ICP-MS Analysis of Heavy Metals in Cannabis Sativa

1. Introduction

Hemp (or: industrial hemp) and cannabis are strains of the Cannabis sativa plant differentiated on the basis of total delta-9-tetrahydrocannabinol (delta-9-THC) content (delta-9-THC and tetrahydrocannabinolic acid (THCA)). Cannabis sativa plant material that exceeds 0.3% wt/wt THC on a dry weight basis is considered to be marijuana (cannabis) under the US Controlled Substances Act. Industrial hemp is Cannabis sativa L. with a THC threshold below 0.3% (= % THCA x 0.877 + % Delta-9-THC). Definitions of hemp and cannabis are regulated in a specific manner in many parts of the world.  In this work hemp material has been utilized, but all descriptions are valid for cannabis as well.

Hemp is known to accumulate heavy metals such as lead, cadmium, arsenic, mercury, chromium, nickel, manganese and cobalt in its roots, shoots, buds and seeds, and has been used for the remediation of contaminated soil (phytoremediation and phytoextraction).
On the other hand, this property may hinder the use of hemp in food or medical industries. As a consequence, all hemp material used in either food or pharma products must be tested for the heavy metal content.

As of February 2020, Canada and 24 US states have issued regulations for the testing of heavy metal content in cannabis, and all have provided limits for arsenic, cadmium, mercury and lead. In addition, four states have also set limits for one or more of these metals: chromium, barium, silver, selenium, antimony, copper, nickel and zinc.

This report describes the analysis of arsenic, cadmium, mercury and lead (elements typically referred to as the “big four”) by inductively coupled plasma coupled to mass spectrometry (ICP-MS) for three different Cannabis sativa varieties of industrial hemp (cannabidiol (CBD) and cannabigerol (CBG) content: information from manufacturer):

• Hemp buds “Felina”, CBD/CBG content 3.8/0.2%
• Hemp buds “Santhica”, CBD/CBG content 3.9/0.2%
• Hemp buds “Finola”, CBD/CBG content 3.8/0.2%

 

2. State regulations

The tables below list the “big four” heavy metals and their limits based on jurisdiction and route of administration for various US states and Canada (all data as of February 2020).

 

State Arkansas* California*  Canada Colorado* Connecticut*,
      Ohio
  Concentration (ppm) Concentration
(µg/kg bodyweight /day)
Intended use All uses Inhalable Other All uses Inhalable Oral
Rectal
Vaginal
Topical All uses
Arsenic 0.200 0.200 1.500 0.200 0.200 1.500 3.000 0.140
Cadmium 0.200 0.200 0.500 0.200 0.200 0.500 3.000 0.090
Lead 0.500 0.500 0.500 0.500 0.500 1.000 10.000 0.290
Mercury 0.100 0.100 3.000 0.100 1.000 1.500 1.000 0.290
 
State Hawaii Iowa, Minnesota Louisiana, Virginia Maryland, Oklahoma Massachusetts, Rhode Island Michigan, Missouri Montana
  Concentration (ppm)  
Intended use All uses All uses All uses All uses All uses oral Inhalable Other Extract Flower
Arsenic
10.000 1.500 10.000 0.400 0.200 1.500 0.200 1.500 10.000 2.000
Cadmium 4.000 0.300 4.100 0.400 0.200 0.500 0.200 0.500 4.100 0.820
Lead 6.000 1.000 10.000 1.000 0.500 10.000 0.500 0.500 6.000 1.200
Mercury 2.000 0.500 0.500 0.200 1.000 1.500 0.100 3.000 2.000
0.400
 
State Nevada* New Hampshire* New Mexico New York* Pennsylvania* Vermont* Washington*
  Concentration (ppm) Concentration (µg/daily dose)
Intended use All uses
All uses All uses All uses All uses All uses All uses
Arsenic 2.000
4.206 2.000 0.200 0.400 10.000 10.000
Cadmium 0.820 2.704 0.800 0.200 0.300 4.100 4.100
Lead 1.200 8.712 1.200 0.200 1.000 10.000 6.000
Mercury 0.400 8.712 0.400 0.200 0.200 2.00 2.000

* State specific certified reference material Heavy metal mixTraceCERT® standard solutions according to state regulation available

 

3. Hemp sample preparation and preparation of internal standard, blank and cannabis sample solutions

Sample preparation
Sample milling One hemp bud (approx. 2.5 g) was manually broken into pieces and placed into a 50 mL stainless steel milling beaker. A 25 mm stainless steel milling ball was added, and the sealed beaker mounted onto a cryo ball mill equipped with a liquid nitrogen filling system for permanent coolant supply. Grinding parameters: Pre-cooling at 5 Hz, two cycles of 90 s at 30 Hz and 30 s at 5 Hz (for intermediate cooling). The final material was collected into a 100 mL glass bottle.
As an alternative, e.g. 25 mL zirconia milling beakers and 15 mm zirconia milling balls can be utilized. This setup has lower throughput due to limited sample capacity and increased milling times.
Preparation of internal standard, blank and sample solutions
General Samples were weighed on a calibrated analytical balance with a readability of 0.1 mg.
Single-channel pipettes with variable volume and gravimetrically tested polypropylene pipette tips were used for reagent, standard and sample transfers.
Indium standard solution (internal standard) 3 mL nitric acid 60% and 1000 µL of indium ICP standard (1000 mg/L) were pipetted into a 100 mL volumetric quartz flask. Subsequently the flask was filled to mark with ultrapure water to obtain a final concentration of 10 µg/mL.
Rhodium ICP standards may also be used as alternative internal standard.
Blank solution 3 mL nitric acid 60%, and 1 mL hydrogen peroxide 31% were pipetted into a 15 mL quartz microwave digestion vial and digested using a microwave digestion system (for conditions see section below). After completion of digestion the solution was quantitatively transferred into a 50 mL polypropylene tube, combined with 50 µL of indium standard solution and filled to 50 mL with ultrapure water.
Sample solution 50 ±1 mg of ground sample was weighed into a 15 mL microwave quartz vial. After addition of 3 mL nitric acid 60%, and 1 mL hydrogen peroxide 31%, the sample was digested utilizing a microwave digestion system. After digestion was completed, the obtained solution was quantitatively transferred into a 50 mL polypropylene tube, combined with 50 µL of indium standard solution and filled to 50 mL with ultrapure water.

 

4. Calibration: preparation of heavy metal standard addition solutions

In order to compensate for sample matrix effects, a standard addition approach utilizing various Certified Reference Material (CRM) Heavy metal mix standard solutions were applied for the preparation of all calibration curves. The final calibration curve was comprised of four data points (three standard addition solutions plus sample solution).

 

Method: general process for the preparation of calibration solutions for ICP
General 50 ±1 mg of ground sample was weighed into a 15 mL microwave quartz vial. After addition of 3 mL nitric acid 60%, 1 mL hydrogen peroxide 31% and different volumes of a CRM Heavy Metal Mix standard solution the sample was digested using a microwave digestion system. After digestion was completed the obtained solution was quantitatively transferred into a 50 mL polypropylene tube, combined with 50 µL of indium standard solution and filled up to 50 mL with ultrapure water. The resulting sample was subsequently subjected to ICP-MS analysis.

 

5. Preparation of standard addition solutions (hemp “Felina”)

5.1 Preparation of standard addition solutions using Heavy metal mix III standard solution (As, Cd, Hg, Pb)

Standard addition solution no. Volume (µL)
(Heavy metal mix III)
Resulting heavy metal concentration (µg/g)
(initial sample weight 50 mg)
    Arsenic Cadmium Lead Mercury
1 25 2.0000 2.0000 5.0000 1.0000
2 50 4.0000 4.0000 10.0000 2.0000
3 100 8.0000 8.0000 20.00000 4.0000

Heavy metal mix III was diluted by a factor of 1:10 using ultrapure water.

 

5.2 Preparation of standard addition solutions using Heavy metal mix IV standard solution (As, Cd, Hg, Pb)

Standard addition solution no. Volume (µL)
(Heavy metal mix IV)
Resulting heavy metal concentration (µg/g)
(initial sample weight 50 mg)
    Arsenic Cadmium Lead Mercury
1 50 4.0020 1.6408 2.4020 0.8004
2 100 8.0040 3.2816
4.8040 1.6008
3 200 16.0080 6.5632 9.6080 3.2016

Heavy metal mix IV was used without further dilution.

 

6. Preparation of standard addition solutions (hemp “Santhica”)

6.1 Preparation of standard addition solutions using Heavy metal mix V standard solution (As, Cd, Hg, Pb)

Standard addition solution no. Volume (µL)
(Heavy metal mix V)
Resulting heavy metal concentration (µg/g)
(initial sample weight 50 mg)
    Arsenic Cadmium Lead Mercury
1 50 1.4000 0.9000 2.9010 2.9010
2 100 2.8000 1.8000 5.8020 5.8020
3 200 5.6000 3.6000 11.6040 11.6040

Heavy metal mix V was diluted by a factor of 1:10 using ultrapure water.

 

6.2 Preparation of standard addition solutions using Heavy metal mix VI standard solution (As, Cd, Hg, Pb)

Standard addition solution no. Volume (µL)
(Heavy metal mix VI)
Resulting heavy metal concentration (µg/g)
(initial sample weight 50 mg)
    Arsenic Cadmium Lead Mercury
1 50 1.4000 0.9000 2.9010 2.9010
2 100 2.8000 1.8000
5.8020 5.8020
3 200 5.6000 3.6000
11.6040 11.6040

Heavy metal mix VI was used without further dilution.

 

7. Preparation of standard addition solutions (hemp “Finola”)

7.1 Preparation of standard addition solutions using Heavy metal mix VII standard solution (As, Cd, Hg, Pb)

Standard addition solution no. Volume (µL)
(Heavy metal mix VII)
Resulting heavy metal concentration (µg/g)
(initial sample weight 50 mg)
    Arsenic Cadmium Lead Mercury
1 10 3.0000 0.6000 2.0000 1.0000
2 20 6.0000 1.2000 4.0000 2.0000
3 50 15.0000 3.0000 10.0000 5.0000

Heavy metal mix VII was used without further dilution.

 

7.2 Preparation of standard addition solutions using Heavy metal mix VIII standard solution (As, Cd, Hg, Pb)

Standard addition solution no. Volume (µL)
(Heavy metal mix VIII)
Resulting heavy metal concentration (µg/g)
(initial sample weight 50 mg)
    Arsenic Cadmium Lead Mercury
1 10 1.0000 0.6000 1.8000 1.8000
2 20 2.0000 1.2000
3.6000 3.6000
3 50
5.0000 3.0000
9.0000 9.0000

Heavy metal mix VIII was used without further dilution.

 

8. Hemp sample digestion

Digestion conditions    
System Microwave digestion system turboWAVE, MLS (Germany) (or equivalent)  
Digestion program Nitric acid digestion at 280 °C  
Microwave vial Quartz glass  
Basic load 110 mL ultrapure water and 5 mL nitric acid or 115 mL ultrapure water  
Charging pressure 40 bar  
Deflation rate 5 bar/min (from T < 80 °C)  
Vessel cooling Yes (> 40 °C)  

 

Digestion program parameters
Time [h] Microwave output (W) Temperature 1 (°C) Temperature 2 (°C) Pressure (bar)
00:03:00 700 70 60 100
00:15:00 1000 180 60 120
00:30:00 1200 280 60 120

After digestion the obtained solution should be clear and particle free. Conditions must be adapted to the specific microwave system used.

 

9. ICP-MS analysis of hemp

ICP-MS conditions  
System ICP-MS Element 2, ThermoScientific (or equivalent)  
Plasma output: approx. 1300 W Nitric acid digestion at 280 °C  
Plasma gas flow approx. 16 L/min  
Sample delivery Peristaltic pump (or equivalent), delivery volume approx. 1 mL/min  
Nebulizer Quartz spray chamber (or equivalent) / Meinhardt nebulizer (or equivalent)  
Nebulizer gas flow approx. 1 L/min  
Assist gas flow approx. 1 L/min  
Mass resolution 4000 + 10000  
Calibration Standard addition  

The analysis was performed in the sequence: Blank, samples 1 – x, addition solutions.

 

10. Calibration data (hemp “Finola”)

Standard addition solutions were prepared utilizing Heavy metal mix VIII. Comparable results were achieved with the other hemp varieties.

 

11. Experimental results

Element Hemp Felina Hemp Santica Hemp Finola
CRM Used Heavy metal mix III Heavy metal mix IV Heavy metal mix V Heavy metal mix VI Heavy metal mix VII Heavy metal mix VIII
Concentration (ppm)
Arsenic < 0.1 < 0.1 < 0.1 < 0.1  0.1  0.1
Cadmium < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1
Lead  0.5  0.5  0.4  0.4    0.7  0.7
Mercury < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1

 

12. Conclusion

This work demonstrates a comprehensive ICP-MS workflow, using the standard addition calibration method, for determination of heavy metals in Cannabis sativa plant materials. Critical elements in the process include homogenization of samples and use of accurate traceable Certified Reference Material mixes that are tailored to state specific regulations for heavy metals in cannabis.  Reproducible samples were prepared by grinding the hemp with a cryo ball mill. Samples were then digested utilizing a specific digestion protocol optimized to provide clear digestion solutions. The resulting solutions were subjected to ICP-MS analysis. Calibration data was obtained by the preparation and analysis of standard addition solutions obtained by diluting six different heavy metal CRM mixes containing arsenic, cadmium, lead and mercury. The final results were consistent and revealed a Cd and Hg concentration of <0.1 ppm for all hemp samples. The Finola variety contained 0.1 ppm arsenic and 0.4 to 0.7 ppm lead was detected in all three hemp varieties.

 

13. Materials