Have you ever worked with new methods and said to yourself “yes I know what these internal standards and surrogates are measuring and when to add them to the samples”? Sounds familiar right? Well, I remember when I first started working in the laboratory and needed to fully understand and follow all method protocols and standard operating procedures (SOPs). One of the questions I asked was, what is the difference between method 525.2 and its internal standard and surrogates versus every other method we extract and analyze?
I want to start by explaining what surrogates and internal standards are. A surrogate is an analyte added to a sample prior to an extraction. The surrogate is pure, unlikely to be found in the sample, and is at a known amount. As a chemist, surrogates measure my ability to perform all steps within a procedure, testing my technique from extraction to final analysis. Whether I was testing clean drinking water samples or complex tissue samples from fish, surrogates were added to measure sample preparation techniques, instrumentation, my performance, and ability to obtain accurate results.
An internal standard is like a surrogate as it is a pure analyte and unlikely to be found in a sample and is at a known amount. However, an internal standard differs with its function. It is used to measure the relative responses of other analytes and surrogates, mostly to confirm the analytical instrumentation that I am using is working properly. One of the most common questions I get about internal standards is when to add them. First, you must follow your method requirements. For instance, most environmental protection agency (EPA) methods will have an internal standard added at the end of an extraction procedure, right before analysis.
However, one method that throws a “stick in the spokes” when talking about the addition of internal standards is EPA method 525.2. This method states that the internal standards are added at the beginning, just like surrogates. With this method, I always made sure to emphasize to new users of EPA method 525.2 that it is not like most EPA methods and internal standards are added directly to the sample, prior to the extraction procedure. I know it can be confusing, that is why it is important to always refer to the method to ensure you are following the requirements.
So, you may be asking yourself, “why is the internal standard addition in EPA method 525.2 different from most of the other methods”? Well, the intent is to correct for any variability in the sample preparation procedure! When implemented this way small errors in technique during the extraction all the way to vialing the final volume can be accounted for in the recoveries (or lack of) by using the internal standard.
Another question that I am commonly asked in relation to 525.2 is if the internal standards are added with the surrogates and spike analytes, then how do the internal standards get monitored? Method 525.2 has that covered by implementing a fortification solution. This solution is added to each sample and will check the internal standard recovery through the entire sample preparation process. Each internal standard must be recovered over 70% and then used to adjust for analyte loss during sample preparation.
Lastly, a question that I am always asked, is if there is another way to improve the workflow to reduce the risk of poor analyte recoveries? Well, I have done this type of testing with groups of chemists. I have found that no matter how well everyone’s techniques are aligned, there is always going to be a difference in recoveries in the end. Some are much better than others. However, I have noticed the loss is much less when utilizing sample prep automation, rather than a full manual (hands-on) process. Although, EPA method 525.2 adjusts for these losses by adding the internal standard prior to sample extraction, it does not allow the analyst to accurately monitor their extraction efficiency. Therefore, EPA method 525.3 has made the change and requires addition of internal standard after the extraction and utilizes the surrogates to monitor extraction efficiency, as done with most EPA methodology.
For more information on internal standard implementation at the start of an extraction and how it is carried out, please click on the button below to download our latest application note.