NACRW Presentation July 29, 2022 Invited Speaker.
Trace Analysis and Pesticides - no simple solution for cleanup.
Steve Lehotay the “gold standard” of all trace residue analysis from the U.S. Department of Agriculture provided an update on the quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method, originally developed for multiresidue pesticide analysis. Like any method, QuEChERS had some deficiencies, which were addressed with the new QuEChERSER (adding “efficient” and “robust” to the acronym) mega-method. The European Union (EU) Reference Laboratories developed a new method for the multiresidue analysis of highly polar pesticides, called the quick polar pesticides (QuPPe) method. What is QuPPe and how does it compare to QuEChERS and how can we learn from previously developed methods?
What is QuEChERS vs. QuEChERSER??
Its a multistep method that helps to remove matrix interfaces, so what is new -sample size, extraction solvent, salting out step, clean-up and final extract, note LCMSMS and GCMSMS technologies in the latest Q…..ERSER method- “efficient and robust,” as to accomodate a wider array of pesticides including polar, non-polar where Lehotay coined the term “mega method” now does this work for hemp ?? Not without specialized instrumentation wher ITSP is online using CTC PAL or Gerstel platform for cleanup and Low pressure GC- a configuration change to lower the GC column pressure- speed up the analysis at a compromise to the efficiency - but hey you have MS to deconvolute- could be a win win configuration. https://www.restek.com/en/technical-literature-library/articles/an-introduction-to-low-pressure-GC-MS-LPGC-MS/
Validation of a high-throughput method for analysis of pesticide residues in hemp and hemp products - Journal of Chromatography A 1645 (2021) 462097
In this study, the QuEChERSER mega-method for sample prepa-ration was validated for the analysis of 93 pesticides and metabo- lites in hemp powders, oil, pellets, and plant using UHPLC-MS/MS and ITSP + LPGC-MS/MS for identification and quantification. How- ever, dried hemp plant and flowers were too complex for analysis, and they should have been further diluted in the study. Up to 49 pesticides could be monitored in both analytical tools to provide additional confidence in the results.
Another R&D project to see if viable as hemp flower is not equivalent to complexity of the cannabis flower, definitely worth exploring since nowadays most modern testing labs will have both LCMSMS and GCMSMS technologies.
LGC Launches CANNLEARN APP - Longboard Scientific Provided Content
Its well recognized that training is lacking in many hemp/cannabis testing laboratories. As a young industry with few standard methods - anything I could do to help educate and provide content that bridges the gap. LGC - Dr. Ehrenstorfer in collaboration with the UK National Measurement Laboratory (hosted at LGC) has developed CannLearn - its an excellent and easy to use platform in the digital age ! Here is the sign up link : https://www.lgcstandards.com/GB/en/CannLearn_app
Topics include:
Introduction to why testing/screening of cannabis is required
Introduction to quality assurance, including method validation, measurement uncertainty, traceability, and quality control
Things to consider before carrying out an analysis
Laboratory set-up, including contamination control
Health and safety
Working with the LGC Team was fantastic- they were a phenomenal group and I enjoyed putting the content together.
Special thanks to Kate Evans at Longboard Scientific Consulting Corporation for sharing your technical expertise on our #CannLearn courses!
Hope you sign up and learn a few tips ! Put in on you phone and go CANNLEARN!
SPE Cleanup
Trying to cleanup botanical matrix is not a slam dunk ! Trace level pesticide analysis requires preservation of compounds -removing interferences without too many steps is extremely challenging aspect but necessary. Want to thank UCT for all the donations of SPE, dSPE and Quechers. Was able to try a few approaches-dSpe, C18 passthroughs, cleanup DPX tips, Quechers, and Spinfiltr (mixed blend of spe materials). Not one method 100% worked - but combination methods appeared to show the best results for select pesticides. This is just a snapshot but definitely have a goal of performing experiments that address an unmet need in over all cleanup goals.
Making sure testing laboratories are Audit Ready for Today and Beyond!
Not one to plug but I enjoyed this course a lot and feel empowered to help testing laboratories be the best they can be to meet the ISO 17025-2017 standard. It is a complex business managing an overall Quality Management system as well as navigating ever changing regulations, as well as scientific gray areas for many- Methods Validation another topic to start off with in 2021. Audit Ready !! Are you?
Making progress!
I really enjoy this process and seeing fruits of so many scientists labor, exciting times.
Expert Review Panels
AOAC CASP now has three (3) vetted, active Expert Review Panels (ERPs). The ERPs review methods that are submitted in response to calls for methods based on CASP SMPRs. The CASP Cannabinoids ERP is chaired by Dr. Melissa Phillips of NIST and recently looked at methods for cannabinoids in chocolate, the Chemical Contaminants ERP is chaired by Andrew Pham of Alkemist Laboratories and will meet on December 16 to review a method for heavy metals in cannabis, and the Microbial Contaminants ERP is chaired by Julia Bramante and will eventually review methods submitted for the SMPRs developed by the Microbial Contaminants Working Group.
ERPs typically meet during AOAC’s Analytical Methods Weeks. In 2021, methods weeks are scheduled for the weeks of April 26, August 2, and December 13. For more information including open calls for methods please see the website.
I am learning so much being a part of the ERP for heavy metals, between sample preparation methods to the method setup/conditions to the validation parameters. Enjoying the process and looking forward to seeing this approved, it will on strengthen testing and improve patient safety.
HPLC Validation - Stability Indicating Methodology Explained, SME Michael Dong, PhD.
Solid paper written by a SME in pharmaceutical analysis, he has a deep understanding on the validation process as it applies to the pharmaceutical industry - in addition stability indicating methodology is a critical step in drug development. LCGC just published this very applicable whitepaper. It was invited to help review and edit, it was so well written I didn’t have much to contribute or change. Although Pharma focused a lot of the information can be applied to other industries.
https://www.linkedin.com/feed/update/urn:li:activity:6731575101592100865/?commentUrn=urn%3Ali%3Acomment%3A(ugcPost%3A6731575101067812864%2C6731593338471911425)
Some takeaways from this paper- include the following on method robustness:
https://cdn.sanity.io/files/0vv8moc6/chroma/3a1fcc45168e578e63bd2393ff21c037ac7854b9.pdf. (available free on LCGC Website)
Typical parameters that can be adopted for any HPLC method being developed and evaluated for robustness. Definitely including solution stability- CRM-certified reference materials and over a course of study period- i.e. 7 days solution stability (sample).
System Suitability- A "check" on fit for purpose. Peak Tailing.
System Suitability - USP 621/CDER FDA - meaning and why would this be a useful to implement? Discussion 1- Peak Tailing one factor in System Suitability.
If I were a betting person - I think I would pick Peak A….however Peak B would probably cause me a bit of angst and Peak C- I would definitely be losing my edge as a chromatographer.
What is this parameter - Peak Tailing ….it can tell you a lot about HPLC method performance. What does it encroach on ? Peak Resolution which will be the next topic.
What is the cause of Peak tailing and non-asymmetrical peak shape?
A) hanging around way too long either in the mobile phase or the stationary phase
B) Column has over 15,000 injections, I think its time to throw it out
C) I think I overloaded the column - like a lot!
D) What was my pH again of my buffer?
E) How old is the guard column again - oh that’s on my maintenance log right?
F) may be my tubing is a big old - and valves a bit dirty - time to clean. Problem with Dead Volume?
A-F would all be the right answer. Column chemistry plays a big role in peak tailing and overall system maintenance, keeping a HPLC column log and activating “injector” number within software systems can help to monitor performance.
which peak here is the “bad actor” - although not super concerning- THCA definitely is “peak tail” vulnerable- why ?
The predicted pKa values are all around 2.9 which is approximately equal to the pH of the mobile phase (if 0.1% formic acid modified) so one might expect the acid groups to be about 50% ionized and more polar- although the high organic content of the mobile phase will tend to stabilize the neutral molecule over the ionized form and thus reduce the effective acidity (increase pKa). The observed elution suggests significant additional lipophilicity for the addition of a carboxylic acid group-where most molecules are neutral in this system.
Always related to Beaker.
The Original Beaker ! Good Facts to know.
Update ASTM D37 Laboratory Standards- Principle Author
Work Item Number: WK71840 Date:09-24-20
Ballot Rational:
Preparing a test sample of hemp/inflorescence is a critical step in the overall analytical measurement and obtainment of accurate results. To date there are many different technologies that cover a broad scope of sample sizes, however within the scope of this standard practice <100gram sample sizes are considered. In general cannabis/hemp samples are heterogenous in nature, thus relying heavily on robust sampling strategy which directly effects the downstream sample processing. An overview of sample comminution processes includes the following: cutting, blending, grinding, chopping, milling, cryomilling- all of which reduces the particle size and the fundamental error (FE). This practice is to provide a general overview of required steps and technologies to obtain a test sample size with uniform particle size as well as how to minimize the loss of volatile components such as terpenoids and pesticide residues.
Standard Practice for Sample Preparation Hemp Inflorescence for the Purpose of Analyses
Agilent Collaboration- Presentation Soon to be Published in LCGC
MJH BRANDS: LCGC, CST
CLIENT: AGILENT
TITLE: How to Build a Hemp Testing Laboratory
Setting up a laboratory is a very complex endeavor. With a strong business plan and the necessary capital, a new laboratory can be in place in as little as a year. Working within the regulatory landscape and your knowledge of analytical testing, you can set up a workflow that will handle your anticipated testing volume and avoid some common pitfalls. Industry partners like Agilent can help you at every step along the way, for the short and long term.
By:
Katherine M. Evans PhD, C (ASCP)CM
CEO
Longboard Scientific Consulting
DEK: Expanding legalization creates opportunities for the analytical lab
Mobile and examples of container laboratory
Love to give a shout out to my colleague and friend from Phenomenex!
Pushing the envelope- my friend and colleague- Sean Orlowicz. I have much respect for him- his scientific approach and his passion/love for the science, it is contagious when around him. I missed him at the Emerald Conference but he was not forgotten, he has a way to excite an audience and knows his stuff, hope to have him onsite more often - working together on the next challenging method. Love the hat—- of course there is a story around this hat - a rush embroidery job before the conference and only a limited number of hats printed, but came out great, couldn’t be happier. Looking forward to working together Mr. !!
Science Unfiltered Podcast - Episode 6, Featuring Sean Orlowicz and yours truly !
HPLC and UHPLC For Practicing Scientists
Book Edit - Chapter 11
November 11, 2019
Updating content and website refresh. It has been some time since I posted on my blog, been a busy year between working for out of state producer and consulting in CA. Its been a transition time as the industry changes are happening fast. The USDA announcing new testing rules (THC-0.3%) being enforced strictly and the recent vaping crisis, providing both safety and concern for the consumer. I will be introducing some new technical content on these topics, stay tuned!!
Free Information
In today's world of social media, internet and all the different was to support super fast information obtainment- why is there still a big question mark as to what this information means and is it really correct? Do you believe everything you read or should you always have a suspicious mind towards digital information- what about information from expert sources- directly from people- is it accurate or just full of fluff? How do we know ? it all comes down to trust and being on the side that you hire people to provide expertise- that expertise costs money- bottom line is the information is not free at this point.
I on several occasions have been seriously generous with my time. In the last 2 months as acting Laboratory Director of a working medical and recreational cannabis testing laboratory I have been able to see first hand the complexities of analyzing such a complex plant material- it is absolutely critical that the scientific approach is solid, methods robust and validated- if not walk away! With no standards or little literature precedence - make sure the methods meet or exceed FDA, USP, AOAC guidance. For example for microbial analysis - follow the FDA BAM- follow that and the methods will stand up to any audit.
So I digress---- keep this in mind if you decide to go off on your own and have Plan B, C...D even.
The worst thing you can ever do is to doubt your level of expertise or ability to help an organization. If an organization is considering utilizing your consulting expertise then obviously they need YOU for what they can't do or can't do as well themselves. Always remember that. I myself have to remember that - as frustrating as it seems - I am the one they are asking to help them because I have the know-how that they don't.
Throwing Monday away is extremely frustrating...and remember time is money!!!
So in order to not go completely off kilter- I contacted my good friends at Orion GMP. They seem to have run into the same road-blocks with their proposals, on much larger and in-depth scale than I. It definitely gave me piece of mind I am not alone and that I need to have more business savviness on the front end of any business deal or conversation- after all information - my information is not FREE!
As the World Turns!
One never know which way the road will take you, the journey is always full of decisions and question marks. Why are decisions so hard to make anyway? This is probably off topic but it certainly does impact family life having a scientific career. The job market just isn't stable anymore and the days of being in one job- one place are pretty much over. This time I want to approach things in a scientific manner- go off my gut.
According to Monster.com:
- Chemists will see 1.1 percent job growth in coming years, which is slightly faster than the 0.7 percent national average. Not exactly stellar growth but it is forward not backward.
Another glaring fact is that unless retooling or re-education happens it will be hard to keep up with current trends - for example:
- Growth of genetics/genomics (NGS)
As the emphasis moves ever-more-tangibly towards a personalized medicine treatment paradigm, expect to see an increase in demand for candidates with a combination of life science and data science education and skills.
At this point in time - I am at a crossroads- not ready to retire although that sounds wonderful, the fact of the matter is- that certainly isn't reality. So what's next? Follow the passion- not the money or salary- that is where it needs to be to have a future with stability in mind.
RIGHT!!
What are Terpenes and why are they so familiar?
Terpenes have very distinct smells, Alpha Pinene for example smells like pine needles & rosemary whereas Beta Pinene smells like dill, parsley and basil, same molecular weight- different spatial arrangement of certain atoms. See below Alpha and Beta Pinene Molecular Structures:
Note the difference between alpha and beta is the double bound, alpha in the ring, beta ortho position (#1) on the 6 membered ring- same but different!! Very hard to chemically separate if present in a mixture.
Some very common Terpenes and interesting smell and health benefits. What is your Fav?
My personal favorite of terpene rich plants is hops- as a craft beer fan- IPA is super aromatic with the following terpenes: monoterpene (C10) myrcene and the sesquiterpenes (C15) caryophyllene and humulene (not pictured above). Most of these terpenes are highly water insoluble and are usually added in as "dry hopped" process. Not to get side tracked but dry hopping involves adding hops to the fermenter or keg after fermentation- in other words not part of the beer boiling process where many essential terpenes are lost.
Besides just smelling fantastic many terpenes have medicinal/health qualities. Aromatherapy uses the medicinal properties of many terpenes to regulate mood, sleep problems, acuity and overall health. it is not a new practice- since 1937 essential oils were known to help burn victims and were thought to have healing powers- however the Egyptians were known to use essential oils long before a French chemist discovered it. Egyptian medical papyrus considered to date back to around 1555 BC contains remedies for all types of illnesses and the methods of application are similar to the ones used in Aromatherapy and Herbal medicine today. The Egyptians used a method known as infusion to extract the oils from aromatic plants and incense was probably one of the earliest ways of using aromatics. We are still practicing the art of infusion today with all types of carrier oils- MCT, Olive oil, Avocado oil, Mac Nut oil...etc. To be continued....next topic is how terpenes are delivered as medicine???
What Smells?
Terpenes- volatile unsaturated hydrocarbons usually found in plants- essential oils which in turn provide the aroma or "smell."
Isoprene itself, a C5H8 gaseous hydrocarbon, is emitted by the leaves of various plants as a natural byproduct of plant metabolism. Next to methane it is the most common volatile organic compound found in the atmosphere. Plants are pretty smart- terpenes provide natural defense against bugs/pests
Classification of terpenes is made on the basis of the number of carbon atoms of which they are composed, terpenes and their associated terpenoids are terpenes that have some rearrangement or oxidation to create assorted functional groups (alcohols, esters etc.).
The prefix which is present in the names, is based on the number of units by which the terpene molecule is built up. For example, the hermiterpenes belong to the half-terpenes with five carbon atoms (C5) and the word mono, in monoterpenes for the number one. Sesqui stands for 1.5, di for 2 and tri for 3. heir associated terpenoids (also known as isoprenoids) are terpenes that have some rearrangement or oxidation to create assorted functional groups (alcohols, esters etc.).
The Building block Isoprene, units of Carbon (C5)
So now we know what terpenes are at least their make up chemically- how are they made? Are they metabolites? Starting with the monoterpenes - what are some of the compounds and aromas. Next post I will discuss.
LCMSMS Analysis of Mycotoxins
Hplc (Reversed Phase Liquid Chromatography) coupled with MS (Mass Spectrometry), in this case tandem Mass Spectrometry or MS/MS can be used for rapid identification and quantitation of mycotoxins in food stuffs.
Mycotoxins can be separated by reversed-phase HPLC and detected using an electrospray ionization interface (ESI) and tandem MS, using MRM (multiple reaction monitoring) in the positive ion mode, The best approach is to have an all in one method that can detect all of the following compounds 4 main aflatoxins (B1,B2,G1, G2) and Ochratoxin A:
Ochratoxin A
The goal is to find a fast method that would not require additional instrumentation such as an HPLC with Fluorescence Detector or ELISA since LCMSMS is used for many other assays including pesticides residue analysis. An example analysis is shown below:
Example TIC of MRM LCMSMS analysis of Mycotoxin Testing.