How Standardized Sample Preparation Can Solve the Inter-Laboratory Variation Problem: Page 2 of 4

March 6, 2020
Volume: 
3
Issue: 
2
Abstract / Synopsis: 

The problem of different cannabis laboratories obtaining different results on the same samples, inter-laboratory variation, has been studied extensively. One of the causes of the problem is different laboratories prepare their samples differently. Another issue is how varying amounts of moisture in plant material can impact potency measurements. Ideas on how to improve the situation are given.

There is almost zero bias in the data set, indicating almost all the error in these measurements is random (10). The standard deviation in the data is almost 0.2 wt.% total THC. This means one laboratory could test a hemp sample and find 0.2 wt.% total THC and declare a crop legal, whereas a different laboratory could find 0.4 wt.% total THC in the same sample and declare the crop illegal. People’s livelihoods and hundreds of thousands of dollars are on the line when hemp is tested for total THC. With data like this, how can we honestly assure hemp farmers that their test results are fair? Clearly, in terms of potency and pesticide testing there is an inter-laboratory variation problem. Other analyses performed by cannabis laboratories, such as terpenes, heavy metals, biocontaminants, and mycotoxins may be just as variable.

The inter-laboratory variation problem threatens the very existence of our industry. If there is no consistency across laboratories, how can regulators know which materials are legal and which are not? If there is no consistency between laboratories, how can cannabis businesses rationally run their businesses in the face of unreliable data? This is a problem that needs to be solved sooner rather than later. In a previous column (2), I gave a laundry list of potential solutions to the problem. One of them was standardizing sample preparation methods. In this column I discuss this idea in detail.

The Current State of Cannabis Sample Preparation

Chromatographic analyses are used to measure cannabinoids (11), terpenes (11), and pesticides in cannabis samples. For these analyses to work properly, the sample must be homogenized and extracted. This involves significant manual sample preparation including grinding, mixing, extracting, and filtering.

I have been involved in cannabis analysis since marijuana first become legal in Colorado in 2013. In that time, I have visited dozens of cannabis laboratories around the U.S. and have observed that no two laboratories prepare their samples for chromatographic analysis the same way.

I have seen laboratories use mortars and pestles, handheld grinders, coffee grinders, spice grinders, herb grinders, and cryo-grinders to prepare samples for analysis. These different methods will produce samples with differing surface area, meaning different amounts of cannabinoids will be extracted from the same sample. Here is a list of the variables that are not controlled and why they matter.

1. Grinding
This is not relevant for liquid samples, but is very important for plant material such as buds, trim, and biomass. Work by myself and others have clearly shown that cannabis is an inhomogenous, naturally variable material (11). This means plant material must be homogenized by grinding prior to analysis. Grinding will produce a sample with a specific particle size distribution, particle shape distribution, and surface area. The latter matters because when performing a solid-liquid extraction, the surface area of the sample effects the rate at which molecules are extracted. Typically, the greater the surface area the faster analytes will be extracted.

2. Moisture Content
Given the nature of cannabis based materials, analyte concentrations are reported as weight percents rather than in moles/liter. In fact, Federal law requires total THC in hemp be measured as a weight percent (8,9). When plant material is harvested it contains significant amounts of moisture, and must be quickly dried to prevent the growth of mold. Since we are using weight percent measurements, and the denominator of these calculations is the weight of the original sample, variations in moisture content can affect the final results. Properly dried plant material contains about 10% moisture, however changes in temperature and humidity can affect the amount of adsorbed moisture on plant material, alter the sample’s weight, and then cause variation in analyte weight percent values. In a perfect world, third party laboratories would measure the moisture content of every plant sample before analysis, and take this into account when calculating weight percents. Some laboratories do not routinely measure the moisture content of incoming plant material, trust the sample submitter to have dried the sample properly, and assume ambient conditions will not affect moisture content. One cannot assume any of this. One way to dry biomass is to perform a loss on drying experiment. The weight of the sample is determined, the sample is thrown in an oven, heated for some time at some temperature, and then the weight is determined again. The assumption is made that all of the loss is from moisture evaporating. Let me be crystal clear: This measurement is useless for marijuana and hemp. We all know that in addition to moisture cannabis contains volatiles such as terpenes. Part of the loss on drying in addition to moisture is loss of terpenes. Also, at the temperatures often used for loss on drying the acid cannabinoids can decarboxylate resulting in additional weight loss. Work by myself and others has shown that only about half of the weight loss upon drying is moisture, the rest is most likely terpenes and other volatiles (12). Thus, the loss of drying values is not a measure of moisture content, but a measure of the loss of total volatiles upon heating. These values should not be used to correct analyte weight percent measurements. A solution to this problem is to measure the moisture content of samples prior to analysis using near infrared absorbance. Moisture meters based on this technology exist that are fast, accurate, and affordable (Google the term “near infrared moisture analyzers” to find a number of manufacturers). These systems measure moisture, not loss on drying. In my opinion, all laboratories should have one of these systems, measure the moisture content of submitted plant material, and then take this into account when calculating analyte weight percent values.

References: 
  1. B.C. Smith, Cannabis Science and Technology 2(2), 12–17 (2019).
  2. B.C. Smith, Cannabis Science and Technology 2(3), 10–14 (2019).
  3. M.O. Bonn-Miller, M.J.E. Loflin, B.F. Thomas, J.P. Marcu, T. Hyke, and R. Vandrey, JAMA, J. Am. Med. Assoc. 318, 1708 (2017).
  4. B. Young, The Seattle Times, January 5, 2016. https://www.seattletimes.com/seattle-news/marijuana/some-pot-labs-in-state-failed-no-pot-at-all-says-scientist/.
  5. L. Wagner, M. Bott, M. Villarreal, and M. Horn, NBC Bay Area, November 16, 2017, https://www.nbcbayarea.com/investigations/Industry-Insiders-Warn-of-Fraud-at-Marijuana-Testing-Labs-458125743.html?_osource=SocialFlowFB_BAYBrand.
  6. B.C. Smith, P. Lessard, and R. Pearson, Cannabis Science and Technology 2(1), 48–53 (2019).
  7. California Bureau of Cannabis Control Regulations, Section 5719.
  8. 115th United States Congress, Senate Bill S.2667, ”Hemp Farming Act of 2018.”
  9. https://www.ams.usda.gov/rules-regulations/hemp
  10. B.C. Smith, Cannabis Science and Technology 1(4), 12–16 (2018).
  11. M. Giese, M. Lewis, L. Giese, and K. Smith, J. AOAC Int. 98(6), 1503–1522 (2015).
  12. B. Smith, M. Giese, and M. Lewis, unpublished results.
  13. B.C. Smith, Cannabis Science and Technology 2(6), 28–33 (2019).
  14. https://www.nist.gov/programs-projects/nist-tools-cannabis-laboratory-quality-assurance

About the Columnist

Brian C. Smith, PhD, is Founder, CEO, and Chief Technical Officer of Big Sur Scientific in Capitola, California. Dr. Smith has more than 40 years of experience as an industrial analytical chemist having worked for such companies as Xeros, IBM, Waters Associates, and Princeton Instruments. For 20 years he ran Spectros Associates, an analytical chemistry training and consulting firm where he improved their chemical analyses. Dr. Smith has written three books on infrared spectroscopy, and earned a PhD in physical chemistry from Dartmouth College.

How to Cite this Article

B.C. Smith, Cannabis Science and Technology 3(2), 10–15 (2020).