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Amines: Topaz or Base Deactivated Liners?

Amines can be difficult to analyze by GC, since they are active and adsorb to surfaces within the chromatographic system, including the inlet liner and the column.  This leads to loss of compound response, and peak tailing.  While deactivations can help to mitigate these effects, the quality of deactivations varies.  Primary amines are especially difficult, due to their susceptibility to interact with active acidic sites like silanols, commonly found in liners and columns.

Restek offers a base deactivation for both liners and columns, designed to passivate glass surfaces with basic sites, lessening the chances of adverse reactions with amines and other basic compounds.  As you may be aware, Restek launched Topaz inlet liners in 2017 as our newest premium liner deactivation.  One question that has come up a few times is “How do Topaz liners compare to base deactivated liners for analysis of difficult amines?” A study was conducted to compare responses of active bases on the two deactivation chemistries.

The method conditions shown in Table 1 were used for the comparison.  A mix containing primary, secondary, and heterocyclic amine groups, as well as an ethanolamine and an aniline, was evaluated on Restek’s base deactivation and Topaz deactivation (See Figure 1).  Two different concentration levels were tested on each liner.  A total of 7 liners from each deactivation were evaluated.  Liners were single taper with wool tested in splitless mode, as this provides a very rigorous test of the deactivation.

Table 1: Method conditions for testing liners for amine response.

Figure 1: Amines that were tested on the liners.  Chemical structures from Wikipedia.

Figure 2 shows how response factors for various amines at 5-10 ng compare on the two deactivations.  Pyridine and 2,6-dimethylaniline had similar performance on both deactivations.  Examining more difficult amines like diethylenetriamine (DETA) and diethanolamine (DEA), the Topaz deactivated liner had higher compound responses, though the relative liner to liner variation for diethanolamine was slightly higher for Topaz compared to the base deactivated liners.  Diethylenetriamine is very difficult to analyze by GC as it contains two primary amine groups and a secondary amine group.  Diethanolamine contains a secondary amine group, but also has two alcohol groups, which can be reactive.

Figure 2: Amines response comparison on Topaz and Base deactivated liners at 5-10 ng splitless.

Figure 3 shows a comparison of the same amines at 2.5-5 ng.  Once again, pyridine and 2,6-dimethylaniline had similar performance on both deactivations.  DETA, on the other hand, was non-detect on the base deactivated liner at 5 ng.  The Topaz liners showed high liner to liner variability for DETA, but did elicit a response on all liners tested.  Topaz also showed better response for DEA compared to the base deactivated liner, though there was more variation from liner to liner.

Figure 3: Amines response comparison on Topaz and Base deactivated liners at 2.5-5 ng splitless. Diethylenetriamine was non-detect on all base deactivated liners tested at 5 ng.

The above study demonstrated that the Topaz liners generally had better responses for the most difficult amine compounds that were tested here, compared to the base deactivated liners.

Chiral separation on a C18 column? Separation of d- and l- Amphetamines, Part I

Did you know that chiral chemistry was discovered by Louis Pasteur, a French chemist and biologist in 1848? However, it took about a century to find that chirality plays a key role not only in the life of plants and animals, but also in several aspects of drug design, and both pharmaceutical and illicit drug development.

As a chemist or a forensic toxicologist, you could be using chiral analysis in biological samples or some street samples to determine legal or illicit drug consumption or to identify illicit drug manufacturing locations. Whatever your field, chiral separation of drug enantiomers is essential in order to show that the active enantiomer is, in fact, present in your specimens. In the past, different techniques like chiral selector in mobile phases, GC-MS and LC-MS chiral columns were used for the chiral separation. However, most LC studies use an expensive chiral column in combination with cyclodextrin additives that can cause ion suppression and contamination in electrospray ionization, a major drawback of chiral stationary phases along with the high cost. Toxicology or forensic labs with strict budgets may not be able to adopt a method with such an expensive component.

So, do you have a C18 column in your lab? If yes, then you are all set!! A cost effective chiral separation can be achieved with a C18 column by adapting a simple pre-column derivatization technique without the need for a costly and specialized chiral column. We evaluated this achiral technique in the tech note “Analysis of Amphetamines by LC-MS/MS for High-Throughput Urine Drug Testing Labs”. Methamphetamine is an old drug with a rich history. Amphetamine and methamphetamine are psychostimulant drugs that occur as two enantiomers, dextrorotary and levorotary, as a result of their chiral center (Figure 1). The dextromethamphetamine (d-isomer) form is highly abused and typically found in illicit preparations. However, detection of abuse is complicated because consumption of over-the-counter and prescription medications containing l-isomer may yield positive results if the analytical method used cannot distinguish between the d- and l- enantiomers.

Figure 1: Structures of d- and l-Amphetamine and Methamphetamine Enantiomers.

The aforementioned enantioselective LC-ESI-MS/MS technique separated the d- and l enantiomers of methamphetamine and its metabolite, amphetamine in human urine, after pre-column derivatization with 1-fluoro-2,4-dinitrophenyl-5-l-alanineamide (Marfey’s reagent) using a Raptor C18 column. Marfey’s reagent is an effective derivatizing reagent for separation of d- and l- amphetamine and methamphetamine isomers by converting them to diastereomers. This method is compatible with any LC-MS/MS instruments. Accurate and reproducible analysis was achieved in 7 minutes of chromatographic analysis time, making the column, sample preparation and chromatographic method well suited for selective, low-cost, high-throughput analysis and improved methamphetamine result interpretation.

Click on the link for more details on the Analysis of Amphetamines by LC-MS/MS for High-Throughput Urine Drug Testing Labs

In part 2, I will discuss more about the sample preparation, derivatization and chromatographic conditions…

Using Raptor ARC-18 for a Rapid Analysis of Vitamin E Acetate

We recently took a look at combining Vitamin E Acetate and cannabinoids into one method https://blog.restek.com/?p=65679.  This route allows labs to diversify their testing services with a few simple modifications, if using our potency method.  We wanted to give labs another option as well.  Therefore, Justin Steimling went back into the lab and developed a standalone rapid analysis for Vitamin E Acetate.

A Raptor ARC-18 30 x 3.0 mm, 2.7 µm column was utilized for this quick 3 minute run.  Justin used methanol instead of acetonitrile since it is actually a stronger solvent for this compound.  Another added benefit to this method is the lower cost associated with methanol compared to acetonitrile.  Whether your lab would like a rapid standalone method or modifying your potency method, we have two routes for analyzing Vitamin E Acetate.

Tetrachlorobenzene isomers added to ProEZGC!

Tetrachlorobenzenes are manufactured as intermediates for the production of fungicides, herbicides, defoliants, insecticides as well as dielectric fluids for transformers (1). There are three isomers with the following chlorine substitutions; 1,2,3,4 / 1,2,3,5 / 1,2,4,5. While the chemical industry measures these compounds for purity they are also found in the environment as the result of leaking transformers and to a lesser extent from the manufacture of paper. In the pulp and paper industry these compounds can be a byproduct of the bleaching process which uses chlorine and chlorine dioxide (2).

 

Image of 1,2,3,4- tetrachlorobenzene.

 

 

https://pubchem.ncbi.nlm.nih.gov/compound/tetrachlorobenzene#section=3D-Conformer

 

 

 

 

 

 

 

 

We have had several requests for these compounds and have added them to the over 500 compounds currently on the Rxi-624Sil MS ProEZGC library.

On the Rxi-624Sil MS, the isomers elute in the order as they are listed below:

  • 1,2,3,5- Tetrachlorobenzene
  • 1,2,4,5- Tetrachlorobenzene
  • 1,2,3,4- Tetrachlorobenzene

 

Figure 1: Shows the Tetrachlorobenzene isomers with alkanes on the Rxi-624Sil MS.

 

  1. Government of Canada. 1993. Tetrachlorobenzene. Canadian Environmental Protection Act Priority Substances List Assessment Report. Environment Canada and Health Canada, Ottawa.
  2. US EPA. 1994. Locating and Estimating Air Emissions from Sources of Chlorobezenes (revised). EPA-454/R-93-044. Office of Air Quality Planning and Standards. Research Triangle Park, NC.

 

Afraid of HILIC? Watch These Videos and Overcome Your Fear!

Hydrophilic Interaction Liquid Chromatography or HILIC is a powerful tool to analyze polar compounds that have limited retention on typical reversed-phase columns. The power of HILIC can be utilized across multiple markets including environmental, food safety, and clinical diagnostics. But to be honest, it can be challenging to implement a HILIC method and more considerations have to be taken compared to the development of a reversed-phase LC method. There are many different types of HILIC columns on the market, but no matter what type of HILIC columns you use, there are some basic principles you need to know to not only save time, but most importantly, be successful! We have made a series of 4 short videos to give you a quick look at HILIC method development.  Hopefully you will be more confident to develop your own HILIC method after watching them!

Video 1:  Understanding the HILIC Separation Method in LC

In the first video, we talk about the separation mechanisms of HILIC and a variety of ways polar analytes can interact with HILIC stationary phases. The good news is that the solvents used in HILIC methods are similar to those used in reversed-phase LC methods! For most of the LC-MS/MS analysis, HILIC methods have significant increases in instrument sensitivity because it’s operated under higher organic solvent conditions which aids in desolvation.

Video 2: Conditioning Your LC Instrument and Column for HILIC

Don’t be surprised when you see inconsistent performance (including retention time and sensitivity) from your new HILIC column upon the first few injections. This video will tell you how to condition your LC instrument and HILIC column especially when you are switching LC systems from reversed-phase to HILIC analysis. Remember that all types of HILIC columns are not built equally, and the conditioning time can vary depending on your analytes and analytical conditions.

Video 3: Buffer Choices for HILIC

Since pH plays a critical role in the performance of a HILIC method, the first step for method development is to determine a suitable pH for your mobile phases. In this video, we explain how to incorporate pH into your HILIC method development and how to fine-tune the buffer concentration in order to obtain optimal peak shapes, resolution, and sensitivity.

Video 4: Injection Solution Conditions for HILIC

During the process of method development described in videos 2 and 3, you also want to pay attention to the injection solution since it can have a great impact on the performance of your HILIC method. Matching the aqueous/organic ratio of the injection solution to the initial mobile phase conditions is the best practice for HILIC-based analysis. Therefore it is important to think ahead to determine if your sample preparation procedure and final sample solution will be compatible with your HILIC method.

So there you have it! Hope you enjoy watching these videos and don’t forget to share your HILIC method development successes with us!

What’s in your mouse feed?

When it comes to different types of GC techniques, headspace (HS) analysis is about as clean as they come. Typically, hundreds of injections can be made without doing any inlet maintenance or column trimming. It is one of my favorite techniques in the lab since it encompasses countless different unique applications. Even though HS is the coolest thing since sliced bread, it can be tricky at times. I recently found this out while working on a collaboration with a researcher at a university. The research is focused on immunometabolism, which I know nothing about, but this researcher needed help testing some mouse feed containing an additive for residual solvents, which I know something about! So, I set out on my task to test their mouse feed for residual solvents, and encountered something that could help other scientists out there doing HS — the importance of running matrix matched calibrations.

 

Sometimes it is assumed a solvent based calibrations will apply well for samples via HS because in theory all of the volatile analytes are driven into the gas phase. To disprove this, we ran a quick test comparing analyte responses at the same concentration; one vial with standard only and the other vial that is matrix matched.

 

 

Comparing the responses for these compounds, methanol in the solvent based vial was ~7x higher than the matrix matched vial and acetone in the solvent based vial was ~3x higher than the matrix matched vial. If we were to calibrate off of a solvent based calibration, we have the potential of reporting a false-negative for the presence of methanol and acetone in the additive. It was decided that we matrix match the calibration standards in order to give a more representative response.

 

Moral of the story when analyzing samples by HS: no matter what type of analysis is being conducted, it is extremely important to run calibration standards as similar as possible to the sample matrix that is being analyzed to ensure that you are producing accurate and reliable results!

A Quick Look at Vitamin E Acetate Screening on the Raptor ARC-18

 

There’s been a lot of focused on vaping crisis and the potential culprit causing dozens of deaths in the United States.  The CDC believes Vitamin E Acetate might be the common linker in these cases.  Vitamin E derived oils are sometimes used as a thickening agent or to dilute THC and increase profits.1 Normally, Vitamin E is not harmful at low levels when ingested or applied as a topical in lotions.  However, when inhaled, this compound could potentially compromise lung functions.2

Therefore, cannabis labs are interested in screening for Vitamin E Acetate to help evaluate the quality of vaping products and protect the safety of the consumer.  Most states that have legalized some form of cannabis have established regulations for potency, pesticides, mycotoxins, and residual solvents.  However, Vitamin E acetate is not regulated within the market YET.

So, we wanted to take a quick look at one potential route to analyzing this compound.   Rather than having to develop another standalone method for just this one compound, we wanted to see if we could keep things simple and easy for you.  Justin Steimling who developed Restek’s 19 cannabinoids workflow (https://www.restek.com/chromatogram/view/LC_GN0601) went back into the lab to see how well this compound could be added to the method.

The total analysis time is 15 minutes.  Since the method ends on 98% acetonitrile, the backpressure was kept below 400 bar even after increasing the flow to 0.8 mL/min after CBT.  The revised method includes both a column flushing procedure into the method and it allows for the simultaneous measurement of 19 cannabinoids and vitamin E acetate.

This analysis still allows for quantification of Vitamin E acetate as part of a modified potency analysis. We’re continuing to look at other methods for rapidly screening for Vitamin E Acetate as interest in this compound continues to develop.

 

  1. “Breakthrough in CDC vaping illness investigation: Vitamin E acetate linked to THC may be to blame”, CNN, Nov. 8th 2019, Jen Christensen, https://www.cnn.com/2019/11/08/health/vaping-injury-vitamin-e-thc-bn/index.html
  2. “In a breakthrough, health officials identify possible culprit behind vaping illnesses: vitamin E acetate”, STAT, Nov. 8th 2019, Megan Thielking, https://www.statnews.com/2019/11/08/vaping-illnesses-vitamin-e-acetate/

Analyzing orange: peel and pulp separately or as whole?

Up to this point, I’ve focused on the optimization of QuEChERS salts and dSPE cleanup with fairly homogenous matrices. So, what about oranges? Should I peel them and analyze only the pulp? After all, that is the edible part. The peel is used as well – pressed into essential oils or scraped into zest. Besides, there are uses where the peel and the pulp are used together in some products; such as orange juice, where squeezed oranges lose the majority of the flavor due to preservation and additional flavor (made from the peel) needs to be added back into the juice. So, there is a need for analysis for individual parts of orange as well as for orange as a whole.

Orange pulp

Let’s start with the orange pulp. It is a matrix that has high water and sugar content and low pH (3.3 – 4.2). Because pH plays a role in pesticide stability, the buffered QuEChERS salts (AOAC or EN) are preferable. When we look at the data collected after spiking orange pulp with the QuEChERS Performance mix (#31152) and extracting with all three types of extraction salts (Fig 1), there are higher responses for several pesticides, especially when buffered salts are used. Overall, the EN salts (#25849) were chosen because most of the pesticides exhibited higher recoveries.

Comparison of total pesticides' recoveries in orange pulp using different QuEChERS salts

Figure 1: Comparison of total pesticides’ recoveries in orange pulp using different QuEChERS salts

Read the rest of this entry »

Glycols…. Tis The Season!

Tis the season to be thinking about glycols…. Why you ask?  Well its currently winter as I’m writing this and glycols, specifically propylene glycol (PG) and ethylene glycol (EG), along with other ingredients, are used in airports for deicing planes.  Propylene glycol based fluids are more common since they are less toxic than ethylene glycol fluids.

Glycols are highly soluble in water and are readily transported to waterways. While these compounds can be quickly degraded by bacteria, this process can use up available oxygen killing animals that rely on oxygen in the water to survive. Due to these effects on the surface waters and marine habitats, it’s crucial to get precise and accurate data, ensuring the analysis of these samples to maintain healthy and habitable waters.

If you’re having trouble with your aqueous glycol injections, as in; symmetrical peak shapes, consistent retention and robust methods, give these helpful tips a try to see if they help your analysis.

  • Rtx-Wax column (cat# 12455)
  • Sky Precision Liner with wool (cat# 23305.1)
  • 1 µL injection volume with 50:1 split
  • 50:50 water/methanol rinse

Because split injection was used for the analysis of glycols in water, it provides rapid sample transfer and a narrow analyte band, which produces symmetrical peaks, retention time consistency, and also improves reproducibility for low-level analysis of glycols in water. In addition, it reduces the amount of water in the injection port, which minimizes the potential for backflash. The 50:1 split injection used in this method effectively prevented sample expansion volume issues. Even though the amount of sample on column was significantly reduced, adequate sensitivity and linearity were still achieved.

Figure 1: Shows propylene and ethylene glycols at 1 ng on column concentration in 50:1 split mode.

References/links:

https://www.restek.com/pdfs/EVAN2873-UNV.pdf

https://www.restek.com/Technical-Resources/Technical-Library/Environmental/enviro_EVAN2873-UNV

Custom Solutions for Your Cannabis Testing Needs

If you’re a cannabis testing lab or manufacturer, you’re probably familiar with the current reference standards available on the market.  However, these off the shelf solutions might not fit your workflow needs.  Wouldn’t it be nice to have an option to dial in your ideal references standards to meet your daily testing tasks?

Well…Restek’s custom standards are here to help!  Did you know that 1/3 of the ampules that leave Restek’s facility are custom ordered reference standards (yes, I took the liberty of “lifting” that from the link below).

If your lab has a need for custom standards, visit our Reference Standards – Search, Select, and Custom Request link https://www.restek.com/Forms/Reference-Standards-Search-Select-and-Custom-Request and fill out the easy to use request form.

This form offers you the capability to add your desired compound list, concentrations, platform for analyzing the mix, and preferred solvent.  Don’t worry, if you don’t know the answers to some of these questions, we’re here to help with that as well.  Our formulations chemists have a LOT of experience with custom reference standards and can provide support.

If this is your first time filling out this form, don’t hesitate to watch the step by step video which will  walk you through the process.  You can find the video right on the search page.