liquid chromatography analysis of hormonesPart 1 of this blog post, LC-MS Hormones Detection Application Kit as per EPA Method 539 (1), presented two tools (kit and webinar) to address the challenges of detecting hormones in drinking water.

A brief recap if this is the first post you are viewing in this two-part series. Last year, the World Health Organization published a 52-page report titled, Pharmaceuticals in drinking water (downloadable PDF) due to increasing concerns over the long-term health impact of hormones in water to humans and aquatic species. Prior to this report, in 2010, the U.S. EPA published the U.S. EPA Method 539 for determination of hormones in drinking water (link to method PDF) defining the application protocol to consist of Solid-Phase Extraction (SPE), Liquid Chromatography, Electrospray Ionization, and Tandem Mass Spectrometry (LC-ESI-MS/MS) for the analysis.

During the course of our webinar, Application Protocol for EPA Method 539, (available on-demand; link to registration page from where you can view the webinar), there were many questions on the kit and using equipment other than ours, and I thought the answers would be an interesting quick read.

Note that the questions and responses have been edited for readability.

Question: Are the internal standards and surrogate standards included in the Hormone Applications Kit?

No, they are not. But, they are readily available from Fisher Scientific. Internal and surrogate stand kits for other U.S. EPA methods are also available from Fisher Scientific. The reason for not including them is because typically the standards are consumed in different amounts plus customers have indicated that they would prefer not to have the standards included in such kits and wish to order them separately.

Question: Only Thermo Fisher Scientific instruments are used in this protocol. Can we use instruments from other manufacturers?

According to the U.S. EPA Method 539, LC columns, LC conditions, MS conditions, internal or surrogate standards can be changed. The reason we developed this kit to be used with our instruments is to ensure reproducibility and accuracy of the analysis, and save your time in method developments. Note that the sample collection, QC requirements and solid-phase extraction steps cannot be changed.

Question: What is Unregulated Contaminant Monitoring Rule (UCMR 3)?

Every 5 years, U.S. EPA issue a new list of no more than 30 unregulated contaminants to be monitored by public water systems (PWSs). This monitoring provides a basis for future regulatory actions to protect public health. The third (UCMR 3) was published on May 2, 2012 and it built on the established structure of UCMR 1 and 2, and includes 28 new chemicals and 2 viruses. Seven hormones described in this application protocol are included in UCMR3.

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Question: What is the difference between manual and automated Solid-Phase Extraction (SPE)?

Automated SPE have many advantages over manual operation. For example, by automating all four steps of SPE, our Thermo Scientific Dionex AutoTrace 280 system requires only about 15-30 minutes of operator time, following by hours of unattended SPE process, while in manual SPE, it takes hours to operate and wait for the sample preparation to be completed. The Dionex AutoTrace 280 system also uses positive pressure to ensure superior reproducibility and precision to manual SPE.


Question: We have been talking about automated and manual SPE. What about online SPE? Have you tried that?

That is an interesting area. Online SPE has been receiving a lot of attention for some time. And, we do offer our Thermo Scientific Dionex UltiMate 3000 RSLC system among others with automated online SPE solutions. They are very popular, specifically for low, trace-level applications. We also have developed software tools to easily automate such applications.

There is one significant drawback for online SPE. The problem is that the whole system typically has to wait for the sample preparation step in the methodology each time an analysis is performed unlike an offline SPE stepup where multiple samples can be processed in parallel. That means when the online SPE step takes much longer than the analysis, you will need to wait to process the sample. Today, when we are all moving towards high-pressure HPLC applications to speed up methods and analysis as much as possible, the online SPE step can be time-limiting. But, each lab needs to make a decision on the best solution for them depending on throughput and workflow. We are pleased to offer both solutions to our customers.

Question: I don’t have a Thermo Scientific Dionex UltiMate 3000 HPLC system; can I use the Hormone Application Kit on my own HPLC instrument?

Yes, you can. But, keep in mind that the conditions have been optimized for our instruments. Hence, you will need to follow the procedures outlined in the U.S. EPA Method 539 Section 9.2 if using your own HPLC instrument. You will then have to verify and validate your method to ensure that it delivers the results as outlined in the U.S. EPA method.

Question: What other tools do I need for method development besides the kit?

The Hormone Application Kit (P/NTF-MKIT0011S) included everything you need for the U.S. EPA Method 539. Beside the product bundle (SPE disks, HPLC column, and vials) and the detailed application protocol, Thermo Scientific TraceFinder methods are also part of this kit. These verified LC/MS/MS methods will dramatic reduce your method development time. Additional methods for hormone analysis can also be downloaded at the link.

Do you have questions after viewing the kit details and the webinar? If so, do let us know via the Comments box below. We look forward to hearing from you!