Insulin is sticky. It requires proper flush methods for the system, column and MS. Are you maintaining your HPLC? Both the HPLC and MS require regular inspections and maintenance to perform well. Sounds like it could be something very simple such as a worn injector valve rotor seal, inadequate needle washing, solubility issue and/or perhaps poor column wash methods. This is a training issue and you should have someone local with experience assist you.

Here is a link to an article, "Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems" which may help you troubleshoot it properly.

https://hplctips.blogspot.com/2015/02/carry-over-carryover-contamination-in.html

I would review the mobile phases used by the USP. But, be sure you use volatile pH buffers like Formic Acid (not Phosphate buffer unless you like cleaning the MS:). Inappropriate mobile phases can cause 'dumping' of the molecular target (like Insulin) into blanks and poor %RSD....

Also, never use 100% solvent in your mobile phases (especially water since it can cause column collapse). Example, use 95% water and 5% ACN.

You need to give us more information such as mobile phase condition. What is your initial condition and so on. The attached method has no issue with carry over and you should use as a guideline. You can put A mobile phase as 100% water and mobile phase B as ACN (all with acid), as long as you pump minimum of 5% B so no phase collapse as Bruce suggested

I agree with Narong, we need more information to help you. Did you use guard colunm? Injection volume? What's your retention time? Did you filter this standard?

" I noticed a reduction of the carry-over when solving insulin in 1% FA." - You may not have completely dissolved the insulin, which was trapped somewhere between the injector and the detector - most likely at the top of the column or the pre-column.

From my point of view, there are many reasons to this happen. Hope this helps.

Thank you for the answers so far.

I'm using a core-shell c18 column without a guard column

retention time: 3,1 min.

gradient program (10-50% B from 0 to 4 min, followed by half a minute at 50% B, then back from 4,5 min to 7 min 50-10% B

injection volume 0,5 ul

flow: 0,200 ml/min

I did not filter the standard.

Exact HPLC column dimensions please. Your return to initial conditions appears too weak and too short. No wash method ??? Your peak elutes at 3.1 minutes, but your method ends at 4.0 minutes. Too short. Probably solubility problems. You should be dissolving the sample FULLY before injecting. What about maintenance? The column. Has it been verified to be clean and test OK? What about the HPLC system?

Sounds like you may be a student using HPLC and LC/MS for the first time (?). Please ask your teacher(s) or the local person in charge of the instrumentation for assistance. The web is not the best place to obtain training for these types of analytical systems.

Michel, William is right. Chromatography is not a simple and straightforward technique like a pH meter, even this one has its tricks.

Just for you to understand, the need to know the size of the column is to compare with the retention time of its compound, that is, if it is coming too close to that of the dead volume.

And try to reduce the concentration of your pattern by 2 to 10 times less, to try to help you anyway. If there is a solubility problem, it may well solve it.

As William Letter already stated your gradient is too short/too steep. At high concentration of ACN proteins may stick to your column. So decrease your final concentration to e.g. 40%B and double the time to 8 min.

If this does not solve your problem try another column, because some types can show memory effects. Look for one that is dedicated to RP separation of proteins.

check my researchgate profile and look for my question on carry-over. I have a diagnostic procedure to figure out what may cause the carry-over a) instrument or b) column chemistry. Do the several gradient up and down on the same injection, if you see insulin in each gradient, it is the column issue. As the previous comment, your gradient is too short. You don't know what is eluted after your peak so you need to clean (flush) the column really well before the next injection. Ask your teacher at your department for help.