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Electronic Cigarettes Part X: Vapor Analysis – Application Note

FFAN2127-UNV_header

With the last installment of this blog series garnering some comments/questions, I thought it would be appropriate to post the following link to our application note, which has been featured in a lot of these blogs: http://www.restek.com/Technical-Resources/Technical-Library/Foods-Flavors-Fragrances/fff_FFAN2127-UNV

 

 

Analysis of Nicotine and Related Compounds in Urine Using Raptor™ Biphenyl

As Applications Chemists in the LC lab, one of the most exciting parts of our jobs is the variety of analyses we are exposed to. One day you are developing a method for potency analysis in cannabis samples, the next you are looking at anti-epileptic drugs in urine.    We’re regularly challenged to think outside the box to provide the best solutions for our customers.

In some recent work, my colleague, Shun-Hsin Liang, was tasked to develop a method for nicotine and related compounds in urine using LC-MS/MS.   The challenge was finding suitable analytical conditions for a rapid, accurate, and reproducible method geared toward high-throughput testing laboratories.    Typically, this analysis uses high-pH chromatography with relatively high concentrations of additives to increase retention, improve peak shape, and reduce peak tailing.   These conditions may lead to some additional non-routine maintenance (like changing pump seals and replacing tubing), and if you’re in a high-throughput laboratory, you know non-routine maintenance events always pop up at the most inopportune time.

Shun-Hsin was able to use the Raptor™ Biphenyl column and standard, low pH, reversed-phase LC-MS mobile phases that are compatible with a variety of LC-MS instrumentation. The method provided excellent performance for the simultaneous analysis of nicotine, two major metabolites (cotinine and trans-3’-hydroxycotinine), two minor metabolites (nornicotine and norcotinine), and a minor tobacco alkaloid, anabasine, in human urine. Accurate and reproducible analysis was achieved in less than 5 minutes of chromatographic analysis time, making the column and method well suited to low-cost, high-throughput analysis of nicotine-related compounds.

Please click on the link for more complete details of Shun-Hsin’s most recent work on the Analysis of Nicotine & Related Compounds in Urine Using Raptor Biphenyl.

All of my peaks are tailing… What should I do?

I get quite a few customer questions concerning peak tailing during LC analysis, and how to best troubleshoot this issue.   Peak tailing may be attributed to a variety of different causes including secondary interactions, contamination, column loading, etc.   This list goes on and on.   I usually ask a few key questions, and generally can give some good advice based on the responses.

  1. Is this a new problem?
  2. What are your analytical conditions?
    1. Mobile phases?
    2. Column?
    3. Gradient?
  3. Which analytes are troublesome?

During some applications work I was doing in the lab, I came across a very familiar issue: All of my peaks are tailing…

Figure 1_c-gramI asked myself the above questions:

  1. Is this a new problem? Yes, I just started method development.
  2. What are your analytical conditions? Flow rate – 0.5 mL/min, Temp – 30 °C
    1. Mobile phases? MPA: 0.1% formic acid in H2O, MPB: 0.1% formic acid in MeOH
    2. Column? Raptor Biphenyl, 2.7 µm 50 mm x 3.0 mm I.D.
    3. Gradient? 10-75%B in 5 minutes, re-equilibrate to 10%B for 2 minutes
  3. Which analytes are troublesome? All of them.   A mixture of 6 beta-blockers.

Nothing struck me as particularly odd with the analytical conditions, all analytes were well retained and eluting during the gradient, and there was sufficient re-equilibration time.  As I looked at the analyte structures, I noticed that these were all very active compounds.

Figure 2_pindololBased on this information, I believed the peak tailing was attributed to secondary interactions caused by silanol activity.   The cartoon below depicts how the secondary amine in the pindolol molecule could be potentially interacting with residual silanols on the surface of the silica.

Figure 3_silanol activity

With the addition of a buffer to my mobile phase, I should be able to mitigate this issue.  Since I’m already using formic acid, choosing a complimentary salt, Ammonium formate, will give the best buffering capacity at the desired pH.

Figure 4_bufferBy changing the composition of my mobile phase, I was able to reduce the secondary interactions, resulting in much more symmetrical peaks, and even got the bonus of some increased resolution between labetalol and oxeprenolol. With a flow rate of 0.5 mL/min, the total volume of mobile phase used per injection is 1.4 mL of MPA and 1.1 mL of MPB – for all intents and purposes these are relatively equal.   The addition of buffer to both aqueous and organic mobile phases ensures that the secondary interactions will be mitigated throughout the entire gradient – solving the peak tailing issues for both early and late eluting compounds.

Figure 5_final c-grams

See http://www.restek.com/chromatogram/view/LC_GN0550 for the final optimized method using 0.1% Formic acid and 5mM Ammonium formate modified mobile phases.

Raptor Biphenyl LC Columns provide the data needed for global antibiotic testing

Because the widespread use of preventative veterinary antibiotics has resulted in increased antimicrobial resistance in humans, the US Food and Drug Administration (FDA) released guidelines to address the issue in food animals (December 11, 2013). Similar constraints are also prevalent in the European Union.  Recently, European Researchers1 developed a validated method for the determination of veterinary antibiotics in bovine urine by LC-MS/MS. The column chosen was a Raptor Biphenyl column (150 x 2.1 mm, 2.7 um), because of superior resolution and sensitivity relative to other columns tested. While Restek scientists claim no affiliation with these researchers, their universities, their methods or results, we respect their efforts and are proud to provide chromatographic products that make their analyses possible. The paper can be found on the Elsevier’s Science Direct website for purchase. View the abstract here: http://www.sciencedirect.com/science/article/pii/S0308814615004707

 

1 Department of Veterinary Science and Public Health and Department of Health, Animal Science, and Food Safety, University of Milan, Italy, the Department of Health Sciences, V. le Europa, Campus S. Venuta, Germaneto, and the Department of Chemistry at the University of Nis, Bulevar in Serbia

 

Electronic Cigarettes Part IX: Vapor Analysis – What does all this mean?

Sorry for the two month blog delay, but by now you know we were utilizing multi-bed thermal desorption (TD) tubes to collect and analyze electronic cigarette vapor (see our last blog here). You also know that we found some interesting volatile organic compounds (VOCs) like formaldehyde, acetaldehyde, acrolein, xylenes, as well as siloxanes in electronic cigarette vapor. It is important to stress that the hazardous air pollutants (HAPS) formaldehyde, acetaldehyde, and acrolein were found in the vapor of four commercially available 1st generation e-cigarettes; however, these compounds were not present in the solutions. It is also important to note that these compounds were not found in the background air. Lastly, I must emphasize that our peers like Goniewicz et al. and Kosmider et al. have made the same observations for e-cig vapor. So we are just one of a few of the messengers (remember that when you are looking to shoot the messenger).

Up until now we have only talked about the presence of carcinogenic and toxic VOCs being identified in electronic cigarette vapor. However, we have not put any of this into a context, which may help make all these blogs more relevant to human health. To expound upon this further, it is important for me to acknowledge that I am more than likely breathing high pptv to low ppbv levels of formaldehyde, benzene, and other toxic VOCs as I type this blog. Therefore it is unjust to merely point out that we identified toxic VOCs in e-cig vapor.

So without further ado… remember that the HAP acrolein was not found in electronic cigarette solutions. In addition, acrolein was not found in the background air. However, acrolein was found in the vapor from all four of the e-cigarettes evaluated in our work. The acrolein concentrations ranged from 1.5 to 6.7 ppmv per 40 mL puff (0.003 to 0.015 µg/mL), which is comparable to the 0.004 µg/mL Goniewicz et al. reported. To put these concentrations into perspective, these levels exceeded the National Institute of Occupational Safety and Health (NIOSH) short-term exposure limit (STEL) of 350 ppbv. It is important to note that although we were not calibrated at the time for formaldehyde and acetaldehyde, the vapor concentrations for these two compounds appeared to be approximately the same as the acrolein concentrations observed. Again, the observation is consistent with what Goniewicz et al. reported.

It then becomes clear to me why end users experience what is often referred to as “throat hit.” These three carbonyls are well known mucous membrane (including eyes, nose, and respiratory tract) irritants, and inhaling ppmv levels (as those observed in the current study and our peers’ studies as well) of these three carbonyls would surely illicit said sensation. And we have not even begun to talk about the other identified and numerous unidentified VOCs we observed.

But as the title begs… WHAT DOES ALL THIS MEAN? Well obviously this means we cannot tell you electronic cigarettes contain no toxic chemicals. In fact, some e-cig manufacturers are already putting out disclaimers about their products.

 

M.L. Goniewicz, J. Knysak, M. Gawron, L. Kosmider, A. Sobczak, J. Kurek, A. Prokopowicz, M. Jablonska-Czapla, C. Rosik-Dulewska, C. Havel, P. Jacob III, N. Benowitz, Levels of selected carcinogens and toxicants in vapour from electronic cigarettes, Tob Control 23 (2014) 133.

Kosmider, A. Sobczak, M. Fik, J. Knysak, M. Zaciera, J. Kurek, M.L., Goniewicz,Carbonyl compounds in electronic cigarette vapors: effects of nicotine solvent and battery output voltage,Nicotine Tob Res 16 (2014) 1319.

 

Maximum temperatures of packed columns – Sulfur Gases

For my fifth and final post in this series, I would like to focus on packed columns for light sulfur gases.  Compounds include (but not limited to) hydrogen sulfide, sulfur hexafluoride, carbonyl sulfide, sulfur dioxide, methyl mercaptan, ethyl mercaptan, etc.

Because some analysts prefer PTFE tubing and/or PTFE frits for their columns, the maximum temperature may be limited to this hardware (which is 200°C).

If the columns below are in SilcoSmooth® tubing, their maximum temperatures are shown below.

Max Temp

Column and/or Packing

(°C)

Rt-XLSulfur

290

1.5% XE-60 / 1% H3PO4 on CarboBlack B

250

Chromosorb® T

250

 

To read my previous posts from this series, please see the links below.  I hope you have found them useful.

Maximum temperatures of packed columns – Porous Polymers

Maximum temperatures of packed columns – Liquid Phases

Maximum temperatures of packed columns – Hydrocarbon Analysis

Maximum temperatures of packed columns – Molecular Sieves

Maximum temperatures of packed columns – Molecular Sieves

For my forth post in this series, I would like to focus on molecular sieve packed columns.   At Restek, our three most common molecular sieve packings are the ShinCarbon, 5A, and 13X.  The ShinCarbon is a carbon molecular sieve, while the 5A and 13X are zeolite molecular sieves.  To read more about these packings/columns, please review Molecular Sieve Packed Columns and Fixed (Permanent) Gas Analysis.

 

Max Temp

Column and/or Packing

(°C)

ShinCarbon

280 / 300*

Molecular Sieve 5A

300 / 350*

Molecular Sieve 13X

350

 

* May be briefly programmed to this temperature.

Maximum temperatures of packed columns – Hydrocarbon Analysis

For my third post in this series, I would like to focus on specialty packed columns for hydrocarbon analysis.  In some cases, the packings in these columns are proprietary, so detailed information cannot be provided.  In other cases, application information may be limited.  However, we can provide maximum temperature limits (see table below).

For other columns used for hydrocarbon analysis, you should be able to find the maximum packing temperature based upon the maximum temperature of the liquid phase(s) and/or the porous polymer(s) in one of the links below.

Maximum temperatures of packed columns – Liquid Phases

Maximum temperatures of packed columns – Porous Polymers

If there is a packed column for hydrocarbon analysis (which is sold by Restek) and not included in this table or in either link above, email us at support@restek.com .  Thank you.

 

Max Temp

Column and/or Packing

(°C)

D3606 Column Set

165

0.19% picric acid on CarboBlack C

120

23% Rt-1700 on Chromosorb PAW

110

n-Octane on Res-Sil C

150

OPN on Res-Sil C

150

2abc Refinery Gas Column Set

110

Alumina F-1

300

Rtx-1 SimDist 2887

350

My GC capillary column was not sealed when I received it!

Traditionally, GC capillary column manufacturers have used several different methods to seal their products while in transit and storage. These sealing options include septa, silicone plugs, flame sealing, and press-fit caps.

 image1

I’m not going to discuss the advantages and disadvantages of these various approaches in this post. However, what I would like to address is a question that comes up periodically here in our technical service group:

What happens if you receive a brand new GC capillary column and one (or both) of the seals are no longer in place?

Is the column compromised? Will it still work? These are good questions to ask and consider.

Most of us have been told that exposing a capillary column to air (oxygen) can damage the phase. We are also aware that moisture or particles might enter an uncapped or unsealed column. What should you do if your column arrives without a proper seal?

Keep in mind that sealing a column is only intended to help protect column ends from damage and to keep particles from physically entering the column. These sealing mechanisms will not keep light or diffusive gases (such as hydrogen or helium) inside the column for very long.

Don’t panic. Although sealing the ends of columns is considered to be a good industry practice, occasionally, septa or silicone plugs will come loose, flame seals will break off, and column caps will disconnect. Yes, the column is now “exposed” and there is the possibility of “stuff” entering the column. However, the column is usually at relatively low (ambient) temperatures when this occurs, so the phase should be fine and it’s highly unlikely that the column has suffered any damage.

So, what should you do?

Install the column, leak-check the installation, and completely purge the column for a minimum of 20 minutes with clean, high quality carrier gas to help remove residual oxygen and moisture that may have entered the column during storage and transport.

This is a critical step. GC capillary columns MUST be thoroughly purged before being heated in the GC oven! Otherwise, stationary phase damage will occur.

Condition the column per the manufacturer’s recommendations. This will stabilize the baseline and minimize bleed. The following links provide detailed information on how to condition your columns:

How to Condition a New Capillary GC Column

PLOT column instruction sheet

Micropacked (0.53mmID) column instruction sheet

How do I condition a new packed or micropacked column?

In summary, it’s very unlikely that a missing end seal (or two) will result in damage to your column.

Thank you for reading!

The Most Useful Pesticide Standard Ever…monitoring GC inlet “dirtiness”

I have spent most of my time over the past several years testing pesticides and there is one standard that I simply can’t live without…QuEChERS Performance Standards Kit.

It is based on a really interesting article* on analyte protectants with a few compounds added.

The standard has 40 compounds housed in three vials for long term stability. The smart folks in our Reference Standards department made each vial 300 ppm in acetonitrile/ acetic acid (99.9:0.1). This makes it simple to blend equal volumes of the three vials to get a mix at 100 ppm.

  • It has compounds from different pesticide classes
  • Covers a range of polarities and volatilities (so good for GC and LC)
  • Has “good” or easy pesticides and problem pesticides
  • Read more at the product page

The mix is a great method development tool and we use it to evaluate both sample preparation and instrument performance.  Certain compounds make excellent probes for certain aspects of instrumental performance and I will show you one here. I monitor deltamethrin to determine the “dirtiness” of my inlet.
From our experience, we know that deltamethrin forms an isomer as nonvolatile material builds up in the GC inlet especially in the liner. It can also happen with high inlet temperatures too.

I am always monitoring the formation of this deltamethrin isomer to keep an eye on my liner performance and can quickly replace my liner when the breakdown is bad enough. In order to monitor the breakdown isomer, I have to make sure to add it to my mass spec method. I have been using GC-MS/MS lately and I simply use the same MS/MS transitions that I use for deltamethrin. The breakdown isomer will elute just before deltamethrin on “5” type columns.

In this example, I am running on an Rxi-5ms column using efficiency optimized flow and optimal heating rate.

You can see that tracking the deltamethrin isomer formation and the decrease in deltamethrin signal is a good indication of inlet performance. As more orange samples are injected, more nonvolatile material deposits on the liner causing a decrease in deltamethrin signal. After some predetermined point…maybe loss of 20% of the signal…I replace the inlet liner.

Stay tuned for more ways I use The Most Useful Pesticide Standard Ever

 

deltameth1

 

 

 *Combination of Analyte Protectants To Overcome Matrix Effects in Routine GC Analysis of Pesticides in Food Matrixes,  Katerina Mastovska, Steven J. Lehotay, and Michelangelo Anastassiades, Anal. Chem. 2005, 77, 8129-8137.