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Shifting Analyte Retention Times or Matrix Interferences?

Occasionally, we will get a Technical Service call about peak retention times moving during a customer analysis.  Early eluting compounds are especially prone to this shifting, but the issue can often be attributed to matrix interferences that are also not well retained.    Pregabalin and Gabapentin are two antiepileptic drugs typically analyzed by LC-MS/MS.  Because of their small, polar nature, they may be difficult to retain using reversed phase chromatography.

Structures

In our example of an antiepileptic drug panel, Pregabalin and Gabapentin are well retained on the Raptor Biphenyl column.

LC_CF0616

See http://www.restek.com/chromatogram/view/LC_CF0616 for full method details.

 These two analytes can be accurately quantitated and results are reproducible when looking across different column lots:

RT info

However, when analyzing urine samples using a simple dilute & shoot method, quantitation becomes a bit more difficult.   Matrix interferences may give the illusion of peaks moving within the chromatogram.     In the example below, a sample chromatogram with 100 ng/mL of antiepileptic drugs in human urine was diluted 5x with 0.1% formic acid in water.  The retention time of the analytes is remaining the same, but there are clear interferences from the matrix components.

If we take closer look, and extract the MRM transitions for pregabalin, we can clearly see matrix interferences that could make quantitation difficult.

The next time you see your analyte “shifting” retention time, I would recommend running a solvent standard to rule out the possibility of matrix interferences.     For more information on this and other challenging analyses, be sure to come to our Monday morning workshop at MSACL 2015 US in San Diego, CA (MSACL Workshop).

 

Great Internal Standards for Pesticide Residues…SRMs too!

lady face

 

In just about all of my pesticide residue work, I use a convenient multicomponent internal standard mix that contains several PCBs as well as popular internal standard compound triphenylphosphate. The mix contains the six compounds in the chromatogram below. I have been working on a method for our GC Multiresidue Pesticides Kit which has about 200 compounds. We will be publishing the method…retention times and SRMs soon!

I am just about to put the kit into action with some citrus and celery samples. Luckily, I realized I had forgotten to add my internal standards to my method. I have provided a downloadable spreadsheet with run conditions and optimized SRM transitions for the six internal standard compounds. I hope this helps shortcut your own method development.

GCMS IS Mix cgram

Electronic Cigarettes Part VI: Vapor Analysis – Have we made our point? (hint: look for the bold)

In the last electronic cigarette blog we revealed that e-cig vapor has many more constituents than the propylene glycol, glycerin, nicotine, and flavorings on the manufacturer’s solution list. In fact, we found 82 compounds [i.e., unidentified and identified (some only tentatively)] in the vapor of one e-cigarette vendor. Now if you recall from e-cig blog cuatro, we found 45 compounds in the e-juice. Well since that blog we went over those e-cigarette liquid results with a fine tooth comb and actually found 64 compounds. So… that means our vapor analysis (of the exact same juice) exposed an additional 18 compounds.

Vapor B

Of particular interest was the presence of formaldehyde, acetaldehyde, acrolein, xylenes, and several siloxanes. The observation of these compounds in the vapor is significant for the two following reasons: 1) all three of the aforementioned carbonyls are acutely toxic; in addition, formaldehyde is a known human carcinogen and acetaldehyde is a probable human carcinogen. 2) None of these compounds were present in the e-juice, which indicates they were generated during the vaporization process and/or from the e-cigarette materials. This is exactly what we were hypothesizing about in part I of this blog series. If you recall, this is also why we chose to rock out the thick film volatiles column. Our observations are consistent with the fact that pyrolysis of both propylene glycol and glycerin (something taking place in e-cigarettes) results in the formation of formaldehyde, acetaldehyde, and acrolein. Our observations are also consistent with the fact that polysiloxanes are often used as plastic additives and the majority of the 1st generation e-cigarettes, like those evaluated in this study, are made with plastic bodies. All of the abovementioned have profound implications for how e-cigarettes should be evaluated, especially when considering the fact that the e-cigarette vapor is ultimately what end users are exposed to. So have we made our point by now? I hope so, but just in case… test the vapor not the juice. Stay tuned for the next blogs to see just how we tested the e-cig vapor, what concentrations were observed, and all things e-cig related.

Swagelok® and Parker® Tube Fitting Manuals

When asked by customers for what I consider the best reference guides on Swagelok® and Parker® fittings, I send them the following links.  I thought our blog readers may also benefit from these links, so here they are (below).  I hope you find them useful.

Swagelok® Tube Fitter’s Manual

Parker® Instrument Tube Fitting Installation Manual

 

If you are looking for fittings, we have a large selection for our chromatography customers.

Swagelok® fittings

Parker® fittings

 

Did you know that you can type in the Swagelok® or Parker® part number into the Search box at the top right of our webpage (shown by the red arrow below) and if we have the item, it will provide you a link under the Products (tab) search result?

 

SearchBox2

 

Medical Cannabis Terpenes Standards now available from Restek!

As many of you dedicated ChromaBLOGraphy readers know, Restek has supported the medical marijuana market for years with reference materials, and GC and LC consumables.  We find the field fascinating, so much so that we try to anticipate the upcoming needs of medical cannabis analysts through method development for compounds in addition to cannabinoids that may be therapeutic, for example, terpenes.  Method development and ongoing calibration and quantification efforts are easier with access to Certified Reference Materials.  That’s why I’m excited to announce that Restek now has Medical Cannabis Terpenes Standards!

The standards were formulated to contain the major medical cannabis terpenes and were divided into two separate mixes for maximum stability.  Concentrations are high (2500 µg/mL each compound) to provide value, while keeping solubilities-in-solvent in mind.  The analyte lists and catalog numbers are provided below as Terpene (CAS #).

Catalog Number 34095

(-)-Isopulegol (89-79-2)

(-)-alpha-Bisabolol (23089-26-1)

alpha-Humulene (6753-98-6)

alpha-Pinene (80-56-8)

alpha-Terpinene (99-86-5)

beta-Caryophyllene (87-44-5)

beta-Myrcene (123-35-3)

(-)-beta-Pinene (18172-67-3)

Camphene (79-92-5)

delta-3-Carene (13466-78-9)

d-Limonene (5989-27-5)

gamma-Terpinene (99-85-4)

Geraniol (106-24-1)

(-)-Guaiol (489-86-1)

Linalool (78-70-6)

Nerolidol (7212-44-4)

Ocimene (13877-91-3)

p-Cymene ( 99-87-6)

Terpinolene (586-62-9)

 

Catalog Number 34096

(-)-Caryophyllene oxide (1139-30-6)

1,8-Cineole (470-82-6)

Comparison of Phencylidine and Prazepam as Internal Standards in Medical Cannabis Potency Analysis with GC-FID

Recently in the ChromaBLOGraphy posts below I proposed the use of Phencylidine (PCP) as an internal standard (ISTD) for cannabinoids analysis with GC-FID when using the Rxi-35Sil MS GC column.  I demonstrated that the RSD% of Average Response Factors (Avg RFs) and Correlation Coefficients (CCs) for the calibration curves generated using the ISTD technique were excellent, and that ISTD calibration/quantification could reduce quantitative error associated with sample introduction into the GC.

Possible Internal Standards for Medical Cannabis Potency Testing by GC

Calibration Curves for Cannabinoids Based on PCP Internal Standard – Medical Cannabis GC-FID

Internal Standard versus External Standard Quantification in Medical Cannabis Potency Analysis with GC-FID

You may remember that in addition to PCP, I added Prazepam to each calibration standard (see chromatogram below).  Prazepam worked about as well as PCP as an ISTD, with slightly larger RSD% values for select cannabinoids and very slightly lower CCs (see table below).  The absolute RF differences for cannabinoids using ISTDs PCP or Prazepam are explained simply by having larger peak areas for PCP versus Prazepam, a function of the better FID response for PCP.

So…should we use two internal standards for this work?  We could.  One strategy is to use PCP as an ISTD for earlier eluting cannabinoids (e.g., CBDV, THCV, CBC, CBD) and Prazepam for later eluting cannabinoids (e.g., delta-8-THC, delta-9-THC, CBN, CBG).  Why?  It’s possible that PCP response is more representative for the more volatile cannabinoids (earlier eluters) and that Prazepam is better for the less volatile compounds, although the current data set is not definitive in that regard.

My preference at this point?  Consider PCP as an internal standard for all the cannabinoids and Prazepam as a potential indicator for determining when to change the GC inlet liner.  Prazepam, as the last eluting compound and potentially the least volatile, should be an early indicator of non-volatile “dirt”, like chlorophyll,  building up over repeated cannabis extract injections and eventually leading to poor quantitative transfer of cannabinoids from the GC inlet to the GC column.  Think of Prazepam as a “control chart” compound that upon declining in area count in a continuous fashion signals the need for GC inlet maintenance.

In summary, the addition of a combination of PCP and Prazepam to every cannabinoids standard and every cannabis extract prior to analysis will lead to better potency determinations when using GC-FID.

Chromatogram with ISTDsDifferent ISTDs Table

Optimization of Cannabis Analyses from the Emerald Conference

Good news, everyone!

I’ve added some comprehensive speaker notes to the talk I delivered at the Emerald Conference about the optimization of cannabis analyses. In this talk, I outlined some easy ways to improve your potency, terpenes, and residual solvents analyses for cannabis. I added the notes in the hope that the talk can be an educational tool about some of the theory behind HPLC and GC optimization. There are also some Star Wars references for your enjoyment.

Simply click the link below to download the presentation and notes in PDF format. As always, feel free to comment  or email me if you have any questions. I will quiz you on the notes, though.

2015 January Emerald Conference Cannabis Optimization with speaker notes

Cannabis Analysis – We’ve Come a Long Way, Baby!

A little less than a year ago at Pittcon in Chicago, my colleague Frank Dorman from the Pennsylvania State University and I sat down over beers with Ken Snoke and Wes Burk from Emerald Scientific, a small startup distribution business for cannabis labs. We were joined by Bill and Christi Schroeder and Ted Flood from Cal-Green Solutions, a startup cannabis testing lab. Our discussion revolved around the analytical side of the cannabis industry, current methods, and challenges facing both new and established labs. A couple of rounds later, we had landed on the idea that the cannabis analytical industry needed a robust proficiency testing program starting with cannabis potency analysis for GC and LC…and a conference.  The idea was that this conference would be an opportunity for scientific members of the cannabis industry to network, learn, and collaborate.

Cannabis Summit 2014

A couple of days later, Frank and I returned to Pennsylvania to resume our ongoing projects, but Ken and Wes, with the help of Cliff Beneventi, seized on the idea of a conference and ran with it (they also ran with the PT program, but that’s not what this post is about). Less than a year later, on January 23rd, the first annual Emerald Conference was held. Just before the conference we were all a little nervous. Would we get more than two dozen attendees? Will the speakers give interesting presentations? Will attendees be engaged? By the afternoon of the 23rd, we all knew that the conference was a smashing success. In-depth and insightful presentations were given to a sold-out audience of over one hundred and twenty engaged, attentive, and professional attendees. Panel discussions were lively and immensely valuable. Ideas were being exchanged, and the atmosphere was one of friendly collaboration both between labs and with vendors. It was incredible! Here’s a sampling of some of the talks and that were included in the conference:

Dr. Jeffrey Raber: “Scientific Frontiers in Cannabis”

Dr. Rodger Voelker: “Sampling Variability among Cannabis Flower”

Dr. Frank Dorman: “Potency with Simultaneous Incurred Pesticide Testing”

 Emerald conference 2015

The Emerald Conference reflects the increasing legitimacy of this industry, and its rapid growth and maturation from a scientific standpoint. I’d like to thank Ken, Wes, and Cliff for putting together such a great conference for the cannabis community. I think everyone who attended is already looking forward to next year!

Before presenting my talk at the conference, I noted that I had been working with the cannabis analytical industry for several years now, and that the progress I’ve seen from the industry in terms of analytical technique and good science has been incredible. Can the industry improve? Of course. But in my experience, I’ve never met a group of scientists so driven, passionate, and hungry for knowledge and good science. It’s been a real pleasure. I don’t want to be bossy, but cannabis scientists need to take a minute and congratulate themselves on their work to date…now get back to work!

 

Electronic Cigarettes Part V: Vapor Analysis – Yes… we found formaldehyde too!

It has been over 3 months since the last electronic cigarette blog. I will spare you the excuses. However, I can proudly say that we will be releasing a full application note on electronic cigarettes in the very near future (i.e., 1 to 2 months hopefully). In the meantime I feel obliged to respond to all the various inquiries I have received from colleagues about the presence of formaldehyde in electronic cigarettes. Lately I hear things like “oh I just heard on NPR about formaldehyde in electronic cigarettes… is this true… did you know that?”  Well… obviously you already know my answer to this hot topic question. So allow me to indulge…

In parts I II III and IV of this blog series we covered analyzing the e-juice for nicotine and impurities. In the last e-cig blog we saw that electronic cigarette solutions contained numerous impurities. At that point in time we did not go into a lengthy discussion on what compounds were found and their potential implications for human health. Why you ask? Well because theoretically no one is drinking the e-juice… nor is anyone bathing in the e-liquid… and lastly I doubt anyone is injecting the e-solution. The main route of exposure to e-cigs and any compounds of interest is the direct result of the vaporization process, whereby with the use of a heated filament, the liquid is turned into a vapor which the end user inhales. Despite the aforementioned, the majority of e-cig research has focused on analyzing the e-juice. However, now that the knowledge of formaldehyde in the vapor has hit the mainstream media, perhaps that paradigm will change. FYI – Anyone interested in further reading or references should just reach out. Now… after much delay and teasing here is what we have seen in the e-cig vapor:

E-Cig Vapor A

I know… boring. So how about we zoom in:
E-Cig Vapor B

Based on the above chromatogram our e-cig vapor has a lot more constituents than the manufacturer’s juice listing of propylene glycol, glycerin, and nicotine. No surprise here, we already saw this trend when analyzing the raw solutions. NOTE: these vapor results are for the exact same e-juice results presented in our last e-cig blog. So what are all of these peaks? Well… the following table will hopefully help break down all the unknown compounds into several more digestible sections. Note: We only included tentative compounds with a mass spectral quality of 80% or greater according to the NIST 2005 database. Compounds with 100% quality have been confirmed with standards.

E-Cig Vapor Table A

We found 82 unidentified and identified (some only tentatively) compounds in the e-cigarette vapor for this manufacturer. Of particular interest was the presence of formaldehyde, acetaldehyde, acrolein, as well as several siloxanes. All of which we did not find in the raw e-juice.

So… what does this all mean? Oh… and how did we measure the vapor? This time I promise to come back sooner than three months to answer these questions.

Restek’s EZGC Online Suite that includes the Method Translator and Flow Calculator, and a Chromatogram Modeler, wins a TASIA Award – Chris Nelson, One of the Suite Builders

The Analytical Scientist is a very smartly produced scientific magazine full of interesting articles, including many on chromatography.  I’ve had the pleasure of working with two of the minds behind this publication, Rich Whitworth and Frank van Geel, on a TAS GCxGC contribution, and have been impressed with the volume of quality work they’ve put out.  I’m sure they’d be the first to admit that it’s not just the two of them; it’s the people that work with them that make their efforts stronger.  And that’s one of the reasons I’m posting this blog, to acknowledge the contribution of Restek’s Chris Nelson, one of the chief architects behind our EZGC™ Online Suite, which recently won one of The Analytical Scientist Innovations Awards for 2014.

Restek’s EZGC™ Online Suite, which includes the Method Translator and Flow Calculator, and a Chromatogram Modeler, speeds gas chromatography method development, and we’ve benefitted greatly from these tools in our own work as you can see from the list of ChromaBLOGraphy posts below.  We couldn’t have done any of this without the efforts of Chris Nelson to make the program accurate and user-friendly.  So…many thanks to Chris for his work on EZGC™ and to The Analytical Scientist for recognizing the power of this GC method development platform.  Try the tools in your lab today and let me know what you think of them.  You can get some ideas from the posts below (note that those are links that you can click in to get the post).

Peak Capacity in Capillary GC

Alternate GC Carrier Gas: Helium to Nitrogen, 30m x 0.25mm x 0.25µm Column to 20m x 0.15mm x 0.15µm Column

Switching from Helium to Nitrogen Carrier Gas for GC by Switching from a 30m x 0.25mm x 0.25µm Column to a 20m x 0.15mm x 0.15µm Column

Chemstation 101: Quickly Update Your Retention Time Windows Using the Run Times Provided by the EZGC Method Translator

Electronic Cigarettes Part III: We’re gonna vape on the EZGC bandwagon.

Calculating Splitless Valve Time for Splitless Injection GC with the New EZGC Method Translator and Flow Calculator – Pesticides

Calculating your GC Column Length with the Restek EZGC Method Translator and Flow Calculator

Using the Restek EZGC Method Translator and Flow Calculator to Support Shoot-and-Dilute GC Method Development – Going from GC-ECD to GC-MS

Half the GC Column, Three Times Faster Analysis, Same Chromatogram!

Using Hydrogen Carrier at a Lower Flow Rate for GC-MS – Separations Compromised?

Believe to Achieve: Evaluating the Accuracy of the EZGC™ Chromatogram Modeler

Faster GC Analysis of Medical Cannabis Terpenes with Same 624Sil MS Selectivity

Setting the splitless injection purge valve time in gas chromatography

“I Can’t Drive 55” — The Pure Power of EZGC

EZGC Made Even Easier – Free web version now available!