Ibuprofen carry over issue

<p> Hi peers,</p><p></p><p>I have a tough issue with the carry over of ibuprofen on UPLC. This sample contains very concentrated Ibuprofen (2.4mg/ml) and the standard contains only 2.4ppm Ibuprofen (it is  RS method). The carry over is about 0.1% and so caused system suitability failed. I changed different solvents for strong needle wash, no luck. When I injected 2 ul or 4 ul Diluent with partial loop over filled mode, the peak area is same (about 60000), while 8 ul Diluent injection got 1940108 of peak area, which is 32 times bigger than 2ul and 4ul injections. Anybody can explain that? In additional, Ibuprofen seems stuck in the system forever, even if after a lot of injections of other samples or Diluent. Any ideas?</p><p></p><p>Thank you in advance! </p><p></p><p>lorus</p>


  • Hello

    Please end the details of your method, including injection mode, washes, gradient and sample diluent. I think it will be possible to optimize further.

    I suspect that ultimately the root of the issue is the very high concentration of active. I suspect that there are now two issues on-going, one is the CO and the other is contamination. Based upon your observation it appears that the system is saturated with the active, that is being stocked on the system and then released with the subsequent injections. Please be aware that concentration is the root cause of the problem, any solubility issues with this high concentration, and the compound will drop out of solution and remain in the system. Let’s just do the maths…2µL of 2.4 mg/mL   is 2.4 µg/uL  and in fact your 2 µL injection is in fact 4,800 ng on column.

    To explain your observations, in PLNO mode, the sampling needle draws extra sample to “rinse” the needle ensuring that the volume drawn into the sample loop is homogeneous. The practice if pre-wetting sample pipettes before delivering the measured volume is a good analogy. Additional sample which has fallen out of solution and is now coating the fluid path surface, will re-dissolve when it encounters the additional diluent. The re-dissolved precipitated sample trapped in the system, will then add to your peak area.

    I think, that the system will need to get cleaned up. It may be necessary to inspect and replace some parts. First of all, flush to waste with the column off line. Flush with a solvent that will dissolve the compound. Make some injections of strong sample diluent and even consider DMSO to clean the injection pathway. For example, first inject in PLNO mode, with maximum overfill factor, with loop offline programmed and then do several Full loop mode injections with maximum overfill factor (for small loops e.g. 2 µL automatic is the highest overfill factor you can choose).

    Please check the following parts in order.

    • Sample Loop. Take off line and wash and consider replacing
    • Pre-Piercing Needle
    • Sample Needle

    If this does not work call a FSE to assist in the cleanup process.

    After this we can take a look at the method. Can the sample diluent be made stronger? Can the weak wash be improved, is the strong wash as strong as possible? If solubility is an issue consider starting the gradient at a higher organic level  20% instead of 5%. Consider shallowing out the gradient and can the  gradient hold at the end of the run be extended? All of these tools should be considered one at a time to see which can assist. Ultimately a considerable drop in concentration will be the most practical.

  • Thank you for your analysis and suggestions.

    There are 2 actives in this sample to be analyzed, one is very polar and insoluble in organic and has 7 times lower concentration than Ibuprofen. Ibuprofen dissolved in organic solvent.

    In order to analyse RS, the sample has to be so concentrated. The start gradient can not be higher with the organic otherwise the polar compound can not be well resolved. I changed both strong and weak needle washes to methanol with the maximun volume 2500 ul from weak as water strong as Acetonitrile. It seems it is much better: There are no carry over for a while but appeared again in last sample set.

    Do you know if the needle wash solvent goes through prepiercing needle? Because we changed a new needle (I don't know if the needle we changed included prepiercing needle or not.) and the carry over is still there.

    Thank You!

  • Hello

    I will answer the last first. If you use a Preventative Maintenance kit you would get a new Pre-piercing needle, but it does not need to be chnage every time, but rather jut annually, as can get dull. If it gets dull then it can stick in the vial cap and get contaminated. For this analysis not overfilling the vials would be really important as again that will contaminate the pre-piercing needle. You can make an injection with and w/o a vial cap and see if there is any difference to assess if the PP needle is contributing to CO.

    Otherwise the PP needle gets a cleaned when at the needlewash port at the back. The both needles are washed at a separate position from the injection port which is a deign benefit for ACQUITY not present in other injectors. The WW bubbles up and washes the whole assembly, especially as you are washing for so long.

    I think if the CO is coming back, if only in later sample sets I have two other recommendations, I would put DMSO in the Strong wash say 10% as it will solubilize the ibuprofen and clean it up. Now we know that stronger methanol cleans up the fluidics better. Then you could cut down on the 2,500 uL that will make your cycle time pretty long. As you are using PLNO it should not interfere with the chromatography.

    The other thought is to add a solvent blank injection after every 20 samples. The blank injection should inject the strong wash solvent, using a injection volume that matches the maximum appropriate for the loop. Use PLNO mode, selecting the maximum pre-injection volume, ensure “loop offline” is programmed. Follow this injection with a Full loop mode solvent blank, using the maximum overfill factor (note for small loop e.g. 2 µL automatic is the highest overfill factor you can choose). Again, put the DMSO in the blank.

    Try one thing at a time!

    Hope that one of thee suggestions helps, I attached a document about the wash sequence.