Part 5: Contamination and Errors Associated with the ICP-MS Testing Procedure
The first installment of the series gave an overview of why testing cannabis and hemp for heavy metal contaminants is so important and how the pharmaceutical industry can play a critical role in preparing the cannabis industry for federal oversight.
Part two focused on how growers and cultivators need to actively investigate all potential sources of elemental contamination before they can even hope to minimize them. And, three highlighted how potential sources from the extraction and manufacturing processes can contribute to the problem. Part four provided evidence that without having complete control over extraction and production variables it can lead to the potential of high levels of heavy metals in some products, which has led to recalls and, in some cases, litigation
Part five will take a look at how analytical testing procedures can often lead to contamination and sources of errors if not adequately addressed.
Note: The series has been summarized from two chapters in Robert Thomas’ upcoming book, “Measuring Heavy Metal Contaminants in Cannabis and Hemp: A Practical Guide,” which will be published by CRC Press this September. The book, including its table of contents, is now available for preordering from the publisher’s website.
The potential for elevated levels of heavy metals being present in some hemp and cannabis products has meant that testing for elemental contaminants is absolutely critical. The most suitable and widely used technique is considered to be Inductively Coupled Plasma Mass Spectrometry (ICP-MS), which is a very sophisticated multielement analytical technique that can easily measure down to ppt (parts per trillion) detection levels. However, it does not preclude the use of other techniques such as ICP-OES (Optical Emission Spectrometry), Flame atomic absorption (FAA), electrothermal atomization atomic absorption (ETAA) or even atomic fluorescence (AF) as long as the detection capability is low enough for the required maximum limits defined by the state. It is also worth emphasizing that ICP-MS, with its superior sensitivity over other atomic spectroscopic approaches, is probably the best suited, because it is a multielement technique and even though today there are just four heavy metals required by most states there is a strong possibility that when the FDA has oversight of the cannabis industry, that list will be expanded to at least 10 elemental contaminants, and maybe more if the pharmaceutical industry is any indicator.
Benefits of ICP-MS for testing cannabis products
There are approximately, 20,000 ICP-MS systems being used around the world for a wide and diverse range of trace element applications. However, even though it is very powerful technique with unparalleled sensitivity, it requires a skilled analytical chemist with a reasonably high level of knowledge and expertise to develop and run methods and to fully-understand the nuances of ultra-trace elemental analysis. Of particular importance in the real-world applicability of ICP-MS is a full understanding of lab cleanliness, sources of contamination, sample preparation, digestion techniques, instrumental method development, interference corrections, calibration routines, use of reference materials and validation procedures. In other words, in the hands of a novice or inexperienced user it could easily generate erroneous results1.
For that reason, the expertise of the testing lab and the personnel running the instrumentation is of prime consideration, and in particular to have an intimate knowledge of working in the ultra-trace environment, to be aware of all the potential sources of elemental contaminants and to use robust validation procedures to ensure high integrity of the data1. This is exactly what the pharmaceutical industry was faced with in the US when they abandoned the old USP Chapter 231 sulfide precipitation colorimetric test2 and wrote two brand new methods – USP Chapter 2323 to define 24 elemental impurities of toxicological concern and USP Chapter 2334 to address the most suitable analytical procedure including plasma spectroscopic technique, sample digestion procedure and robust validation protocols using standardized spike recovery testing procedures. The backbone of this new USP Chapter 233 was USP Chapter 7305, which describes the use of plasma spectrochemistry and USP Chapter 1225 for the Validation of Compendial Procedures6 for pharmaceutical-related samples.
Note: A risk assessment strategy was also allowed by the USP and other global pharmacopeias, described in ICH Q3D guidelines7as long as compelling evidence could be demonstrated that an elemental impurity should be excluded from the list (refer to part three for details).
Laboratory testing procedures
Before we venture into what is required from a cannabis testing lab to be proficient in using highly sophisticated analytical equipment like ICP-MS for measuring ultra-trace levels of heavy metals in cannabis, let us take a closer look at the inherent limitations in the way it is carried out today. Consumers are asked to trust product ingredients, dosing suggestions and claims based on what the producers tell them. An informed consumer might ask for a Certificate of Analysis (CoA) from the producer to show third-party lab test results.
However, the problem with this is that there is very little required standardization across different testing facilities. This was exemplified in a recent case I mentioned in part four of the series where a CBD manufacturer claimed their product was “heavy metal” free but a concerned consumer got it tested and found to be over the state’s legal limit for lead, copper, and nickel. This case, which is currently going through litigation highlights the problems that cannabis testing labs are not being seriously regulated, meaning there is no set of standardized protocols for equipment, operating procedures, certifications or qualifications of lab personnel. So one lab might get results that are below the state-based maximum allowable limits, while another lab shows higher results - which one is correct?
Cannabis testing labs currently do not come under the umbrella of the FDA, so they have to be guided by state regulators who often do not have the necessary inspection expertise. Without standardization across all facilities, test results can be wildly inconsistent. What is the value of a CoA from a third-party lab that does not meet any kind of standards themselves?
However, as they begin to gain more experience, state regulators are attempting to put in place basic accreditation measures for cannabis labs to be licensed. One such approach is to implement the International Organization for Standardization (ISO) laboratory competence certification accreditation system, which in conjunction with the International Electrotechnical Commission (IEC), is responsible for ISO/IEC 17025:2017, a standard for calibration and testing laboratories across the globe, ensuring technical competence and the ability to produce precise and accurate test and calibration data. In addition, ISO/IEC 17043:2010, specifies general requirements for the competence of providers to develop and operate proficiency testing schemes using well-established interlaboratory comparison studies.
But the testing of cannabis and cannabis products requires specialized sample preparation and methods of analysis that can be extremely challenging for new labs and novice users due to its complex biological and biomolecular composition. ISO/IEC accreditation standards can provide confidence to cannabis consumers that testing is being performed correctly and to a universally accepted standard. To qualify for the accreditation, laboratories must conform to the ISO standards in all areas, including analytical procedures, calibration of instruments and equipment, as well as properly staffed and trained technicians who have met specific academic credentials. Overall, these required qualifications can be costly and time consuming, which may deter many of the start-up cannabis labs to invest in the necessary infrastructure to ensure they meet these high standards. So, to satisfy the demands of the industry, specialized organizations like Emerald Scientific and others are developing Inter-Laboratory Comparison and Proficiency Test (ILC/PT) programs specifically for cannabis and hemp testing facilities, establishing much needed standardized protocols for testing8.
More recently, the National institute of Standards and Technology (NIST) is spearheading a multi-phase project to encourage best practices in lab testing. The program called CannaQAP (Cannabis Quality Assurance Program) is meant to help laboratories accurately measure key chemical compounds in cannabis and cannabis products including oils, edibles, tinctures and balms9. The program aims to increase accuracy in product labeling and help consumers to be adequately informed about cannabis products being sold on the market today. The initial phase will focus on potency testing, but a future direction will also include contaminants such as heavy metals and pesticides. It’s through the involvement of recognized and well-established standards’ organizations like NIST that will help to raise the industry standard for cannabis and hemp testing.
Ways to reduce sources of errors associated with sampling and testing
So let us now take a closer look at some of the most important factors for a testing lab to consider when measuring elemental contaminants in cannabis and hemp, which should help them better understand the major analytical issues and to become more comfortable with working in the ultra-trace element environment10.
• It might seem obvious, but ensure the sample being tested is representative of the batch of cannabis being grown, or the product lot of the cannabinoid being sold or consumed.
• Make sure the cleanliness of the area where the incoming samples are being received. Check for sources of dirt, dust or particulates, which might contain elemental contaminants. In particular avoid metallic surfaces that might be in contact with the samples, such as stainless or galvanized steel.
• ICP-MS offers unparalleled sensitivity and extremely low detection limits, so the instrument should be operated in an area free of environmental contamination. If possible it should be installed in a special positive-pressure, thermostatted room where air can leave the room without circulating back in and fluctuating air temperatures will not negatively impact the thermal stability of instrument components, which could lead to signal drift.
• You cannot operate an instrument in a warehouse and expect to get high quality data. Consider installing the instrument in a class 1000 (ISO 6) or class 10,000 (ISO 7) room, which refers to the number of particles of size 0.5 µm or larger per cubic foot of air. As a point of reference, the semiconductor industry (which is typically chasing zero elemental contaminants in its computer chips) will usually install their instruments in a class 10 (ISO 4) or 100 (ISO 5) room. Note: ISO clean room classifications are shown in parenthesis11.
• Avoid metallic grinding equipment in preparing the sample for digestion and analysis, because it could potentially end up in the sample solution you are presenting to the instrument. Cryogenic grinding equipment is very useful, but ensure that polymer-based internal components are used.
• Personal use of jewelry, cosmetics, lotions, perfumes and shampoos can have a negative impact on the analysis as they are all have various elemental components including nickel, chromium, silver, gold, platinum, zinc, silicon, titanium, iron, cadmium.
• An operator that smokes (tobacco or cannabis products) will elevate the background level, particularly for an element like cadmium.
• Sample digestion can be a major source of contamination from used microwave vials, containers and vessels. Have a robust acid cleaning process that ensures your reagent blanks are clean and free of elemental contaminants (a commercial acid cleaning system for vessels, beakers and volumetric ware is an excellent investment).
• All analytical reagents, chemicals, acids, and deionized water should be ultra-high purity grade.
• Use multielement calibration standards designed for use with ICP-MS and not ones made for AA or ICP-OES, which are typically of lower purity.
• When carrying out the measurement of heavy metals using the instrumental technique, it is important to adopt a robust quality assurance program (QAP) based on recognized quality management systems such as ISO, and GLP which utilize official mandated methods defined by federal agencies such as FDA, EPA, NIST and USDA or consensus methods put out by independent standards’ organizations like ASTM, AOAC, USP, AHPA and Emerald. This QAP aspect is absolutely critical, because without currently having a choice of certified reference materials of cannabis and cannabis extracts to validate the accuracy of the method, the analytical procedures must involve recognized spike recovery procedures where all standards, blanks and samples are spiked with known concentrations based on the regulated maximum limits for the cannabis product to ensure that no false positive or negative results are generated.
• One such recognized method to follow is outlined in the validation protocols defined in USP Chapter 233 which should be a critical component of all heavy metal testing of cannabis products. Meeting these performance requirements described in these tests must be demonstrated experimentally using an appropriate system suitability procedure and reference materials to demonstrate detectability, accuracy, specificity, stability, ruggedness and drift. The suitability of the method, known as the J-value validation protocol must be determined by conducting studies with the material under test supplemented/spiked with known concentrations of each target element of interest at the appropriate acceptance limit concentration. It should also be emphasized that the materials under test must be spiked before any sample preparation steps are performed. This spike recovery procedure is a very important part of validating the method and the generated data has to be shown to the regulatory agency as proof that the analysis has been carried out correctly.
• Understand the errors and uncertainty involved with your analytical methodology including volumetric glassware, analytical balances, calibration standards, reference materials, sample preparation procedures and precision/repeatability of the instrumental technique used.
• Routine maintenance of the instrument components (sample introduction parts, cones, ion optics etc.) should be carried out on a regular basis to minimize contamination from the sample matrices.
• If you are tasked with characterizing cannabis vaping pens, get familiar with methodology for measuring toxic metals in vaping liquids, as well as determining toxic metals in the aerosol being delivered to the consumer, because the requirements are very different It is relatively straight forward to aspirate the liquid into the spectrochemical measuring technique, using a conventional sample introduction system and quantitate the elemental contaminants, as long as suitable calibration standards are used. However, how do you aspirate an aerosol from the vaping device directly into the instrument and be able to quantitate each puff or spray unless you have specialized sampling tools and/or smoking machines. This becomes very challenging unless you have had experience in carrying out this type of analysis. However, Halstead and Gray and co-workers have recently published two very pertinent papers, which measure a suite of toxic metals in both liquids and aerosols in nicotine-based electronic cigarettes12, 13.
In summary, understand all the practical real-world detection capability of the entire analytical methodology and in particular the potential sources of contamination that can impact the limit of quantitation (LOQ), some of which are outlined below:
o Cleanliness of sample preparation test area and equipment
o Laboratory dust/dirt of unknown origin (eg. old Pb-based paint)
o Cleanliness of sample digestion procedure
o Quality of the deionized water
o Purity of analytical reagents, acids and solvents
o Impurities in laboratory glassware and plastic vessels/containers
o Purity of the plastic tubing used in delivering the sample to the instrument
o Contaminants from the analyst, including clothing, cosmetics, lotions, perfumes, shampoo, jewelry, smoke
Finally, I’d like to offer a word of advice for regulators who are tasked with inspecting testing labs for heavy metals analysis. You should become familiar with analytical procedures described in USP Chapter 233 and, in particular, the strict validation protocols described in the chapter. These protocols, which are based on strict spike recoveries have been developed and tested by the USP over the past 20 years. They are the very essence of the FDA inspection process of pharmaceutical labs to ensure the data generated is of the highest quality. If a lab is not carrying out these procedures thoroughly and correctly, there is a very good chance the sample data will be flawed.
However, providing data for regulatory inspection and product release documentation in any industry is only part of the challenge facing the modern analytical laboratory. For example in the pharmaceutical industry, external inspections of facilities and procedures means that a great deal of infrastructure is required to support the actual analytical operation including (but not limited to) people, equipment specifications, GLP capability, lab facilities, SOPs, validation and data management. The pharmaceutical analytical laboratory has taken this aspect very seriously, because they are a part of a highly regulated industry. It is unlikely that cannabis state regulators will have the necessary background and experience to carry out this kind of detailed inspection of cannabis testing labs. However, it is a given that when the FDA eventually comes around, they will know exactly what they are looking for and will leave no stone unturned, having had a number of years of experience of regulating the pharmaceutical industry. My previous publication, “Measuring Elemental Impurities in Pharmaceuticals” covers this in greater detail14, but an excellent resource for getting ready for an FDA inspection can also be found in these references15, 16.
Note: There have recently been a number of public announcements from federal and other standards organizations on regulating heavy metals in cannabis and hemp, which are in various stages of publication. Please check out these references for their current positions (on 7/28/20): FDA17, EPA18, USDA19, NIST20, USP21, AHPA22, ASTM23, and AOAC24.
There are many factors that influence the ability to get the correct result with any trace element technique, particularly in today’s high throughput cannabis testing laboratories. Unfortunately with ICP-MS the problem is magnified even more because of its extremely low detection capability and potential for serious contamination from a multitude of laboratory sources. So, in order to ensure that the data reported is an accurate reflection of the material being sampled, the analyst must be aware of all the potential sources of errors that could negatively impact the quality of the measurements. It is therefore critical that the analyst is suitably trained and has the required knowledge and expertise to be working in the ultra-trace element environment.
The author of this article, Rob Thomas, organized a fantastic, focused workshop on this topic in October 2019. If you are interested in finding out more about the use of ICP-MS for heavy metal testing in cannabis you can view nine talks from that workshop below.
1. Holding Data to a Higher Standard, Part II: When Every Peak Counts-A Practical Guide to Reducing Contamination and Eliminating Error in the Analytical Laboratory, P. Atkins, Cannabis Science and Technology, 1 (4), 40–49 (2018), https://www.cannabissciencetech.com/view/holding-data-higher-standard-part-ii-when-every-peak-counts-practical-guide-reducing-contamination
2. United States Pharmacopeia General Chapter <231> Heavy Metals Test in USP National Formulary (NF), https://www.usp.org/chemical-medicines/elemental-impurities-updates
3. United States Pharmacopeia General Chapter <232> Elemental Impurities – Limits: First Supplement to USP 40–NF 35, 2017, https://www.usp.org/chemical-medicines/elemental-impurities-updates
4. United States Pharmacopeia General Chapter <233> Elemental Impurities – Procedures: Second Supplement to USP 38–NF 33, 2015, Pharmacopeia Online, https://www.usp.org/chemical-medicines/elemental-impurities-updates
5. USP General Chapter <730> Use of Plasma Spectrochemistry for Pharmaceutical Analysis, Pharmacopeia Online, http://www.uspbpep.com/usp31/v31261/usp31nf26s1_c730.asp
6. USP General Chapter <1225> for Validation of Compendial Procedures , Pharmacopeia Online, http://www.uspbpep.com/usp29/v29240/usp29nf24s0_c1225.html
7. ICH Q3D Step 5 Guidelines, ICH Website: http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html (Q3D)
8. Emerald Proficiency Test, Emerald Scientific, https://pt.emeraldscientific.com/
9. Feds Launch Cannabis Testing Program To Help Consumers Know What They’re Buying, K. Jarger, Marijuana Moment, July 22, 2020; https://www.marijuanamoment.net/feds-launch-cannabis-testing-program-to-help-consumers-know-what-theyre-buying/
10. Clean Chemistry for Elemental Impurities Analysis of Pharmaceuticals in Compliance with USP Chapter <232>, Chunguang Jin, AAPS PharmSciTech, 17, 1141–1149 (2016); https://link.springer.com/article/10.1208/s12249-015-0452-4
11. ISO Clean Room Particle Count Classifications, https://www.mecart-cleanrooms.com/learning-center/cleanroom-classifications-iso-8-iso-7-iso-6-iso-5/
12. Analysis of Toxic Metals in Liquid from Electronic Cigarettes; N. Gray et. al., Int. Journal Environ Res Public Health. 2019 Nov; 16(22): 4450.
13. Analysis of Toxic Metals in Electronic Cigarette Aerosols Using a Novel Trap Design, M. Halstead et.al. Journal of Analytical Toxicology, 2019; 001: 1-7
14. Measuring Elemental Impurities in Pharmaceuticals: A Practical Guide, R. J. Thomas, CRC Press, Boca Raton. FL, US, 2018, ISBN: 9781138197961, https://www.routledge.com/Measuring-Elemental-Impurities-in-Pharmaceuticals-A-Practical-Guide/Thomas/p/book/9781138197961?utm_source=crcpress.com&utm_medium=referral
15. The Food and Drug Newsletter, 2013, https://www.fdanews.com/ext/resources/files/The_Food_And_Drug_Letter/2013/Inspection-Readiness-ExecSeries.pdf
16. Q3D Elemental Impurities Guidance for Industry, U. S. Department of Health and Human Services, FDA, Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER), March 2020, http://www.fda.gov/regulatory-information/search-fda-guidance-documents/q3dr1-elemental-impurities
17. FDA Regulation of Cannabis and Cannabis-Derived Products, Including Cannabidiol (CBD), https://www.fda.gov/news-events/public-health-focus/fda-regulation-cannabis-and-cannabis-derived-products-including-cannabidiol-cbd
18. EPA Seeks Public Comment on Pesticide Applications for Hemp, https://www.epa.gov/newsreleases/epa-seeks-public-comment-pesticide-applications-hemp
19. U.S. Domestic Hemp Production Program, USDA Website; https://www.ams.usda.gov/rules-regulations/hemp
20. NIST Tools for Cannabis Laboratory Quality Assurance, https://www.nist.gov/programs-projects/nist-tools-cannabis-laboratory-quality-assurance
21. Cannabis Inflorescence for Medical Purposes: USP Considerations for Quality Attributes, N. D. Sarma, et.al., ACS Journal of Natural Products, 2020 83 (4), 1334-1351, https://pubs.acs.org/action/showCitFormats?doi=10.1021/acs.jnatprod.9b01200&ref=pdf
22. AHPA Cannabis Committee, http://www.ahpa.org/AboutUs/Committees/CannabisCommittee.aspx
23. ASTM Committee D37 on Cannabis, https://www.astm.org/COMMITTEE/D37.htm
24. AOAC Cannabis Analytical Science Program https://www.aoac.org/scientific-solutions/casp/
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