Tag Archives: sample prep

A More Effective and Efficient Approach to Purer Cannabidiol Production Using Centrifugal Partition Chromatography

By Lauren Pahnke
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Many physicians today treat their patients with cannabidiol (CBD, Figure 1), a cannabinoid found in cannabis. CBD is more efficacious over traditional medications, and unlike delta-9 tetrahydrocannbinol (THC), the main psychoactive compound in cannabis, CBD has no psychoactive effects. Researchers have found CBD to be an effective treatment for conditions such as cancer pain, spasticity in multiple sclerosis, and Dravet Syndrome, a form of epilepsy.

CBD is still considered an unsafe drug under federal law, but to meet the medical demand, 17 states in the US recently passed laws allowing individuals to consume CBD for medical purposes. A recent survey found that half of medicinal CBD users rely on the substance by itself for treatment. As doctors start using CBD to treat more patients, the demand for CBD is only expected to rise, and meeting that demand can pose challenges for manufacturers who are not used to producing such high quantities of CBD. Furthermore, as CBD-based drugs become more popular, the US Food and Drug Administration (FDA) will likely require manufacturers to demonstrate they can produce pure, high-quality products.

cannabidiol
Figure 1. The structure of cannabidiol, one of 400 active compounds found in cannabis.

Most manufacturers use chromatography techniques such as high performance liquid chromatography (HPLC) or flash chromatography to isolate compounds from natural product extracts. While these methods are effective for other applications, they are not, however, ideal for CBD isolate production. Crude cannabis oil contains some 400 potentially active compounds and requires pre-treatment prior to traditional chromatography purification. Both HPLC and flash chromatography also require silica resin, an expensive consumable that must be replaced once it is contaminated due to irreversible absorption of compounds from the cannabis extract. All of these factors limit the production capacity for CBD manufacturers.

Additionally, these chromatography methods use large quantities of solvents to elute natural compounds, which negatively impacts the environment.

A Superior Chromatography Method

Centrifugal partition chromatography (CPC) is an alternative chromatography method that can help commercial CBD manufacturers produce greater quantities of pure CBD more quickly and cleanly, using fewer materials and generating less toxic waste. CPC is a highly scalable CBD production process that is environmentally and economically sustainable.

The mechanics of a CPC run are analogous to the mechanics of a standard elution using a traditional chromatography column. While HPLC, for instance, involves eluting cannabis oil through a resin-packed chromatography column, CPC instead elutes the oil through a series of cells embedded into a stack of rotating disks. These cells contain a liquid stationary phase composed of a commonly used fluid such as water, methanol, or heptane, which is held in place by a centrifugal force. A liquid mobile phase migrates from cell to cell as the stacked disks spin. Compounds with greater affinity to the mobile phase are not retained by the stationary phase and pass through the column faster, whereas compounds with a greater affinity to the stationary phase are retained and pass through the column slower, thereby distributing themselves in separate cells (Figure 2).

Figure 2- CPC
Figure 2. How CPC isolates compounds from complex, natural mixtures. As the column spins, the mobile phase (yellow) moves through each cell in series. The compounds in the mobile phase (A, B, and C) diffuse into the stationary phase (blue) at different rates according to their relative affinities for the two phases.

A chemist can choose a biphasic solvent system that will optimize the separation of a target compound such as CBD to extract relatively pure CBD from a cannabis extract in one step. In one small-scale study, researchers injected five grams of crude cannabis oil low in CBD content into a CPC system and obtained 205 milligrams of over 95% pure CBD in 10 minutes.

Using a liquid stationary phase instead of silica imbues CPC with several time and cost benefits. Because natural products such as raw cannabis extract adhere to silica, traditional chromatography columns must be replaced every few weeks. On the other hand, a chemist can simply rinse out the columns in CPC and reuse them. Also, unlike silica columns, liquid solvents such as heptane used in CPC methods can be distilled with a rotary evaporator and recycled, reducing costs.

Environmental Advantages of CPC

The solvents used in chromatography, such as methanol and acetonitrile, are toxic to both humans and the environment. Many environmentally-conscious companies have attempted to replace these toxic solvents with greener alternatives, but these may come with drawbacks. The standard, toxic solvents are so common because they are integral for optimizing purity. Replacing a solvent with an alternative could, therefore, diminish purity and yield. Consequently, a chemist may need to perform additional steps to achieve the same quality and quantity achievable with a toxic solvent. This produces more waste, offsetting the original intent of using the green solvent.

CPC uses the same solvents as traditional chromatography, but it uses them in smaller quantities. Furthermore, as previously mentioned, these solvents can be reused. Hence, the method is effective, more environmentally-friendly, andeconomically feasible.

CPC’s Value in CBD Production

As manufacturers seek to produce larger quantities of pure CBD to meet the demand of patients and physicians, they will need to integrate CPC into their purification workflows. Since CPC produces a relativelyduct on a larger scale, it is equipped to handle the high-volume needs of a large manufacturer. Additionally, because it extracts more CBD from a given volume of raw cannabis extract, and does not use costly silica or require multiple replacement columns, CPC also makes the process of industrial-scale CBD production economically sustainable. Since it also uses significantly less solvent than traditional chromatography, CPC makes it financially feasible to make the process of producing CBD more environmentally-friendly.

Suggested Reading:

CPC 250: Purification of Cannabidiol from Cannabis sativa

Introduction to Centrifugal Partition Chromatography

dSPE cleanups

The Grass Isn’t Always Greener: Removal of Purple Pigmentation from Cannabis

By Danielle Mackowsky
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dSPE cleanups
strains
Cannabis strains used (clockwise from top left): Agent Orange, Tahoe OG, Blue Skunk, Grand Daddy and Grape Drink

Cannabis-testing laboratories have the challenge of removing a variety of unwanted matrix components from plant material prior to running extracts on their LC-MS/MS or GC-MS. The complexity of the cannabis plant presents additional analytical challenges that do not need to be accounted for in other agricultural products. Up to a third of the overall mass of cannabis seed, half of usable flower and nearly all extracts can be contributed to essential oils such as terpenes, flavonoids and actual cannabinoid content1. The biodiversity of this plant is exhibited in the over 2,000 unique strains that have been identified, each with their own pigmentation, cannabinoid profile and overall suggested medicinal use2. While novel methods have been developed for the removal of chlorophyll, few, if any, sample preparation methods have been devoted to removal of other colored pigments from cannabis.

QuEChERS
Cannabis samples following QuEChERS extraction

Sample Preparation

Cannabis samples from four strains of plant (Purple Drink, Tahoe OG, Grand Daddy and Agent Orange) were hydrated using deionized water. Following the addition of 10 mL acetonitrile, samples were homogenized using a SPEX Geno/Grinder and stainless steel grinding balls. QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) non-buffered extraction salts were then added and samples were shaken. Following centrifugation, an aliquot of the supernatant was transferred to various blends of dispersive SPE (dSPE) salts packed into centrifugation tubes. All dSPE tubes were vortexed prior to being centrifuged. Resulting supernatant was transferred to clear auto sampler vials for visual analysis. Recoveries of 48 pesticides and four mycotoxins were determined for the two dSPE blends that provided the most pigmentation removal.

Seven dSPE blends were evaluated for their ability to remove both chlorophyll and purple pigmentation from cannabis plant material:

  • 150 mg MgSO4, 50 mg PSA, 50 mg C18, 50 mg Chlorofiltr®
  • 150 mg MgSO4, 50 mg C18, 50 mg Chlorofiltr®
  • 150 mg MgSO4, 50 mg PSA
  • 150 mg MgSO4, 25 mg C18
  • 150 mg MgSO4, 50 mg PSA, 50 mg C18
  • 150 mg MgSO4, 25 mg PSA, 7.5 mg GCB
  • 150 mg MgSO4, 50 mg PSA, 50 mg C18, 50 mg GCB

Based on the coloration of the resulting extracts, blends A, F and G were determined to be the most effective in removing both chlorophyll (all cannabis strains) and purple pigments (Purple Drink and Grand Daddy). Previous research regarding the ability of large quantities of GCB to retain planar pesticides allowed for the exclusion of blend G from further analyte quantitation3. The recoveries of the 48 selected pesticides and four mycotoxins for blends A and F were determined.

dSPE cleanups
Grand Daddy following various dSPE cleanups

Summary

A blend of MgSO4, C18, PSA and Chlorofiltr® allowed for the most sample clean up, without loss of pesticides and mycotoxins, for all cannabis samples tested. Average recovery of the 47 pesticides and five mycotoxins using the selected dSPE blend was 75.6% were as the average recovery when including GCB instead of Chlorofiltr® was 67.6%. Regardless of the sample’s original pigmentation, this blend successfully removed both chlorophyll and purple hues from all strains tested. The other six dSPE blends evaluated were unable to provide the sample clean up needed or had previously demonstrated to be detrimental to the recovery of pesticides routinely analyzed in cannabis.


References

(1)           Recommended methods for the identification and analysis of cannabis and cannabis products, United Nations Office of Drugs and Crime (2009)

(2)            W. Ross, Newsweek, (2016)

(3)            Koesukwiwat, Urairat, et al. “High Throughput Analysis of 150 Pesticides in Fruits and Vegetables Using QuEChERS and Low-Pressure Gas Chromatography Time-of-Flight Mass Spectrometry.” Journal of Chromatography A, vol. 1217, no. 43, 2010, pp. 6692–6703., doi:10.1016/j.chroma.2010.05.012.

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Quality From Canada

Near Infrared, GC and HPLC Applications in Cannabis Testing

By Tegan Adams, Michael Bertone
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When a cannabis sample is submitted to a lab for testing there is a four-step process that occurs before it is tested in the instrumentation on site:

  1. It is ground at a low temperature into a fine powder;
  2. A solution is added to the ground powder;
  3. An extraction is repeated 6 times to ensure all cannabinoids are transferred into a common solution to be used in testing instrumentation.
  4. Once the cannabinoid solution is extracted from the plant matter, it is analyzed using High Pressure Liquid Chromatograph (HPLC). HPLC is the key piece of instrumentation in cannabis potency testing procedures.

While there are many ways to test cannabis potency, HPLC is the most widely accepted and recognized testing instrumentation. Other instrument techniques include gas chromatography (GC) and thin layer chromatography (TLC). HPLC is preferred over GC because it does not apply heat in the testing process and cannabinoids can then be measured in their naturally occurring forms. Using a GC, heat is applied as part of the testing process and cannabinoids such as THCA or CBDA can change form, depending on the level of heat applied. CBDA and THCA have been observed to change form at as low as 40-50C. GC uses anywhere between 150-200C for its processes, and if using a GC, a change of compound form can occur. Using HPLC free of any high-heat environments, acidic (CBDA & THCA) and neutral cannabinoids (CBD, THC, CBG, CBN and others) can be differentiated in a sample for quantification purposes.

Near Infrared

Near infrared (NIR) has been used with cannabis for rapid identification of active pharmaceutical ingredients by measuring how much light different substances reflect. Cannabis is typically composed of 5-30% cannabinoids (mainly THC and CBD) and 5-15% water. Cannabinoid content can vary by over 5% (e.g. 13-18%) on a single plant, and even more if grown indoors. Multiple NIR measurements can be cost effective for R&D purposes. NIR does not use solvents and has a speed advantage of at least 50 times over traditional methods.

The main downfall of NIR techniques is that they are generally less accurate than HPLC or GC for potency analyses. NIR can be programmed to detect different compounds. To obtain accuracy in its detection methods, samples must be tested by HPLC on ongoing basis. 100 samples or more will provide enough information to improve an NIR software’s accuracy if it is programmed by the manufacturer or user using chemometrics. Chemometrics sorts through the often complex and broad overlapping NIR absorption.

Bands from the chemical, physical, and structural properties of all species present in a sample that influences the measured spectra. Any variation however of a strain tested or water quantity observed can affect the received results. Consistency is the key to obtaining precision with NIR equipment programming. The downfall of the NIR technique is that it must constantly be compared to HPLC data to ensure accuracy.

At Eurofins Experchem , our company works with bothHPLC and NIR equipment simultaneously for different cannabis testing purposes. Running both equipment simultaneously means we are able to continually monitor the accuracy of our NIR equipment as compared to our HPLC. If a company is using NIR alone however, it can be more difficult to maintain the equipment’s accuracy without on-going monitoring.

What about Terpenes?

Terpenes are the primary aromatic constituents of cannabis resin and essential oils. Terpene compounds vary in type and concentration among different genetic lineages of cannabis and have been shown to modulate and modify the therapeutic and psychoactive effects of cannabinoids. Terpenes can be analyzed using different methods including separation by GC or HPLC and identification by Mass Spectrometry. The high-heat environment for GC analysis can again cause problems in accuracy and interpretation of results for terpenes; high-heat environments can degrade terpenes and make them difficult to find in accurate form. We find HPLC is the best instrument to test for terpenes and can now test for six of the key terpene profiles including a-Pinene, Caryophyllene, Limonene, Myrcene, B-Pinene and Terpineol.

Quality Systems

Quality systems between different labs are never one and the same. Some labs are testing cannabis under good manufacturing practices (GMP), others follow ISO accreditation and some labs have no accreditation at all.

From a quality systems’ perspective some labs have zero or only one quality system employee(s). In a GMP lab, to meet the requirements of Health Canada and the FDA, our operations are staffed in a 1:4 quality assurance to analyst ratio. GMP labs have stringent quality standards that set them apart from other labs testing cannabis. Quality standards we work with include, but are not limited to: monthly internal blind audits, extensive GMP training, yearly exams and ongoing tests demonstrating competencies.

Maintaining and adhering to strict quality standards necessary for a Drug Establishment License for pharmaceutical testing ensures accuracy of results in cannabis testing otherwise difficult to find in the testing marketplace.

Important things to know about testing

  1. HPLC is the most recommended instrument used for product release in a regulated environment.
  2. NIR is the best instrument to use for monitoring growth and curing processes for R&D purposes, only if validated with an HPLC on an ongoing basis.
  3. Quality Systems between labs are different. Regardless of instrumentation used, if quality systems are not in place and maintained, integrity of results may be compromised.
  4. GMPs comprise 25% of our labour costs to our quality department. Quality systems necessary for a GMP environment include internal audits, out of specification investigations, qualification and maintenance of instruments, systems controls and stringent data integrity standards.
OGanalytical instruments.

New Cannabis Lab Rules In Oregon Aim to Curb Fraud

By Aaron G. Biros
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The Oregon Health Authority (OHA) recently implemented a set of temporary rules effective through June 28th of this year with the goal to establish a set of regulations for cannabis testing by October 1st. An investigation by The Oregonian highlighted some of the previous problems with cannabis testing in the state.

The most impactful rule changes include The NELAC Institute (TNI) mandatory standards for laboratories that the Oregon Environmental Laboratory Accreditation Program (ORELAP) will use to accredit labs. Initial rules in the Oregon medical cannabis program, HB 3460 from 2013, did not specify accreditation rules for cannabis testing.

oganalyticalworkstation
The OG Analytical laboratory in Eugene, Oregon is working to comply with new regulations, including new sample collection rules

ORELAP currently performs accreditation for lab testing under the Clean Air Act, Clean Water Act, Resource Conservation and Recovery Act and the Safe Drinking Water Act. The new cannabis testing rules will give ORELAP the authority to accredit and regulate cannabis labs in the state of Oregon.

Rodger Voelker, Ph.D., laboratory director of OG Analytical in Eugene, OR, believes these rules are monumental in establishing legitimacy in cannabis testing. “These new rules have major repercussions mainly because they require not only getting accreditation, but maintaining it with very strict requirements,” says Voelker. “That also includes procedural guidelines that very carefully outline the quality of laboratory practices and establishes a set of criteria for method validation.”

Roger Voelker
Rodger Voelker, lab director at OG Analytical laboratory

Voelker notes that two of the biggest changes are in quality control and data management. “The documentation they require is very thorough and strict with the idea that any aspect of an analysis can be replicated,” adds Voelker. “This is a real win for us in my opinion because now we have an agency that can issue the appropriate credentials as well as have the authority to make punitive measures.”

The timeline for implementation with temporary rules allows state regulators to work with laboratories to perform accreditation and bring laboratories up to speed. According to Shannon Swantek, ORELAP compliance specialist, products that dispensaries sell in medical and recreational markets are required to be tested under the new rules and in the analyte lists by an ORELAP accredited laboratory, starting on October 1st.

Swantek’s job is to accredit cannabis labs to the TNI standards, which is essentially very similar to ISO 17025, just with more prescriptive measures and the ability to pair with state agencies to enforce rules after accreditation. “The timeline for accreditation is dependent on how ready the lab is and how compliant they are to the TNI standard already,” says Swantek. “The culture had gotten so fraudulent that the legislature felt Oregon needed some serious, more strict rules in place.”

OGanalytical instruments.
Labs need very expensive instruments to perform all of the testing required by OHA

One of the biggest changes coming to Oregon cannabis testing is the new sampling requirement. “An accredited laboratory employee must take the sample because sampling is where a lack of training or outright fraud is skewing results, which occurs when a grower brings in a sample not representative of the batch,” adds Swantek. Sample preparation methods will also be required to be more robust to meet the action limits of pesticide testing in particular, helping to identify lower levels like parts-per-billion, according to Swantek.

Reports were also lacking key information in the past. The new rules will require more information such as the procedure used, the analyst carrying it out, dilution factors and any other information you need to theoretically reproduce the result. This will result in more accurate labels on products.

Many are concerned that the new lab testing requirements will raise the price of testing too much. In reality, those current prices are not realistic for accurate data, which points to the rampant fraud that ORELAP is trying to eradicate. “The old rules were written in such an ambiguous way that the prices were set by laboratories without a proper quality program or even without proper instrumentation,” says Swantek.

oganalyticalsign
OG Analytical had to close its doors briefly to meet accreditation

The accreditation process will require particularly robust quality control systems in labs. “Accreditation to the TNI Standard means that lab quality systems will require a documentation system, training procedures, record keeping, personnel requirements, organization details, proof of no conflicts of interest and corrective actions if noncompliant,” adds Swantek. “We single out each method or procedure, look at their raw data and proficiency testing and determine if they are meeting the technical requirements.”

According to Voelker, other industries have learned to adjust their costs with stringent lab testing rules. “I get that no one wants to pay more for lab testing, but the reality is that joining the world of commodities comes with additional costs to ensure consumer safety,” says Voelker. These rule changes will undoubtedly bring more consistency to Oregon’s cannabis industry with accurate lab testing and help the OHA shed more light on issues surrounding consumer safety.