Why should I filter my samples instead of doing dilute-and-shoot?

Let’s face it, the whole method development process can be time consuming and expensive. Using solid phase extraction, while a much cleaner extraction method, can require a bit more time and evaluation. So dilute-and-shoot methods end up being the method of choice in some laboratories. The problem is that these methods are dirty and lead to a lot of other issues (dirty mass spec, clogged injection ports, frequent changing of LC columns,…). In this blog post, I’ll be discussing filtration methods and how those are a superior option to dilute and shoot.

Many companies make filter devices that can be used as an additional sample clean up step. Biotage makes ISOLUTE FILTER+ plates. For these plates, you load the sample into the plate and apply positive pressure or a vacuum to filter the sample through the plate. One benefit of the FILTER+ plates is that the filter is 0.2 um filter, which is recommended for LC-MS/MS analysis. This means that the bigger particulates are being filtered out of the sample, which leads to a cleaner sample and a cleaner LC column. These FILTER+ plates can be used with sample volumes from 50 uL to 1.5 mL, which is great for a range of sample types and LOQs.

So now that we know the idea behind filtration devices, how do we actually filter our samples with them? For the FILTER+ plates, you can filter urine, serum, plasma, or even blood samples! For urine samples, if hydrolysis is necessary, you add your sample, enzyme, hydrolysis buffer, and internal standard and incubate the sample (incubation time varies depending on the enzyme that you use. If you need some enzyme tips, check out my previous blog post on hydrolysis!). You can then do one of two things. You can dilute your sample with water and apply it to the FILTER+ plate or you can just apply the sample to the FILTER+ plate! Just remember, when you dilute your sample with water, your area counts for your analytes of interest will be lower. However, the samples are cleaner than they would have been without the dilution or in a dilute-and-shoot method. Dilute-and-shoot methods also tend to have greater variation between sample replicates, as was shown by ARUP Institute in this article.

Using a filtration device like the FILTER+ plates can save a lot of sample preparation time when compared to dilute-and-shoot. There is no need to centrifuge the sample to get rid of any particulates, nor is there a sample transfer step. Just filter with the ISOLUTE FILTER+ plates and you’re good to go!

While an extraction method like solid phase extraction or supported liquid extraction are the preferred techniques for sample clean up, sometimes they are not immediately practical due to cost constraints in a lab. Using filtration methods are always a good alternative to dilute-and-shoot.

 

When trying to develop a new method, how do I do a literature search?

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.  Continue reading When trying to develop a new method, how do I do a literature search?

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?