to ask if the rotor seal can be the problem? because other compounds such as fatty acid chain do not have such carryover issue, it only happens to my plasmalogen samples

I'm not sure whether a particular compound can be 'sticky' to the seal while the rest don't.

[Gerhard Kratz]

If you have scratches on the rotor seal and viscous samples carryover is possible. Slight scratches and low viscosity samples and it might happen that you don't see carryover at that stage. I would open the valve and check the rotor seal. Rotor seal should be part of maintenance and changed minimum every 6 months.

[biggirl20]

Hi Gerhard Kratz,

the LC-MS just had its maintenance performed last month with the parts clean and changed

not sure whether my wash solvent is 'strong' enough, 90% H2O with 10% ACN is used for seal wash and 'washing' of autosampler.

[HippyLabRat]

Are you using a binary or quaternary pump? What are your run times?

Use this gradient as a rinse: 90% [ACN only] on one line, and 10% [0.5% Formic Acid in water] on another line, and let it go linearly over about 5 minutes until its 50/50. Then return it to initial conditions over the next 2 minutes, and then let it run at those conditions for 2 more minutes.

Full disclosure: I haven't used a Waters microflow column, and these times are what I'd use for a standard 2.5-2.7um 2.1x100mm HPLC column. You should be able to adjust the times based on your void time and volume, but I think you get the idea.

[biggirl20]

Hi HippyLabRat,

Thanks for the suggestion! would try that out once im using the machine again..

the run setting is as below:

Time, Flowrate, %A, %B, Curve
Initial, 0.1mL/min, 99, 1, -
1, 0.1, 99, 1, 6 (linear)
10, 0.1, 17.5, 82.5, 6
10.01, 0.15, 1, 99, 6
15, 0.15, 1, 99, 6
15.01, 0.15, 99, 1, 6
16.7, 0.15, 99, 1, 6
16.71, 0.1, 99, 1, 6
17.2, 0.1, 99, 1, 6