I am guessing you are using reversed phase chromatography (e.g C8, C18 analytical column). Lipids are non-polar, hence they will strongly interact with C8, C18 analytical columns. (what is your mobile phase?)

There is no easy solution for this if you keep using such columns. I would run several cleaning gradients after each sample to avoid carryover, but still depending on the amount of lipid you are using you might see a strong interfering signal.

If you can, I would take a step back and redesign my experiment.

You provide no information about your HPLC method so no useful advice or suggestions can be offered. With lipids, poor sample solubility in the mobile phase or injection solvent are the main causes of contamination or carry-over problems. Lipid analysis also requires regular column flushing with wash solutions to remove residual material over time. The choice of wash solution depends on the lipid, the mobile phase composition and the exact column type being used. *Please contact an experienced on-site chromatographer for assistance with your method.

HPLC "Carry-over" is a term used to describe a type of sample contamination which results in sample peaks re-appearing in later runs, which do not actually contain the sample (e.g. such as 'blank' runs). The contamination can last for several sequential runs, often decreasing in amount after each injection (which is a key observation when troubleshooting this issue). When proper HPLC (LC-MS) instrument training has been provided, most modern HPLC system designs make carryover extremely rare, but when it does appear, the contamination is often due to one or more of the following: (1) A lack of HPLC system maintenance (esp the A/S rotor seal); (2) Overloading samples which foul the column (very common new user reason); (3) Poor Wash Vial Usage, Needle Wash and/or Sample Vial Selection; (4) Inadequate operator training in how to set-up and use the chromatography system. *Note: Proper operator training greatly reduces the chances of contamination and is the most overlooked reason for the problem. A minimum of five years industrial training for HPLC and ten years industrial training for LC-MS is usually needed to develop a basic level of proficiency (you can not learn these techniques by taking a "class").

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

Dear William and Pabon,

Thanks for your kind response.

I cleaned the CSH column by IPA: ACN: MeOH: H2O (1:1:1:1) overnight, and we ran a serial four injections as the attached figure shows The blank solvent still has the PA carryover after the PA standard injection.

I uploaded a figure that contained the LC chromatogram and other useful information, please check it and give me suggestions, many thanks.

Best,

Bingpeng

We appreciate the additional information, but lots of basic chromatography info is still missing (e.g. Column Temp, sample volume/solution/concentration, a chromatogram of the std etc). Please get the help of an experienced local chromatographer with industrial experience. Lipids are complex to analyze by HPLC (LC-MS). They stick to everything (esp S.S.) so it is important to use strong solvents to flush them away. Your "wash" solution is far too weak to clean the column or system (25% IPA?). C18 for PA is not ideal at all and the CSH column you selected is not well suited for this application (the treated Waters PREMIER CSH column is better). However, while we have no idea what HPLC system you are using, it is probably a SS based system so all of the tubing, valves and parts will allow the PA to stick to it (resulting in carryover), esp when an aqueous method AND aqueous wash routine are used. *Suggestions: Replace the injector's rotary seal, switch to a stronger wash solution and use after every analysis run. Perhaps try a NP method in Neg ESI mode too which will allow you to use better solvents for lipids. Most of all, contact an experienced chromatographer with lipid analysis experience via LC-MS in industry to take a look at your specific instrument setup. ON-site help will allow you to move forward faster (Web forums are poor at assisting new and intermediate level users with these types of questions).

William Letter

Thank you very much for your kind response and useful comments.

I could also supplement some information for you and hope it will be useful.

My used column temp is 55dc, and the sample volume is around 500ul. the concentration of PA standard is 3uM in the solvent mixture of IPA:ACN:Water 2;1:1. My using the LC system is waters ACQUITY uplc system and the current using wash buffer is (weak buffer: 10% ACN and strong buffer :IPA: ACN=1:1). In addition, may I know what is a NP method?

Finally, as you mentioned, the on-site check and help may be very important. I also sought help from the local company (waters) and they are also trying to solve it. I will convey your suggestions to them and hope it's helpful to solve this problem, thanks again.

Best,

Bingpeng

Bingpeng Yan : You need the help of a professional chromatographer to perform this analysis. Using an HPLC system with little to no formal training is unwise and will lead to the collection of invalid data using invalid methods. HPLC is a complex technique. it takes the average scientific five years of full time, industrial training just to achieve a basic level of proficiency in HPLC. "Basic" level... * Having the samples analyzed by an industrial chromatographer with experience in the analysis of lipids will save you time, money and increase your chances of collecting valid data to use.

You wrote: "and the sample volume is around 500ul". - I hope that is a typo because the estimated column volume for your tiny column is around 240 ul, so the maximum injection volume (~ 3%) would be about 7ul. Please hire someone to run the analysis.

William Letter

Thanks a lot for your suggestions. I am not the professional chromatographed guy as you mentioned but I am also an experienced user of LC-MS instruments. My main purpose is to utilize the LC-MS method to measure the lipids substrate or product so that it could characterize the enzyme activity. So if the LC method does not work, I prefer to choose another approach to evaluate the enzyme activity.

Additionally, I thought you mentioned that the sample volume is the liquid volume in the glass vial in the sample manager. Now I realise you meant it should be the injection volume, and my injection volume is 3uL.

Thank you for your professional advice!

Best wishes,

Bingpeng

The technique of chromatography is appropriate for lipids, but someone with more experience is needed to develop and optimize the method of analysis.