OR WAIT null SECS
A robust method for identifying heavy metals in cannabis samples using ICP-MS is discussed as well as highlights of the importance of sample preparation methods for a wide range of cannabis products.
Heavy metals are some of the most important contaminants to test for in cannabis analysis. Many heavy metals, such as arsenic, mercury, and lead, can be introduced into the cannabis plant-and therefore the flowers-through cultivation in contaminated soil. Heavy metals can also be introduced later on in the manufacturing process from contaminated processing equipment or raw materials. Because of the health risks associated with these metals, it is imperative that they are detected before entering the market place. This article discusses a robust method for identifying heavy metals in cannabis samples, using inductively coupled plasma-mass spectrometry (ICP-MS). It also highlights the importance of sample preparation methods that can be applied to a wide range of cannabis products. Both will help scientists and technicians better understand how to uncover contamination and help protect consumers from harm.
Numerous studies have identified that cannabis and hemp plants are active accumulators of heavy metals from the soil and water they grow in, which have been contaminated through anthropogenic activities such as mining and smelting (1). Concentrations of these heavy metals are in the parts per billion (ppb) or parts per trillion (ppt) region. Although considered to be at a trace level, the heavy metals may still cause harm to consumers and would therefore require sensitive testing capabilities.
As the use of legalized cannabis across many forms and products continues to increase, it is also critical that robust methods are likewise developed to detect and quantify trace levels of these heavy metals. This is particularly important for long-term users of cannabis products, including patients that may have compromised immune systems or use cannabis-based medicine products because heavy metals can accumulate in the body and cause serious harm (2).
Furthermore, because regulatory limits for heavy metals can vary between jurisdictions and more elements and lower limits are being added to regulations all the time, it is essential that laboratories leverage methods of detection that have the flexibility and sensitivity required for this fast-changing industry.
For example, Canada and U.S. states such as California, Oregon, and Colorado have published limits for heavy metals. Though regulations can vary between geographic regions where cannabis is permitted, many of the set limits are based on United States Pharmacopeia (USP) “General Chapter <232>” guidance (3). The key metals of interest are cadmium (Cd), lead (Pb), arsenic (As), and mercury (Hg). These heavy metals fall within the U.S. Food and Drug Administration (FDA) Class 1 category for substances that are toxic to humans with use allowed in the manufacture of pharmaceuticals (4). Table I provides a list of heavy metal limits based on jurisdiction and route of administration.
To help combat the issues surrounding the frequent prevalence of heavy metals in cannabis, inductively coupled plasma-mass spectrometry (ICP-MS) can be used for trace metal analysis. ICP-MS provides effective detection limits for elemental impurities down to ppb or ppt levels.
The technology works by combining a high temperature ICP source and a mass spectrometer. Samples are introduced into an argon plasma as aerosol droplets, which are dried, dissociated into atoms, and then ionized. Quadrupoles are often used to rapidly scan the mass range with one mass-to-charge ratio being allowed to pass through the mass spectrometer at a given time. The number of ions hitting the detector for a given mass is related to the concentration of the element in that sample (5).
In comparison to other techniques such as inductively coupled plasma-optical emission spectrometry (ICP-OES), ICP-MS can achieve greater sensitivity and many orders of magnitude, enabling a reduction in the amount of sample required. The greater sensitivity is ideal for the determination of trace metals in cannabis samples since the usual levels for some analytes are extremely low (sub-ppb).
The analysis of heavy metals in cannabis remains challenging because of the low level of contaminants in complex matrices, the wide variety of cannabis types, and homogeneity issues that can exist in certain samples. Cannabis flowers are a natural, heterogeneous substance that must be homogenized prior to preparation. When a sample is heterogeneous, say in cannabis brownies or gummy bears, the concentration of heavy metals can vary widely within the sample and the accuracy of analysis is reduced. Though the process of homogenization ensures consistency within the sample, it can also introduce contamination. It is, therefore, vital that preparation protocols are produced that can effectively homogenize cannabis samples while avoiding contamination. The homogenization of cannabis flowers is particularly challenging because the sample requires an optimal digestion protocol to ensure that there are no particulates left in the final digest state.
The following experiment tested the ability of ICP-MS to perform accurate and reproducible analyses of cannabis samples. To account for the wide variety of cannabis product types including flower, edibles, oils, and extracts a robust sample preparation scheme must be used. This was achieved using microwave digestion. The technique works by using microwave energy to increase the temperature and pressure within the vessel and break down everything in the vessel to its constituent elements. For testing flowers, prior to microwave digestion, 3–5 g of cannabis flower were first homogenized with 0.50±0.05 g of each sample weighed and then transferred into a standard 75 mL digestion vessel. The sample amount was chosen based on the proposed California regulations, which require a minimum 0.5 g of the representative cannabis sample. Microwave digestion must be performed using high pressure technology that is capable of processing the 0.5 g required by the proposed California regulations.
The California limits on “all inhaled cannabis goods” were used for this analysis because they are the most stringent compared to other jurisdictions and are particularly suitable for testing cannabis flowers, see Table I. Once the sample had been placed in the digestion vessel, 7 mL of nitric acid (70%) and 3 mL of hydrogen peroxide (30%) were added. The vessels were left uncapped for 10 min to enable any prereactions to occur safely prior to the digestion program.
All samples were digested using PerkinElmer’s Titan MPS system. Spikes of known heavy metal standard concentrations were added to the microwave vessel prior to the addition of reagents, permitting an evaluation of how sample preparation affects analyte recovery. A total of 200 ppb gold (Au) was also added to each sample to stabilize mercury. The digestion program for dissolution of cannabis samples requires three steps with target temperatures of 160 °C, 200 °C, and 50 °C, respectively for each step. The full digestion program can be found in Table II.
It is important for samples from volatile extracts to be given time after digestion to enable the evaporation of residual solvents. All samples were then diluted with deionized water to a volume of 50 mL, resulting in a total dilution factor of 100x with a reagent matrix of 14% HNO3. Calibration was developed by using a blank and four calibration standards to cover the wide range of concentrations for all cannabis sample types.
The analysis was performed using PerkinElmer’s NexION ICP-MS, combined with the All Matrix Solution (AMS) system. The AMS system minimizes the need for dilution of samples with high levels of total dissolved solids. In previous ICP-MS systems, there was a limit to the amount of material that could be delivered to the plasma before degradation of ionization efficiency. Long analytical runs would therefore be constrained because of the subsequent matrix suppression and deposition on the nebulizer cones (6). The AMS system adjusts the flow of the dilution and nebulizer argon gases while maintaining a constant flow to the torch. This permits dilutions of up to 200 times, without requiring off-line dilutions of high-matrix samples. The dilution of the aerosol stream allows for more efficient ionization, reduced matrix suppression, and less deposition on the nebulizer cones. For this analysis, robust conditions for the high sample matrix were created and matrix loading in the plasma was reduced by setting the AMS dilution factor to approximately three times.
The NexION ICP-MS includes a universal cell that can be used in both collision and reaction mode. The collision mode was used to acquire all the analytes in this analysis. This methodology reduces common polyatomic interferences by passing both interfering and analyte ions through a cloud of inert helium molecules. As all polyatomic molecules are larger than analyte ions of the same mass, more energy is lost as the larger ions more frequently collide with the inert gas molecules. The resulting lower energy ions can then be excluded to reduce spectral interference.
The results of the analysis found that all quantitative sample data were less than the target limits for heavy metals in inhalable cannabis products, see Table III. The accuracy of the method was tested by following the USP “General Chapter <233>” requirements, which state that the matrix must be spiked with target elements at concentrations of 50%, 100%, and 150% of the maximum permitted daily exposure (PDE) (7). Method validation requires for the mean spike recoveries of each target element to be within 70–150% of actual concentrations. Table IV shows that the spike recovery test for target elements at concentrations of 50%, 100%, and 150% of the PDE were well within the 70–150% acceptance criteria range, with the lowest mean recovery percentage of 81% for samples spiked with lead.
The repeatability test requires six independent samples spiked with target elements at concentrations that are 100% of the PDE. USP “General Chapter <233>” method validation requires the measured percentage relative standard deviation (%RSD) to not exceed 20% for each target element. Table V shows that the %RSDs for all target elements were within 3% and therefore under the acceptance limit.
The ruggedness of the method was assessed by testing the six repeatability solutions, which were prepared by two different analysts. The USP “General Chapter <233>” requires that the %RSD of the 12 replicates must be less than 25% for each target element. Table VI shows that all 12 measurements had RSDs <2.5%, below the 25% required for method validation.
The performance of this method for the cannabis industry has been further validated by The Emerald Proficiency Test for Heavy Metals. The Emerald Test is an inter-laboratory comparison and proficiency test program for cannabis testing laboratories (8). The results from the PT inter-laboratory samples passed the test to meet the requirements for inter-laboratory reproducibility and accuracy.
The analytical tests show that ICP-MS is capable of producing reproducible quantitative data at sensitivity levels less than the California target limits for “all inhaled cannabis goods.” The results also confirm that the method passes the acceptance criteria for the testing protocols described in USP “General Chapter <233>.”
The analysis demonstrated that ICP-MS is a suitable technique for performing accurate and reproducible analyses of cannabis flower samples. The initial digestion and sample handling procedure can be applied to a wide variety of sample types including flowers, extractions, and edibles, providing complete digestion with no particulate residue. Moreover, the procedure can be implemented across different cannabis laboratories and jurisdictions.
ICP-MS is particularly suitable for heavy metal analysis in cannabis because it provides greater sensitivity than other techniques. The application of a universal cell with collision capabilities enables greater certainty of the identification of an element as the system can be implemented in multiple ways. If there is anything within the sample that could lead to a false positive, the technology can provide verification through the application of different gases in the universal cell.
As the demand for testing cannabis increases and more elements are added to the regulations, there will be a greater need for robust analytical methodologies that have the flexibility to analyze a wide variety of cannabis forms and products. ICP-MS provides a critical analytical tool that will be able to meet the growing needs of the cannabis industry.
Aaron Hineman is the Inorganic Product Line Leader, Americas at PerkinElmer in Blaine, Washington. Toby Astill is the Global Market Manager – Cannabis & Hemp Markets at PerkinElmer in Downers Grove, Illinois. Direct correspondence to: Aaron.firstname.lastname@example.org and Toby.email@example.com
A. Hineman and T. Astill, Cannabis Science and Technology 2(5), 66-70 (2019).