How to perform accurate and reproducible analyses of cannabis flower samples

Heavy metals are widely recognized as being toxic, so as cannabis use becomes more commonplace, it is imperative that cannabis flowers and other cannabis derivatives are tested to ensure that patient and consumer safety is maintained.

There is increasing demand for accurate testing of cannabis flowers and other cannabis derivatives for toxins such as the heavy metals lead (Pb), arsenic (As), cadmium (Cd) and mercury (Hg).

A number of states, including California,6 Oregon and Colorado, have published action limits for heavy metals in line with wider federal pharmaceutical and nutraceutical requirements in the US,1,2,3,4,5

Jurisdictions that permit cannabis use to stipulate the maximum allowable limits of heavy metals in cannabis and related products, many of which are in line with USP <232> and ICH Q3D recommendations.

Limits vary depending on the route of administration, largely in line with the limits set out in ICH Q3D recommendations. Canada does not currently impose specific requirements around the presence of metals in cannabis products, but it does refer analysts to USP <232> and <233> for guidance.

Table 1 details a series of currently known limits for heavy metals.

Table 1. A list of the heavy metals and their limits based on jurisdiction and route of administration. Source: PerkinElmer Cannabis & Hemp Testing Solutions

  Canada
(Based on
USP <232>)
California6 Colorado Connecticut,
Maryland,
Nevada,
New Mexico
Massachusetts Minnesota Washington
Heavy Metal Inhaled
Cannabis
Goods
(μg/g)
All Inhaled
Cannabis
Goods
(μg/g)
Other
Cannabis
Goods
(μg/g)
Inhaled
Products
(ppm)
“μg/kg of
body weight
per day"
All Uses
(μg/kg)
Ingestion
Only
(μg/kg)
PPM in
Final
Product
μg/Daily
Dose
(5 grams)
Cadmium (Cd) 0.2 0.2 0.5 0.2 0.09 200 500 0.3 4.1
Lead (Pb) 0.5 0.5 0.5 0.5 0.29 500 1000 1.0 6.0
Arsenic (As) 0.2 0.2 1.5 0.2 0.14 200 1500 1.5 10.0
Mercury (Hg) 0.1 0.1 3 0.1 0.29 100 1500 0.5 2.0

 

The study described here employed limits required by California on “all inhaled cannabis goods” because these were the most strict and therefore the most applicable to analysis of cannabis flowers.

There are a number of challenges to consider when performing elemental analysis of cannabis, particularly the complex requirements around sample preparation and digestion.

A robust sample preparation scheme must be utilized in order to account for the diverse array of cannabis sample types (concentrates, edibles, extracts, tinctures, flower, waxes and oils).

Sample preparation generally involves the use of homogenization and microwave digestion in order to break down the complex matrix and extract any heavy metals present.

This required the development and employment of specific sample preparation protocols, microwave digestion conditions and ICP Mass Spectrometry (ICP-MS) methodologies to ensure a robust method suitable for the full range of cannabis sample types.

ICP-MS is a highly effective technique that is well suited to the analysis of trace metals. Its capacity to observe low levels in complex matrices makes ICP-MS an excellent tool for determining trace metals in cannabis samples, particularly because the normal levels of certain analytes are exceptionally low (sub-ppb).

This article presents a range of data illustrating the suitability of the Titan MPS™ Microwave Sample Preparation System and the NexION® ICP-MS for the determination of heavy metals in cannabis flowers.

Analyses are verified in line with the validation protocols defined in USP General Chapter <233>. These protocols are frequently employed when evaluating levels of elemental impurities in samples.

Experimental

Sample preparation procedure

All samples analyzed in the examples presented were digested in standard 75 mL TFM vessels using microwave digestion (Titan MPS System: PerkinElmer Inc., Shelton, Connecticut, USA).

Around 3-5 grams of cannabis flower was ground and homogenized. This was in line with California-proposed regulations that state that “the laboratory shall analyze at minimum 0.5 grams of the representative sample of cannabis goods or cannabis product to determine whether heavy metals are present.”6

To meet this requirement, 0.50 ± 0.05 g of each sample was weighed using a weight boat before being transferred into a digestion vessel. Next, 7 mL of nitric acid (70%) and 3 mL of hydrogen peroxide (30%) were added and vessels were left uncapped for a total of 10 minutes to allow any pre-reactions to occur safely.

Table 2. Titan MPS System microwave digestion program for dissolution of cannabis samples. Source: PerkinElmer Cannabis & Hemp Testing Solutions

Step Target
Temp
(°C)
Pmax
(bar)
Ramp
(min)
Hold
(min)
Power
(%)
1 160 30 5 5 90
2 200 30 5 20 100
3 50 30 1 30 0

 

Vessels were then capped and digested according to a pre-defined program (Table 2). Spikes were added to the microwave vessel prior to the addition of the reagents to evaluate the effect of the sample preparation on analyte recovery. A total of 200 ppb gold (Au) was added to each sample in order to stabilize the mercury content.

Once digestion was completed, samples were diluted to a final volume of 50 mL using deionized water, resulting in a total dilution factor of 100x with a reagent matrix of 14% HNO3.

This same matrix was used to prepare calibration standards. Figure 1 illustrates the cannabis flower and the resulting clear solution once this had been digested and prepared for analysis.

Cannabis flower before and after digestion.

Figure 1. Cannabis flower before and after digestion. Image Credit: PerkinElmer Cannabis & Hemp Testing Solutions

Instrumentation

A PerkinElmer NexION ICP-MS was used to complete the analysis. This instrument included the company’s proprietary Universal Cell Technology™ (UCT) and the All Matrix Solution (AMS) system.

The NexION ICP-MS was configured with the standard SMARTintro™ sample introduction module. This was comprised of a glass cyclonic spray chamber, MEINHARD® glass concentric nebulizer and a quartz torch with 2 mm id injector.

Table 3. NexION ICP-MS Operating Conditions. Source: PerkinElmer Cannabis & Hemp Testing Solutions

Parameter Value
RF Power (W) 1600
Nebulizer Flow (L/min) 0.88
Dilution Gas Flow (L/min) 0.11
Sample Uptake Rate (mL/min) 0.20
Collision (He) Gas Flow (mL/min) 4

 

Table 3 details the instrument’s operating parameters. An AMS dilution factor of approximately 3x was used to help reduce matrix loading in the plasma and ensure robust plasma conditions in the high sample matrix. Collision mode using helium was employed in the acquisition of all analytes.

This straightforward methodology allows the UCT to reduce or completely eliminate common polyatomic interferences via kinetic energy discrimination (KED).

Calibration

A calibration was developed using a blank and four calibration standards. This allowed the method to accommodate a diverse array of concentrations for all cannabis sample types, including extracts and concentrates.

Table 4. Elements and standard concentrations. Source: PerkinElmer Cannabis & Hemp Testing Solutions

Analyte Mass Standard 1
(μg/L)
Standard 2
(μg/L)
Standard 3
(μg/L)
Standard 4
(μg/L)
Cadmium (Cd) 110.90 0.5 1 5 10
Lead (Pb) 207.98 1.25 2.5 12.5 25
Arsenic (As) 74.92 0.5 1 5 10
Mercury (Hg) 201.97 0.1 0.2 1 2

 

Table 4 lists the masses, elements and standard concentrations used. Both the calibration blank and standard were prepared in 14% nitric acid to matrix match with the samples, as per the previous section.

A total of 200 ppb gold (Au) was also added to the calibration blank and each standard in order to stabilize the mercury. Internal standards (Ge, In, and Tb) were added on-line to monitor instrument response between samples.

Results and discussion

Method validation

Specific requirements for method validation are defined in USP General Chapter <233>.

Accuracy

It is necessary to spike the materials and matrix being evaluated with target elements at concentrations of 50%, 100% and 150% of the maximum permitted daily exposure (PDE).

Mean spike recoveries for each target element are required to be within 70% and 150% of the actual concentrations.

California inhalational limits for all inhaled cannabis goods were utilized in the calculation of spike levels, while the 50%, 100% and 150% spike levels were calculated based on a nominal preparation factor of 100.

Table 5 details the limits and spike levels employed in this study.

Table 5. PDEs and Spike Levels. Source: PerkinElmer Cannabis & Hemp Testing Solutions

Analyte PDE for Inhaled
Products
Spike Level (μg/L)
50% PDE 100% PDE 150% PDE
Cadmium (Cd) 0.2 1.00 2.00 3.00
Lead (Pb) 0.5 2.50 5.00 7.50
Arsenic (As) 0.2 1.00 2.00 3.00
Mercury (Hg) 0.1 0.50 1.00 1.50

 

Repeatability

To ensure repeatability, a total of six independent samples should be spiked at 100% of the defined target limits before being analyzed. Measured percent relative standard deviation (%RSD) must remain within 20% for each individual target element.

Ruggedness

Repeatability measurement testing involved analyzing the six repeatability test solutions - on different days by different analysts, using a different instrument. The %RSD of the 12 replicates is required to be less than 25% for each individual target element.

Sample analysis

Quantitative sample data was consistently found to be less than the lowest calibration standard. This was also less than the regulated target limits for heavy metals in inhalable cannabis products.

Table 6. Sample Results. Source: PerkinElmer Cannabis & Hemp Testing Solutions

Element Sample Results Units (μg/g) Pass/Fail
1 2 3 Mean SD Limit
Cadmium (Cd) 0.029 0.037 0.042 0.036 0.006 0.2 Pass
Lead (Pb) 0.009 0.021 0.010 0.013 0.007 0.5 Pass
Arsenic (As) 0.027 0.030 0.045 0.034 0.010 0.2 Pass
Mercury (Hg) 0.056 0.044 0.044 0.048 0.007 0.1 Pass

 

Meeting the Validation Criteria

Quantitative sample data was found to be consistently below the lowest calibration standard and therefore below target limits for heavy metals in inhalable cannabis products.

Accuracy

Table 7 exemplifies the methodology’s potential to yield accurate data. It reveals that pre-digestion spike recovery tests performed on the sample matrix successfully pass at every spike level - 50%, 100%, and 150% of the target limits.

Mean spike recoveries for individual target elements fell comfortably within the acceptance criteria of 70% to 150%.

Table 7. Accuracy Test Results. Source: PerkinElmer Cannabis & Hemp Testing Solutions

Element Mean
Unspiked
Sample
(μg/g)
Mean Recovery (%) Pass/Fail
50% 100% 150%
Cadmium (Cd) 0.036 87 94% 91 Pass
Lead (Pb) 0.013 81 85% 84 Pass
Arsenic (As) 0.034 94 96% 98 Pass
Mercury (Hg) 0.005 97 95% 107 Pass

 

Repeatability

A total of six independently prepared cannabis flower samples were digested before being spiked at 100% of the target limit. These were then analyzed.

The %RSDs for every target element was within 3% - comfortably below the 20% acceptance limit (Table 8).

Table 8. Repeatability Test Results. Source: PerkinElmer Cannabis & Hemp Testing Solutions

Repeatability
Element Sample
1
Sample
2
Sample
3
Sample
4
Sample
5
Sample
6
Mean %RSD
(μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g)
Cadmium (Cd) 0.22 0.21 0.23 0.22 0.23 0.23 0.23 2.90%
Lead (Pb) 0.43 0.43 0.44 0.43 0.45 0.47 0.44 1.10%
Arsenic (As) 0.23 0.22 0.22 0.23 0.24 0.24 0.23 1.10%
Mercury (Hg) 0.10 0.10 0.10 0.10 0.10 0.11 0.10 1.20%

 

Ruggedness

Table 7 shows the six samples used for the repeatability study, as prepared by two different analysts. The RSDs for each of these measurements were all found to be < 2.5% (Table 9) – comfortably below the method requirement of 25%.

Table 9. Ruggedness Test Results. Source: PerkinElmer Cannabis & Hemp Testing Solutions

Element Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Sample 12 Mean %RSD
(μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g)
Cadmium (Cd) 0.22 0.21 0.23 0.22 0.23 0.23 0.20 0.21 0.19 0.21 0.19 0.21 0.21 7.07%
Lead (Pb) 0.43 0.43 0.44 0.43 0.45 0.47 0.38 0.43 0.38 0.42 0.38 0.43 0.42 6.85%
Arsenic (As) 0.23 0.22 0.22 0.23 0.24 0.24 0.20 0.22 0.21 0.22 0.21 0.22 0.22 5.26%
Mercury (Hg) 0.10 0.10 0.10 0.10 0.10 0.11 0.09 0.10 0.09 0.11 0.09 0.10 0.10 4.96%

 

Conclusion

The examples presented in this article clearly demonstrate the capacity of PerkinElmer’s NexION ICP-MS to perform accurate, reproducible cannabis flower analyses when coupled with the Titan MPS Sample Preparation System.

By leveraging PerkinElmer’s AMS and Universal Cell Technology, it was possible to develop a robust method.

Quantitative sample data was found to be well within the target limits for heavy metals in the guidance around inhaled cannabis goods, confidently meeting acceptance criteria for testing protocols outlined in USP General Chapter <233>.

References

  1. United States Pharmacopeia (USP) and The National Formulary (NF) Online (USP-NF): http://www.usp.org/usp-nf/key-issues/ elemental-impurities;
  2. ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) Q3D Step 4 - Guideline for Elemental Impurities; http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/ Guidelines/Quality/Q3D/Q3D_Step_4.pdf;
  3. United States Pharmacopeia General Chapter <232> Elemental Impurities in Pharmaceutical Materials – Limits: Second Supplement to USP 39–NF 34, May, 2016, Updates Published in Pharmacopeial Forum 42(2);
  4. United States Pharmacopeia General Chapter <233> Elemental Impurities in Pharmaceutical Materials – Procedures: Second Supplement to USP 38–NF 33, December, 2015;
  5. Elemental Impurities in Drug Products: Guidance for Industry: Food and Drug Administration Document: https://www.fda.gov/downloads/Drugs/Guidances/ UCM509432.pdf, August, 2018;
  6. Bureau of Cannabis Control, California Code of Regulations under Division 42 of Title 16 § 5723: https://bcc.ca.gov/law_regs/cannabis_order_of_adoption.pdf (Accessed January 17, 2019);
  7. Thirty-Minute Guide to ICP-MS, PerkinElmer Technical Note. http://www.perkinelmer.com/lab-solutions/resources/docs/ TCH-30-Minute-Guide-to-ICP-MS-006355G_01.pdf, 2017;
  8. The Emerald Test: Inter-laboratory Comparison and Proficiency Test for Cannabis Testing Labs, https://pt.emeraldscientific.com.

Acknowledgments

Produced from materials originally authored by Aaron Hineman, Ryan Purcell-Joiner, and Toby Astill from PerkinElmer and Anresco Laboratories.

About PerkinElmer Cannabis & Hemp Testing Solutions

With the cannabis and hemp markets continuing to grow rapidly and regulations strengthening, labs increasingly need streamlined access to best-in-class testing solutions geared toward the unique requirements of the industry. Whether your lab is well established or just starting up, PerkinElmer is a single-source vendor for instruments, methods, reagents, and consumables on hand to help enhance your testing capacity and get ahead of the competition.

They help drive analytical best practices and operating procedures and commit to ensuring your laboratory has maximum uptime. Learn about their various instruments, testing methods, and applications for cannabis analyses. Let them work with you to build an efficient workflow, so you can focus on growing your business.


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Last updated: Sep 24, 2021 at 10:51 AM

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