Reproducible Sample Homogenization of Cannabis

Cannabis is classified as a “soft” drug which can lower the inhibition threshold for consuming “hard” drugs like cocaine or heroin. In 1925 possession of cannabis was prohibited worldwide. Today, restricted consumption is legal in some countries such as Canada, the Czech Republic and Israel. The first country to legalize the cultivation, sale, and distribution of cannabis was Uruguay in 2013. Several studies1 have verified the benefits of using cannabis for medical treatment, so some countries have begun the process of making the use of cannabis, under strictly controlled conditions, legal. In 29 of the American states, the sale of cannabis for medical reasons has been permitted since 2016. Germany legalized it a year later.

Benefits of cannabis on human health can be grouped into five clusters:

  • Gastro-intestinal
  • Pain/sleep
  • Mood/behavior
  • Neurological
  • Other

The main components of cannabis are cannabinoids like cannabinol (CBN), tetrahydrocannabivarin (THCV), or tetrahydrocannabinol (THC) and these can act as psychoactive, appetite suppressant, or sedative compounds. These have beneficial effects, e. g. on metabolic disorders like diabetes, pain relief, anti-inflammatory processes, and even on the treatment of bacteria like MRSA. It also has a positive influence on diseases such as; asthma, cancer, Alzheimer’s, arthritis, HIV, multiple sclerosis and Tourette’s syndrome

Another major group of ingredients are the so-called terpenoids like D-Limonene, Linalool, or α-Pinene. The terpenoids produce an “entourage effect” which means they can alter/enhance cannabinoid uptake in the Blood-Brain-Barrier. Terpenoids also act as antioxidants or anti-inflammatory agents.

Cannabis occurs in a variety of edible forms, e.g. concentrate, ground leaves or related products such as cookies or jelly bears. The principle areas of interest in the quality control of cannabis-related products are the concentrations of terpenoids and cannabinoids, which are normally detected by HPLC analysis. The amount of pesticide is also of interest. The cannabis sample preparation process has to be adaptable to the significant complexity of the various matrices, to ensure reliable analytical results. There are a few challenges to overcome: samples may be fibrous, sticky, or fatty and the sample amount may vary strongly.

Pesticide Extraction with the Mixer Mill MM 400 (QuEChERS Method)

To make sample preparation to pesticide residue analysis more efficient, the QuEChERS method (“quick, easy, cheap, effective, rugged and safe”) has been developed. It consists of three steps:

  • Homogenization
  • Extraction
  • Analysis

Care has to be taken that the sample does not get too warm during the homogenization process, as some pesticides are volatile. 10 g of the pulverized hemp sample is extracted with 10 ml acetonitrile after the homogenization. Next, the organic phase is dried and tested for pesticides with chromatographic analysis.

The sample is extracted in the presence of a salt mixture (e. g. sodium chloride and magnesium sulphate in a 1:2 ratio) to avoid ghost peaks in the chromatograms. The mixture is agitated for 1 to 3 minutes with acetonitrile and salt to transfer the pesticides from the sample into the organic phase. The mixture can be agitated in a laboratory mill such as RETSCH’s Mixer Mill MM 400. It shakes the sample with a frequency of up to 30 Hz in a 50 ml Falcon tube, which ensures thorough mixing of the sample to improve subsequent extraction.

Pre-Cutting of Dried Hemp Plants in the Cutting Mill SM 400 XL

The Cutting Mill SM 400 XL processes sample pieces with a maximum size of 170 mm x 220 mm and has a grinding chamber volume of 7.5 l. Manual pre-cutting is not required, so large sample volumes are fed and fully homogenized in short periods of time.

The throughput is significantly higher than that of smaller models because of the wide opening of the hopper, large 240 mm x 240 mm surface of the bottom sieves, and considerable grinding chamber volume. Additionally, the SM 400 XL can reach grind sizes down to 1 mm, depending on the sample material.

Using a 20 mm bottom sieve, 100 kg dried hemp with an initial particle size of up to 60 mm was cut batchwise to a fineness <20 mm within an hour. The only mill strong enough to quickly process such large amounts of fibrous material with no blockages of the hopper by wedged pieces is the SM 400 XL. Using RETSCH’s Ultra Centrifugal Mill ZM 300, the pre-cut sample can now be pulverized. The SM 400 XL is suitable for milling all parts of the plant for easy ‘rolling’ or filling of material into a variety of objects for smoking.

Pulverization of Hemp in the CryoMill for Subsequent Pesticide Analysis

The extraction via QuEChERS can be improved by reducing the particle size to <0.5 mm. Due to the sticky and oily sample properties, the Ultra Centrifugal Mill is unable to achieve this. An effective way of making oily materials break easily is embrittlement of the sample, e.g. with liquid nitrogen. Cryogenic grinders such as RETSCH’s CryoMill are specially designed for these applications, as the grinding jar, and thus the sample, is constantly cooled with LN2.

The CryoMill generates grind sizes <0.1 mm, which means that higher pesticide amounts are detected in the hemp sample homogenized with the CryoMill after extraction than in the sample milled with the ZM 300. 5 g pre-cut hemp sample was ground in a 50 ml stainless steel grinding jar with one 25 mm stainless steel grinding ball. Ball and sample were filled in the grinding jar, the lid was closed tightly, lastly the jar was clamped into the CryoMill.

The grinding process does not begin before a temperature of -196 °C is reached and maintained thanks to an automatic pre-cooling function. Due to the autofill system of the CryoMill, the operator never comes into contact with liquid nitrogen and the machine maintains the temperature at -196 °C during grinding. The pre-cooling time was set to 3 minutes at 5 Hz. Grinding was done at 30 Hz for 3 min. The embrittled sample can now be ground to much smaller particle sizes than in the ZM 300, but for larger sample quantities, the ZM 300 is an appropriate option.

Sample Homogenization in the Ultra Centrifugal Mill ZM 300

The ideal mill for pulverizing granules like grains or fibrous samples like hemp plants is the Ultra Centrifugal Mill ZM 300. It can be equipped with a large range of accessories, allowing for adaption to the sample´s requirements and reaches a maximum speed of 23,000 min-1.

Hemp contains oil which makes it a temperature-sensitive material; to reduce heat build-up during grinding, it is recommended to use a distance sieve. The shearing forces between ring sieve and rotor enable successful size reduction of fibrous materials. The shearing forces and the formation of heat are reduced because of the small gap between the sieve and the rotor. By using a 0.5 mm distance sieve at a speed of 23,000 min-1, 20 g of the precut hemp flowers can be ground to a particle size smaller than 0.5 mm. The utilization of a cyclone has a cooling effect on the sample and helps to efficiently discharge the material from the grinding chamber. The pulverized sample is now primed for extraction of pesticides with the QuEChERS method.

Homogenization of Edibles in Ball Mills like MM 400 or CryoMill

Food samples which are sticky or fatty may block the mill when processed at room temperature. Consequently, cryogenic grinding either in the CryoMill or the MM 400 is the best option to avoid loss of volatile ingredients and caking of the sample.

Sample volumes like 1-2 cookies or a few jelly bears are best homogenized in the MM 400. This ball mill is perfectly suited for homogenizing sample volumes up to 2 x 20 ml in under 1-2 minutes. It is crucial to fill the jar with the grinding ball(s) first and then with the sample and close it tightly before embrittling.

The evaporation of LN2 would result in a significant pressure increase inside the grinding jar, so care must be taken that no LN2 is enclosed in the grinding jars. The closed grinding jars containing the sample are embrittled in a LN2 bath for 2-3 minutes. Due to the high energy input and the consequent frictional heat, the grinding process should not take longer than 2 minutes. This prevents the sample from heating up and preserves its breaking properties. If longer grinding times are needed, these should be paused by intermediate cooling of the closed grinding jars in the MM 400.

Using a 25 mm stainless steel grinding ball, 5 pieces of jelly bears were pulverized in the MM 400 in a 50 ml grinding jar. The closed jar was immersed in a liquid nitrogen bath for approximately 4 min, then clamped into the mill. After 90 seconds at 30 Hz, the sample was fully pulverized to a fineness of 0.3 mm. Suitable grinding jars for cryogenic grinding are made of PTFE or steel.

Cryogenic grinding in the CryoMill gives the advantage of continuous cooling of the grinding jar with LN2. This constant temperature is provided without the requirement for intermediate cooling breaks, even for long grinding times. A zirconium oxide grinding jar should be used for heavy-metal-free grinding. One praline, a few pieces of liquorice, or similar samples are generally pulverized in the CryoMill.

Homogenization of Cookies in the Knife Mill GM 200

The Knife Mill GM 200 accepts sample volumes up to 700 ml. It is designed for thorough homogenization of samples with high fat, oil, sugar or water content. The powerful 1000 W drive means the mill can homogenize even difficult samples very quickly and efficiently without the need for more than two grinding steps or blockages. The innovative Boost function provides a temporary speed increase to 14,000 min-1, allowing extra power for the homogenization of difficult samples in a very short time.

The mill may be operated in three different modes to optimize the homogenization process with regard to the material properties:

  • Standard mode = cutting
  • Reverse mode = impact
  • Interval mode = improved sample mixing

The function to save 4 program sequences is helpful when combining two grinding steps, for example, pre-crushing in impact mode followed by fine grinding in cutting mode, or if two different speeds are required. Up to 8 programs can be stored for routine applications.

Pulverization of 8 large cookies was achieved in two steps. To improve mixing of the sample, the interval mode was used for 10 seconds in the pre-grinding step at 4,000 min-1. The use of the standard lid ensures that the sample can move freely in the container, thus reducing the heat development and subsequent fat release.

In the homogenization step, which takes 35 seconds at 10,000 min-1, the use of the reduction lid 0.5 l is preferable to force the sample towards the blades and consequently increase the grinding efficiency. Using this method, the entire sample was homogenized to <0.5 mm.

Cutting Mill SM 400 XL

Figure 1. Cutting Mill SM 400 XL

Initial hemp sample (left) and after size reduction in the SM 400 XL (right)

Figure 2. Initial hemp sample (left) and after size reduction in the SM 400 XL (right)

Ultra Centrifugal Mill ZM 300

Figure 3. Ultra Centrifugal Mill ZM 300

fter pre-cutting in the SM 400 XL (left) the sample is pulverized to <0.5 mm in the ZM 300

Figure 4. After pre-cutting in the SM 400 XL (left) the sample is pulverized to <0.5 mm in the ZM 300

Mixer Mill MM 400 with adapter for 8 conical centrifuge tubes

Figure 5. Mixer Mill MM 400 with adapter for 8 conical centrifuge tubes

CryoMill with 50 l dewar

Figure 6. CryoMill with 50 l dewar

The hemp sample milled in the CryoMill (left) yields a higher pesticide amount after extraction than the sample milled in the ZM 300 (right).

Figure 7. The hemp sample milled in the CryoMill (left) yields a higher pesticide amount after extraction than the sample milled in the ZM 300 (right).

Jelly bears before (left) and after cryogenic grinding in the Mixer Mill MM 400

Figure 8. Jelly bears before (left) and after cryogenic grinding in the Mixer Mill MM 400

Knife Mill GRINDOMIX GM 200

Figure 9. Knife Mill GRINDOMIX GM 200

Cookies before (left) and after pulverization in the Knife Mill GM 200 (right)

Figure 10. Cookies before (left) and after pulverization in the Knife Mill GM 200 (right)

References

1. http://www.cannabis-med.org/german/studies.htm

This information has been sourced, reviewed and adapted from materials provided by RETSCH GmbH.

For more information on this source, please visit RETSCH GmbH.

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