Why should I choose a protein precipitation 96-well plate for my method instead of doing a traditional protein crash?

Most clinical chemists have developed a blood, serum or plasma assay using a protein crash because it is inexpensive and generally removes proteins that interfere with detection or the analysis in some way.   This is usually done by adding a “crash” solvent like methanol or acetonitrile to the blood sample, vortex mixing, then centrifuging and removing the supernatant to remove the crashed proteins from the sample.  Sometimes the crash solvent is acidified to aid solubility and begin the denaturation process.  The crash solvent is usually added at a ratio of 3:1, but can sometimes require up to 10:1 solvent:specimen.  For more information on the process of doing a protein crash I recommend this article. Continue reading Why should I choose a protein precipitation 96-well plate for my method instead of doing a traditional protein crash?

In-well hydrolysis plates: Do they really work?

In-well hydrolysis means hydrolyzing urine samples in the well of the extraction plate instead of outside of the extraction plate (I talked about why we hydrolyze in this blog post). This can be a time saver for high throughput labs, which makes it look very appealing. In this blog post, I will be discussing some of the advantages of using an in-well hydrolysis plate and some of the results that I’ve gotten when compared to an out-of-well hydrolysis plate.

Continue reading In-well hydrolysis plates: Do they really work?

Putting it together: what are the first steps to building a proper method?

Last time, I discussed how to gather information from the literature that would best suit our application for the detection of naloxone, buprenorphine, norfentanyl, and methadone in urine. Let’s quickly recap: we have our matrix and our analytes and a Shimadzu NexeraX2 LC with a 5500 Sciex MS. We did some searching and eventually settled on a poster we found on the Biotage website titled “Sample Preparation Strategies for Urine Panels with 50 or More Drugs and Metabolites Analyzed by LC-MS/MS.” Now what?

Continue reading Putting it together: what are the first steps to building a proper method?

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?