View source for Journal:Development and validation of a fast gas chromatography–mass spectrometry method for the determination of cannabinoids in Cannabis sativa L

{{DISPLAYTITLE:Journal:Development and validation of a fast gas chromatography–mass spectrometry method for the determination of cannabinoids in ''Cannabis sativa'' L}}
{{Infobox journal article
|name         = 
|image        = 
|alt          = <!-- Alternative text for images -->
|caption      = 
|title_full   = Development and validation of a fast gas chromatography–mass spectrometry method for the determination<br />of cannabinoids in ''Cannabis sativa'' L
|journal      = ''Journal of Food and Drug Analysis''
|authors      = Cardenia, Vladimiro; Toschi, Tullia G.; Scappini, Simona; Rubino, Rosamaria C.; Rodriguez-Estrada, Maria T.
|affiliations = University of Bologna, Enecta B.V.
|contact      = Email: tullia dot gallinatoschi at unibo dot it
|editors      = 
|pub_year     = 2018
|vol_iss      = '''26'''(4)
|pages        = 1283–92
|doi          = [http://doi.org/10.1016/j.jfda.2018.06.001 10.1016/j.jfda.2018.06.001]
|issn         = 1021-9498
|license      = [https://creativecommons.org/licenses/by-nc-nd/4.0/ Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International]
|website      = [https://www.sciencedirect.com/science/article/pii/S1021949818301066 https://www.sciencedirect.com/science/article/pii/S1021949818301066]
|download     = [https://www.sciencedirect.com/science/article/pii/S1021949818301066/pdfft?md5=b3164ee2bf934547b92b0e48f241ee9b&pid=1-s2.0-S1021949818301066-main.pdf https://www.sciencedirect.com/science/article/pii/S1021949818301066/pdfft] (PDF)
}}
{{ombox	 
| type      = notice	 
| image     = [[Image:Emblem-important-yellow.svg|40px]]	 
| style     = width: 500px;	 
| text      = This article should be considered a work in progress and incomplete. Consider this article incomplete until this notice is removed.	 
}}
==Abstract==
A routine method for determining [[wikipedia:Cannabinoid|cannabinoids]] in ''Cannabis sativa'' L. [[wikipedia:Inflorescence|inflorescence]], based on fast [[gas chromatography]] coupled to [[mass spectrometry]] (Fast GC-MS), was developed and validated. To avoid the [[wikipedia:Decarboxylation|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 [[wikipedia:Diazomethane|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 [[wikipedia:Hemp|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 [[wikipedia:Cannabidiol|cannabidiol]] (CBD; 1.56 ± 0.03 g/100 g) and CBGA (0.83 g/100 g). [[wikipedia:Tetrahydrocannabivarin|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===
[[File:Fig0 Cardenia JofFoodDrugAnal2018 26-4.jpg|600px]]
{{clear}}

==Introduction==
Recently, the interest on ''Cannabis sativa'' L. has drastically increased. However, attention has primarily been given to addressing its psychoactive<ref name="FengNew17">{{cite journal |title=New psychoactive substances of natural origin: A brief review |journal=Journal of Food and Drug Analysis |author=Feng, L.Y.; Battulga, A.; Han, E. et al. |volume=25 |issue=3 |pages=461–71 |year=2017 |doi=10.1016/j.jfda.2017.04.001 |pmid=28911631}}</ref> and non-psychoactive compounds, such as Δ9-[[wikipedia:Tetrahydrocannabinol|tetrahydrocannabinol]] (Δ9-THC) and [[wikipedia:Cannabidiol|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.<ref name="BrighentiDevelop17">{{cite journal |title=Development of a new extraction technique and HPLC method for the analysis of non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp) |journal=Journal of Pharmaceutical and Biomedical Analysis |author=Brighenti, V.; Pellati, F.; Steinbach, M. et al. |volume=143 |pages=228–36 |year=2017 |doi=10.1016/j.jpba.2017.05.049 |pmid=28609672}}</ref> More recently, the different ''Cannabis'' species have been divided into two broad types: ''C. sativa'' or “[[wikipedia:Hemp|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.<ref name="CittiPharma18">{{cite journal |title=Pharmaceutical and biomedical analysis of cannabinoids: A critical review |journal=Journal of Pharmaceutical and Biomedical Analysis |author=Citti, C.; Braghiroli, D.; Vandelli, M.A. et al. |volume=147 |pages=565-579 |year=2018 |doi=10.1016/j.jpba.2017.06.003 |pmid=28641906}}</ref> However, hemp contains more than 500 different [[wikipedia:Cannabinoid|cannabinoids]], of which about 10 have been classified according to their chemical structure, such as [[wikipedia:Tetrahydrocannabivarin|Δ9-tetrahydrocannabivarin]] (THCV), cannabidiol (CBD), [[wikipedia:Cannabigerol|cannabigerol]] (CBG), Δ8-tetrahydrocannabinol (Δ8-THC), Δ9-tetrahydrocannabinol (Δ9-THC), [[wikipedia:Cannabichromene|cannabichromene]] (CBC), [[wikipedia:Cannabinol|cannabinol]] (CBN), cannabidiolic acid (CBDA), Δ9-tetrahydrocannabinolic acid (THCA), and cannabigerolic acid (CBGA).<ref name="LeghissaARev18">{{cite journal |title=A review of methods for the chemical characterization of cannabis natural products |journal=Journal of Separation Science |author=Leghissa, A.; Hildenbrand, Z.L.; Schug, K.A. et al. |volume=41 |issue=1 |pages=398-415 |year=2018 |doi=10.1002/jssc.201701003 |pmid=28986974}}</ref>

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).<ref name="McPartlandAreCann15">{{cite journal |title=Are cannabidiol and Δ(9) -tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review |journal=British Journal of Pharmacology |author=McPartland, J.M.; Duncan, M.; Do Marzo, V. et al. |volume=172 |issue=3 |pages=737-53 |year=2015 |doi=10.1111/bph.12944 |pmid=25257544 |pmc=PMC4301686}}</ref> The application of CBD for intractable pediatric epilepsy has also been recently studied.<ref name="TzadokCBD16">{{cite journal |title=CBD-enriched medical cannabis for intractable pediatric epilepsy: The current Israeli experience |journal=Seizure |author=Tzadok, M.; Uliel-Siboni, S.; Linder, I. et al. |volume=35 |pages=41–4 |year=2016 |doi=10.1016/j.seizure.2016.01.004 |pmid=26800377}}</ref> On the other hand, CBC, which is particularly present in freshly harvested ''C. sativa'', normalizes ''in vivo'' intestinal motility when intestinal inflammation occurs.<ref name="IzzoInhib12">{{cite journal |title=Inhibitory effect of cannabichromene, a major non-psychotropic cannabinoid extracted from Cannabis sativa, on inflammation-induced hypermotility in mice |journal=British Journal of Pharmacology |author=Izzo, A.A.; Capasso, R.; Aviello, G. et al. |volume=166 |issue=4 |pages=1444–60 |year=2012 |doi=10.1111/j.1476-5381.2012.01879.x |pmid=22300105 |pmc=PMC3417459}}</ref> 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 [[wikipedia:Decarboxylation|decarboxylation]] by heating or drying and thus exhibit their corresponding biological effects.<ref name="CittiPharma18" /> 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<ref name="CarcieriCanna18">{{cite journal |title=Cannabinoids concentration variability in cannabis olive oil galenic preparations |journal=Journal of Pharmacy and Pharmacology |author=Carcieri, C.; Tomasello, C.; Simiele, M. et al. |volume=70 |issue=1 |pages=143–49 |year=2018 |doi=10.1111/jphp.12845 |pmid=29057480}}</ref> 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–mass spectrometry|liquid chromatography coupled to mass spectrometry]] (LC-MS) or an ultraviolet detector (LC-UV).<ref name="BrighentiDevelop17" /><ref name="CittiPharma18" /><ref name="LeghissaARev18" /><ref name="CittiAnal18">{{cite journal |title=Analysis of cannabinoids in commercial hemp seed oil and decarboxylation kinetics studies of cannabidiolic acid (CBDA) |journal=Journal of Pharmaceutical and Biomedical Analysis |author=Citti, C.; Pacchetti, B.; Vandelli, M.A. et al. |volume=149 |pages=532-540 |year=2018 |doi=10.1016/j.jpba.2017.11.044 |pmid=29182999}}</ref><ref name="TsaiSolid99">{{cite journal |title=Solid-phase extraction and gas chromatography-mass spectrometry analysis of 11-nor-9-carboxy-δ9-tetrahydrocannabinol in urine for monitoring marijuana abuse |journal=Journal of Food and Drug Analysis |author=Tsai, Y.-Y.; Tai, S.-J.; Huang, M.-C. et al. |volume=7 |pages=177–84 |year=1999}}</ref><ref name="LinUrine18">{{cite journal |title=Urine specimen validity test for drug abuse testing in workplace and court settings |journal=Journal of Food and Drug Analysis |author=Lin, S.Y.; Lee, H.H.; Lee, J.F. et al. |volume=26 |issue=1 |pages=380–84 |year=2018 |doi=10.1016/j.jfda.2017.01.001 |pmid=29389577}}</ref>

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.<ref name="AngeliANovel18">{{cite journal |title=A novel single-step GC-MS/MS method for cannabinoids and 11-OH-THC metabolite analysis in hair |journal=Journal of Pharmaceutical and Biomedical Analysis |author=Angeli, I.; Casati, S.; Ravelli, A. et al. |volume=155 |pages=1–6 |year=2018 |doi=10.1016/j.jpba.2018.03.031 |pmid=29602054}}</ref><ref name="PacificiEval17">{{cite journal |title=Evaluation of cannabinoids concentration and stability in standardized preparations of cannabis tea and cannabis oil by ultra-high performance liquid chromatography tandem mass spectrometry |journal=Clinical Chemistry and Laboratory Medicine |author=Pacifici, R.; Marchei, E.; Salvatore, F. et al. |volume=55 |issue=10 |pages=1555–1563 |year=2017 |doi=10.1515/cclm-2016-1060 |pmid=28207408}}</ref> As reported in literature, LC-MS sensitivity is lower than that of GC-MS.<ref name="LeghissaARev18" /> 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).<ref name="MudgeLeaner17">{{cite journal |title=Leaner and greener analysis of cannabinoids |journal=Analytical and Bioanalytical Chemistry |author=Mudge, E.M.; Murch, S.J.; Brown, P.N. |volume=409 |issue=12 |pages=3153–63 |year=2017 |doi=10.1007/s00216-017-0256-3 |pmid=28233028 |pmc=PMC5395585}}</ref><ref name="CasiraghiExtract18">{{cite journal |title=Extraction Method and Analysis of Cannabinoids in Cannabis Olive Oil Preparations |journal=Planta Medica |author=Casiraghi, A.; Roda, G.; Casagni, E. et al. |volume=84 |issue=4 |pages=242–49 |year=2018 |doi=10.1055/s-0043-123074 |pmid=29202510}}</ref> Different silylation procedures have been reported for this scope; Purschke ''et al.''<ref name="PurschkeDevelop16">{{cite journal |title=Development and validation of an automated liquid-liquid extraction GC/MS method for the determination of THC, 11-OH-THC, and free THC-carboxylic acid (THC-COOH) from blood serum |journal=Analytical and Bioanalytical Chemistry |author=Purschke, K.; Heinl, S.; Lerch, O. et al. |volume=408 |issue=16 |pages=4379-88 |year=2016 |doi=10.1007/s00216-016-9537-5 |pmid=27116418 |pmc=PMC4875941}}</ref> 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.<ref name="EscriváAnal17">{{cite journal |title=Analysis of cannabinoids by liquid chromatography-mass spectrometry in milk, liver and hemp seed to ensure food safety |journal=Food Chemistry |author=Escrivá, Ú.; Andrés-Costa, M.J.; Andreu, V. et al. |volume=228 |pages=177–85 |year=2017 |doi=10.1016/j.foodchem.2017.01.128 |pmid=28317711}}</ref> However, no data are reported about [[wikipedia:Ester#Preparation|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<ref name="CardeniaAnal12">{{cite journal |title=Analysis of cholesterol oxidation products by Fast gas chromatography/mass spectrometry |journal=Journal of Separation Science |author=Cardenia, V.; Rodriguez-Estrada, M.T.; Baldacci, E. et al. |volume=35 |issue=3 |pages=424-30 |year=2012 |doi=10.1002/jssc.201100660 |pmid=22258809}}</ref>, phytosterols and phytostanols in milk dairy products in less than 10 min<ref name="InchingoloAnal14">{{cite journal |title=Analysis of phytosterols and phytostanols in enriched dairy products by Fast gas chromatography with mass spectrometry |journal=Journal of Separation Science |author=Inchingolo, R.; Cardenia, V.; Rodriguez-Estrada, M.T. |volume=37 |issue=20 |pages=2911-9 |year=2014 |doi=10.1002/jssc.201400322 |pmid=25069661}}</ref>, and heroin and cocaine in 3 min.<ref name="AmiravFast17">{{cite journal |title=Fast Heroin and Cocaine Analysis by GC–MS with Cold EI: The Important Role of Flow Programming |journal=Chromatographia |author=Amirav, A. |volume=80 |issue=2 |pages=295–300 |year=2017 |doi=10.1007/s10337-017-3249-7}}</ref>

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).

{| 
 | STYLE="vertical-align:top;"|
{| class="wikitable" border="1" cellpadding="5" cellspacing="0" width="50%"
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;" colspan="3"|'''Table 1.''' Characteristic ions of cannabinoids obtained by silylation (TMS) and methylation-silylation (MET-TMS) reactions
 |- 
  ! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Cannabinoid
  ! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Quantifier ion (''m''/''z'')
  ! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Qualifier ions (''m''/''z'')
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|THCV-1TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|343
  | style="background-color:white; padding-left:10px; padding-right:10px;"|358, 315, 275
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|CBD-2TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|390
  | style="background-color:white; padding-left:10px; padding-right:10px;"|458, 301, 337
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|CBC-1TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|303
  | style="background-color:white; padding-left:10px; padding-right:10px;"|371, 386, 246
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|Δ8-THC-1TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|386
  | style="background-color:white; padding-left:10px; padding-right:10px;"|303, 265, 330
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|Δ9-THC-1TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|371
  | style="background-color:white; padding-left:10px; padding-right:10px;"|386, 315, 303
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|Δ9-THCd3-1TMS (IS<sub>2</sub>)
  | style="background-color:white; padding-left:10px; padding-right:10px;"|374
  | style="background-color:white; padding-left:10px; padding-right:10px;"|389, 315, 73
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|CBG-2TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|337
  | style="background-color:white; padding-left:10px; padding-right:10px;"|321, 460, 391
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|CBN-1TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|367
  | style="background-color:white; padding-left:10px; padding-right:10px;"|310, 382, 295
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|CBDA-3TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|491
  | style="background-color:white; padding-left:10px; padding-right:10px;"|453, 559, 492
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|CBDA-1MET-2TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|433
  | style="background-color:white; padding-left:10px; padding-right:10px;"|434, 501, 73
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|THCA-2TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|487
  | style="background-color:white; padding-left:10px; padding-right:10px;"|488, 550, 413
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|THCA-1MET-1TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|429
  | style="background-color:white; padding-left:10px; padding-right:10px;"|430, 431, 73
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|CBGA-3TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|561
  | style="background-color:white; padding-left:10px; padding-right:10px;"|562, 417, 453
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|CBGA-1MET-3TMS
  | style="background-color:white; padding-left:10px; padding-right:10px;"|503
  | style="background-color:white; padding-left:10px; padding-right:10px;"|417, 518, 73
 |-
  | style="background-color:white; padding-left:10px; padding-right:10px;"|5α-Cholestane (IS<sub>1</sub>)
  | style="background-color:white; padding-left:10px; padding-right:10px;"|217
  | style="background-color:white; padding-left:10px; padding-right:10px;"|218, 372, 357
 |-
|}
|}

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

<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===


==References==
{{Reflist|colwidth=30em}}

==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.

<!--Place all category tags here-->
[[Category:LIMSwiki journal articles (added in 2019)‎]]
[[Category:LIMSwiki journal articles (all)‎]]
[[Category:LIMSwiki journal articles on cannabis research]]
[[Category:LIMSwiki journal articles on cannabis testing]]