Improvements in Processing Drinking Water Samples by Method 525, Part 2: Extraction Procedure

In the first part of this 2-part blog series, I highlighted the improvements made by the EPA regarding the preparation and preservation of samples.  In this post, I will focus more on the changes the EPA has made to Method 525 which affect the analysis of the prepared samples.

Specifically, I will address:

  • The use of internal standards and their addition at different points during the extraction
  • Improving results with the use of a different sorbent material

Internal Standards

EPA Method 525.2 outlines a quantitation method which they refer to as an “internal standard calibration procedure.”  This term makes the procedure sound complicated, but don’t let it fool you.  This method essentially requires that you add your internal standards to your blanks and standards prior to analyzing them on your GC/MS.  This builds the internal standard responses into your calibration curve, and sets it up for use when analyzing your samples.  The method requires an external standard to be measured as an unknown sample, so you can be sure your calibration curve is accurate.

This can be a very powerful technique for quantitation by GC/MS, but there is one very strange aspect to the way this technique is implemented in Method 525.2.  The method requires you to add your internal standards to the sample solutions prior to extraction.

For those of you who may be unfamiliar with the purpose of an internal standard, let’s recap and clarify.  Internal standards are compounds that you add to all your analytical solutions (blanks, standards, QC standards, samples) to correct for analyte losses that might occur during your sample analysis.  Your analyte responses get calculated based on the ratio of the analyte response and the internal standard response.  Then if your method performance suffers (let’s say your instrument drifts partway through your sample analysis), the signal for both your analyte and your internal standard should be affected by the same magnitude and the response ratio should remain unaffected.

The keys to success when using internal standards are:

  • The internal standard compounds should not be naturally present in any of your samples
  • The internal standards should behave in a similar manner to your target analytes
  • The same concentration of internal standards should be present in every single solution

Now, if you recall, I mentioned that Method 525.2 requires internal standards to be added prior to your extractions.  This is an odd approach to internal standardization because, in doing this, you are making an assumption that every single extraction is going to recover 100% of your analytes.  Those of us who live in the real world know that this isn’t true.  In most methods where internal standardization is used for quantitation, the internal standards are added to the final concentrated extract.  This ensures that 100% of the internal standard is in the sample, and changes in the response for the internal standard are due to analyte losses and not due to artifacts of the extraction process.  The EPA corrected the approach to internal standardization when they published Method 525.3, which recommends that internal standards be added to the final extracts – allowing for a more accurate quantitation of your target analytes.

Alternate Solid Phase Extraction Sorbents

Another difference between Methods 525.2 and 525.3 is the type of media used for extraction.  Method 525.2 recommends the use of a C18 silica-based media for extraction, whereas Method 525.3 recommends the use of a polymer-based divinylbenzene (DVB) media.  Both media types allow for efficient reversed-phase separation, however, DVB media offers a few added benefits.  Polymeric-based SPE media, such as DVB, usually consists of highly cross-linked polymers which allows them to be used in almost any solvent.

DVB polymers are made up of only carbon and hydrogen atoms.  This structure means there are no functional groups (such as silanols) hanging around and causing unwanted interactions (check out this blog post for a refresher on retention mechanisms in SPE).  The benefit to a structure like this is it can be used in all pH ranges, as opposed to C18 media which has a very specific pH range over which it can be used effectively.  Another advantage of DVB over C18 is its additional surface area.  With DVB media, the vinyl groups are much less sterically hindered than the octadecyl functional groups in C18 media.  Media with greater surface area results in media with more active sites, which gives DVB a higher capacity to retain analyte compounds compared to C18.

As you can tell, the EPA is making a number of improvements to existing methods protocols, which is resulting in performance-based methodology, as well as improved recoveries for troublesome compounds.  Check out this application note to see an example of the data you can expect to generate when you process drinking water samples per EPA Method 525.3.

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