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.