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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. Keywords: HPLC, Method development, Validation, Cannabinoids * Correspondence: email@example.com Sanja Zivovinovic and Ruth Alder contributed equally to this work. Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH), Vladimir-Prelog-Weg 4, 8093 Zürich, Switzerland © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Zivovinovic et al. Journal of Analytical Science and Technology (2018) 9:27 Page 2 of 10 Introduction are of lower pharmacological importance (Pavlovic et al. Since centuries, Cannabis sativa L. (C. sativa)isusedfor 2018). Cannabigerolic acid (CBGA) is the starting point in industrial purposes but it is better known as illegal drug the biosynthetic pathway of all cannabinoids, which are possessing psychotropic properties. However, C. sativa is synthesized in vivo in a carboxylated form (Fig. 1). In the also a highly decorated medicinal plant for the use as anti- plant, CBDA and THCA are synthesized by enzymatic cat- cancer agent, for neuroprotection and as bone marrow alyzed reactions. However, ex vivo stress conditions like stimulants (Velasco et al. 2016; Machado Rocha et al. heat and UV light decompose these precursors to their 2008). With the legalization of cannabis for therapeutic decarboxylated form: CBGA ➔ cannabigerol (CBG), use, the demand for pure and characterized samples has CBDA➔ CBD and THCA ➔ THC, respectively (Citti et al. grown significantly (Corroon and Phillips 2018). Therefore, 2018a; Sirikantaramas and Taura 2017). Under UV light, currently new pharmacopeial monographs are in develop- Δ9-THC is further aromatized to cannabinol (CBN). THC ment for quality control of C. sativa-based medicinal prod- and CBD are two main biomarkers in commercial available ucts (Pavlovic et al. 2018). Besides the medical use, there is hemp samples. THC is mostly responsible for psychotropic an enormous interest from consumers/patients in the activities whereas CBD is more anxiolytic and sleep indu- utilization of low Δ9-tetrahydrocannabinol (THC) hemp cing. In comparison to THC, CBD is not considered a con- for recreational use. In recent years, a kind of gold-rush trolled substance. Numerous reports have been published fever is observed in Europe and all over the world and for the qualitative and quantitative analysis of cannabinoids many new suppliers entered the market (Pellechia 2018). in cannabis and its preparations. This study will therefore Since there is a complicated and different legislation for C. focus on those substances for possible therapeutic use such sativa products all over Europe, caution for quality control as CBD, Δ9-THC, CBG, CBN, cannabidivarin (CBDV), has to be taken. Although there is no upper limit for the cannabichromene, and tetrahydrocannabivarin (Amada et cannabidiol (CBD) or cannabidiolic acid (CBDA) content al. 2013;Thomas et al. 2007). Several comprehensive in most European countries, maximum limits of THCor reviews of the chemical analysis of cannabis plants, Δ9-tetrahydrocannabinolic acid (THCA) contents vary corresponding preparations, and forensic specimens were between 0.1 and 1% within Europe. presented in the past (Citti et al. 2018b;Wangetal. 2017; Cannabinoids belong to terpenophenolic compounds and Patel et al. 2017; ElSohly and Salem 2000). The most are the main constituents of the cannabis plant. Terpenoids widespread techniques applied for separation were gas and phenols were also identified in the cannabis plant but chromatography (GC) with and without derivatization, Fig. 1 Biosynthetic pathway of selected cannabinoids Zivovinovic et al. Journal of Analytical Science and Technology (2018) 9:27 Page 3 of 10 high-performance liquid chromatography (HPLC), and to Chromatographic analysis a lesser extend supercritical fluid chromatography (Wang HPLC conditions et al. 2016; U.N.O.o. Drugs, Crime 2013). The GC method Reversed-phase chromatography was done using a is still officially employed by the authorities for the deter- LaChrom Elite System (Hitachi, Ltd., Tokio, Japan) HPLC mination of cannabinoids. But it is obvious that acidic system consisting of a LaChrom Elite L-2200 autosampler, forms are not accessible without prior derivatization, and a LaChrom Elite L-2130 pump, a LaChrom Elite L-2350 further conversion of THCA to THC is not quantitative at column oven, and a LaChrom Elite L-2420 UV-VIS all (Dussy et al. 2005). Some researchers postulate that an detector. For peak integration, Agilent EZChrom Elite was accurate cannabinoid profile should be evaluated by deter- used. The final liquid chromatography analysis was mining the acid and neutral forms separately (Pavlovic et performed on a Phenomenex Kinetex XB-C18 column al. 2018; Citti et al. 2018b; Calvi et al. 2018;Ambachet al. (150 × 4.6 mm, 2.6 μm) applying gradient elution, using 2014). On the other hand, LC-based procedures render pure-water (with 0.1% FA) and acetonitrile (with 0.1% FA) the derivatization step superfluous and enable the detec- as the organic phase. The injection volume was 15 μL, and tion of the heat-labile acid precursor in less time. How- the dwell volume of the HPLC system was 1.8 mL. The ever, determining chromatographic conditions is more column-oven temperature was set to 50 °C, and the flow challenging. Additionally, the pre-analytical phase has to rate was 0.8 mL/min. Monitoring of all cannabinoids was be taken into account for method development and valid- done at λ = 220 nm. ation. Extraction, storage conditions, and stability deter- mination play a pivotal role in the analysis of C. sativa UHPLC conditions L.-derived products (Dussy et al. 2005; Brighenti et al. Reversed-phase chromatography was done using a 2017; Mudge et al. 2017). HITACHI ChromasterUltra UHPLC system consisting The main scope of this study was the development and of a 6270 autosampler, a 6310 column oven, a 6170 bin- validation of a fast and convenient UV-detector-based ary pump, and a 6430 Diode Array Detector. For peak RP-HPLC method for the fast quantification of cannabi- integration, Agilent EZChrom Elite was used. The final noids in CBD samples and forensic cannabis samples. The liquid chromatography analysis was performed on a Phe- present study examines further pre-analytical conditions nomenex Kinetex XB-C18 column (150 × 2.1 mm, and the analytical stability of cannabinoids under different 1.7 μm) applying gradient elution, pure-water (with 0.1% stress conditions. Eight authentic CBD-hemp materials formic acid), and acetonitrile (with 0.1% formic acid) as and 12 forensic cannabis samples offering a wide range of the organic phase. The injection volume was 5 μL, and cannabinoid patterns were analyzed. Results of the overall the dwell volume of the UHPLC system was 0.7 mL. The THC-content of forensic samples were compared with gas column-oven temperature was set to 50 °C, and the flow chromatographic method (U.N.O.o. Drugs, Crime 2013), rate was 0.8 mL/min. Monitoring of all cannabinoids the formerly gold standard in cannabinoid analysis. Add- was done at λ = 220 nm. itionally, a modified loss on drying method was applied to determine the moisture content of all cannabis samples. Extraction Finally, the developed method was transferred easily to an All preliminary extraction experiments were performed ultra-high performance liquid chromatography (UHPLC) using sample (A). Twenty milligrams of sample was ex- device using know metrics, thus further reducing analysis tracted with 2.5 mL solvent in a cooled ultrasonic bath. time from 10 to less than 5 min. 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 di- Materials and methods luted with solvent. Recovery effect (RE) was tested at QC Analytical standards were obtained from Lipomed (Rein- low level using three independent spiked hop samples. ach, Switzerlanf). Formic acid (FA), methanol (MeOH), Response ethanol (EtOH), and acetonitrile (ACN) were obtained extracted sample with analytes %Recovery effectðÞ RE ¼ −1 100 Response from Merck (Darmstadt, Germany) and were of LCMS post−extracted spiked sample grade. Pure-water was generated from an in-house water purification system from Labtec (Villmergen, Switzerland). Extraction efficiency (EE) was determined in triplicate For all experiments, Gilson DIAMOND tips were used. extracting CBD and THC rich samples three times. Hop strobiles (Humulus lupulus L.) were obtained from local pharmacies. CBD-hemp tobacco samples were pur- Validation chased from several licensed producers within Switzerland. Commercially available 1 mg/mL methanolic solutions of The Forensic Institute Zurich (Zurich, Switzerland) all analytes were used as stock solutions for calibration provided 12 forensic cannabis samples. and QC spiking solutions. Four different concentrations Zivovinovic et al. Journal of Analytical Science and Technology (2018) 9:27 Page 4 of 10 Fig. 2 UV chromatogram of Cal 2 recorded at λ = 220 nm of the analytes in the range of 1 –100 μg/mL were were analyzed in duplicate. The final amount of analyte chosen. Working solutions were prepared by serial [%] was calculated using the dilution factor given by the dilution from each stock solution in methanol. QC low procedure and the weighed amount of plant sample. The and QC high samples were analyzed in duplicate on each determined concentration of the authentic forensic sam- of 6 days. Accuracy was given in terms of bias as the ples was compared to those obtained by established percent deviation of the mean calculated concentration GC-FID-based method as described previously (U.N.O.o. compared to the theoretical value. Intra-day and Drugs, Crime 2013). (Details can be found in inter-day imprecision was calculated as relative standard Additional file 1). deviation (RSD) according to Peters et al. (2009). Phen- procoumon was used as internal standard (IS) at a final Loss on drying concentration of 200 μg/mL. Loss on drying experiment of hop was performed in an oven (VD20 Binder, Huber) 105 °C for 2 h (Pharmacopoea Stability studies europaea (Ph. Eur.) 2.2.32) (Ph. Eur., Loss on Drying Stability of cannabinoids was tested in an oven (VD20 (2.2.32) 2018). Cannabis samples were placed in weighing Binder, Huber) at 100 °C and under UV light (Honle, Sol 2, flasks and were dried to constant mass at 60 °C. 350–700 nm). For the heat stability experiment, the sample was placed in weighing flasks. For the UV stability, one Results and discussion weighing flask was covered with aluminum foil and the Chromatographic analysis other was exposed to UV light. For both stability experi- Reversed phase chromatography RP-HPLC was chosen ments, 20 mg of sample was taken from each of the flasks for the separation of eight cannabinoids. The focus was at indicated time points and was analyzed. At indicated set on C18 columns, since these were the most com- time points, 20 mg of sample was taken from each of the monly used in the literature. Several C18 columns with vials and analyzed. All stability experiments were different eluent compositions, flow rates, and column performed in duplicate. temperatures were tested (Additional file 1: Table S1). A baseline separation of all analytes was finally achieved Quantification using the Kinetex XB-C18 HPLC column (2.6 μm, 150 × Authentic samples were extracted and quantified applying 4.6 mm,) with H O/0.1% FA and ACN/0.1% FA as solv- the developed and validated method. All samples (20 mg) ent. Flow rate and temperature was set to 0.8 mL/min Zivovinovic et al. Journal of Analytical Science and Technology (2018) 9:27 Page 5 of 10 and 50 °C, respectively (Additional file 1: Table S2). Se- two were ACN/H O in a ratio of 1:1 and ACN 99.9%. lected cannabinoids were separated within 10 min under All extraction experiments were performed using CBD HPLC conditions (Fig. 2). After cleaning and reequilibra- hemp sample (A). The mass to volume ratio was chosen tion, complete run time of this method was 20 min. The as proposed previously (Mudge et al. 2017). As shown in resolution of all analytes was at least R >1.7 and therefore Fig. 3a, ACN 99.9% yielded the lowest amount of ex- in the acceptable range for quantification. The asymmetry tracted target analytes, especially for CBG, CBGA, THC, factor of all peaks is between 1.2 and 1.5 (Table 1). Al- and THCA. However, it could be shown that MeOH/ though for peaks 3 and 4, resolution and asymmetry factor H O (4:1) and ACN/H O (1:1) provided similar per- 2 2 were not in the optimal range, validation data in terms of formance than the standard method. These two alterna- bias and imprecision for CBGA(3) and CBG(4) were tive solvents were even able to extract more CBGA, acceptable. The same column was used by Mudge et al. CBG, and CBD than MeOH/CHCl (9:1). Finally, ACN/ (2017)and De Backer et al. (2009)(Cittiet al. 2018b)to H O (1:1) was chosen because of better compatibility separate the same number of cannabinoids, but with with starting conditions of the HPLC method. Further- separation times of 14 min and 20 min, respectively. As more, higher aqueous content renders this extraction internal standard (IS), phenprocoumon was used. Under system more environmentally friendly. According to selected chromatographic conditions, a clear separation UNODC, homogenized plant samples should be ex- between the IS and all cannabinoids was achieved. Finally, tracted with solvent in an ultrasonic bath for 15 min in- the developed HPLC method was transferred to an cluding several vortexing steps (10 s each) (U.N.O.o. UHPLC system coupled to a diode array detector (DAD). Drugs, Crime 2013). Several different extraction tech- The chemistry of the column (Kinetex C18, 1.7 μm, 150 × niques were tested in duplicate. The performance of the 2.1 mm) was similar to the HPLC column, and the same four procedures was compared and shown as relative ex- mobile phases were used. The injection volume was tracted yield of cannabinoids. Different extracting reduced to 5 μL. Target analytes were separated in less methods were plotted against the standard method than 5 min (Additional file 1: Figure S5). The resolution of (Fig. 3b). Samples extracted without vortexing yielded the all peaks was above 1.5 and the asymmetry (10%) between same amount of cannabinoids as the standard method. 0.9 and 1.1. Vortexing alone or shortening ultra-sonication time to 5 min lowered the yield to around 50% for CBGA, THCA, Extraction CBGD, CBG, and THC and to about 70% for CBDA. The most commonly used extracting agent for cannabi- These results clearly indicate the need for 15 min in the noids according to literature (Patel et al. 2017; U.N.O.o. ultrasonic bath whereas the influence of vortexing was Drugs, Crime 2013; De Backer et al. 2009; Zoller et al. insignificant. Extraction efficiency (EE) was controlled by 2000) is a mixture of methanol (MeOH) and chloroform repeating extraction process three times on samples (F) (CHCl ) in a ratio of 9:1. Considering the volatility and and (P5) showing highest contents in CBD/CBDA and toxicity of chloroform and taking green chemistry guide- THC/THCA, respectively. In general, the EE shows the lines into consideration, this halogenated solvent should completeness of an extraction procedure of authentic not be the solvent of choice. Performance of other samples. For CBD-rich cannabis samples with low extracting solvents and solvent mixtures were compared amounts of THC/THCA, the EE for THC, CBDA, and to the standard MeOH/CHCl (9:1) mix. One of these CBD was greater than 94.4% after one extraction step. For mixtures was MeOH/H O (4:1) which is also used by all other analytes, an EE > 99% was observed in the first Mudge and coworkers (Mudge et al. 2017). The other step. For THC- and THCA-rich samples, first extraction yielded in an EE of around 99% for THC/THCA whereas after the second extraction step, all target analytes were Table 1 Retention times and chromatographic values of each extracted completely (Fig. 4). Interestingly, in THC-/ analyte obtained by the HPLC method THCA-rich samples, no CBD or CBDA was found. EE for R (min) Analyte Resolution R Asymmetry (10%) THC and THCA was only around 90% for the first extrac- 4.2 1 0 1.3 tion step. However, after the second extraction, no more 5.4 2 8.9 1.2 target analytes were found in the samples. 5.7 3 1.8 1.3 5.9 4 1.7 1.3 Method development and validation Since there is no cannabinoid-free cannabis matrix, ex- 6.3 5 2.1 1.2 tracts from a closely related plant were used as surrogate 8.3 6 13.7 1.3 matrix. The use of hop (Humulus lupulus L.) appeared to 8.9 7 5.2 1.2 be most appropriate, because it belongs to the same family 9.6 8 5.9 1.5 (Cannabacea) as cannabis (Nuutinen 2018). Therefore, it Zivovinovic et al. Journal of Analytical Science and Technology (2018) 9:27 Page 6 of 10 Fig. 3 a Data of different extraction solvents (a) and procedures (b) is assumed that the general and non-specific composition 1. Since there was a full recovery of all analytes observed and structure of hop is similar to cannabis. Blank extract (Table 2) and no co-eluting substance in the blank matrix of hop samples were investigated in detail, but no was observed, a matrix-matched calibration was not co-eluting substances in relation to any target analytes of necessary. As surrogate matrix for calibrants and quality cannabis were detected. The recovery effect (RE) of an control (QC) samples, the solvent mix ACN/H 0(1:1) is analyte is the ratio of the detector response obtained from used. Four different concentrations of the analytes were an amount of the analyte added before and after the ex- chosen for the calibrations: 1, 10, 50, and 100 μg/mL. The traction process to blank matrix or its extract, respectively. dilutions were made using ACN/H O 1:1. For six follow- To calculate the RE, blank material was spiked with refer- ing days, the analysis of four calibrants and two QC sam- ence standards and re-extracted as described above (N = ples (QC and QC ) were performed. QC samples Low High 3). The extraction recovery effect was calculated using Eq. were analyzed in duplicate. Validation data is shown in Fig. 4 Extraction efficiency on CBD-rich (F) and THC-rich (P5) cannabis samples Zivovinovic et al. Journal of Analytical Science and Technology (2018) 9:27 Page 7 of 10 Table 2 Method validation data: recovery effect, bias, intra-day precision (RSD ), interday-precision (RSD ) R T Analyte Cal. model EE (%), (CV, %) RE (%), (CV, %) QC High QC Low Bias (%) RSDT (%) RSDR (%) Bias (%) RSDT (%) RSDR (%) CBDV 1/x > 99.9 103.1 − 0.2 4.7 5.6 2.9 4.6 11.9 CBDA 1/x 95.3 (1.2) 105.9 − 2.3 2.6 6.1 3.1 4.3 11.3 CBGA 1/x > 99.9 104.3 0.4 3.3 4.4 1.4 9.4 16.3 CBG 1/x > 99.9 106.2 2.8 2.4 5.7 − 1.3 3.9 20.0 CBD 1/x 94.4 (1.3) 106.8 3.9 2.1 5.7 5.2 4.3 9.9 CBN 1/x > 99.9 104.3 − 3.9 3.3 5.8 3.2 5.3 11.1 THC Non-weighted 94.4 (1.5) 102.7 8.6 1.1 1.9 10.3 6.2 12.7 THCA 1/x > 99.9 103.0 − 3.7 1.3 6.0 1.2 4.6 16.8 Table 2.Biasfor QC and QC for all analytes varied observed. Starting from 0.14 μg/mL concentration of Low High between 1.3 and − 10.3% and 3.9 and 8.6%, respectively. CBN increased to around 4.25 μg/mL after 72 h. There- Blank samples were injected after the highest calibrant, fore CBN concentration may be useful as quality marker and carry-over was not observed. Across the calibration for storage conditions of cannabis samples and should range, 1/x was found to be the best fit for all cannabi- be further take into account for estimation of initial noids, whereas a non-weighted calibration was used for THC content. THC (Table 2). The decision on weighted calibration was For the UV stability experiment, sample (B) was placed made by comparing the deviations of the back-calculated in a weighing flask and exposed to UV light (λ =350– concentrations from the respective nominal concentra- 750 nm, Fig. 5b). Since the temperature under the UV tions of the calibrators. Limit of quantification (LoQ) for lamp can slightly increase (up to 36 °C), a control, cov- all analytes was set to 1 μg/mL because of low bias and ac- ered with aluminum foil, was also analyzed (Additional ceptable imprecision data. The limit of detection was not file 1: Figure S6A). The samples were quantified after in- investigated systematically. Due to the early elution of the dicated time points of exposure. Data in Fig. 5b showed IS phenprocoumon, 60 different small molecules were a time-dependent decrease of all target analytes under screened for a second internal standard for late eluting UV-light exposure. A fivefold increase in CBN content peaks. Sixty different small molecules were screened for a could be detected, from approximately 0.2 to 1 μg/mL, second internal standard for use as a second internal after 8 h. Interestingly, CBGA and CBG were more sus- standard for late eluting peaks. Under given chromato- ceptible to UV light compared to heat conditions. In the graphic conditions, co-elution was observed with one of control, only a slight increase in CBN and decrease of the target analytes. The list of selected compounds is given THCA was observed. in the supporting information (Additional file 1: Table S3). Because of the acceptable bias and imprecision data, Quantification phenprocoumon was accepted as IS for all target analytes. THC and THCA concentrations of the 12 forensic hemp samples were analyzed using the developed HPLC-UV Stability method. Results were compared with results obtained by CBD-hemp sample (A) was analyzed after indicated time the forensic institute Zurich using standard GC-FID points. All analyses were performed in duplicate. In method. In general, correlation of both methods was Fig. 5a, a time-dependent decline of the concentrations high (R = 0.956, Fig. 6). For all samples, the difference is observed for all three acidic cannabinoids (CBDA, between the expected and the found THC+THCA con- CBGA, and THCA) and at the same time the formation tent was between − 0.3 and 2.4% (Additional file 1: Table of their decarboxylated forms (CBD, CBG, and THC) is S4). The slope of the regression line and y-intercept in detected. The decarboxylation process of THCA oc- both cases was 1.033 and 0.447, respectively. In general, curred more rapidly than for the other two acidic pre- THC/THCA were quantified higher by the HPLC-based cursors. The concentrations of CBD, CBG, and THC method than the GC method. This could be explained show a saturation at different time points (Fig. 5a). by incomplete conversion of THCA to THC or conver- Afterwards, the curves start to decline, indicating further sion of THC to CBN at higher temperatures. Dussy et conversion of these compounds. For CBD and CBG, the al. showed that at 150 °C THC reaches its stability decline of the analyzed concentrations appeared to start optimum for an accurate analysis, and above that roughly after 30 h; however, for THC, at about 4 h. At temperature, the conversion to CBN takes place (Dussy the same time, an increase in CBN content could be et al. 2005). Interestingly, in forensic samples, only Zivovinovic et al. Journal of Analytical Science and Technology (2018) 9:27 Page 8 of 10 Fig. 5 Heat stability (a) and UV-stability (b) studies on seven cannabinoids THC/THCA and CBD/CBDA were detected. Other tar- certificate, reported cannabinoid values were not batch get analytes were found only in traces. The cannabinoid specific. For nearly all samples, CBD content was far content of eight different legally sold CBD-hemp below the declared values. Only for sample (C), CBD tobacco samples obtained in Switzerland were analyzed. content was higher than indicated. In general, it can be For only two out of eight samples (B, C), a certificate of observed that the THC+THCA contents of all tobacco analysis was available online. As indicated in the samples were under the maximal legally allowed limit of 1%. CBDV and CBN were below the detection limit. Nevertheless, our analysis also showed that samples (F) and (H) with a concentration of 0.91% and 0.90% re- spectively were very close to this threshold. Since CBD tobacco is sold in whole plant pieces, the content of the cannabinoids can vary depending on which part of the flower is ripped and ground and used for analysis, because different parts of the plant produce different amount of substances. To minimize such fluctuations, all batches should be homogenized before being analyzed and sold. Loss on drying The content of volatile compounds in crude drugs is an important factor since it influences the concentrations of the substances with pharmacological activity. Through manipulation, a lower content of target compounds Fig. 6 Method comparison of overall THC/THCA content could be reported. The current monograph on cannabis determination by GC-FID and LC-UV presented in the German Pharmacopoeia does not Zivovinovic et al. Journal of Analytical Science and Technology (2018) 9:27 Page 9 of 10 Table 3 Content of cannabinoids in commercial available hemp samples. Declared values were given in parenthesis. Values written in italics are below the LoQ [%] (A) (B) (C) (D) (E) (F) (G) (H) CBDA 4.5 12.5 16.1 (13.7) 12.6 (14) 5.8 13.3 13.2 17.3 CBD 0.3 1.6 1.0 (0.4) 0.5 (0.4) 9.8 6.5 1.6 2.5 CBD+CBDA 4.8 (18) –– – 15.6 (21) – 14.8 (18) 19.8 (23) THC 0.1 0.2 0.1 (0.05) 0.05 (0.05) 0.6 0.7 0.2 0.3 THCA 0.4 0.4 0.6 (0.6) 0.5 (0.6) 0 0.2 0.5 0.6 THC+THCA 0.5 (< 1) 0.6 (< 1) –– 0.6 (< 1) 0.9 (< 1) 0.7 (0.8) 0.9 (0.9) CBGA < nd 1.49 0.90 0.56 0.24 0.14 0.3 0.15 CBG < nd 0.24 0.17 0.24 <nd 0.15 0.06 0.12 CBDV < nd < nd < nd < nd < nd < nd < nd < nd CBN [%] < nd < nd < n.d < nd < nd < nd < nd < nd Loss on drying 5.8 4.3 6.5 4.4 8.3 7.7 8.8 5.9 nd not detected provide any information about loss on drying (BfArM legalized products and to provide characterized products 2017). The monograph of hop includes drying the sample for therapeutic use. at 105 °C for 2 h. The loss on drying of hop was performed as described in the Ph. Eur. and additionally till constant Additional file mass was achieved after drying at 60 °C. Both procedures Additional file 1: Table S1. Use d RP-HPLC columns. Table S2. Gradient resulted in loss of 3.8% (Table 3). Since the cannabinoids elution HPLC method. Table S3. Used compounds for use as internal stand- are heat instable, hemp samples were dried till constant ard. Table S4. Results obtained by GC- and LC-based methods. Figure S5. mass at 60 °C. The loss on drying of all tested CBD-hemp UHPLC-UV chromatogram of Cal 2 recorded at λ = 220 nm. Figure S6. UV experiment with covered samples (A) and non-covered samples (B). (DOCX samples is shown in Table 3. The highest loss was deter- 246 kb) mined in samples (E) and (G) and was higher than 8%. The lowest loss of 4.5% was determined in sample (B). In Abbreviations general, all hemp samples showed a higher loss on drying ACN: Acetonitrile; C. sativa: Cannabis sativa; Cal: Calibrator; CBD: Cannabidiol; compared to hop samples. CBDA: Cannabidiolic acid; CBDV: Cannabidivarin; CBG: Cannabigerol; CBGA: Cannabigerolic acid; CBN: Cannabinol; CHCl : Chloroform; EE: Extraction efficiency; EtOH: Ethanol; FA: Formic acid; FID: Flame ionization Conclusion detector; GC: Gas chromatography; HPLC: High-performance liquid We developed a fast and reproducible HPLC-UV method chromatography; IS: Internal standard; LC: Liquid chromatography; LoD: Limit of detection; LoQ: Limit of quantification; ME: Matrix effect; MeOH: Methanol; for the quantification of hemp samples. Method validation Ph. Eur.: Pharmacopoea europaea; QC: Quality control; RE: Recovery effect; confirmed that the method produces repeatable and RP: Reversed phase; RSD: Relative standard deviation; THC: Δ9- accurate results for eight different cannabinoids in less Tetrahydrocannabinol; THCA: Δ9-Tetrahydrocannabinolic acid; UHPLC: Ultra-high-performance liquid chromatography; UV: Ultraviolet time. The use of a water to acetonitrile mixture (50%) for extraction instead of chlorinated organic solvent mixtures Acknowledgements renders this method more ecologically friendly The pre- The authors want to acknowledge Danielle Luethi for excellent support in sented procedure is universally applicable in a wide range laboratory handling of all samples. We further thank the Forensic Institute of Zurich for providing and analyzing confiscated hemp samples. of settings from pharmacopeial monographs, research, quality control, and regulatory evaluation of this emerging Funding field of herbal industry. Additionally, the transfer to an This work has not been financially supported. UHPLC-DAD system reduced the analysis time to less Availability of data and materials than 5 min providing additional ecological benefits. An al- Research data have been provided in the manuscript and supporting ternative loss on drying experiment was further described information. and showed similar results when applied on closely related Authors’ contributions hop samples. CBN content could be used as a marker for This study was designed by CS. SZ and RA equally performed the storage control of cannabis samples. The results obtained experimental work. The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the from the analysis of authentic samples highlight the need manuscript. for accurate determination of the cannabinoids concentra- tions in regularly time intervals of different C. sativa L. Competing interests strains to limit the risk of increased THC content in The authors declare that they have no competing interests. Zivovinovic et al. 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"Journal of Analytical Science and Technology" – Springer Journals
Published: Dec 1, 2018
Keywords: Analytical Chemistry; Characterization and Evaluation of Materials; Monitoring/Environmental Analysis
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