Setting analyte identification parameters is important and will play a large role in just about everything else especially determination of limit of detection, limit of quantitation and false positive/false negative rates. This seems straightforward because, of course, you must be able to identify your pesticides. Although parameters include retention time matching and mass spectral data, setting the actual criteria for your parameters will allow you to do or not do certain things…like meet regulatory maximum residue levels. This will largely be dictated by mass spectral criteria like ion ratio specifications.
But I want to discuss retention time, a chromatographic parameter, because care still needs to be taken when using it for compound identification. Why retention time? Because I know that it’s critical for pesticide analysis, has important consequences and it’s something many clients struggle with when developing pesticide methods. Let me walk you through a gap I see. Let’s set our criteria using the reference documents below (Ref 1 and 2). For example, retention times of an analyte in known standards and in samples should match, within a certain window like ±X.X minutes. This is fairly straightforward.
But first another retention time parameter is needed. To match retention times, you need a retention time. So, analytes should have a retention time longer than that of the time it takes an unretained compound to elute from the column. It is important that the analyte is actually retained on the column, not simply flowing through the column to the detector. The degree of retention is related to chemical interactions; this is what is specific about chromatographic data that makes it useful for compound identification. The elution time of an unretained compound is often called the “elution time of the void volume,” “void time” or casually referred to as the “solvent front.” There is little specificity in this because no retention, or chemical interaction, occurs making this type of data unsuitable for compound identification.
Lack of retention is a problem with polar pesticides, like daminozide or chlormequat chloride, when using a typical reversed phase LC separation popularly used for multi-residue methods. This is why some of these pesticides often require single analyte methods specifically designed to test difficult to retain pesticides. I see many cannabis pesticide testing methods published that have known polar pesticides in their multi-residue methods. It is important to ask about true retention of these polar pesticides. Look for void times in publications of these methods and ask your technology provider for this information if they are supplying you a method that includes polar pesticides. You can determine the void time experimentally or even use online calculators to get an estimate. After you know the void time of your system, you can confirm if your early eluting analytes are retained. You may have some more work to do otherwise…or at least know you are identifying compounds with only half of your parameters.
Guidelines for the Validation of Chemical Methods in Food, Feed, Cosmetics, and Veterinary Products by the U.S. Food and Drug Administration
Analytical Quality Control And Method Validation Procedures For Pesticide Residues Analysis In Food And Feed Document Nº SANTE/12682/2019 produced by the European Union Reference Laboratories.