It happens to all of us. We’re getting a new method developed and validated and then it comes time to run our negative urines. And everything comes up as positive! There are peaks for our analytes of interest in every urine that we run! How is that possible? In this blog post, I’m going to discuss some of the more troublesome analytes that I’ve encountered as far as finding an actual blank sample and what I’ve done to try and fix the issue.
Continue reading How can we extract and analyze for ever-present contaminants of interest?
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.
Continue reading How to Monitor and Prevent Sample Carryover during Method Development
In my last blog post we talked about LogP and its role in sample prep. Today we are going to discuss the acid dissociation constant, pKa, and how it affects method development. Knowing and understanding the pKa of your compounds tells you if the compound can be ionized, and under what conditions, so you can use this property to develop better sample prep methods.
Continue reading Why is pH adjustment important for sample prep methods?
The goal of sample preparation is to create cleaner samples, collecting the compounds of interest and eliminating interferences – that “junk” we don’t care about the can cause ion suppression and matrix effects that affect sensitivity, accuracy and precision. The ideal sample prep method removes all interfering compounds and produces 100% recovery of all analytes of interest. The problem is that many interfering compounds have properties that are similar to the compounds we need to detect and quantitate. It takes some skill and knowledge to develop a method that washes interfering compounds away and elutes the analytes of interest in a separate step. You need to make sure your washes don’t elute the compounds you care about, and you don’t want interfering junk eluting with your analytes.
Understanding the chemical properties of the compounds in your sample matrix, both the ones you want to detect and the ones you want to eliminate, is necessary for successful method development. Is the molecule acidic or basic? What functional groups are present? Can they be ionized? Is the molecule hydrophilic or hydrophobic? Polar or non-polar? In this post, I am going to discuss the octanol-water partition coefficient, or LogP and its role in sample preparation using supported liquid extraction (SLE) and solid phase extraction (SPE).
Continue reading What is the role of LogP in sample prep methods?
Urine hydrolysis is a part of sample preparation that we do to eliminate glucuronides. So first, what are glucuronides? And why do we want to get rid of them? Glucuronides are formed during metabolism. These glucuronide compounds attach to drugs to make them more water-soluble. This allows for easier excretion of the drugs in urine. However, this is not what we want to analyze when looking for our drugs of interest. We would rather look for just morphine or just oxazepam instead of morphine-3-β-D glucuronide or the oxazepam glucuronide. The process of removing these glucuronides from our analytes of interest is called hydrolysis.
In this post, I’ll tell you about my experience with urine hydrolysis and my success (or lack of success) when using different enzymes.
Continue reading Urine hydrolysis: how did I choose which enzyme to use?
For many of us developing an analytical assay requires numerous experiments in addition to lots of data review, and yet despite the feeling of confidence in our success, how reliable is our assay really? How do we measure reliability of our assay? Well, there are many ways but here I’ll explain the most common approach using a theoretical analyte called compound X, which, in this case, is readily prescribed for arthritis. And like many drugs, compound X is excreted in large quantities, unmodified, in human urine. So, for arguments sake, let’s say we’ve already defined our LC/MS-MS method and we’ve ironed out an extraction method using SLE+ (Thanks Bruce!)
Continue reading How to calculate percent recovery and matrix effects for your analytical assays
There is often confusion as to how to choose the proper SPE ion exchange media. How do you determine if you should be looking at anion or cation, weak or strong? In this post, I will give some simple guidelines on making the best choice. You will see that ion exchange is all about acids and bases.
Continue reading Choosing the best ION Exchange Mode for Solid Phase Extraction
If you are frustrated with low recoveries caused by protein binding during supported liquid extraction (SLE) and solid phase extraction (SPE), this blog post will give you a few tips. Proper sample pre-treatment is the key to solving this issue.
Continue reading Techniques for disrupting protein binding in sample prep
Often the question arises asking how can I extract both acids and bases with Supported Liquid Extraction (SLE). In this blog post I will discuss some ways to extract both acidic and basic analytes in the same method.
Continue reading Extracting both acidic and basic analytes in a single supported liquid extraction (SLE) procedure
When it comes to sample cleanup the question occasionally comes up asking what technique is best for my application? Solid Phase Extraction (SPE) or Supported Liquid Extraction (SLE). Well, what’s right for you depends on your application and the characteristics of your analytes. Both techniques offer high recoveries, are reproducible, and are easily automated. In this post I would like to outline some of the advantages, disadvantages and limitations of each technique.
Continue reading When should I choose SLE or SPE for sample cleanup?