Previously, I spoke about the sources of information available for method development. We discussed some of the resources we could use to figure out how to build a method for the detection of naloxone, buprenorphine, norfentanyl, and methadone in urine. For this post, we’ll go through the search process with these analytes using urine as our matrix of focus. For this assay we’re running a Sciex 5500 triple quadrupole mass spectrometer with a Shimadzu NexeraX2 UHPLC. Since we’re not out to reinvent the wheel here, let’s assume this assay, in some form, has already been done. It’s likely Sciex has an application note with these analytes detected in urine. Perhaps all of them in are in a large, complex urine panel or they’re located in various other app notes with other vendors? Either way, the easiest approach is the simplest: first look for an application note from Sciex or other vendors that have as much similarity to our assay as possible.
A brief search with the term “Drugs of Abuse” within the Sciex knowledge base doesn’t turn up much – strike one. So, I decided to contact the technical support via Live Chat – strike two. To be honest, they never answered, probably because they were not awake yet. No problem, I know it’s out there. What about PubMed? I quickly cue up the following “buprenorphine urine Mass Spectrometry” and get a number of hits. As I scroll down, I see a title of great interest “Quantitation of Buprenorphine, Norbuprenorphine, Buprenorphine Glucuronide, Norbuprenorphine Glucuronide, and Naloxone in Urine by LC-MS/MS.” It has a couple of key ingredients for our assay, plus some bonus information regarding their glucuronides, which, fortunately, my colleague Jillian has posted about before! But, this article is not free and I’m not at the point where I want to scour PubMed for an open access article that suits my needs. So, let’s have a look at the vendors, perhaps Biotage?
Okay, so we head over to the Biotage website and go straight to the search box and, again, cast a wide net with the search term “drugs of abuse.” We see that 89 Support Documents & Applications are available along with 24 News & Events. Let go with the support and applications. That’s still pretty broad, but if we look to the left we see a list of options. Remember, we’re looking for an LC/MS method for the detection of naloxone, buprenorphine, norfentanyl, and methadone in urine. On the left side of the page, we see the term “Matrix.” Under the dialog box, we can refine our search to the matrix we are interested in, urine. Now we should be down to 34 results. However, we can narrow our search even further when we explore the “Analyte” feature located just below “Matrix.” From the drop down box, we search to find one of our analytes, buprenorphine, and immediately we get seven results. I always look at the most recent, and after filtering by year, we come upon a posters and application notes ranging from 2012 to 2017. Looking at the most recent titled “Sample Preparation Strategies for Urine Panels with 50 or More Drugs and Metabolites Analyzed by LC-MS/MS.” We find it’s a poster from ASMS 2017. We wanted an application note, but it’s from this year and looks like a good resource. Looking a little closer we see all of our analytes in table 3, along with some of their physical constants like pKa and Log P, and their MRM transitions, LC parameters, along with three different options for extraction – all in urine and with enzyme hydrolysis – pay dirt! Although we weren’t able to quickly locate a good piece of literature in PubMed, we gathered a lot of information from a single source by searching among a vendors knowledge base. This is probably the quickest and most efficient way to gather basic information about your assay’s analytes. For more nuanced information about analytes, white papers are an excellent resource, as are blogs or webinars on-demand. Next time, we’ll put our collected information together and go over the steps to build our LC/MS method.