When should I choose a mixed-mode SPE?

 

Mixed mode SPE phases have become very popular for sample clean-up prior to analysis using mass spectrometry.  Having the capability to retain compounds by two modes of interaction during solid phase extraction is useful when a large number of analytes with different properties are of interest.  Most mixed mode phases are bonded silica or polymeric reversed phase materials with an ion-exchange group bonded to it.  Continue reading When should I choose a mixed-mode SPE?

Should we use vacuum or positive pressure for processing samples in our lab?

Solid-phase extractions (SPE) can be a long and sometimes complicated process. So, we want to make sure that everything works the first time we extract. There are several different techniques we can use to extract. We can use a manual vacuum extraction or we can use manual or automated positive pressure to extract. In this blog post, I will be discussing some of the benefits that we see when using positive pressure for SPE.

Continue reading Should we use vacuum or positive pressure for processing samples in our lab?

What are some good sources of reference for sample prep method development?

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. Continue reading What are some good sources of reference for sample prep method development?

What’s the Best Way to do SLE?

SLE (supported liquid extraction) is a sample prep technique that has been in use for over ten years now, but many analytical chemists don’t know about, or understand the best way to do an SLE extraction.  In this post, I’m going to talk about how SLE works and the proper way to do an SLE extraction for sample clean up.

Continue reading What’s the Best Way to do SLE?

How can we extract and analyze for ever-present contaminants of interest?

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?

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.

Continue reading How to Monitor and Prevent Sample Carryover during Method Development

Why is pH adjustment important for sample prep methods?

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?

What is the role of LogP in 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: how did I choose which enzyme to use?

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?

How to calculate percent recovery and matrix effects for your analytical assays

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