Difference between revisions of "Journal:Development and validation of a fast gas chromatography–mass spectrometry method for the determination of cannabinoids in Cannabis sativa L"

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<tt>R = 2(t<sub>R2</sub> – T<sub>R1</sub>) / (''w''<sub>1</sub> + ''w''<sub>2</sub>)</tt>
<tt>R = 2(t<sub>R2</sub> – T<sub>R1</sub>) / (''w''<sub>1</sub> + ''w''<sub>2</sub>)</tt>
where t<sub>R</sub> is the retention time of the chromatographic peak and ''w'' is the peak width at its base level. The sensitivity and repeatability precision (intraday and interday) of the method on both standard mixture and matrix were determined. The reproducibility and recoveries on hemp inflorescences were estimated.
The limit of detection and quantification (LOD and LOQ, respectively) were calculated by the signal-to-noise ratio (S/N); LOD was expressed as S/N of 3.3:1, whereas an S/N of 10:1 was used for LOQ. The intraday and interday precision of fast GC–MS, expressed as relative standard deviation (RSD; %) was calculated by manually injecting samples (n = 3) in the same day (intraday precision) for three consecutive days (interday precision, n = 9). The injections were performed by different operators for testing the method precision. The recoveries of cannabinoids were estimated at two spiking levels of phytocannabinoids standard mixture (25 ng/mL [A]; 25.0 μg/mL [B]) in hemp inflorescence, using the following equation:
<tt>% recovery = [C<sub>fc</sub> - C<sub>c</sub>) / C<sub>f</sub>] x 100</tt>
where C<sub>fc</sub> is the cannabinoid amount found in the spiked sample, C<sub>c</sub> is the cannabinoid amount present in the unspiked sample, and C<sub>f</sub> is the spiked amount of cannabinoid standards. Three independent replicates (''n'' = 3) were run for spiked and non-spiked hemp.
The method robustness (RSD; %) was assessed by determining cannabinoids in hemp inflorescence (from their extraction to the fast GC-MS analysis) in triplicates by two different operators.
===Statistical analysis===
Statistical analysis of data was performed by SPSS 21.0 (IBM-SPSS Inc., Chicago, Illinois, USA). Analysis of variance (ANOVA) was carried out to investigate the effect of derivatization reagents, solvents, and analytical conditions. Tukey's honest significance test and T-test were carried out at a 99% confidence level to separate means of parameters. P-values under the significance level of 0.001 were considered statistically significant.
==Results and discussion==
===Derivatization study===





Revision as of 16:14, 22 October 2019

Full article title Development and validation of a fast gas chromatography–mass spectrometry method for the determination
of cannabinoids in Cannabis sativa L
Journal Journal of Food and Drug Analysis
Author(s) Cardenia, Vladimiro; Toschi, Tullia G.; Scappini, Simona; Rubino, Rosamaria C.; Rodriguez-Estrada, Maria T.
Author affiliation(s) University of Bologna, Enecta B.V.
Primary contact Email: tullia dot gallinatoschi at unibo dot it
Year published 2018
Volume and issue 26(4)
Page(s) 1283–92
DOI 10.1016/j.jfda.2018.06.001
ISSN 1021-9498
Distribution license Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Website https://www.sciencedirect.com/science/article/pii/S1021949818301066
Download https://www.sciencedirect.com/science/article/pii/S1021949818301066/pdfft (PDF)

Abstract

A routine method for determining cannabinoids in Cannabis sativa L. inflorescence, based on fast gas chromatography coupled to mass spectrometry (Fast GC-MS), was developed and validated. To avoid the decarboxylation of the carboxyl group of cannabinoids, different derivatization approaches—i.e., silylation and esterification (diazomethane-mediated) reagents and solvents (pyridine or ethyl acetate)—were tested. The methylation significantly increased the signal-to-noise ratio of all carboxylic cannabinoids, except for cannabigerolic acid (CBGA). Since diazomethane is not commercially available, is considered a hazardous reactive, and requires one-day synthesis by specialized chemical staff, the process of silylation was used along the entire validation of a routine method. The method gave a fast (total analysis time < 7.0 min) and satisfactory resolution (R > 1.1), with a good repeatability (intraday < 8.38%; interday < 11.10%) and sensitivity (LOD < 11.20 ng/mL). The suitability of the fast GC-MS method for detection of cannabinoids in hemp inflorescences was tested; a good repeatability (intraday < 9.80%; interday < 8.63%), sensitivity (LOD < 58.89 ng/mg), and robustness (< 9.52%) was also obtained. In the analyzed samples, the main cannabinoid was cannabidiolic acid (CBDA; 5.19 ± 0.58 g/100 g), followed by cannabidiol (CBD; 1.56 ± 0.03 g/100 g) and CBGA (0.83 g/100 g). Δ9-tetrahydrocannabivarin (THCV) was present at trace levels. Therefore, the developed fast GC-MS method could be a valid, routine alternative for a fast, robust, and highly sensitive determination of the main cannabinoids present in hemp inflorescences.

Keywords: cannabinoids, cannabis, decarboxylation, fast gas chromatography, methylation

Graphical abstract

Fig0 Cardenia JofFoodDrugAnal2018 26-4.jpg

Introduction

Recently, the interest on Cannabis sativa L. has drastically increased. However, attention has primarily been given to addressing its psychoactive[1] and non-psychoactive compounds, such as Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). In the past, the genus Cannabis was allocated into three main species: a drug-type (C. indica) with high levels of Δ9-THC, a fiber-type (C. sativa L.) with low levels of Δ9-THC, and an intermediate type C. ruderalis Janish.[2] More recently, the different Cannabis species have been divided into two broad types: C. sativa or “hemp” when referring to industrial use (fiber-type), and therapeutic “marijuana” (drug-type) when referring to varieties with a high level of Δ9-THC (>0.6%; w/w). To date, the main use for hemp is largely related to food; in fact, hemp seeds are generally used for producing oil and flour, and, depending on the country's local regulations, they may also be employed on the basis of their pharmacological properties.[3] However, hemp contains more than 500 different cannabinoids, of which about 10 have been classified according to their chemical structure, such as Δ9-tetrahydrocannabivarin (THCV), cannabidiol (CBD), cannabigerol (CBG), Δ8-tetrahydrocannabinol (Δ8-THC), Δ9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC), cannabinol (CBN), cannabidiolic acid (CBDA), Δ9-tetrahydrocannabinolic acid (THCA), and cannabigerolic acid (CBGA).[4]

Hemp cannabinoids exhibit diverse biological effects. THCV displays various pharmacological profiles according to the type of molecular target (in vitro antagonistic/inverse agonistic effects and an in vivo agonism effect in an antinociception model).[5] The application of CBD for intractable pediatric epilepsy has also been recently studied.[6] On the other hand, CBC, which is particularly present in freshly harvested C. sativa, normalizes in vivo intestinal motility when intestinal inflammation occurs.[7] It should be pointed out that C. sativa does not produce Δ9-THC, CBD, CBG, and CBC, but their respective carboxylic acid forms (precursors) Δ9-THCA, CBDA, CBGA, and CBCA can undergo decarboxylation by heating or drying and thus exhibit their corresponding biological effects.[3] The galenic preparations of cannabis (such as medicinal oils), which are important for their possibility of being employed as a whole set of cannabinoids, are characterized by a high variability[8] and require a robust, simple quality control method for their titration.

Considering these and other biological effects of cannabinoids, their analysis in cannabis is of great interest and importance. There are several analytical methods for determining cannabinoids, most of which use gas chromatography coupled to mass spectrometry (GC-MS) or a flame ionization detector (GC-FID), or high-performance liquid chromatography coupled to mass spectrometry (LC-MS) or an ultraviolet detector (LC-UV).[2][3][4][9][10][11]

When GC-MS is used, the electron impact ionization (EI) generates mass spectra, which can be compared with those present in compound libraries for their identification. However, with LC-MS and electrospray (ESI) and atmospheric pressure chemical ionization (APCI), only molecular ions are generated, without other useful fragments for compound characterization; as such, expensive equipment able to perform tandem mass spectrometry (MS-MS) experiments is required.[12][13] As reported in literature, LC-MS sensitivity is lower than that of GC-MS.[4] However, there is a lot of criticism around the use of GC for cannabinoid analysis, since the high temperature of both injector and detector lead to decarboxylation of cannabinoid acids if not previously derivatized (e.g., using a process such as silylation).[14][15] Different silylation procedures have been reported for this scope; Purschke et al.[16] utilized N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), while other researchers have used the combination of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) or MSTFA, with either trimethylchlorosilane (TMCS) or ethyl acetate.[17] However, no data are reported about esterification of cannabinoid carboxylic acids by diazomethane and the use of fast GC-MS for cannabinoid determination. Fast GC-MS has been demonstrated to provide the advantages of mass spectrometry boosted by the utilization of fast chromatography. In fact, the use of fast GC-MS drastically reduces the time of analysis without impairing sensitivity, resolution, and other analytical parameters (such as repeatability and reproducibility). Fast GC-MS has been successfully utilized for the determination of cholesterol oxidation products in 3.5 min[18], phytosterols and phytostanols in milk dairy products in less than 10 min[19], and heroin and cocaine in 3 min.[20]

To the best of our knowledge, no previous works have been published on the determination of cannabinoids in hemp inflorescences by fast GC-MS. The aim of this work was to develop and validate a fast GC-MS method for determining the main cannabinoids (CBD, CBDA, CBG, CBGA, Δ9-THC, Δ8-THC, THCA, THCV, CBC, and CBN) in hemp inflorescences, as related to different derivatization reagents (through silylation and esterification).

Materials and methods

Reagents and solvents

Chloroform, n-hexane, methanol, and ethanol were purchased from Merck (Darmstadt, Germany). N° 1 filters (70 mm diameter) were used (Whatmann, Maidstone, England). N,O-Bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA:TMCS, 99:1, v/v), N-methyl-N-(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane (MSTFA:TMCS, 99:1, v/v), and (trimethylsilyl)diazomethane solution were supplied by Sigma Aldrich (Germany). Certified phytocannabinoid mixture 1 (1 mg/mL in acetonitrile; containing CBD, CBDA, CBG, CBGA, Δ9-THC, Δ8-THC, THCA, and CBC at 100 μg/mL of each compound); (−)-Δ9-THC-D3 (THCd3, 0.1 mg/mL in methanol); THCA (1.0 mg/mL in methanol); and Δ9-THC (0.1 mg/mL in methanol) were purchased from LGC Standards S.r.L. (Milano, Italy). Millipore membrane filters (0.45 μm and 0.20 μm) were supplied by Merck (Germany).

Sampling

Three different batches of hemp inflorescences (E.U.-registered Cannabis sativa L. Futura 75 variety; fiber-type), harvested at different growing times (from middle July to the end of August 2017), were supplied by a local company (Green Valley Società Agricola S.R.L., Castelvecchio Subequo, Italy). Each batch was comprised of three independent samples (n = 3), where each sample included 10 inflorescences from 10 different plants (n = 10). Before performing cannabinoids extraction, the collected samples were dried by natural ventilation at 32 ± 1 °C for 60 h. Afterwards, dried hemp's apical and lateral peaks were sifted. The seeds with diameter >1 mm were removed by sieving. Finally, the material was ground with an analytical mill to obtain a homogeneous sample. The powdered samples were stored at −22 °C under nitrogen atmosphere until the analysis.

Extraction

Twenty five milligrams of ground sample were weighed into a glass test tube and 1.5 mg of 5α-cholestane (internal standard 1, IS1) were added. The extraction was performed using 10 mL of a 9:1 (v/v) methanol/chloroform mixture. The sample was stirred for 15 min (350 oscillations/min), sonicated for 10 min, centrifuged (5 min at 1620 g) and the solvent was collected. The extraction was repeated twice, and the surnatants were transferred into a 25 mL flask, which was made up to the flask volume with the same solvent. The extract was then filtered through a millipore filter (0.45 μm). One mL of the filtered extract was transferred to a glass tube that contained 0.5 μg of THCd3 (internal standard 2, IS2), taken to dryness under nitrogen flow and then derivatized.

Derivatization

Two different derivatization reactions were compared: methylation and silylation. For methylation, 1 mL of the filtered extract was added with IS2, dried under nitrogen flow, methylated with 300 μL of diazomethane, vortexed for 30 s and then dried under nitrogen flow. Silylation was then performed at 60 °C for 15 min, using 50 μL of pyridine and 150 μL of n-methyl-n-trimethylsilyltrifluoroacetamide + 1% of chlorotrimethylsilane (MSTFA-TMCS); the silylated sample was then dried at 40 °C and dissolved in 100 μL of n-hexane.

Fast gas chromatography–mass spectrometry (fast GC-MS) analysis

The cannabinoids were determined with a fast GC-MS Shimadzu QP 2010 Plus instrument (Kyoto, Japan) equipped with a Restek RTX 5 column (0.1 μm film thickness, 10 m × 0.1 mm); helium was used as the carrier gas (constant flow; linear velocity of 47.4 cm/s). The oven temperature was programmed from 180 °C (30 s) to 250 °C at 10 °C/min, and then to 350 °C (at 60 °C/min); final temperature was maintained for 5 min. The injector, interface, and ion source temperatures were 300, 330, and 200 °C, respectively, while the filament voltage was 70 eV (electronic impact). One μL of derivatized sample was manually injected (split 1:30).

Validation of the method

The response linearity was evaluated by means of calibration curves. For each compound, a calibration curve in the concentration range of 0.25 ng/mL–25 μg/mL was built using the internal standard method. Six different concentration levels were tested in triplicates. The cannabinoids were recognized by their mass spectra and were quantified by single ion monitoring (SIM). In particular, one quantifier ion and three qualifier ions were used (Table 1) for both derivatization methods (methylation and silylation).

Table 1. Characteristic ions of cannabinoids obtained by silylation (TMS) and methylation-silylation (MET-TMS) reactions
Cannabinoid Quantifier ion (m/z) Qualifier ions (m/z)
THCV-1TMS 343 358, 315, 275
CBD-2TMS 390 458, 301, 337
CBC-1TMS 303 371, 386, 246
Δ8-THC-1TMS 386 303, 265, 330
Δ9-THC-1TMS 371 386, 315, 303
Δ9-THCd3-1TMS (IS2) 374 389, 315, 73
CBG-2TMS 337 321, 460, 391
CBN-1TMS 367 310, 382, 295
CBDA-3TMS 491 453, 559, 492
CBDA-1MET-2TMS 433 434, 501, 73
THCA-2TMS 487 488, 550, 413
THCA-1MET-1TMS 429 430, 431, 73
CBGA-3TMS 561 562, 417, 453
CBGA-1MET-3TMS 503 417, 518, 73
5α-Cholestane (IS1) 217 218, 372, 357

The chromatographic peak resolution was determined on a critical pair (CBC and Δ8-THC), according to the following expression:

R = 2(tR2 – TR1) / (w1 + w2)

where tR is the retention time of the chromatographic peak and w is the peak width at its base level. The sensitivity and repeatability precision (intraday and interday) of the method on both standard mixture and matrix were determined. The reproducibility and recoveries on hemp inflorescences were estimated.

The limit of detection and quantification (LOD and LOQ, respectively) were calculated by the signal-to-noise ratio (S/N); LOD was expressed as S/N of 3.3:1, whereas an S/N of 10:1 was used for LOQ. The intraday and interday precision of fast GC–MS, expressed as relative standard deviation (RSD; %) was calculated by manually injecting samples (n = 3) in the same day (intraday precision) for three consecutive days (interday precision, n = 9). The injections were performed by different operators for testing the method precision. The recoveries of cannabinoids were estimated at two spiking levels of phytocannabinoids standard mixture (25 ng/mL [A]; 25.0 μg/mL [B]) in hemp inflorescence, using the following equation:

% recovery = [Cfc - Cc) / Cf] x 100

where Cfc is the cannabinoid amount found in the spiked sample, Cc is the cannabinoid amount present in the unspiked sample, and Cf is the spiked amount of cannabinoid standards. Three independent replicates (n = 3) were run for spiked and non-spiked hemp.

The method robustness (RSD; %) was assessed by determining cannabinoids in hemp inflorescence (from their extraction to the fast GC-MS analysis) in triplicates by two different operators.

Statistical analysis

Statistical analysis of data was performed by SPSS 21.0 (IBM-SPSS Inc., Chicago, Illinois, USA). Analysis of variance (ANOVA) was carried out to investigate the effect of derivatization reagents, solvents, and analytical conditions. Tukey's honest significance test and T-test were carried out at a 99% confidence level to separate means of parameters. P-values under the significance level of 0.001 were considered statistically significant.

Results and discussion

Derivatization study

References

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Notes

This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added. Everything else remains true to the original article, per the "NoDerivatives" portion of the distribution license.