In my previous post, I briefly mentioned the process of method development. Today, I’ll go into a bit more detail and will explain how to start the process so we can get a global view of what we’re doing, and more importantly, why we do it. The why: the sole purpose for method development is to construct a robust and analytically sound method that will not just pass the barriers of validation, but provide physicians and patients with sound and reliable results. Now, let’s discuss the “how.” For example, your employer has asked you to build a method for the detection of a small group of analytes in patient urine. The task-at-hand entails building a LC/MS method and, perhaps, a sample clean-up method (SPE, SLE,…) for the detection of a small group of analytes used to combat opioid addiction: naloxone, buprenorphine, norfentanyl, and methadone. It seems straightforward and only involves four analytes. But, we need more information, and your employer expects you to pick that up.
So where to begin? Whether a veteran or a newbie, there is always one place to begin – the literature. Chances are extremely high that someone, somewhere, has already looked into these four analytes in the matrix you’re interested in. Perhaps not all in the same analytical suite, but we might get lucky given there are only four and they’re very similar in function. Rather than reinvent the analytical wheel, it’s in your best interest (and easier) to use your resources and cut down your time in the lab. As one of my mentors in graduate school told me “you can save yourself days, months, or longer, by spending a couple of hours in the library.”
On that note, let’s look into some resources. You may not have access to the paid subscriptions like Elsevier, Wiley, or Sage, as these are usually accessible to government and academic institutions. So, we’ll err on the side of caution and assume we haven’t these resources. Instead, we will first look at our analytes in the context of their physical and intramolecular properties. While Wikipedia is often relied up, be wary of the information that website carries as it is truly, an open resource. Websites that are more reliable are RSC’s ChemSpider, the Human Metabolome Database, DrugBank, PubChem, and Chemicalize.org. The last website actively applies algorithms to calculate your analytes theoretical physical-properties, including pKa and LogP, and plots them with visual representations of your analyte across the physiological pH range (Stephanie talked about LogP and pKa in her blog posts). While they provide you with 100 free credits, they go fast, and after that, it’s pay-for-play, or pay for each individual compound you look up. Now that you know a little bit more about your analytes, you need to see what others have done with them under the same pretenses.
The first place I look is at PubMed. Although PubMed isn’t a primary source for analytical or forensic chemistry, it does have a decent amount of clinical chemistry resources. Moreover, they can also provide similar or suggested literature and sometimes will direct you to articles that are open access or free without a subscription. Google’s Scholar is another decent search engine, but for me it isn’t always helpful. Forensic applications are somewhat easier to locate. Most of the articles from The Journal of Analytical are free, at least legacy articles, and if you’re a member of the Society of Forensic Toxicologists you’ll enjoy a free subscription! These articles generally host information specific to LC/MS parameters, SPE extraction, and statistical analyses, which is all quite helpful when trying to build a new method. They also provide insight into the current trends and applications for licit and illicit drugs of abuse. But, should you find a paper you absolutely must have, you might consider paying the $30 or so for the manuscript, even if it’s just for a 24 hour rental.
The sources mentioned above are just a small sampling of the resources that are available and can help road-map your method development approach. I also want to mention that, when looking into drugs of abuse (DOA’s), both The National Institute on Drug Abuse (NIDA) and Substance Abuse and Mental Health Services Administration (SAMHSA) are excellent resources as they’re the leading research bodies responsible for administering and organizing research focused on drugs of abuse. One last resource to mention is the vendors. Vendors go through a lot of in-house method development and post their results as application notes or white papers on their websites. While these sources of information are not designed to be a full solution to your assay, they are invaluable as road-maps – I can’t understate the value these note and papers possess. While you might have to register your information, the small amount of time it takes can cut down on your literature search dramatically. Next time, we’ll use a few of our sources to investigate our analytes and construct a road-map for our method. I hope that this information will help you figure out where to start when developing a new method! I listed just a few literature resources that I find helpful, but the list is truly limitless.