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Spring is finally here! Unfortunately so are the ticks…

After a long and cold winter here at the Restek headquarters in Bellefonte, PA, USA it seems that spring is finally here!  It is so nice this time of year to get outside and enjoy the weather and the beautiful outdoors that Pennsylvania has to offer.   Unfortunately after a hike in the woods there is always the required tick check.  Ticks are nasty little arachnids and can carry a number of diseases, including Lyme disease.  I’ve had to pull a few off of myself, but animals seem to be even more susceptible to getting ticks since they are closer to the ground and walk through the grass and brush where ticks like to hide out.  Repellants like DEET (N,N-diethyl-m-toluamide) can be applied on exposed skin and clothing to protect from ticks.   Permethrin containing products can also be used to treat clothing and other outdoor gear that may be exposed.   It is always important to read the application instructions carefully and apply the product only as directed (for you and your animals!).

Another thing to consider is that many of these topical treatments and other personal care products eventually end up in our streams and lakes.  Jack Cochran and I, with the help of Cory Fix, worked on a project where we were using GCxGC-TOFMS to evaluate the Las Vegas Wash, an urban river that flows into Lake Mead.  In order to see very low levels of both targeted and non-targeted analytes we took 4L of wash through a disk extraction and concentrated to a final volume of 1 mL.  We found a lot of interesting things in that water and of course DEET was one of them!

The sample preparation for the Las Vegas Wash sample followed EPA Method 527.  The combination of a large sample volume (4L) and combining and concentrating the extracts to 1 mL, allowed low ppt detection.

The sample preparation for the Las Vegas Wash sample followed EPA Method 527. The combination of a large sample volume (4L) and combining and concentrating the extracts to 1 mL, allowed low ppt detection.

We found hundreds of chemicals in the sample.  These included prescription drugs, illicit drugs, flame retardants and other personal care products.

This is only a very small list of what we found. We actually detected hundreds of chemicals in the sample. These included prescription drugs, illicit drugs, flame retardants and other personal care products.

Are fatty acids overwhelming your QuEChERS dSPE PSA cleanup and causing issues in your GC analysis? Get more cleanup capacity with cartridge SPE cleanup!

Fatty acids are important molecules in the human body because they are used as a source of fuel.  There are many food sources of both “healthy” and “unhealthy” fatty acids.  Many sources of dietary fatty acids come from fruits, vegetables, seeds, nuts, and animal fats.  The QuEChERS methodology was developed to analyze pesticides in fruits and vegetables, however many scientists (including Restek) have adopted this approach to analyze pesticides in other commodities (tobacco, dietary supplements) or a further deviation of looking at other residues (PAHs, PCBs, PBDEs) in other food types (milk, tea, seafood).  My colleague, Julie Kowalski, wrote a really nice 3 part series on QuEChERS for Separation Science that starts with the basics and finishes with using QuEChERS as a concept, or tool, instead of a direct method (A Primer, Beyond the Basics, The Concept).

Let’s get back to fatty acids.  They are in foods, we want to test those foods, and they don’t play nicely with the GC injection port, and can completely overwhelm your target analytes. In the QuEChERS method, PSA (primary secondary amine) sorbent is used in the dispersive solid phase extraction (dSPE) cleanup to remove fatty acids.  However, sometimes the capacity of the dSPE format is just not enough to provide an effective cleanup of the extract for analysis.  I found this out first hand when I was trying to develop a method for determining PCBs and PBDEs in human milk using the QuEChERS concept.  In my initial method development I was using GCxGC-ECD, but found that my target analytes were shifting retention times by almost 30 seconds!  A quick look on the TOFMS and we confirmed that large amounts of fatty acids were still present in the extract.

We then moved to a 500 mg PSA cartridge SPE (cSPE) cleanup step to better remove the fatty acids present in human milk.  The process was actually pretty simple.  After a quick conditioning step with acetone, I loaded 2.5 mL of my extract on the cartridge, for this project my extract was in 1:1 hexane:acetone, and simply pulled it through the cartridge.  After pulling vacuum for 2 min to dry the cartridge, I eluted with 5 mL hexane.  Since human milk also has a significant amount of fat, I needed to further clean up the extract with a 500 mg silica cartridge.  The cSPE PSA did a really great job of removing all of those fatty acids as you can see in the GCxGC contour plots of the NIST SRM below.

The top contour plot shows the NIST SRM with a silica cSPE cleanup only.  It is clear that large amounts of interferences (fatty acids) are present in the extract.  The bottom contour plot shows that the addition of a PSA cSPE pass through then the silica SPE cleanup provides a much cleaner extract!

The top contour plot shows the NIST SRM with a silica cSPE cleanup only. It is clear that large amounts of interferences (fatty acids) are present in the extract. The bottom contour plot shows that the addition of a PSA cSPE pass through then the silica SPE cleanup provides a much cleaner extract! Both GCxGC chromatograms are on the same scale.

The Rtx-1614 separates MORE halogenated flame retardants than just Polybrominated Diphenyl Ethers (PBDEs)!

Halogenated flame retardants (HFRs) are used in numerous household and office products, including electronics, carpeting and furniture.  Polybrominated diphenyl ethers (PBDEs), a subset of HFRs, have been found to be persistent and bioaccumulative in the environment.  While the main technical mixtures of PBDEs have mostly been phased out of production and use, the concentrations in the environment have not been declining and are currently still widely monitored.  Other halogenated flame retardants are now being used to replace the PBDEs that have been phased out.  There is still debate whether these replacements will be more environmentally friendly than their PBDE counterparts and monitoring the levels and occurrences of these HFRs are important to understand potential environmental and biological implications.  A recent PBS episode of “To The Contrary” highlighted some of these issues.

Achieving GC separations for such a large group of compounds can be difficult. In addition to shared quantification ions making chromatographic resolution necessary, the thermal stability of the compounds must also be carefully addressed.  Decabromodiphenyl ether (BDE 209) is a notably difficult analyte since it can thermally degrade in the GC inlet and on the GC column. A 15 m x 0.25 mm x 0.10 µm Rtx-1614 GC column with a fast elution profile limits thermal degradation of BDE 209 while maintaining resolution of BDE 49 and BDE 71.

Thermodynamic modeling software like Pro ezGC, can aid method development for closely eluting compounds.  This modeling program was used to determine the best separation of all target analytes, keeping in mind that increased residence time in the column would reduce response to thermally labile compounds. Check out the chromatograms below for the actual separations achieved from the modeled program.  What’s even better now is that all of these compounds are available here on the Web EZGC Chromatogram Modeler.  That means that if you only want to model a subset of these compounds, you can see what the best instrument conditions are for your specific separation!!

Halogenated Flame Retardants on Rtx-1614

Figure 1: Instrument conditions from Pro eZGC modeling maximizes resolution while keeping the analysis time to 25 min. Separation is increased between BB 153 and BDE 154, however EHTBP and DP syn are now coeluting.
Oven: 75°C (1 min) to 210°C at 18°C/min, to 310°C (4 min) at 8°C/min
Flow: 1.6 mL/min

    Figure 2: Alternative instrument conditions based on best efficiency flow and optimal heating rate. This results in a fast analysis time, but compromises resolution between BB 153 and BDE 154.     Oven: 75°C (1 min) to 330°C (2.8 min) at 25°C/min     Flow: 1.4 mL/min

Figure 2: Alternative instrument conditions based on best efficiency flow and optimal heating rate. This results in a fast analysis time, but compromises resolution between BB 153 and BDE 154.
Oven: 75°C (1 min) to 330°C (2.8 min) at 25°C/min
Flow: 1.4 mL/min

If you are planning on using a subset of this list for your own modeling in the Web EZGC Chromatogram Modeler you can copy and paste from the original Excel file for this table (Halogenated Flame Retardants Excel Table).HFR Table

 

Analyzing pesticides in herbal tea using QuEChERS and GCxGC-TOFMS of course!

Herbal tea, a non-caffeinated drink made from plants, herbs, or spices has been used throughout history for its potential medicinal benefit.   Various herbal material is mixed depending on the desired medicinal or flavor properties for the tea.   As with any plant based commodity, there is the potential for pesticide residues to remain in the final product.   Herbal tea also falls into a gray area of pesticide regulation because it can also be viewed as a dietary supplement.

It can be very challenging to detect trace levels of pesticide residues in dried plant material found in herbal tea.  The extract, even after cleanup can contain a large amount of coextractive material that can completely overwhelm the target pesticides, making trace detection very difficult. Furthermore, nonvolatile material not removed during extract cleanup deposit onto the inlet and column requiring more frequent maintenance to be performed (Figure 1). Luckily we have been down this road before and came out the other side successfully (See Dietary Supplements and Tobacco application notes).  When encountered with trace level pesticides and difficult food based commodities we employ the QuEChERS methodology and evaluate and quantitate the samples using GCxGC-TOFMS.

The tea samples were first ground to a powder and then we weighed 1 g of tea and added 10 mL of water to hydrate the sample.  After letting that sit for 30 min, we went through the EN QuEChERS extraction procedure.  The extracts were cleaned up using dSPE tubes containing 150 mg MgSO4, 50 mg PSA, 50 mg C18, 7.5 mg GCB.

The primary column was a 1 m x 0.25 mm Rxi Guard column connected to a 30m x 0.25mm x 0.25µm Rxi-5ms.  The guard column (aka retention gap) allowed for better solvent focusing and improved the peak shape of the early eluting pesticides while protecting the analytical column (Figure 2). The secondary column was a 1 m x 0.25 mm x 0.25 µm Rtx-200.  All of the columns were connected using the SGE SilTite µ-Union.  For quantification, I used our new QuEChERS Performance mixes which cover a range of pesticide volatility and overlapped with many of the GC amenable pesticides commonly found in tea or the other ingredients included in the herbal teas.  The herbal tea “Wildberry Zinger” had the most incurred pesticides of the teas that we evaluated (Figure 3 and Table I).

After 28 injections of tea samples, nonvolatile residue was present on the liner.  After changing the liner GC performance was restored.

After 28 injections of tea samples, nonvolatile residue was present on the liner. After changing the liner, GC performance was restored.

The use of the 1 m retention gap allows for better solvent focusing and improves the peak shape of the early eluting pesticides while protecting the analytical column.

The use of the 1 m retention gap allows for better solvent focusing and improves the peak shape of the early eluting pesticides while protecting the analytical column.

Although the herbal tea is very complex, the GCxGC-TOFMS helps to separate the matrix from the pesticides of interest.

Although the herbal tea is very complex, the GCxGC-TOFMS helps to separate the matrix from the pesticides of interest.

Percent recoveries for a subset of the pesticides evaluated.  We achieved good recoveries at both spike levels and found a few incurred pesticides too.

Percent recoveries for a subset of the pesticides evaluated. We achieved good recoveries at both spike levels and found a few incurred pesticides too.

From Glass to Fused Silica in Corning, NY, USA

I almost hate to admit it, but oftentimes when I am enjoying a vacation, I can’t help but think about chromatography and how it touches so many aspects of life.   Last week I was in Corning, NY and went to the Corning Museum of Glass which has a fantastic display of all facets of glass.  The museum started with artistic sculptures, then a historical perspective of glass making, including a display on borosilicate glass used to make scientific glassware (including the Kuderna-Danish Concentrator and the glassware used for soxhlet extractions).  I’m sure my family loved hearing how all of the pieces of glassware are used! At Restek, we acquired Glastron, a manufacturer of specialized glassware, to expand our glassware products.

The final area in the museum is the Innovation Center which is an interactive science and technology exhibit that houses the optics gallery.  I couldn’t ignore the connection to chromatography when the exhibit started explaining how Corning chemist J. Franklin Hyde made fused silica in 1934 from pure liquid chemicals instead of melting dry mineral ingredients like other glass products. Fused silica therefore, has a much higher melting temperature than other traditional glasses. It took another 45 years until fused silica was used for capillary GC columns by adapting a process for the manufacturing of fiber optics. The development of fused silica capillary GC columns changed the entire field of gas chromatography and continues to be the chosen platform for efficient, fast separations.  It makes me wonder, what is the NEXT invention that will change the face of chromatography like fused silica capillary columns did decades ago?

We attended a glass blowing demonstration at the museum that was really great!

Watching the glass blowing demonstration at the museum was really neat!

The 33rd International Symposium on Halogenated Persistent Organic Pollutants, Dioxin 2013

The last week of August I attended the POP symposium in the steamy Daegu, Korea.  With over 500 attendees representing 42 countries the technical content was both informative and interesting.  The conference mainly focuses on persistent organic pollutants named in the Stockholm convention.  These include several organochlorine pesticides, polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), polychlorinated biphenyls (PCB), along with some of the newer additions of perfluorooctane sulfonic acid (PFOS), polybrominated diphenyl ethers (PBDEs) and the proposed chemicals, hexabromocyclododecane (HBCD), short-chained chlorinated paraffins, chlorinated naphthalenes, hexachlorobutadiene and pentachlorophenol.  While those POPs alone can fill a week full of material ranging from analytical, toxicological, fate, transport and levels in the environment, other organic pollutants were added to this year’s program.  Several presentations focused on an emerging issue of pharmaceuticals and personal care products (PPCPs) in water and also on the analysis of polycyclic aromatic hydrocarbons (PAHs).  Speakers from Brazil, Vietnam, Korea, Australia, Canada, Belgium and the USA, just to name a few all discussed the issues of organic pollutants in our environment and the levels that are found in humans and animals.  It really highlights the importance of persistent organic pollutants and that environmental and food safety are truly global issues!

We were serenated by some very talented traditional Korean musicians during the opening reception!

We were serenaded by some very talented traditional Korean musicians during the opening reception!

Do you measure the length of your GC column?

When purchasing a GC column the length that you buy isn’t necessarily the exact length that you get.  In fact, it is typical that you will receive 0.5 to 1.0 meter more than what you bought!  Knowing the exact length of your column and inputting the correct value into the GC instrument software is important so that the electronic pressure control (EPC) can accurately deliver your desired carrier gas flow.   When you need to trim your GC column for maintenance, the new column length needs to be determined to properly translate the current method and maintain analyte resolution.

So how do you measure the length of your GC column? Well, one way to NOT measure your column is by unraveling the column from the cage and measure using a measuring tape.  That would be pretty messy! There are actually two ways to get an accurate column length measurement.

The first way is to calculate the column length by using the following equation:

Column length = π * diameter of column cage * number of loops on the cage

For example:   π *0.175m * 56 loops = 30.8m

Accuratly determine column length by measuring the diameter of the column cage and counting how many times the column loops around the cage

The second way to determine accurate column length is by first measuring a holdup time (aka dead time, or dead volume) of a non-retained peak.  Then increase or decrease your GC column length in a column pressure/flow calculator until the calculated holdup time matches the experimental holdup time.

Calculating column length using the GC pressure/flow calculator

So do you determine the accurate length of your GC column?  If so, what method do you use?

Testing for pesticides in tobacco… QuEChERS makes it easy!!

Whenever it is time to analyze pesticides in complex matrices I always hope that we can use the QuEChERS sample preparation approach and not something more laborious!  The Quick-Easy-Cheap-Effective-Rugged-Safe method was developed for multiresidue pesticides in fruits and vegetables, however we have been using this approach outside of the typical fruit and vegetable matrices with success.   When compared to more traditional sample prep techniques (liquid-liquid and solid phase extraction) QuEChERS significantly decreases the time spent and solvent used.

Tobacco can be a very complex sample, but with a few modifications to the EN QuEChERS method we were able to get good spike recoveries (92%)  for  a wide range of pesticides.   Some of the modifications that we made were to use a 2 g sample and add 10 mL of water to hydrate the sample, then after the addition of 10 mL of acetonitrile we vortexed the sample for 30 min, instead of the typical 1 min shake.  We also used the power of GCxGC-TOFMS to separate some of the matrix interferences away from our analytes of interest (Figure below).  In a 1D GC-MS analysis we would have needed to perform cartridge SPE instead of the dispersive SPE to further remove matrix interferences.

If you want all of the details check out the full application note that was recently published! Evaluation of Dispersive and Cartridge Solid Phase Extraction (SPE) Cleanups for Multiresidue Pesticides in QuEChERS Extracts of Finished Tobacco Using GCxGC-TOFMS

 

GCxGC separation of matrix components from the incurred pesticide piperonyl butoxide in the second dimension that would have coeluted in a one-dimensional analysis.

GCxGC separation of matrix components from the incurred pesticide piperonyl butoxide in the second dimension that would have coeluted in a one-dimensional analysis.

 

This isn’t your average CSI, the International Network of Environmental Forensics (INEF)

Next week (June 10-12) at the Pennsylvania State University (PSU) the International Network of Environmental Forensics conference is being held.  This isn’t your typical crime scene investigation but, as defined by the INEF, “environmental forensics is the use of scientific techniques to identify the source, age, and timing of a contaminant into the environment.”  We may not be learning how to catch the killer, but maybe the contaminator! This conference is essentially next door to Restek (10 min drive) so it is a great opportunity to attend presentations from some of the international leaders in the field.  I know that I am looking forward to learning more about environmental forensics.  A few Restek scientists will also be presenting at the conference.  Chris English will be presenting work that he did using purge and trap to analyze BTEX in crude oils.  He did a blog on the topic if you are interested.   I will be presenting some work that I did using GCxGC-TOFMS to fingerprint crude oils and tarballs using petroleum biomarkers.  We used the NORDTEST oil spill identification system to determine if some of the tarballs we received from Florida matched the oil from the Deepwater Horizon Oil Spill.  The full application note was just published if you are interested in more detail. Last, but certainly not least, Jack Cochran will present about true peak capacity increase using GCxGC for environmental forensic applications.  Jack also blogged about true peak capacity increase for GCxGC in the past.  The list of speakers, topics and visiting my alma mater has me excited for a great week next week!

Here is the INEF program if you are interested.

Mining through complex data and non-target analysis is made easier with GCxGC-TOFMS

I am currently working on a project looking for pesticides in herbal tea.  At this point, I am using the contents of one tea bag (~1.5g) and 10mL of freshly boiled water with a QuEChERS extraction.  I am still working through the details on the impact of the hot water and what type of cleanup will be necessary for pesticide determination, so that will be a blog for another day. I first decided to do some split injections of the raw extract to see what type of natural products are in some of the herbal teas I picked up at the grocery store.

Using GCxGC-TOFMS with a 30m x 0.25mm x 0.25µm Rxi-5Sil MS in the first dimension and a 1m x 0.25mm x 0.25µm Rtx-200 in the second, I processed the data by doing a “peak find” and library search for anything with a S/N of at least 100.  I then get my peak list and sort according to the NIST library similarity number, so that the highest matches are at the top of the list.  I start scanning through the list and see what names sound either like a pesticide or something else interesting.  For example, I came across the name niacinamide with a similarity of 912, and a spectra that seemed fairly unique.  I did a quick google search on the name and (according to Wikipedia) it is a main ingredient in an acne medication and has shown to have anti-anxiety properties among other potential medicinal uses.  This type of peak find is made possible by the spectral deconvolution of the time-of-flight mass spectrometer.

I also mine through the data by taking advantage of the ordered chromatograms produced by GCxGC.  This means that compounds with a similar structure, or a homologous series, will elute in a band across the chromatogram.  I found another compound, lupeol, which is a triterpenoid that has been noted to have several medicinal properties.  By looking at peaks that are eluting near lupeol, I found sitosterol which is a phytosterol that may reduce cholesterol.

While I don’t have standards to positively confirm peak identity, a high NIST match similarity with a spectra that contains abundant high m/z ions increases my confidence in the peak assignments.  I really enjoy digging through the data and seeing if anything interesting pops up.  Using GCxGC-TOFMS can give you A LOT of data to mine through, but with spectral deconvolution and ordered chromatograms, it makes it much easier.

The NIST library spectra, the peak true (spectral deconvoluted) and caliper spectra all show a high match similarity for Niacinamide

The NIST library spectra, the peak true (spectral deconvoluted) and caliper spectra all show a high match similarity for Niacinamide

 Sitosterol and Lupeol have a similar structure and elute in a band in the GCxGC-TOFMS chromatogram

Sitosterol and Lupeol have a similar structure and elute in a band in the GCxGC-TOFMS chromatogram