Tag Archives: matrix

3 Essential Components of Microbial Safety Testing

By Heather Ebling
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Microbial contamination on cannabis products represents one of the most significant threats to cannabis consumers, particularly immunocompromised patients who are at risk of developing harmful and potentially fatal infections.

As a result, regulatory bodies in the United States and Canada mandate testing cannabis products for certain microbes. The two most popular methods for microbial safety testing in the cannabis industry are culture-based testing and quantitative polymerase chain reaction (qPCR).

When considering patient safety, labs should choose a method that provides an accurate account of what is living on the sample and can specifically target the most harmful microbes, regardless of the matrix.

1. The Method’s Results Must Accurately Reflect the Microbial Population on the Sample

The main objective of any microbial safety test is to give the operator an indication of the microbial population present on the sample.

Figure 1: MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.

Culture-based methods measure contamination by observing how many organisms grow in a given medium. However, not all microbial organisms grow at the same rate. In some cases, certain organisms will out-compete others and as a result, the population in a post-culture environment is radically different than what was on the original sample.

One study analyzed fifteen medicinal cannabis samples using two commercially available culture-based methods. To enumerate and differentiate bacteria and fungi present before and after growth on culture-based media, all samples were further subjected to next-generation sequencing (NGS) and metagenomic analyses (MA). Figure 1 illustrates MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.

The results demonstrate substantial shifts in bacterial and fungal growth after culturing on the 3M petrifilm and culture-based platforms. Thus, the final composition of microbes after culturing is markedly different from the starting sample. Most concerning is the frequent identification of bacterial species in systems designed for the exclusive quantification of yeast and mold, as quantified by elevated total aerobic count (TAC) Cq values after culture in the total yeast and mold (TYM) medium. The presence of bacterial colonies on TYM growth plates or cartridges may falsely increase the rejection rate of cannabis samples for fungal contamination. These observations call into question the specificity claims of these platforms.

The Live Dead Problem

Figure 2: The enzyme is instantaneously inactivated when lysis buffer is added

One of the common objections to using qPCR for microbial safety testing is the fact that the method does not distinguish between live and dead DNA. PCR primers and probes will amplify any DNA in the sample that matches the target sequence, regardless of viability. Critics claim that this can lead to false positives because DNA from non-viable organisms can inflate results. This is often called the Live-Dead problem. However, scientists have developed multiple solutions to this problem. Most recently, Medicinal Genomics developed the Grim Reefer Free DNA Removal Kit, which eliminates free DNA contained in a sample by simply adding an enzyme and buffer and incubating for 10 minutes. The enzyme is instantaneously inactivated when lysis buffer is added, which prevents the Grim Reefer Enzyme from eliminating DNA when the viable cells are lysed (see Figure 2).

2. Method Must Be Able to Detect Specific Harmful Species 

Toxic Aspergillus spp., which is responsible for at least one confirmed death of a cannabis patient, grows poorly in culture mediums and is severely underreported by current culture-based platforms. And even when Aspergillus does grow in culture, there is a certain non-pathogenic Aspergillus species that look remarkably similar to their pathogenic cousins, making it difficult to speciate using visual identification alone.

Figure 3: The team spiked a known amount of live E. coli into three different environments

Conversely, qPCR assays, such as the PathoSEEK, are designed to target DNA sequences that are unique to pathogenic Aspergillus species, and they can be run using standard qPCR instruments such as the Agilent AriaMx. The primers are so specific that a single DNA base difference in the sequence can determine whether binding occurs. This specificity reduces the frequency of false positives in pathogen detection, a frequent problem with culture-based cannabis testing methods.

Additionally, Medicinal Genomics has developed a multiplex assay that can detect the four pathogenic species of Aspergillus (A. flavus, A. fumigatus, A. niger, and A. terreus) in a single reaction.

3. The Method Must Work on Multiple Matrices 

Figure 4: The team also placed TSB without any E. coli onto a petrifilm to serve as a control.

Marijuana infused products (MIPs) are a very diverse class of matrices that behave very differently than cannabis flowers. Gummy bears, chocolates, oils and tinctures all present different challenges to culture-based techniques as the sugars and carbohydrates can radically alter the carbon sources available for growth. To assess the impact of MIPs on colony-forming units per gram of sample (CFU/g) enumeration, The Medicinal Genomics team spiked a known amount of live E. coli into three different environments: tryptic soy broth (TSB), hemp oil and hard candy. The team then homogenized the samples, pipetted amounts from each onto 3M™ Petrifilm E. coli / Coliform Count (EC) Plates, and incubated for 96 hours. The team also placed TSB without any E. coli onto a petrifilm to serve as a control. Figures 3 and 4 show the results in 24-hour intervals.

Table 1: DNA was spiked into various MIPs

This implies the MIPs are interfering with the reporter assay on the films or that the MIPs are antiseptic in nature.

Many MIPs use citric acid as a flavoring ingredient which may interfere with 3M reporter chemistry. In contrast, the qPCR signal from the Agilent AriaMx was constant, implying there is microbial contamination present on the films, but the colony formation or reporting is inhibited.

Table 3: SenSATIVAx DNA extraction can successfully lyse the cells of the microbes
Table 2: Different numbers of DNA copies spiked into chocolate

This is not an issue with DNA-based methods, so long as the DNA extraction method has been validated on these matrices. For example, the SenSATIVAx DNA extraction method is efficient in different matrices, DNA was spiked into various MIPs as shown in Table 1, and at different numbers of DNA copies into chocolate (Table 2). The SenSATIVAx DNA extraction kit successfully captures the varying levels of DNA, and the PathoSEEK detection assay can successfully detect that range of DNA. Table 3 demonstrates that SenSATIVAx DNA extraction can successfully lyse the cells of the microbes that may be present on cannabis for a variety of organisms spiked onto cannabis flower samples.

Heavy Metals Testing: Methods, Strategies & Sampling

By Charles Deibel
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Editor’s Note: The following is based on research and studies performed in their Santa Cruz Lab, with contributions from Mikhail Gadomski, Lab Manager, Ryan Maus Technical Services Analyst, Laurie Post, Director of Food Safety & Compliance, and Charles Deibel, President Deibel Cannabis Labs.


Heavy metals are common environmental contaminants resulting from human industrial activities such as mining operations, industrial waste, automotive emissions, coal fired power plants and farm/house hold water run-off. They affect the water and soil, and become concentrated in plants, animals, pesticides and the sediments used to make fertilizers. They can also be present in low quality glass or plastic packaging materials that can leach into the final cannabis product upon contact. The inputs used by cultivators that can be contaminated with heavy metals include fertilizers, growing media, air, water and even the clone/plant itself.

The four heavy metals tested in the cannabis industry are lead, arsenic, mercury and cadmium. The California Bureau of Cannabis Control (BCC) mandates heavy metals testing for all three categories of cannabis products (inhalable cannabis, inhalable cannabis products and other cannabis and cannabis products) starting December 31, 2018. On an ongoing basis, we recommend cultivators test for the regulated heavy metals in R&D samples any time there are changes in a growing process including changes to growing media, cannabis strains, a water system or source, packaging materials and fertilizers or pesticides. Cultivators should test the soil, nutrient medium, water and any new clones or plants for heavy metals. Pre-qualifying a new packaging material supplier or a water source prior to use is a proactive approach that could bypass issues with finished product.

Testing Strategies

The best approach to heavy metal detection is the use of an instrument called an Inductively Coupled Plasma Mass Spectrometry (ICP-MS). There are many other instruments that can test for heavy metals, but in order to achieve the very low detection limits imposed by most states including California, the detector must be the ICP-MS. Prior to detection using ICP-MS, cannabis and cannabis related products go through a sample preparation stage consisting of some form of digestion to completely break down the complex matrix and extract the heavy metals for analysis. This two-step process is relatively fast and can be done in a single day, however, the instruments used to perform the digestion are usually the limiting step as the digesters run in a batch of 8-16 samples over a 2-hour period.

Only trace amounts of heavy metals are allowed by California’s BCC in cannabis and cannabis products. A highly sensitive detection system finds these trace amounts and also allows troubleshooting when a product is found to be out of specification.

For example, during the course of testing, we have seen lead levels exceed the BCC’s allowable limit of 0.5 ppm in resin from plastic vape cartridges. An investigation determined that the plastic used to make the vape cartridge was the source of the excessive lead levels. Even if a concentrate passes the limits at the time of sampling, the concern is that over time, the lead leached from the plastic into the resin, increasing the concentration of heavy metals to unsafe levels.

Getting a Representative Sample

The ability to detect trace levels of heavy metals is based on the sample size and how well the sample represents the entire batch. The current California recommended amount of sample is 1 gram of product per batch.  Batch sizes can vary but cannot be larger than 50 pounds of flower. There is no upper limit to the batch sizes for other inhalable cannabis products (Category II).

It is entirely likely that two different 1 gram samples of flower can have two different results for heavy metals because of how small a sample is collected compared to an entire batch. In addition, has the entire plant evenly collected and concentrated the heavy metals into every square inch of it’s leaves? No, probably not. In fact, preliminary research in leafy greens shows that heavy metals are not evenly distributed in a plant. Results from soil testing can also be inconsistent due to clumping or granularity. Heavy metals are not equally distributed within a lot of soil and the one small sample that is taken may not represent the entire batch. That is why it is imperative to take a “random” sample by collecting several smaller samples from different areas of the entire batch, combining them, and taking a 1 g sample from this composite for analysis.


References

California Cannabis CPA. 12/18/2018.  “What to Know About California’s Cannabis Testing Requirements”. https://www.californiacannabiscpa.com/blog/what-to-know-about-californias-cannabis-testing-requirements. Accessed January 10, 2019.

Citterio, S., A. Santagostino, P. Fumagalli, N. Prato, P. Ranalli and S. Sgorbati. 2003.  Heavy metal tolerance and accumulation of Cd, Cr and Ni by Cannabis sativa L.. Plant and Soil 256: 243–252.

Handwerk, B. 2015.  “Modern Marijuana Is Often Laced With Heavy Metals and Fungus.” Smithsonian.com. https://www.smithsonianmag.com/science-nature/modern-marijuana-more-potent-often-laced-heavy-metals-and-fungus-180954696/

Linger, P.  J. Mussig, H. Fischer, J. Kobert. 2002.  Industrial hemp (Cannabis sativa L.) growing on heavy metal contaminated soil: fibre quality and phytoremediation potential. Ind. Crops Prod. 11, 73–84.

McPartland, J. and K. J McKernan. 2017.  “Contaminants of Concern in Cannabis: Microbes, Heavy Metals and Pesticides”.  In: S. Chandra et al. (Eds.) Cannabis sativa L. – Botany and Biotechnology.  Springer International Publishing AG. P. 466-467.  https://www.researchgate.net/publication/318020615_Contaminants_of_Concern_in_Cannabis_Microbes_Heavy_Metals_and_Pesticides.  Accessed January 10, 2019.

Sidhu, G.P.S.  2016.  Heavy metal toxicity in soils: sources, remediation technologies and challenges.   Adv Plants AgricRes. 5(1):445‒446.

IR Spectrum of 2,4-Dichlorophenol in different physical states
From The Lab

Gas Chromatography/Infrared Spectroscopy: A Tool For the Analysis of Organic Compounds in Cannabis

By John F. Schneider
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IR Spectrum of 2,4-Dichlorophenol in different physical states

Editor’s Note: The author will be teaching a 1/2 day short course on this topic at PITTCON in Philadelphia in March 2019.


The combination of gas chromatography and infrared spectroscopy (GC/IR) is a powerful tool for the characterization of compounds in complex mixtures. (1-5) Gas chromatography with mass spectroscopy detection (GC/MS) is a similar technique, but GC/MS is a destructive technique that tears apart the sample molecules during the ionization process and then these fragments are used to characterize the molecule. In GC/IR the molecules are not destroyed but the IR light produced by molecular vibrations are used to characterize the molecule. IR spectrum yields information about the whole molecule which allows the characterization of specific isomers and functional groups. GC/IR is complementary to GC/MS and the combination results in a powerful tool for the analytical chemist.

A good example of the utility of GC/IR vs GC/MS is the characterization of stereo isomers. Stereo isomers are mirror images such as a left hand and a right hand. In nature, stereo isomers are very important as one isomers will be more active then its mirror image. Stereo isomers are critical to medicinal application of cannabis and also a factor in the flavor components of cannabis.

GC/MS is good at identifying basic structure, where GC/IR can identify subtle differences in structure. GC/MS could identify a hand, GC/IR could tell you if it is a left hand or right hand. GC/MS can identify a general class of compounds, GC/IR can identify the specific isomer present.

Why GC/IR?

Gas chromatography interfaced with infrared detection (GC/IR), combines the separation ability of GC and the structural information from IR spectroscopy. GC/IR gives the analyst the ability to obtain information complementary to GC/MS. GC/IR gives the analyst the power to perform functional group detection and differentiate between similar molecular isomers that is difficult with GC/MS. Isomer specificity can be very important in flavor and medical applications.

 IR Spectrum of 2,4-Dichlorophenol in different physical states

IR Spectrum of 2,4-Dichlorophenol in different physical states

Gas chromatography with mass spectrometry detection (GC/MS) is the state-of-the-art method for the identification of unknown compounds. GC/MS, however, is not infallible and many compounds are difficult to identify with 100 % certainty. The problem with GC/MS is that it is a destructive method that tears apart a molecule. In infrared spectrometry (IR), molecular identification is based upon the IR absorptions of the whole molecule. This technique allows differentiation among isomers and yields information about functional groups and the position of such groups in a molecule. GC/IR complements the information obtained by GC/MS.

Interfaces

Initial attempts to couple GC with IR were made using high capacity GC columns and stopped flow techniques. As GC columns and IR technology advanced, the GC/IR method became more applicable. The advent of fused silica capillary GC columns and the availability of Fourier transform infrared spectrometry made GC/IR available commercially in several forms. GC/IR using a flow cell to capture the IR spectrum in real time is known as the “Light Pipe”. This is the most common form of GC/IR and the easiest to use. GC/IR can also be done by capturing or “trapping” the analytes of interest eluting from a GC and then measuring the IR spectrum. This can be done by cryogenically trapping the analyte in the solid phase. A third possibility is to trap the analyte in a matrix of inert material causing “Matrix Isolation” of the analyte followed by measuring the IR spectrum.

Infrared Spectroscopy

The physical state of the sample has a large effect upon the IR spectrum produced. Molecular interactions (especially hydrogen bonding) broadens absorption peaks. Solid and liquid samples produce IR spectra with broadened peaks that loses much of the potential information obtained in the spectra. Surrounding the sample molecule with gas molecules or in an inert matrix greatly sharpens the peaks in the spectrum, revealing more of the information and producing a “cleaner” spectrum. These spectra lend themselves better to computer searches of spectral libraries similar to the computer searching done in mass spectroscopy. IR spectral computer searching requires the standard spectra in the library be of the same physical state as the sample. So, a spectrum taken in a gaseous state should be searched against a library of spectra of standards in the gaseous state.

IR of various phases:

  • Liquid Phase – Molecular interactions broaden absorption peaks.
  • Solid Phase – Molecular interactions broaden absorption peaks.
  • Gas Phase – Lack of molecular interactions sharpen absorption peaks.
  • Matrix Isolation – Lack of molecular interactions sharpen absorption peaks.

IR Chromatograms

GC/IR yields chromatograms of infrared absorbance over time. These can be total infrared absorbance which is similar to the total ion chromatogram (TIC) in GC/MS or the infrared absorbance over a narrow band or bands analogous to selected ion chromatogram. This is a very powerful ability, because it gives the user the ability to focus on selected functional groups in a mixture of compounds.

Conclusion

Gas chromatography with infrared detection is a powerful tool for the elucidation of the structure of organic compounds in a mixture. It is complementary to GC/MS and is used to identify specific isomers and congeners of organic compounds. This method is greatly needed in the Cannabis industry to monitor the compounds that determine the flavor and the medicinal value of its products.


References

  1. GC–MS and GC–IR Analyses of the Methoxy-1-n-pentyl-3-(1-naphthoyl)-Indoles: Regioisomeric Designer Cannabinoids, Amber Thaxton-Weissenfluh, Tarek S. Belal, Jack DeRuiter, Forrest Smith, Younis Abiedalla, Logan Neel, Karim M. Abdel-Hay, and C. Randall Clark, Journal of Chromatographic Science, 56: 779-788, 2018
  2. Simultaneous Orthogonal Drug Detection Using Fully Integrated Gas Chromatography with Fourier Transform Infrared Detection and Mass Spectrometric Detection , Adam Lanzarotta, Travis Falconer, Heather McCauley, Lisa Lorenz, Douglas Albright, John Crowe, and JaCinta Batson, Applied Spectroscopy Vol. 71, 5, pp. 1050-1059, 2017
  3. High Resolution Gas Chromatography/Matrix Isolation Infrared Spectrometry, Gerald T. Reedy, Deon G. Ettinger, John F. Schneider, and Sid Bourne, Analytical Chemistry, 57: 1602-1609, 1985
  4. GC/Matrix Isolation/FTIR Applications: Analysis of PCBs, John F. Schneider, Gerald T. Reedy, and Deon G. Ettinger, Journal of Chromatographic Science, 23: 49-53, 1985
  5. A Comparison of GC/IR Interfaces: The Light Pipe Vs. Matrix Isolation, John F. Schneider, Jack C. Demirgian, and Joseph C. Stickler, Journal of Chromatographic Science, 24: 330- 335, 1986
  6. Gas Chromatography/Infrared Spectroscopy, Jean ‐ Luc Le Qu é r é , Encyclopedia of Analytical Chemistry, John Wiley & Sons, 2006
Dr. Ed Askew
From The Lab

Quality Plans for Lab Services: Managing Risks as a Grower, Processor or Dispensary, Part 4

By Dr. Edward F. Askew
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Dr. Ed Askew

In the last three articles, I discussed the laboratory’s responses or defenses used to reply to your questions about laboratory results that place stress on the success of your business. The Quality Control (QC) results can cause this stress if they are not run correctly to answer the following questions:

  1. Are the laboratory results really true?
  2. Can the laboratory accurately analyze sample products like my sample?
  3. Can the laboratory reproduce the sample results for my type of sample?

Now let’s discuss the most important QC test that will protect your crop and business. That QC sample is the Matrix Sample. In the last article in this series, you were introduced to many QC samples. The Matrix Sample and Duplicate were some of them. Take a look back at Part 3 to familiarize yourself with the definitions.

The key factors of these QC sample types are:

  1. Your sample is used to determine if the analysis used by the laboratory can extract the analyte that is being reported back to you. This is performed by the following steps:
    1. Your sample is analyzed by the laboratory as received.
    2. Then a sub-sample of your sample is spiked with a known concentration of the analyte you are looking for (e.g. pesticides, bacteria, organic chemicals, etc.).
    3. The difference between the sample with and without a spike indicates whether the laboratory can even find the analyte of concern and whether the percent recovery is acceptable.
    4. Examples of failures are from my experiences:
      1. Laboratory 1 spiked a known amount of a pesticide into a wastewater matrix. (e.g. Silver into final treatment process water). The laboratory failed to recover any of the spiked silver. Therefore the laboratory results for these types of sample were not reporting any silver, but silver may be present. This is where laboratory results would be false negatives and the laboratory method may not work on the matrix (your sample) correctly. .
      2. Laboratory 2 ran an analysis for a toxic compound (e.g. Cyanide in final waste treatment discharge). A known amount of cyanide was spiked into a matrix sample and 4 times the actual concentration of that cyanide spike was recovered. This is where laboratory results would be called false positives and the laboratory method may not work on the matrix (your sample) correctly.
  2. Can the laboratory reproduce the results they reported to you?
    1. The laboratory needs to repeat the matrix spike analysis to provide duplicate results. Then a comparison of the results from the first matrix spike with its duplicate results will show if the laboratory can duplicate their test on your sample.
      1. If the original matrix spike result and the duplicate show good agreement (e.g. 20% relative percent difference or lower). Then you can be relatively sure that the result you obtained from the laboratory is true.
      2. But, if the original matrix spike result and the duplicate do not show good agreement (e.g. greater than 20% relative percent difference). Then you can be sure that the result you obtained from the laboratory is not true and you should question the laboratory’s competence.

Now, the question is why a laboratory would not perform these matrix spike and duplicate QC samples? Well, the following may apply:

  1. These matrix samples take too much time.
  2. These matrix samples add a cost that the laboratory cannot recover.
  3. These matrix samples are too difficult for the laboratory staff to perform.
  4. Most importantly: Matrix samples show the laboratory cannot perform the analyses correctly on the matrix.

So, what types of cannabis matrices are out there? Some examples include bud, leaf, oils, extracts and edibles. Those are some of the matrices and each one has their own testing requirements. So, what should you require from your laboratory?

  1. The laboratory must use your sample for both a matrix spike and a duplicate QC sample.
  2. The percent recovery of both the matrix spike and the duplicate will be between 80% and 120%. If either of the QC samples fail, then you should be notified immediately and the samples reanalyzed.
  3. If the relative percent difference between the matrix spike and the duplicate will be 20% or less. If the QC samples fail, then you should be notified immediately and the samples should be reanalyzed.

The impact of questionable laboratory results on your business with failing or absent matrix spike and the duplicate QC samples can be prevented. It is paramount that you hold the laboratory responsible to produce results that are representative of your sample matrix and that are true.

The next article will focus on how your business will develop a quality plan for your laboratory service provider with a specific focus on the California Code Of Regulations, Title 16, Division 42. Bureau Of Cannabis Control requirements.