You’ve just decided to develop a solid phase extraction (SPE) method for a new assay in your laboratory. However, there are so many decisions to be made! Which company do I choose? Which product do I use? Polymer based sorbent or silica based? Cation or anion exchange? While this seems incredibly difficult, it doesn’t have to be that hard! Today, we’re going to walk through this process of figuring out how to develop a starting point for our SPE method.
LC/MS/MS seems to be the new gold standard in sample analysis nowadays. However, some labs (like forensic labs) are a little slower to get LC/MS/MS instruments and are still analyzing samples using GC/MS. The majority of published articles in peer-reviewed journals are for LC/MS/MS so those working in laboratories with GC/MS instrumentation may not know how to change the sample preparation to better fit GC/MS.
First and foremost, GC/MS methods tend to need a higher sample volume than LC/MS/MS methods. GC/MS is a less sensitive instrument, which means more sample is needed to achieve lower limits of quantitation (LOQs). Many LC/MS/MS methods only require 50-200 uL of sample. GC/MS methods usually require 1-3 mL of sample.
Because of the higher sample volume needed for GC/MS analysis, higher mass sorbent beds in the extraction cartridge are usually needed as well. The larger sorbent bed allows for a higher amount of the compounds of interest to bind to the sorbent bed. Because of the larger sorbent bed, higher volumes of wash solvent and elution solvent are needed as well.
GC/MS also can’t be used for super volatile compounds because of the temperature of the injection port. For many methods, the injection port can be anywhere from 150⁰C to 300⁰C. Many compounds can’t hold up to these temperatures. Because of this, derivatization becomes necessary for many compounds when analyzing by GC/MS. Very simply, derivatization involves adding a structure onto your compound of interest that acts as a stabilizer. This adds time to your sample preparation as an incubation period is necessary in order to make sure that the compounds in your sample are fully derivatized.
When reconstituting samples for LC/MS/MS analysis after the extraction and dry down are completed, the mobile phase starting conditions are usually used as a reconstitution solvent. For example, if the LC gradient in a method starts as 90% aqueous mobile phase A and 10% organic mobile phase B, the reconstitution solvent would be that same 90:10 mix. In GC/MS analysis, the reconstitution solvent needs to be 100% organic, usually ethyl acetate or acetonitrile. The reconstitution volume is usually much lower in GC/MS than in LC/MS/MS. Since GC/MS is a less sensitive instrument, the less solvent used to reconstitute the sample, the more concentrated the compounds are as they are injected into the GC/MS.
These are some of the biggest differences between sample preparation methods for GC/MS and LC/MS/MS analysis. As can be seen above, the differences between methods are minimal, which makes it a fairly simple process to develop a new method for one or the other instrument type. If any questions come up during this process, reach out and ask us!
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.
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.