Developing a new sample prep method should be systematic to ensure ruggedness, and can be done quickly with the right tools. The days of using a vacuum manifold are gone, along with the variability and long extraction times incurred. Today’s options provide an array of positive pressure systems that process a wide range of sample matrices, varying in viscosities.
In our lab for example, we work via a three-steps process to develop a new sample prep method:
- We use a semi-automated positive pressure system in the hood to quickly scout the extraction techniques that we think will work, based on previous applications and knowledge of chemistries.
- We then optimize the extraction steps that give us the best performance.
- Once we are close, we transfer the method to an automated sample prep workstation. By simply creating the program and storing it, we can be sure to run it the same way every time.
We also appreciate that the automation allows us to leave and do something else. It seems that we are always multi-tasking, so the automation allows us to get more done in the day.
The systematic process of method development means that we spend a good deal of time planning our experiments. First, we want to consider what our goals are and what our limitations are in terms of instruments and samples.
Because we want to incorporate as few steps as possible, we first ask ourselves: Can we do this with protein crash or supported liquid extraction?
For details on how to determine this, I suggest that you attend a current method development courses given by scientists either at an industry event or online.
Then we consider solid phase extraction as a good option to compare with. It requires a few more steps, but will tell us if we need this for analyte recovery. Usually, we will scout with a technique that can do a wide range of acids, bases, and neutrals, and if we know enough about the analytes, we will add a cation or anion exchanger.
Once I have the extraction options identified, then I consider the desired workflow for our lab. For every industry, there are advantages to using smaller volumes of solvents and generating less solvent waste. If you can eliminate steps in the cleanup then you can reduce time and increase sample throughput. In some cases where sample volume is limited, you can increase sensitivity by concentrating with a lower elution volume and potentially eliminate evaporation steps.
Of course, if I am going to go through the trouble of developing a new method, I want it to take advantage of technological advances that will meet my needs into the future. I need a workflow that is simple to use, gives me the results required by the assay, and is reliable over time. Both instrumentation and consumables need to be fit for purpose and consistent for the life of the assay.
After all the planning, it’s time for the hands-on part. Put together a scouting plate or array of scouting columns.
Again, the method development training can show you exactly how to do this if you haven’t done it before.
Then develop the method using a manual/semi-automated positive pressure system. Try to use small sample volumes, down to 5-10 µL, and extraction sorbents as small as 2 mg to minimize solvent usage throughout the method. See if you can eliminate transfer steps and evaporation. Once you have the method close, transfer it to the platform that you will be using routinely. Optimize settings and store the program. If possible, lock the method so that others cannot change it on the fly.
Do you want to know more about how to successfully develop your Low Volume Extractions? My colleague Stephanie Marin goes through all details in “Less is More – Microelution Extraction for High Throughput Bioanalytical Workflows”.