How to Monitor and Prevent Sample Carryover during Method Development

When running through the exhaustive process of method development, most of us put the majority of our focus on validation and how to complete our crazy validation checklists. Throughout this process, the last thing we want to see is some random hiccup in our workflow. But a whole validation without a hiccup is just wishful thinking, right? Whether your analytes are on back order or you have the misfortune of catastrophic instrument failure, we all experience some type of complication or mishap. One in particular, sample carryover, seems rather innocuous, but without some type of preventive action, it can really ruin a good method. Nonetheless, if you have preventative measures in place, this can be easily preventable.

For this post, I will talk about how to actively monitor and prevent carryover. First, what protocols or strategies can we have in place to detect the presence of sample carryover? The simplest, and often overlooked, is a blank injection. Always run a blank in your LC/MS set. Always. And multiple blanks are even better! It can be water, for reverse-phase, or your LC’s initial conditions – anything that doesn’t have your matrix or standards in it. While most of us run sets of samples, QC’s, and standards, we should also have our blanks strategically placed to detect any potential carryover. For example, when running my calibrators, I inject them from lowest to highest in concentration. Immediately following my highest calibrator, I place a blank. I also tend to have my first and last injections be blanks, with some interspersed where I suspect carryover could occur.

Next, let’s direct our attention to actively preventing carryover. The current venue of HPLC’s (or UHPLC’s) have vendor specific approaches to combating carryover.  Shimadzu has a good look at carryover here. If we look at the Shimadzu Nexera X2 UHPLC system, we have the ability to wash just about everything our sample or standards meet within the injection system. You can dip and back-flush the exterior of the needle or rinse the inside of it too. Is there a chance we need different solvents for different wash steps? Yep, they let you do that if necessary – up to four of them. However, without a good combination of solvents, even the best system will not help us avoid the inevitable. Since most of us inject compounds with highly variable polarities or “stickiness,” we need to cover the spectrum of analyte polarity. My go-to is 25:25:25:25 [v/v] methanol/acetonitrile/IPA/water with 1% formic acid. This strong wash system covers both protic and aprotic systems and verges on the non-polar with the IPA. I have also had success with 45:45:10 [v/v] acetonitrile/IPA/Acetone with 1% formic acid. With either of these as our strong wash solutions, we should be able to avoid injection carryover for our method. Now, you might ask, “why the acid”? For most of my runs, I analyze a lot basic compounds and they can adsorb to the metallic surfaces within the autosampler. So, to minimize this, I add the acid, which helps eliminate any ionic coordination between my basic compounds and the metal surfaces of the system, like the needle.

Similarly, for hydrophobic or “sticky” compounds, I increase the amount of organic I add to the wash. Of course, we can always refine the ratios or substitute solvents to meet the needs of our method. For example, I have a panel of sixteen steroids in serum that I’m analyzing. I decided to go with some form of SPE to clean up the sample, to concentrate my steroids for analysis. Steroids are quite non-polar and are “late-eluters” on reverse-phase systems – most require their LC gradients to start around 40-60% organic. So, will the aforementioned wash systems work for me? No idea, so lets find out! First, I set up my wash solvent(s) and prep a blank composed of my initial gradient conditions. I run the blank followed by a no-matrix standard injection – this is a standard(s) prepped in the initial conditions of the LC gradient (I talked about no matrix standards in a previous blog post). It serves as our reference point for clean injection where zero interference’s are present. Next, I inject three blanks also comprised of my initial conditions. If I don’t see any of my steroid peaks in the injections when comparing it to the no-matrix injection, success! If peaks are present, then I need to know where these peaks are coming from. Since I haven’t injected any of our SPE extracted samples, (I just started method development) then I don’t have any reason to suspect the SPE or the matrix itself.

Therefore, I will first reexamine the wash system. A careful note on this: if the peak area steadily decreases with each injection then we need to alter the polarity of the wash as this indicates carryover. Increase the non-polar character of the solvent, to better match that of our analytes, and run the test again. If the peak area is steady after each blank injection, or even increasing, then we have contamination issues. Put simply, the analytes could have found a way into the LC system, either through solvent contamination or column contamination or something else. At this point, assume nothing is clean. Diagnosing contamination is quite a process and is a blog for another time.

Also, if you have a service contract, use it! There’s no sense in struggling to determine if it’s carryover or contamination on your own! If you don’t have a service contract, your life could get way more complicated…

Please post your tips in addressing carryover if they differ.

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