A few thoughts:

I do not see anywhere in your list that you performed basic maintenance on the injection valve. What you describes sounds a lot like "carryover" and most of the time, worn parts such as injection valve rotary seals are responsible. Valve maintenance should be carried out, inspecting the valve parts (stator, rotor etc) for damage and wear and cleaning/replacing parts as needed. The valve rotor seal is the wear item and should be replaced on a regular basis (some labs, every 6 months, others maybe longer). Additionally, you did not say what brand and model of HPLC you have, but sometimes we also see contamination originating from one or more Wash vials used in the Autoinjector. Wash solution, if used, matters. Some instruments do not need any wash vial at all, others do.

Another source of general contamination observed on an LC-MS system originates from worn and contaminated HPLC vacuum degasser modules (stand-alone or integrated). They require regular professional service to maintain in operating condition. As they wear, they may introduce contamination into the flow path. People often forget about them. Here is a link to an article on the topic:

• "An Often Ignored HPLC & LC/MS Contamination Source. Did you check your Vacuum Degasser?"; https://hplctips.blogspot.com/2015/08/an-often-ignored-hplc-lcms.html

Article that may be of interest:

• "Troubleshooting HPLC Injectors (Manual and Automated)"; https://hplctips.blogspot.com/2013/06/troubleshooting-hplc-injectors-manual.html

• "Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems"; https://hplctips.blogspot.com/2015/02/carry-over-carryover-contamination-in.html

Better to call Service Engg.

Hi William,

Thanks for this information, I cleaned the rotor seal and replaced all wash bottles I'm using an Agilent 1290 HPLC coupled with an Agilent 6470 MS/MS. I additionally tried a 50 degrees C water rinse followed by a IPA wash (not going into column or MS) going through the main-pass and bypass the injector. Our instrument isn't connected to a degassing unit so most of our solvents are frequently sonicated instead. I will have a look into the papers you have sent though, thanks!

The 1290 pumps have integrated vacuum degassers so "you are" using a degasser and it may require service (the degasser lasts a max of ~ 5 years before requiring service). *Sonicating solvent in bottles is often very poor at degassing. Gas permeates the solution quickly over time. **Use continuous degassing as designed for HPLC.

What are "wash bottles"? Are you referring to the pump head seal wash system?

The injector rotor seal should not be cleaned, it needs to be replaced. They are inexpensive consumable parts. Also, be sure to replace the inline pump frit (you did not say which 1290 pump you have, but they have porous filters installed which should be replaced every 1 month. Again, basic maintenance).

Most importantly, use the MS to ID the contamination, then take action to remove the material. Have the HPLC professionally serviced. Contact someone local with professional experience to help you so you do not waste time or money.

Hi William, thank you for this! I will have a look into changing the parts you mentioned. The wash bottles I was addressing were the ones that are linked to the multi wash system, but yes I also changed the seal wash solvent. I will additionally contact Agilent in response to your recommendation.