Separation of cannabinoids for clinical use

PrepChrom C-700: Isolation of cannabidiol (CBD) and tetrahydrocannabinol (THC) from other cannabinoids extracted from cannabis

PrepChrom WordPressThe separation of cannabinoids is an important process which is necessary for clinical studies. The aim of these studies is to investigate the effect of the isolated cannabinoids on patients suffering from chronic illnesses and side effects caused by chemotherapy. The relaxation of laws concerning medical use of cannabis all around the world, especially the United States, has increased the interest in determining the effect of cannabinoids on the human body. Clinical studies require large amount of highly pure separated cannabnnoids. This can easily be achieved with the PrepChrom C-700.

Introduction

Cannabis is an extremely versatile plant and has been used for many purposes throughout history. Some specific sorts are used for fibers in the textile and paper industry because of their long stems. It is a useful source of foodstuffs, such as hemp oil and seed. Due to the cannabinoids, cannabis is also a popular recreational drug. These substances can cause mental and physical effects when consumed. The impact of the cannabinoids on the human body is of great interest for the pharmaceutical sector. The research emphasis lies in treating the side effects of chemotherapy, inflammatory diseases like multiple sclerosis or degenerative illnesses such as Parkinson’s disease.[3] AI FAME GmbH, a Swiss company, is a pioneering company to extract the plant-based active substances and make them water-soluble for improved, further processing.

Here, we aim to separate three cannabinoids, i.e. cannabidiol, tetrahydrocannabinol and tetrahydrocannabinolic acid in high purity from the extract.

Experimental

Equipment: PrepChrom C-700

Sample: Water-soluble cannabis extract. Cultivated, extracted and provided by AI FAME GmbH

Preparation: The chromatographic separation is performed with 1 mL of the water-soluble cannabis extract diluted in 1 mL methanol : water (1:1)

Separation: Method parameters
Column:  Sepacore® C-18 80 g
Particle size:  40-63 μm
Flow rate:  40 mL/min
Sample loop:  2 mL
Detection:  253, 270 nm and SCAN 200-600 nm

Gradient: methanol (A) : water(B)
50 % – 90 % methanol (A) in 20 min
90 % – 90 % methanol (A) in 10 min
90 % – 95 % methanol (A) in 5 min
95 % – 95 % methanol (A) in 10 min

Results

Cannabidiol (CBD) bearing a 1,3-diol functional group on its benzene ring, is the first substance to be eluted when applying reversed phase conditions (Figure 1), hence, is the most polar cannabinoid in the sample. The second compound, eluting after 27 minutes, is tetrahydrocannabinol (THC). The last substance to be eluted is tetrahydrocannabinolic acid (THCA), a form of cannabinoid which easily decarboxilates in to THC when exposed to light or heat. Assignment of the signals and identification was done by comparison with reference HPLC data provided by AI FAME GmbH.

Cannabinoide SN finalFigure 1: Methanol : water separation of the extracted cannabinoids using the PrepChrom C-700. Detection at 270 nm, 254 nm, and 200-600 nm SCAN.

 

 

Conclusion

The three cannabinoids extracted by AI FAME GmbH could easily be separated and separately collected with the PrepChrom C-700.

The water-solubility of the extract enables the use of aqueous solvents that have inherent economic and ecologic advantages over common organic solvents.

The growing interest in the clinical use of cannabinoids is unbreakable. Furthermore, cannabis is decriminalized and it is allowed to exploit the potential of its valuable substances. BUCHI offers straight forward solutions to separate and purify small to large size batches of the precious extracts.

Acknowledgment

AI FAME GmbH, Switzerland, is thanked for providing the extract and for the fruitful analytical discussion.

References

[1] Sutton, IR. Daeniken, P. (2006). Cannabinoids in the management of intractable chemotherapy-induced nausea and vomiting and cancer-related pain. The Journal of Supportive Oncology.

[2] State Medical Marijuana Laws. National Conference of State Legislatures. (16.3.2015). http://www.ncsl.org/research/health/state-medical-marijuana-laws.aspx

[3] Kogan, NM. Mechoulam, R. (2007) Cannabinoids in health and disease. Dialogues Clin Neurosci. 2007 Dec; 9(4): 413–430.

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Short Note #66: Sulfur Dioxide Determination in Beer

SO2 Determination in Beer by Distillation

SO2 Determination in Beer by Distillation

Short Note #66 introduces a novel procedure for the determination of Total sulfur dioxide (SO2) contents in beer using distillation. The most important advance stems from a calibration by means of a stabilized SO2 standard containing acetaldehyde simulating a beer matrix. A calibration equation is derived from a linear correlation of a series of SO2 determinations with SO2 standards and the calibration equation is applied in order to adjust measured SO2 results in samples. Total SO2 contents in a series of 5 beers were evaluated and results compared to the DTNB SO2 Method.

Request this Kjeldahl Distillation Short Note to see the full details and results of the study.

Introduction:
The DTNB SO2 Method is based on a spectrophotometric determination of the reaction product of SO2 with DTNB (5,5-dithiobis(2-nitrobenzoic acid). SO2 is entrained by a nitrogen stream into a buffered DTNB solution and the absorbance is measured at 415 nm. Steam distillation, as a possible alternative, does not fully recover SO2 from the sample without further optimization and adaption. The most important improvements stem from acidification of the sample with an acid mixture of methanol, water and orthophosphoric acid and a calibration by means of a stabilized
SO2 standard solution. The calibration reveals an excellent linear relationship between the determined SO2 amounts and the corresponding known amounts of standard solution. The linear equation is applied in the calculations to correct for Total SO2.

Beer Samples:
− Calanda Lager
− Heineken 1
− Heineken 2
− Prix Garantie Lager
− Heineken 3

Equipment:
− Distillation Unit K-355 with SO2 absorption glass (order number 048680)
− Metrohm DMP 785 Titrino
− Metrohm Pt-Titrode 6.0431.100
− Volumetric pipette 5 ml
− Glass beaker 500 ml

Short Note #65: SO2 Determination in Wine

Distillation Unit K-355

Distillation Unit K-355

Short Note #65 on the distillation of wine describes a novel procedure for the determination of Total SO2 contents in wine. The most important advance stems from a calibration by means of a stabilized SO2 standard containing acetaldehyde simulating a wine matrix. A calibration equation is derived from a linear correlation of a series of SO2 determinations with SO2 standards and the calibration equation is applied in order to adjust measured SO2 results in samples. Total SO2 contents in a series of 11 wines were evaluated and results compared to the OIV SO2 Method.

Introduction:
The OIV SO2 Method is based on the entrainment of SO2 from the wine sample into a titration vessel by means of a nitrogen stream.

Simple steam distillation, as a possible alternative, does not produce results comparable to the OIV SO2 Method without further optimization and adaption.

The most important improvements stem from acidification of the sample with an acid mixture of methanol, water and ortho-phosphoric acid and a calibration by means of a stabilized SO2 standard solution containing acetaldehyde simulating a wine matrix. The calibration reveals an excellent linear relationship between the determined SO2 amounts and the corresponding known amounts of standard solution. The linear equation is applied in the calculations to correct for Total SO2.

Experimental:
The distillation unit should preferably be equipped with an acid resistant pump as available in the BUCHI K-355 (picture above) and the K-360.

The sample is acidified with the acid mixture and steam distilled into the specially designed BUCHI SO2 absorption vessel in which the SO2 reacts with a defined volume of iodine standard solution. Subsequently the distillate is back-titrated with Na-thiosulfate standard solution using a titrator suitable to carry out redox titrations.

>> Download the entire Short Note #65

Short Note #47: Protein Determination in Eggs According to Kjeldahl

Protein Determination in Eggs

Protein Determination in Eggs Using Kjeldahl

A simple and fast procedure for protein determination in eggs, as described in the AOAC 925.31 and LFGB § 64 L05.00-15, is introduced within this Short Note #47.

The sample is digested with sulfuric acid using the SpeedDigester K-436 or K-439, followed by distillation and titration with the Kjeldahl Sampler System K-370/K-371. The determined protein contents correspond to the values from literature (see Short Note #47 for details).

Introduction:
Protein determination is one of the key analyses performed in the food industry. The samples require digestion with sulfuric acid to convert nitrogen into ammonium sulfate. After conversion to ammonia through the alkalization with sodium hydroxide, the ammonia is distilled into a boric acid solution by steam distillation, followed by a titration with sulfuric acid solution. The nitrogen content is multiplied by a sample specific factor (6.25 for egg) to obtain the protein content.

Experimental:
Instrumentation: SpeedDigester K-436, K-439, Kjeldahl Sampler System K-370/K-371

Samples: Whole chicken egg, protein content 12.5 g/100g

Determination: Approx. 1.2 g of the homogenized sample were weighed in directly into a sample tube. A portion of 20 ml of sulfuric acid and 2 Kjeldahl tablets were added, and the digestion was performed using the “egg” method (K-439) or the parameters specified. After digestion the ammonia of the sample was distilled into a boric acid solution by steam distillation and titrated with sulfuric acid (See Short Note #47 for details).

The method was verified by using 0.13 g glycine as the reference substance.

Short Note #45: Nitrogen and Protein Determination in Pharmaceuticals

A simple and fast procedure for nitrogen determination in pharmaceuticals according to the Kjeldahl method (semi-micro), as described in the European Pharmakopeia 6.0 / 2.05.08, is introduced in this study. Protein determination is calculated The sample is digested with sulfuric acid using the SpeedDigester K-436 or K-439, followed by distillation and titration with the KjelFlex K-360. There are no differences between the results obtained with the K-436 and the K-439 respectively.

Introduction: Nitrogen determination is one of the key analyses performed in quality control. The samples require digestion with sulfuric acid to convert nitrogen into ammonium sulfate. After conversion to ammonia through the alkalinization with sodium hydroxide, the sample is distilled into a boric acid receiver by steam distillation, followed by a titration with hydrochloric acid solution. The nitrogen content is multiplied by a sample-specific factor to obtain the protein content.

Sleeping Pill Samples

Sleeping Pill Samples


Experimental Instrumentation: SpeedDigester K-436, K-439, KjelFlex K-360

Samples: Sleeping pills and tranquilization drops

Conclusion: The determination of nitrogen contents in pharmaceuticals according to Kjeldahl using SpeedDigester K-436, K-439, and KjelFlex K-360 provides reliable and reproducible results with low relative standard deviations.

Short Note #60: Cleaning Up Of alpha-Methylstyrene by Flash Chromatography

This short note details the isolation of alpha-Methylstyrene from a crude reaction mixture. This may sound easy, but the reaction mixture contains some dimethyl sulfoxide and the oily wetting agent from the sodium hydride. Both of them are not UV-active and therefore in the TLC invisible. Download or view this Flash Chromatography short note and visit our Chromatography Applications page for other synthetic reaction mixture notes.

Sepacore configuration:
40 X 150mm Cartridge prepacked with silica gel 60
2 C-605 Pump Modules
C-660 Fraction Collector
C-620 Control Unit with SepacoreControl Software
C-635 UV Photometer

Separation Conditions:
Eluent: n-hexane with 0%, 2% und 15% ethyl acetate, step gradient
Flow rate: 100ml/min
Sample: 3 g crude mixture, dissolved in toluene
Injection volume: 4.5 ml (although the mixture is liquid, the sample is not fully miscible with n-hexane and must be dissolved in toluene and
injected directly onto the cartridge)

Sepacore Flash Chromatography System

Sepacore Flash Chromatography System

Short Note #24: Protein Determination in Beer According to Kjeldahl Method

Protein determination in beer according to kjeldahl

Protein Determination in Beer

This short note describes a simple and fast procedue for protein determination in beer according to the Kjeldahl method, as detailed in in the AOAC 920.53 regulations. Visit our Kjeldahl Applications section to download this short note and review all our other available application studies.

Introduction:
Protein determination is one of the key analyses performed in the food industry. The samples require digestion with sulfuric acid to convert nitrogen into ammonium sulfate.

After conversion to ammonia through the alkalinization with sodium hydroxide, the sample is distilled into a boric acid receiver by steam distillation, followed by a titration with sulfuric acid solution. The nitrogen content is multiplied by a sample-specific factor (6.25 for beer) to obtain the protein content.

Instrumentation:
Buchi SpeedDigester K-436, K-439, Buchi Kjeldahl Sampler System K-370, K-371

Samples:
Draft beer and Wheat beer. The protein content from draft beer is 0.50%.

Conclusion:
The determination of protein contents in chocolate according to Kjeldahl using SpeedDigester K-436, K-439, and Kjeldahl Sampler System K-370/K-371 provides reliable and reproducible results that correspond to the labeled values and literature [See Note for table data references] with low relative standard deviations. The total digestion time is approximately 90 min.