INTRODUCTION:

Cannabis sativa L. is an important herbaceous species native to Central Asia¹ that has been used in traditional medicine and as a source of textile fibers since the dawn of time. This fast-growing plant has recently received renewed interest due to its versatile applications: it is truly a treasure trove of phytochemicals and a rich source of cellulose fibers and woody stems.² Over time, different parts of the plant have been used for therapeutic and recreational purposes³, for example, extracting medicinal oils from the seeds or using the inflorescences for psychoactive effects.⁴ Many 19th-century practitioners explain the healing skills of cannabis after the drug was found in Europe during the period of colonial development in Africa and Asia.

The genera Cannabis and Humulus (hops) belong to the Cannabinaceae family.⁵ In general, cannabis that is considered single-specific (Cannabis sativa L.) is divided into several subspecies (C. sativa subsp. Sativa, C. sativa subsp. Indica, C. sativa subsp. Indica, C. sativa subsp. thôlỗ, C. sativa subsp. naturala, C. sativa subsp. kafiristanca.⁶ There are more than 400 chemical compounds in cannabis, with more than 100 phytocannabinoids identified, including Δ9tetrahydrocannabinol (THC) and cannabidiol (CBD).^7,8 These phytocannabinoids work by binding to cannabinoid receptors, as well as other receptor systems. Cannabinoids exert their effects by interacting with specific endogenous cannabinoid receptors, discovered by Devane et al.⁹ Neuronal cannabinoid receptors are known as CB1 receptors and have been found in the brain and peripheral nerves of rats, guinea pigs, dogs, monkeys, pigs, and humans.¹⁰ A second cannabinoid receptor, the CB2 receptor, was identified by Munro et al. in spleen macrophages and in other immune cells.¹¹ Cannabis actions are dose dependent in the body.¹² It combines many of the properties of alcohol, tranquilizers, opiates and hallucinogens; it is anxiolytic, sedative, analgesic, hallucinogen; it stimulates the appetite and has many systemic effects. Tolerance has been shown to extend too many of the effects of cannabis, including many systemic and high effects, and cannabis withdrawal has been well demonstrated in controlled animal studies.^13,14 The withdrawal syndrome shares similarities with withdrawal from alcohol, opiates, and benzodiazepines and includes restlessness, insomnia, anxiety, increased aggression, anorexia, tremor, and autonomic effects.¹³

THC is generally present in relatively low amounts in fresh plant materials and is thought to be artificially produced from THCA during storage and consumption (e.g. smoking).¹⁵ In terms of analysis approach, one must decide whether THCA and THC are analysed independently or as "Total THC" (i.e. the combined amount of THC and THCA). National legislation can occasionally force this choice. Although neither method is required by law, it is common practise to test total THC because it better depicts the material's pharmacological action. The decarboxylation of THCA to THC yields total THC. This can happen either during or before the analysis. THC preliminary analysis is performed using a colour test and thin layer chromatography. Cannabis colour tests are among the most specific available. Only a few plants, such as henna, nutmeg, mace, and agrimony, provide false-positive findings.¹⁶ A positive colour test, on the other hand, only indicates the likely presence of cannabis-containing material and does not offer a clear identification of cannabis. As a result, it is necessary for the analyst to confirm such results using additional, often more discriminative methodologies. TLC is a presumptive screening method based on which we can selectively go for the other final confirmation methods¹⁷ whereas UV visible and infrared spectroscopy are screening techniques based on the interaction between matter and light.¹⁸There are several methods in TLC for qualitative and semi-quantitative cannabis analysis that use a variety of different stationary phases (TLC plates) and solvent systems, as well as somewhat varied sample preparation and spot viewing approaches.

This study broadly focuses on qualitative identification of cannabis using color test, thin layer chromatography, UV visible spectroscopy and Infrared spectroscopy to determine the limit of detection of these conventional methods for plant materials.

MATERIALS AND METHODS:

Fresh Cannabis sample was received from Centre of Excellence for Research and Analysis of Narcotic Drugs and Psychotropic Substances, NFSU, Gandhinagar. Sodium chloride, vanillin, hydrochloric acid 35%, potassium hydroxide was obtained from Merck Life science Pvt. Ltd. Ethanol 95.2% v/v from Ureca consumers co. op. stores ltd., Chloroform extra pure AR from SRL and 4-aminophenol from Oxford laboratory were obtained. Working standard was prepared by adding a known quantity of cannabis (control) in known quantity of four different solvents i.e., dichloromethane, petroleum benzene, methanol, and hexane: acetone (8:2). After that vortex it and filtered with the help of Whatmann filter paper. The filtrate was collected and used as a stock solution. Stock solution was further diluted by serial dilution from (i) to (ix) and then examined by colour tests. [(i) 10mg + 1mL solvent, (ii) 1mg + 1mL solvent, (iii) 0.5mg + 1mL solvent, (iv) 0.25 mg + 1mL solvent, (v) 0.125mg + 1mL solvent, (vi) 0.05mg + 1mL solvent, (vii) 0.025mg + 1mL solvent, (viii) 0.01mg + 1mL solvent, (ix) 0.001mg + 1mL solvent]. For thin layer chromatography dilutions was made from (i) to (iii). [(i) 5mg + 1mL solvent, (ii) 1mg + 1mL solvent, (iii) 0.5mg + 1mL solvent].

The chemical analysis of cannabis was performed by Fast Blue B test and Duquenois Levine test. The procedure followed for color test is given below:

(A) Fast Blue B test - Small amount of sample + mixture of 2.5gm fast blue b and 100gm anhydrous sodium sulphate + 1ml chloroform → shake well for 1 minute + 1ml 0.1N aqueous sodium hydroxide → shake well for 2 minutes and stand test tube for 2minutes. Development of red color in lower layer of chloroform indicates possible presence of cannabis.

(B) Duquenois- Levine test - Small amount of sample + 2ml reagent → shake well for 1minute + 2ml concentrated hydrochloric acid → shake well for 1 minute and stand test tube for 10minutes + 2ml chloroform. Development of violet color in chloroform layer indicates possible presence of cannabis.

[Reagent: 5drops acetaldehyde and 0.4gm vanillin are dissolved in 20ml of 95% ethanol]

Thin layer chromatography of cannabis was performed in which silica plate was used as a stationary phase and hexane: acetone was used as mobile phase in 80:20 ratios. Fast Blue B reagent was used for chemical development of chromatogram which was prepared by adding 50mg of Fast Blue B salt in 20ml of 0.1 N sodium hydroxide. For UV visible spectroscopic analysis Thermo Fisher Evolution 201 UV VIS Spectrophotometer was used and Bruker Invenio ATR-FTIR spectrometer was used for examination of functional group present in cannabis.

RESULT:

Color tests are a part of preliminary analysis of drugs which are based on simple chemical reaction mechanism and give results based on functional group present in the analyte. These tests are not very precise and can produce false positive or negative results but are very helpful to narrow down the further investigation. The formation or change in color in such test is due to change in electron properties such as their orientation, location, etc.¹⁹ It may be addition, removal, substitution, etc. mechanism followed by the electron either from analyte or from reagent. Each color test gives excellent results in suitable solvent or solvents. In the present analysis methanol extract of cannabis gives comparatively better results for Fast Blue B test whereas hexane: acetone extract in 8:2 ratios give better results for Duquenois Levine test which are shown in table 1.

Though it is very common to analyze the cannabis by color test qualitatively but very are focused on its quantitative analysis and sensitivity of that color test in the particular solvent. Methanol extract of cannabis gives positive results for Fast Blue B test up to very less concentration which is 0.001 mg whereas in case of Duquenois Levine test hexane: acetone (8:2) extract gives positive results up to 0.125 mg concentration. The results of both tests are shown in table 2 and 3, respectively.

Table 1: Color test of cannabis in four different extracts

Extract	Dichloromethane	Petroleum benzene	Methanol	Hexane: Acetone (8:2)
Test applied: Fast Blue B test
Test applied: Duquenois Levine test

Table 2: Quantitative analysis of cannabis in Fast Blue B test

Sr. No.	1	2	3	4	5
Concentration of working standard	(i)	(ii)	(iii)	(iv)	(v)
Test applied	A
Results
Sr. No.	6	7	8	9	10
Concentration of working standard	(vi)	(vii)	(viii)	(ix)	Negative Control
Results

Table 3: Quantitative analysis of cannabis in Duquenois Levine test

Sr. No.	1	2	3	4	5	6
Concentration of working standard	(i)	(ii)	(iii)	(iv)	(v)	Negative Control
Test applied	B
Results

Chromatography is a separation technique in which analyte gets separated into its components based on interaction between stationary phase which is made up of silica gel and mobile phase which is either single of mixture of two or more organic solvents. Depending upon the nature of mobile phase and stationary phase the separation varies. In the present work cannabis was analyze by using two different mobile phase to determine better which one gives better separation. In both the solvent systems cannabis was separated successfully but hexane: acetone in 80:20 ratios give comparatively better separation which is shown in table 4.

Table 4: Identification of suitable mobile phase for cannabis

Solvent system

Hexane: Acetone (80:20)

Petroleum Benzene: Diethyl ether (80:20)

Similar to color tests it is necessary to determine the sensitivity of the separation which was determined by spotting the cannabis extract of different concentrations. The chromatogram displayed in figure 1 contained spot of cannabis at different concentration and volume. It consists of 7 spots among which 1^st, 2^nd and 3^rd spots are of extract (i) having 5μl, 10μl and 20μl spotting volume respectively. 4^th, 5^th and 6^th spot are of extract (ii) having 5μl, 10μl and 20μl spotting volume respectively whereas 7^th spot is of extract (iii) having 20μl spotting volume. The Rf value of each spot was calculated successfully which is shown in table 5. It is a ratio of distance travelled by solute to the distance travelled by mobile phase.²⁰ It will be used as reference value for analysis of seized cannabis sample analysis by similar methodology.

Figure No. 1: Chromatogram of cannabis at different concentration and spotting volume

Table 5: Observations and Rf Values

Concentration	5mg/mL			1mg/mL			0.5mg/mL
Spotting volume	5 uL	10 uL	20 uL	5 uL	10 uL	20 uL	20 uL
Spot No.	Distance travelled in cms
1	--	--	1.76	--	--	--	--
2	--	3.5	3.5	--	--	--	--
3	6.7	6.7	6.8	6.6	6.7	6.6	--
4	--	--	9	--	9.1	9.2	9.2
5	11.8	11.9	11.9	11.8	11.7	11.8	11.7
6	--	--	12.2	--	12.1	12.1	12
Distance travelled by mobile phase: 12.4 cm
Spot No.	Rf Value
1	--	--	0.14	--	--	--	--
2	--	0.28	0.28	--	--	--	--
3	0.54	0.54	0.54	0.53	0.54	0.53	--
4	--	--	0.72	--	0.73	0.74	0.74
5	0.95	0.95	0.95	0.95	0.94	0.95	0.94
6	--	--	0.98	--	0.97	0.97	0.96

The spectrophotometric identification of cannabis shows maximum absorbance at 270 nm. Figure 2 shows scatter plot of Rf values obtained by thin layer chromatography and Figure 3 shows UV visible spectra of cannabis sativa of different concentrations.

Figure No. 2: Scatter plot of Rf values obtained

Figure No. 3: UV spectra of Cannabis sativa

Table 6: Absorbance in UV visible spectroscopy

S. No	Concentration	Absorbance
1	0.10 mg/ml	0.022
2	0.25 g/ml	0.065
3	0.50 g/ml	0.208
4	1.0 g/ml	0.377

Table 6 shows UV absorbance of cannabis sample at different concentrations which shows that absorbance declines with decrease in concentration. In ATR-FTIR analysis cannabis shows IR absorbance at 3284 cm^-1, 2919 cm^-1, 1614 cm^-1 and 1032 cm^-1. Absorbance at 3284 cm^-1 shows the presence of O-H group in the sample whereas C-H bond can be determined by the peak at 2919 cm^-1. 1614 cm^-1 depicts the presence of C=C bond while 1032 cm^-1 depicts the presence of C-O bond group in the sample. All the above observations comply with the different functional groups present in the Cannabis which is shown in figure 4.

Figure No. 4: Functional group characterization of cannabis using ATR-FTIR

DISCUSSION:

1 Colour test identification of cannabis indicates that Fast Blue B test is comparatively more sensitivity than Duquenois Levine test.

2 Thin Layer Chromatographic analysis shows that petroleum benzene extract of cannabis can be separation successfully up to 500 ppm concentration by using hexane: acetone (80:20) as a mobile phase which proves that hexane acetone is comparatively more suitable solvent system for separation of cannabis.

3 UV visible spectrophotometric analysis showed the significant absorbance of cannabis till concentration of 0.10mg/ml in methanol as a suitable solvent whereas ATR-FTIR successfully identified the presence of O-H group along with C=C and C-O bonds.

CONCLUSION:

Based on the observations drawn from the study, it can be inferred that Chemical Analysis of Cannabis can be effective for exoneration of the illicit product. Color Tests and Thin Layer Chromatography are sensitive and specific enough to ensure the positive results even with low availability of the testing sample. From the color tests performed, it can be concluded that color tests are highly sensitive and specific to cannabinoidal content in the sample. Although from the study, Fast Blue B Test (Test A) is found to be much more sensitive and specific compared to Duquenois- Levine Test (Test B). Test A gave positive and observable results till lowest concentration i.e. 0.001mg/ml solution while Test B showed sensitivity till 0.125mg/ml solution. From the observations drawn from Thin layer chromatography (TLC), it can be concluded that Hexane: Acetone (8:2) solvent system is best suitable for separation of cannabinoids and gave observable results till concentration of 0.5mg/ml solution involving different spotting volumes. UV visible spectrophotometric analysis showed the significant absorbance of cannabis till concentration of 0.10mg/ml in methanol as a suitable solvent whereas ATR-FTIR successfully identified the presence of O-H group along with C=C and C-O bonds.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Centre of Excellence for Research and Analysis of Narcotic Drugs and Psychotropic Substances, NFSU, Gandhinagar for their kind support during all lab studies.

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Received on 21.10.2022 Modified on 03.02.2023

Research J. Pharm. and Tech 2024; 17(1):413-418.

DOI: 10.52711/0974-360X.2024.00065

S. No

Concentration

Absorbance

1

0.10 mg/ml

0.022

2

0.25 g/ml

0.065

3

0.50 g/ml

0.208

4

1.0 g/ml

0.377