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cannabis chromatography

Determination of cannabinoids in Cannabis sativa L. samples for recreational, medical, and forensic purposes by reversed-phase liquid chromatography-ultraviolet detection

Abstract

Background

Currently, an increasing demand of cannabis-derived products for recreational and medical use is observed. Therefore, the reliable and fast quantification of cannabinoids in hemp samples is essential for the control of product from Cannabis sativa, L. strains. In general, gas chromatography (GC) is the method of choice for the quantification of cannabinoids whereas this method is time consuming and the detection of acidic precursor is not feasible without derivatization.

Methods

We report the successful development and validation of an accurate and broadly applicable reversed-phase high-performance liquid chromatography (RP-HPLC) method coupled to an ultra violet (UV) detector including an optimized extraction procedure for the separation and quantification of eight different cannabinoids.

Results

The optimized method is able to separate cannabidivarin, cannabidiolic acid, cannabigerolic acid, cannabigerol, cannabidiol, cannabinol, Δ9-tetrahydrocannabinol, and tetrahydrocannabinolic acid within 10 min. For all target analytes, the %-Bias at the lower and upper calibration range varied from − 1.3 to 10.3% and from − 3.9 to 8.6%, respectively. The most suitable agent for extracting cannabis plant samples was evaluated to be a mixture of acetonitrile and water in a ratio 1:1. The extraction efficiency was more than 95% for all analytes in recreational hemp samples. Stability studies on acidic cannabinoids showed a high likeliness of decarboxylation at 100 °C and aromatization after exposure to UV light, respectively. A modified loss on drying method revealed a moisture content between 4 and 10%. The developed method was successfully applied to measure the cannabinoid content in recreational and forensic hemp samples representing broad range of cannabinoid amounts and patterns.

Conclusion

The present work proposes validated methods for the determination of cannabinoids in cannabis samples. The use of RP-HPLC-UV renders this method broadly applicable and allows the detection of acidic precursor in even less time compared to GC-based methods.

Introduction

Since centuries, Cannabis sativa L. (C. sativa) is used for industrial purposes but it is better known as illegal drug possessing psychotropic properties. However, C. sativa is also a highly decorated medicinal plant for the use as anticancer agent, for neuroprotection and as bone marrow stimulants (Velasco et al. 2016; Machado Rocha et al. 2008). With the legalization of cannabis for therapeutic use, the demand for pure and characterized samples has grown significantly (Corroon and Phillips 2018). Therefore, currently new pharmacopeial monographs are in development for quality control of C. sativa-based medicinal products (Pavlovic et al. 2018). Besides the medical use, there is an enormous interest from consumers/patients in the utilization of low Δ9-tetrahydrocannabinol (THC) hemp for recreational use. In recent years, a kind of gold-rush fever is observed in Europe and all over the world and many new suppliers entered the market (Pellechia 2018). Since there is a complicated and different legislation for C. sativa products all over Europe, caution for quality control has to be taken. Although there is no upper limit for the cannabidiol (CBD) or cannabidiolic acid (CBDA) content in most European countries, maximum limits of THCor Δ9-tetrahydrocannabinolic acid (THCA) contents vary between 0.1 and 1% within Europe.

Cannabinoids belong to terpenophenolic compounds and are the main constituents of the cannabis plant. Terpenoids and phenols were also identified in the cannabis plant but are of lower pharmacological importance (Pavlovic et al. 2018). Cannabigerolic acid (CBGA) is the starting point in the biosynthetic pathway of all cannabinoids, which are synthesized in vivo in a carboxylated form (Fig. 1). In the plant, CBDA and THCA are synthesized by enzymatic catalyzed reactions. However, ex vivo stress conditions like heat and UV light decompose these precursors to their decarboxylated form: CBGA ➔ cannabigerol (CBG), CBDA➔ CBD and THCA ➔ THC, respectively (Citti et al. 2018a; Sirikantaramas and Taura 2017). Under UV light, Δ9-THC is further aromatized to cannabinol (CBN). THC and CBD are two main biomarkers in commercial available hemp samples. THC is mostly responsible for psychotropic activities whereas CBD is more anxiolytic and sleep inducing. In comparison to THC, CBD is not considered a controlled substance. Numerous reports have been published for the qualitative and quantitative analysis of cannabinoids in cannabis and its preparations. This study will therefore focus on those substances for possible therapeutic use such as CBD, Δ9-THC, CBG, CBN, cannabidivarin (CBDV), cannabichromene, and tetrahydrocannabivarin (Amada et al. 2013; Thomas et al. 2007). Several comprehensive reviews of the chemical analysis of cannabis plants, corresponding preparations, and forensic specimens were presented in the past (Citti et al. 2018b; Wang et al. 2017; Patel et al. 2017; ElSohly and Salem 2000). The most widespread techniques applied for separation were gas chromatography (GC) with and without derivatization, high-performance liquid chromatography (HPLC), and to a lesser extend supercritical fluid chromatography (Wang et al. 2016; U.N.O.o. Drugs, Crime 2013). The GC method is still officially employed by the authorities for the determination of cannabinoids. But it is obvious that acidic forms are not accessible without prior derivatization, and further conversion of THCA to THC is not quantitative at all (Dussy et al. 2005). Some researchers postulate that an accurate cannabinoid profile should be evaluated by determining the acid and neutral forms separately (Pavlovic et al. 2018; Citti et al. 2018b; Calvi et al. 2018; Ambach et al. 2014). On the other hand, LC-based procedures render the derivatization step superfluous and enable the detection of the heat-labile acid precursor in less time. However, determining chromatographic conditions is more challenging. Additionally, the pre-analytical phase has to be taken into account for method development and validation. Extraction, storage conditions, and stability determination play a pivotal role in the analysis of C. sativa L.-derived products (Dussy et al. 2005; Brighenti et al. 2017; Mudge et al. 2017).

Biosynthetic pathway of selected cannabinoids

The main scope of this study was the development and validation of a fast and convenient UV-detector-based RP-HPLC method for the fast quantification of cannabinoids in CBD samples and forensic cannabis samples. The present study examines further pre-analytical conditions and the analytical stability of cannabinoids under different stress conditions. Eight authentic CBD-hemp materials and 12 forensic cannabis samples offering a wide range of cannabinoid patterns were analyzed. Results of the overall THC-content of forensic samples were compared with gas chromatographic method (U.N.O.o. Drugs, Crime 2013), the formerly gold standard in cannabinoid analysis. Additionally, a modified loss on drying method was applied to determine the moisture content of all cannabis samples. Finally, the developed method was transferred easily to an ultra-high performance liquid chromatography (UHPLC) device using know metrics, thus further reducing analysis time from 10 to less than 5 min.

Materials and methods

Analytical standards were obtained from Lipomed (Reinach, Switzerlanf). Formic acid (FA), methanol (MeOH), ethanol (EtOH), and acetonitrile (ACN) were obtained from Merck (Darmstadt, Germany) and were of LCMS grade. Pure-water was generated from an in-house water purification system from Labtec (Villmergen, Switzerland). For all experiments, Gilson DIAMOND tips were used. Hop strobiles (Humulus lupulus L.) were obtained from local pharmacies. CBD-hemp tobacco samples were purchased from several licensed producers within Switzerland. The Forensic Institute Zurich (Zurich, Switzerland) provided 12 forensic cannabis samples.

Chromatographic analysis

HPLC conditions

Reversed-phase chromatography was done using a LaChrom Elite System (Hitachi, Ltd., Tokio, Japan) HPLC system consisting of a LaChrom Elite L-2200 autosampler, a LaChrom Elite L-2130 pump, a LaChrom Elite L-2350 column oven, and a LaChrom Elite L-2420 UV-VIS detector. For peak integration, Agilent EZChrom Elite was used. The final liquid chromatography analysis was performed on a Phenomenex Kinetex XB-C18 column (150 × 4.6 mm, 2.6 μm) applying gradient elution, using pure-water (with 0.1% FA) and acetonitrile (with 0.1% FA) as the organic phase. The injection volume was 15 μL, and the dwell volume of the HPLC system was 1.8 mL. The column-oven temperature was set to 50 °C, and the flow rate was 0.8 mL/min. Monitoring of all cannabinoids was done at λ = 220 nm.

UHPLC conditions

Reversed-phase chromatography was done using a HITACHI ChromasterUltra UHPLC system consisting of a 6270 autosampler, a 6310 column oven, a 6170 binary pump, and a 6430 Diode Array Detector. For peak integration, Agilent EZChrom Elite was used. The final liquid chromatography analysis was performed on a Phenomenex Kinetex XB-C18 column (150 × 2.1 mm, 1.7 μm) applying gradient elution, pure-water (with 0.1% formic acid), and acetonitrile (with 0.1% formic acid) as the organic phase. The injection volume was 5 μL, and the dwell volume of the UHPLC system was 0.7 mL. The column-oven temperature was set to 50 °C, and the flow rate was 0.8 mL/min. Monitoring of all cannabinoids was done at λ = 220 nm.

Extraction

All preliminary extraction experiments were performed using sample (A). Twenty milligrams of sample was extracted with 2.5 mL solvent in a cooled ultrasonic bath. Afterwards, samples were centrifuged at 10 °C for 15 min at 4000 rpm. Supernatant was filtered using a PFTE filter (0.45 μm, Machery Nagel) prior to analysis and tenfold diluted with solvent. Recovery effect (RE) was tested at QC low level using three independent spiked hop samples.

Extraction efficiency (EE) was determined in triplicate extracting CBD and THC rich samples three times.

Validation

Commercially available 1 mg/mL methanolic solutions of all analytes were used as stock solutions for calibration and QC spiking solutions. Four different concentrations of the analytes in the range of 1 –100 μg/mL were chosen. Working solutions were prepared by serial dilution from each stock solution in methanol. QC low and QC high samples were analyzed in duplicate on each of 6 days. Accuracy was given in terms of bias as the percent deviation of the mean calculated concentration compared to the theoretical value. Intra-day and inter-day imprecision was calculated as relative standard deviation (RSD) according to Peters et al. (2009). Phenprocoumon was used as internal standard (IS) at a final concentration of 200 μg/mL.

Stability studies

Stability of cannabinoids was tested in an oven (VD20 Binder, Huber) at 100 °C and under UV light (Honle, Sol 2, 350–700 nm). For the heat stability experiment, the sample was placed in weighing flasks. For the UV stability, one weighing flask was covered with aluminum foil and the other was exposed to UV light. For both stability experiments, 20 mg of sample was taken from each of the flasks at indicated time points and was analyzed. At indicated time points, 20 mg of sample was taken from each of the vials and analyzed. All stability experiments were performed in duplicate.

Quantification

Authentic samples were extracted and quantified applying the developed and validated method. All samples (20 mg) were analyzed in duplicate. The final amount of analyte [%] was calculated using the dilution factor given by the procedure and the weighed amount of plant sample. The determined concentration of the authentic forensic samples was compared to those obtained by established GC-FID-based method as described previously (U.N.O.o. Drugs, Crime 2013). (Details can be found in Additional file 1).

Loss on drying

Loss on drying experiment of hop was performed in an oven (VD20 Binder, Huber) 105 °C for 2 h (Pharmacopoea europaea (Ph. Eur.) 2.2.32) (Ph. Eur., Loss on Drying (2.2.32) 2018). Cannabis samples were placed in weighing flasks and were dried to constant mass at 60 °C.

Results and discussion

Chromatographic analysis

Reversed phase chromatography RP-HPLC was chosen for the separation of eight cannabinoids. The focus was set on C18 columns, since these were the most commonly used in the literature. Several C18 columns with different eluent compositions, flow rates, and column temperatures were tested (Additional file 1: Table S1). A baseline separation of all analytes was finally achieved using the Kinetex XB-C18 HPLC column (2.6 μm, 150 × 4.6 mm,) with H2O/0.1% FA and ACN/0.1% FA as solvent. Flow rate and temperature was set to 0.8 mL/min and 50 °C, respectively (Additional file 1: Table S2). Selected cannabinoids were separated within 10 min under HPLC conditions (Fig. 2). After cleaning and reequilibration, complete run time of this method was 20 min. The resolution of all analytes was at least Rs > 1.7 and therefore in the acceptable range for quantification. The asymmetry factor of all peaks is between 1.2 and 1.5 (Table 1). Although for peaks 3 and 4, resolution and asymmetry factor were not in the optimal range, validation data in terms of bias and imprecision for CBGA(3) and CBG(4) were acceptable. The same column was used by Mudge et al. (2017)and De Backer et al. (2009) (Citti et al. 2018b) to separate the same number of cannabinoids, but with separation times of 14 min and 20 min, respectively. As internal standard (IS), phenprocoumon was used. Under selected chromatographic conditions, a clear separation between the IS and all cannabinoids was achieved. Finally, the developed HPLC method was transferred to an UHPLC system coupled to a diode array detector (DAD). The chemistry of the column (Kinetex C18, 1.7 μm, 150 × 2.1 mm) was similar to the HPLC column, and the same mobile phases were used. The injection volume was reduced to 5 μL. Target analytes were separated in less than 5 min (Additional file 1: Figure S5). The resolution of all peaks was above 1.5 and the asymmetry (10%) between 0.9 and 1.1.

UV chromatogram of Cal 2 recorded at λ = 220 nm

Currently, an increasing demand of cannabis-derived products for recreational and medical use is observed. Therefore, the reliable and fast quantification of cannabinoids in hemp samples is essential for the control of product from Cannabis sativa, L. strains. In general, gas chromatography (GC) is the method of choice for the quantification of cannabinoids whereas this method is time consuming and the detection of acidic precursor is not feasible without derivatization. We report the successful development and validation of an accurate and broadly applicable reversed-phase high-performance liquid chromatography (RP-HPLC) method coupled to an ultra violet (UV) detector including an optimized extraction procedure for the separation and quantification of eight different cannabinoids. The optimized method is able to separate cannabidivarin, cannabidiolic acid, cannabigerolic acid, cannabigerol, cannabidiol, cannabinol, Δ9-tetrahydrocannabinol, and tetrahydrocannabinolic acid within 10 min. For all target analytes, the %-Bias at the lower and upper calibration range varied from − 1.3 to 10.3% and from − 3.9 to 8.6%, respectively. The most suitable agent for extracting cannabis plant samples was evaluated to be a mixture of acetonitrile and water in a ratio 1:1. The extraction efficiency was more than 95% for all analytes in recreational hemp samples. Stability studies on acidic cannabinoids showed a high likeliness of decarboxylation at 100 °C and aromatization after exposure to UV light, respectively. A modified loss on drying method revealed a moisture content between 4 and 10%. The developed method was successfully applied to measure the cannabinoid content in recreational and forensic hemp samples representing broad range of cannabinoid amounts and patterns. The present work proposes validated methods for the determination of cannabinoids in cannabis samples. The use of RP-HPLC-UV renders this method broadly applicable and allows the detection of acidic precursor in even less time compared to GC-based methods.