To troubleshoot the missing peaks for the four PPCPs, check the integrity of the HPLC column, optimize the mobile phase and gradient settings, and ensure proper sample preparation and detector sensitivity. Consider testing higher concentrations and alternative methods like LC-MS/MS for improved detection.

Please ensure everything is same. I found you mentioned that there were 7 new chemicals. Have you check if these chemicals may cause some changes for the old four chemicals? Another issue is your column is 4.6x 250 mm. It looks like to be produced for HPLC not LC-MS/MS. The flow rate is only 0.3 ml/min. I wonder this is not a recommended flow rate for a column in this size. If the matrix is 5 um, the recommended flow rate will be higher than 0.3 ml and you will need to tune a lot on Mass end. If you do not want to change the mass condition, you may need a splitter to direct the extra to waste.

You need to do method troubleshooting to see if it is the HPLC condition or the MS condition that causes the problem. Please inject your four std that you missed to the MS (with no column) to prove that the MS setting is still good. If you see the signal at the expected response (good signal), then you can focus on the column. If your MS is good, inject the same std with column and use the highest organic mobile phase to elute the compound quickly near the solvent front to prove that your column does not affect your analyses. Let me know the results and we can go from there.

Thanks, Narong Chamkasem for your response. I have checked the response by direct infusion to the MS. The signal intensity is good and as expected. However, I can see only one of these 4 when I ran through the column (Isocratic 95% mobile phase), and that too at very low intensity.

Thanks Jien Wu for your response. No, these 7 new chemicals won't affect any of the 4 compounds.

Unfortunately, we don't have MS/MS. We have HPLC-MS (Single quadrupole detector). So, the column is well suited for our instrument. Flow rate and MS tune method are optimized. So 0.3mL is appropriate for the selected MS parameters. Changing the method will be very tedious at this stage.

I asked you to inject std say 10 uL of mid concentration , not infusing the std solution. This to prove that without column, you have ok response. If not, then working on the MS condition, not the column yet.

What is 90% mobile phase?

Sorry, I misunderstood it. I will bypass the column and try. I tried combined infusion as well and the response reduced significantly.

The organic phase (90%) is Methanol.

Mobile phase A is 0.1% FA in H2O.

Thanks Narong Chamkasem

I installed a union and bypassed the column. I can see these compounds at a good intensity. So, the issue is probably with the column. However, I'm unable to understand how only these four compounds are affected. Do you recommend any cleaning or regeneration procedure or something? Also, This column has been used mostly on standards and very few environmental samples. (I clean it with 100% organic phase (Methanol) at least once a week and store it in 100% organic after every run. )

Thanks Narong Chamkasem for your help.

If you confirmed that the MS condition was good, then fix the column issue. Use a different column with similar phase and use 90% B to see if you have good intensity. This to find out if the or column is the issue i.e. absorption or degradation

It is interesting. Looks like something in the column bind your molecules. Maybe you need to use a new column to try?

I Googled PPCP and they are relatively active compounds. Some are antibiotics. Some antibiotic may react with trace metal (iron) from the flow path, column hardware. Please tell me the exact compound you have the issue. My wild guess is that the may bind with the metal. If it is the case I have a few tricks to solve the problem. However, I need to know the name of the compound and how from there.

It was the issue with the column. I tested the new column, and I found all four of them.

However, one thing I'm still unsure about is tetracycline is one of the compounds whose peak disappeared. We used to quantify in ESI positive mode. just for a trial, I tried in ESI negative mode as well with my older column, and I could see it there. (I confirmed with two concentrations and multiple injections) so if there was some issue with the molecules reacting or adsorbing in the column, then it shouldn't be there in the ESI negative run as well( method parameters are the same for both ESI).

I tried reverse flushing my column with pure ACN as well but no change was there in terms of these compounds. So I guess I just have to work with the new column. please suggest the methods for the removal of metals from the column. Compounds such as Tetracycline can bind with the metal strongly.

The compounds are: Ciprofloxacin, Tetracycline, Enrofloxacin, and Ceitrizine.

Also, to increase the lifetime of my column, what should I do?

Currently, store my column in 100% methanol after every run

and our method is at 40 degrees. since we were using it frequently, our column heater was on all the time, and it was at that temperature all the time. but from now on I'm planning to store it at ambient temperature after every run.

What additional steps would you like to recommend for maintenance that, based on your experience, you found great for increasing the lifetime of the column?

Thanks so much for your help Narong Chamkasem and Thanks Jien Wu.

Tetracycline family will bind with metals. To analyse this family, you need to add EDTA or other similar chemicals to inhibit the binding.

I add EDTA in the samples. But this was the case with the standards.

You need to add EDTA in all sample and std solution in excess amount relatively to the analyte concentration to prevent trace metal in the flow path from binding with the target analytes.

Please read my papers in 2015 on glyphosate method in milk by LCMS. It is in my profile.

Or use this as aguide

https://www.unitedchem.com/wp-content/uploads/2023/08/Tetracycline_Antibiotics_in_Milk_Application_Note_2023.pdf

Thanks Narong Chamkasem . I will go through these.