analysis by using radioimmunoassay, high-performance liquid chromatography and capillary electrophoresis to investigate chronic exposure to heroin, cocaine and/or ecstasy in applicants for driving licences. *Forensic Sci Int,* 107(1-3), 121-128,

Transient cocaine-associated behavioral symptoms rated with a new instrument, the scale for assessment of positive symptoms for cocaine-induced psychosis

influence of drugs -- evaluation of analytical data of drugs in oral fluid, serum and urine, and correlation with impairment symptoms. *Forensic Sci Int,* 152(2-3), 149-

Validation of a method to detect cocaine and its metabolites in nails by gas chromatography-mass spectrometry. *Forensic Sci Int,* 159(2-3), 218-222, 0379-0738. Valente, M. J., Carvalho, F., Bastos, M. L., Carvalho, M., & de Pinho, P. G. (2010).

Development and validation of a gas chromatography/ion trap-mass spectrometry method for simultaneous quantification of cocaine and its metabolites benzoylecgonine and norcocaine: application to the study of cocaine metabolism in human primary cultured renal cells. *J Chromatogr B Analyt Technol Biomed Life Sci,*

**9**

*Colombia*

**Gas Chromatography in Forensic Chemistry:**

**Leaves (***Cannabis sativa* **L.) from Colombia**

N.M. Florian-Ramírez1, W.F. Garzón-Méndez2 and F. Parada-Alfonso3

*3Chemistry Department, Science Faculty-Universidad Nacional de Colombia, Bogotá,*

Despite that the narcotics had generated a regrettable stigma for Colombia, our country does not have a deep knowledge about the narcotic chemistry. In this study it is show the analysis of cannabinoids in the Colombian illicit crops of *Cannabis sativa* L. using Gas Chromatography (GC), Figure 1. The main important cannabinoids from *C. sativa* were identified by Gas Chromatography-Mass Spectrometry (GC-MS). The conditions for extraction and quantification of these cannabinoids (cannabidiol (CBD), Δ9 tetrahydrocannabinol (THC) and cannabinol (CBN)) from *C. sativa* leaves were optimized by the response surface analysis-RSA, using as variables the extraction time and the extraction numbers. Then, the better sample amount and the best detection method were determined, after the validation of the GC analytic method was made. Additionally, the cannabinoids of samples of different Colombian geographic regions were analyzed, determining that the THC content vs THC/CBN content can serve as a discriminating factor to distinguish the geographic origin of the samples studied. Finally, it was showed that the GC is a good

The study of extraction process was carried out using RSA, the best response and the less variability for the cannabinoids was obtained with three extraction steps, each one of 7.5 minutes with ethanol as solvent. The quantitative analysis was made by Gas Chromatography-Flame Ionization Detector (GC-FID) with diphenhydramine (DPH) as surrogate standard. In the GC method for quantification of CBD, THC and CBN were assessed their chromatographic parameters; selectivity, linearity, precision, accuracy, Limit

The cannabinoids samples from four different Colombian regions (North region (NR), at Magdalena department, South region (SR) at Cauca department, East region (ER) at Meta department and Central region (CR), at Caldas department) were determined. The content average THC in samples of the NR was 2.81% ± 1.72, in the SR was 10.98% ± 6.70, in the ER was 15.74% ± 2.92 and at the CR was 1.87% ± 1.25. The higher content of THC at cannabis

**1. Introduction**

analytic tool for forensic chemistry in Colombia.

of Detection (LOD) and Limit of Quantification (LOQ).

**Cannabinoids Content in Marijuana**

*2Criminalistic Chemistry Lab, Fiscalía General de la Nación, Bogotá,*

*Departamento Administrativo de Seguridad-DAS, Bogotá,*

*1Scientific Lab, Criminalistic Group,*

## **Gas Chromatography in Forensic Chemistry: Cannabinoids Content in Marijuana Leaves (***Cannabis sativa* **L.) from Colombia**

N.M. Florian-Ramírez1, W.F. Garzón-Méndez2 and F. Parada-Alfonso3 *1Scientific Lab, Criminalistic Group, Departamento Administrativo de Seguridad-DAS, Bogotá, 2Criminalistic Chemistry Lab, Fiscalía General de la Nación, Bogotá, 3Chemistry Department, Science Faculty-Universidad Nacional de Colombia, Bogotá, Colombia*

### **1. Introduction**

194 Gas Chromatography – Biochemicals, Narcotics and Essential Oils

Wylie, F. M., Torrance, H., Seymour, A., Buttress, S., & Oliver, J. S. (2005). Drugs in oral fluid

Yang, W., Barnes, A. J., Ripple, M. G., Fowler, D. R., Cone, E. J., Moolchan, E. T., Chung, H.,

Yarema, M. C., & Becker, C. E. (2005). Key concepts in postmortem drug redistribution. *Clin*

Yonamine, M., & Sampaio, M. C. (2006). A high-performance thin-layer chromatographic

Yonamine, M., & Saviano, A. M. (2006). Determination of cocaine and cocaethylene in urine

Yonamine, M., Tawil, N., Moreau, R. L., & Silva, O. A. (2003). Solid-phase micro-extraction-

Zhang, J. Y., & Foltz, R. L. (1990). Cocaine metabolism in man: identification of four

Zwerling, C., Ryan, J., & Orav, E. J. (1990). The efficacy of preemployment drug screening

0738.

6223.

0232.

201-205, 0146-4760.

2639-2643, 0098-7484.

833(2), 210-218, 1570-0232.

*Toxicol (Phila),* 43(4), 235-241, 1556-3650.

*Biomed Chromatogr,* 20(10), 1071-1075, 0269-3879.

Part II. Investigation of drugs in drivers. *Forensic Sci Int,* 150(2-3), 199-204, 0379-

& Huestis, M. A. (2006). Simultaneous quantification of methamphetamine, cocaine, codeine, and metabolites in skin by positive chemical ionization gas chromatography-mass spectrometry. *J Chromatogr B Analyt Technol Biomed Life Sci,*

technique to screen cocaine in urine samples. *Leg Med (Tokyo),* 8(3), 184-187, 1344-

by solid-phase microextraction and gas chromatography-mass spectrometry.

gas chromatography-mass spectrometry and headspace-gas chromatography of tetrahydrocannabinol, amphetamine, methamphetamine, cocaine and ethanol in saliva samples. *J Chromatogr B Analyt Technol Biomed Life Sci,* 789(1), 73-78, 1570-

previously unreported cocaine metabolites in human urine. *J Anal Toxicol,* 14(4),

for marijuana and cocaine in predicting employment outcome. *JAMA,* 264(20),

Despite that the narcotics had generated a regrettable stigma for Colombia, our country does not have a deep knowledge about the narcotic chemistry. In this study it is show the analysis of cannabinoids in the Colombian illicit crops of *Cannabis sativa* L. using Gas Chromatography (GC), Figure 1. The main important cannabinoids from *C. sativa* were identified by Gas Chromatography-Mass Spectrometry (GC-MS). The conditions for extraction and quantification of these cannabinoids (cannabidiol (CBD), Δ9 tetrahydrocannabinol (THC) and cannabinol (CBN)) from *C. sativa* leaves were optimized by the response surface analysis-RSA, using as variables the extraction time and the extraction numbers. Then, the better sample amount and the best detection method were determined, after the validation of the GC analytic method was made. Additionally, the cannabinoids of samples of different Colombian geographic regions were analyzed, determining that the THC content vs THC/CBN content can serve as a discriminating factor to distinguish the geographic origin of the samples studied. Finally, it was showed that the GC is a good analytic tool for forensic chemistry in Colombia.

The study of extraction process was carried out using RSA, the best response and the less variability for the cannabinoids was obtained with three extraction steps, each one of 7.5 minutes with ethanol as solvent. The quantitative analysis was made by Gas Chromatography-Flame Ionization Detector (GC-FID) with diphenhydramine (DPH) as surrogate standard. In the GC method for quantification of CBD, THC and CBN were assessed their chromatographic parameters; selectivity, linearity, precision, accuracy, Limit of Detection (LOD) and Limit of Quantification (LOQ).

The cannabinoids samples from four different Colombian regions (North region (NR), at Magdalena department, South region (SR) at Cauca department, East region (ER) at Meta department and Central region (CR), at Caldas department) were determined. The content average THC in samples of the NR was 2.81% ± 1.72, in the SR was 10.98% ± 6.70, in the ER was 15.74% ± 2.92 and at the CR was 1.87% ± 1.25. The higher content of THC at cannabis

Gas Chromatography in Forensic Chemistry:

above 20%.

**2.2 Cannabis in Colombia**

the first illegal cultivation of marijuana in this region.

Cannabinoids Content in Marijuana Leaves (*Cannabis sativa* L.) from Colombia 197

Hemphill et al. analyzed the cannabinoids content in different organs of cannabis plants selected from geographical regions by gas-liquid chromatography. The results showed quantitative variations in cannabinoid content in different parts of the plants belonging to the selected varieties. However, each part of the plant presented a profile of cannabinoids that characterized the phenotype of which it comes from chemical, in addition, the accumulation of a specific cannabinoid variety was not found and it was observed that factors such as degree of maturity of body, sex plant, the location of the organ in the plant and the sampling procedures influence the concentration of cannabinoids. Finally,

Gambaro et al. determined THC, CBD and CBN in cannabis preparations by GC-FID and High Performance Liquid Chromatography-Ultraviolet (HPLC-UV) (2002). De Meijer et al. studied the inheritance of chemical phenotype in cannabis varieties, for it they made four

Mechtler et al. analyzed the variation in the distribution of a population of THC in hemp, for it a number of plants from five well-known populations of hemp grown in regions of Austria and Hungary were sampled (2004). ElSohly Slade reported the composition of the

Pellegrini et al. developed a simple and rapid procedure for the determination of cannabinoids in hemp food products derived by GC-MS (2005). Lewis et al. determined the relative amounts of the three most abundant cannabinoids (THC, CBN and CBD) and other abundant compounds present in six compressed bars of cannabis resin by GC-MS analysis (2005).

A global scientific community has recently expressed concern over the detection in the illicit market in cannabis varieties with high content of THC. According to the National Coordination of Drug Policy, which is headquartered in Sweden, in 1961 the levels of THC, the more important psychoactive component, did not exceed 5%, today this number is

Recently, De Backer et al. developed an High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) method for the qualitative and quantitative determination of major cannabinoids in plant material of cannabis (2009). Then, the solubilities of some cannabinoids in supercritical carbon dioxide were determined (Perrotin-Brunel et al. 2010). Milman et al. reported simultaneous quantification of cannabinoids in oral fluid by two dimensional GC-MS (2010). Tipparat et al. studied the cannabinoids composition of

In Colombia, the introduction of marijuana in the Sierra Nevada de Santa Marta was conducted in the early seventeenth century, the use of hemp was quickly displaced by the twine. It is recognizes the uses as intoxicants of this plant since 1927, used by laborers and sailors from the Colombian Caribbean Coast. However, it was until 1945 went it was saw

With the completion of the Vietnam War in 1975, there was an increased cultivation of marijuana encouraged by the intoxicating veterans. Moreover marijuana was used until the

cannabis plants grown in Northern Thailand and its forensic application (2011).

crosses between plants of *C. sativa* with CBDs and THCs pure chemotypes (2003).

cannabinoids were not detected in pollen grains and seeds (1980).

complex mixture of natural cannabinoids from cannabis (2005).

plants of the ER and SR could be indicative of the use of improved varieties, which creates a major concern in potential effects around of the health of drug consumers.

Fig. 1. Cannabis leave sample and a sample gas chromatogram of its extract.

It was established that the value of the Waller phenotype (Turner et al., 1979, 1980), equation 1, did not serve as a differentiation criterion on the geographical origin of the Colombian samples, due to overlapping ranges for three of the regions studied (NR, SR and ER). In order to establish a criterion to discriminate the geographic origin of the Colombian marijuana, different types of correlation among the CBD, THC and CBN were tested, finding that for the four groups of studied samples the ratio THC vs THC/CBN could be used.

$$\text{Waller phenytoype} = \text{(THC} + \text{CBN}) / \text{CBD} \tag{1}$$

### **2. Forensic chemistry of cannabinoids**

GC analysis is a primary analytical technique. It is used in every forensic lab. GC is widely used by forensic scientists, from analysis of body fluids for the presence of illegal substances, for testing of fiber and blood from a crime scene, and to detect residue from explosives (Müller et al., 1999). In the next parts of the chapter cannabinoids works would be reference as well as some aspects of Colombian cannabis.

### **2.1 Cannabinoids analysis by chromatography techniques**

Regarding on the cannabinoids analysis by chromatographic methods, it is worth to mention the following works:

Davis & Farmilo performed an analysis of cannabinoids in leaves and flowering of cannabis by thin layer chromatography and GC. From this analysis it was proposed that the relationship of the THC and CBN content based on the CBD content allowed cannabis differentiation of samples by geographical origin (1963).

plants of the ER and SR could be indicative of the use of improved varieties, which creates a

major concern in potential effects around of the health of drug consumers.

Fig. 1. Cannabis leave sample and a sample gas chromatogram of its extract.

**2. Forensic chemistry of cannabinoids**

mention the following works:

be reference as well as some aspects of Colombian cannabis.

differentiation of samples by geographical origin (1963).

**2.1 Cannabinoids analysis by chromatography techniques**

used.

It was established that the value of the Waller phenotype (Turner et al., 1979, 1980), equation 1, did not serve as a differentiation criterion on the geographical origin of the Colombian samples, due to overlapping ranges for three of the regions studied (NR, SR and ER). In order to establish a criterion to discriminate the geographic origin of the Colombian marijuana, different types of correlation among the CBD, THC and CBN were tested, finding that for the four groups of studied samples the ratio THC vs THC/CBN could be

GC analysis is a primary analytical technique. It is used in every forensic lab. GC is widely used by forensic scientists, from analysis of body fluids for the presence of illegal substances, for testing of fiber and blood from a crime scene, and to detect residue from explosives (Müller et al., 1999). In the next parts of the chapter cannabinoids works would

Regarding on the cannabinoids analysis by chromatographic methods, it is worth to

Davis & Farmilo performed an analysis of cannabinoids in leaves and flowering of cannabis by thin layer chromatography and GC. From this analysis it was proposed that the relationship of the THC and CBN content based on the CBD content allowed cannabis

Waller phenotype = (THC + CBN) / CBD (1)

Hemphill et al. analyzed the cannabinoids content in different organs of cannabis plants selected from geographical regions by gas-liquid chromatography. The results showed quantitative variations in cannabinoid content in different parts of the plants belonging to the selected varieties. However, each part of the plant presented a profile of cannabinoids that characterized the phenotype of which it comes from chemical, in addition, the accumulation of a specific cannabinoid variety was not found and it was observed that factors such as degree of maturity of body, sex plant, the location of the organ in the plant and the sampling procedures influence the concentration of cannabinoids. Finally, cannabinoids were not detected in pollen grains and seeds (1980).

Gambaro et al. determined THC, CBD and CBN in cannabis preparations by GC-FID and High Performance Liquid Chromatography-Ultraviolet (HPLC-UV) (2002). De Meijer et al. studied the inheritance of chemical phenotype in cannabis varieties, for it they made four crosses between plants of *C. sativa* with CBDs and THCs pure chemotypes (2003).

Mechtler et al. analyzed the variation in the distribution of a population of THC in hemp, for it a number of plants from five well-known populations of hemp grown in regions of Austria and Hungary were sampled (2004). ElSohly Slade reported the composition of the complex mixture of natural cannabinoids from cannabis (2005).

Pellegrini et al. developed a simple and rapid procedure for the determination of cannabinoids in hemp food products derived by GC-MS (2005). Lewis et al. determined the relative amounts of the three most abundant cannabinoids (THC, CBN and CBD) and other abundant compounds present in six compressed bars of cannabis resin by GC-MS analysis (2005).

A global scientific community has recently expressed concern over the detection in the illicit market in cannabis varieties with high content of THC. According to the National Coordination of Drug Policy, which is headquartered in Sweden, in 1961 the levels of THC, the more important psychoactive component, did not exceed 5%, today this number is above 20%.

Recently, De Backer et al. developed an High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) method for the qualitative and quantitative determination of major cannabinoids in plant material of cannabis (2009). Then, the solubilities of some cannabinoids in supercritical carbon dioxide were determined (Perrotin-Brunel et al. 2010).

Milman et al. reported simultaneous quantification of cannabinoids in oral fluid by two dimensional GC-MS (2010). Tipparat et al. studied the cannabinoids composition of cannabis plants grown in Northern Thailand and its forensic application (2011).

### **2.2 Cannabis in Colombia**

In Colombia, the introduction of marijuana in the Sierra Nevada de Santa Marta was conducted in the early seventeenth century, the use of hemp was quickly displaced by the twine. It is recognizes the uses as intoxicants of this plant since 1927, used by laborers and sailors from the Colombian Caribbean Coast. However, it was until 1945 went it was saw the first illegal cultivation of marijuana in this region.

With the completion of the Vietnam War in 1975, there was an increased cultivation of marijuana encouraged by the intoxicating veterans. Moreover marijuana was used until the

Gas Chromatography in Forensic Chemistry:

was validated.

cannabinoids detected.

identification of cannabinoids in the extracts.

significant factor for the models.

the analysis model, increased in the extreme values.

Cannabinoids Content in Marijuana Leaves (*Cannabis sativa* L.) from Colombia 199

To optimize the analytic method cannabis leaves from NR were used. The first objective was the optimization of the extraction method and the selection of the analysis method. Then, the sample quantity and quantification method were defined. After, the analytic method

**3.1 Optimization of the extraction method and the selection of the analysis method**  In order to obtain extracts of cannabinoids from plant material samples, De Meijer et al. method was taken as a based because of its quickness, efficiency and reproducibility, this method use extraction with ethylalcohol (EtOH), ultrasound and centrifugation at 4000 rpm (2003). The extraction time (t= 5, 10 and 15 min) and the number of extractions with EtOH (n= 1, 3 and 5) were considered as factors; for each extraction 750 μL were used and graduated to 5.00 mL. Using a 32 factorial model, cannabis leaves were subjected to each one of the extraction conditions, in triplicate, and at the central point (t= 10 min, n= 3) there were done five triplicates. The extracts obtained were analyzed by GC with different forms of detection: GC-FID, GC-MS scan mode and GC-MS Selected Ion Monitoring (SIM) mode. In all cases the response variable was the chromatographic area corresponding to each of the

By comparing the coefficients of variation (C.V.) of the response surfaces for each analyte, based on the detection methods considered, we observed that the detection by GC-FID showed the lowest variation, see Table 1. Therefore, it was decided to use this method for quantitative analysis of cannabinoids, the GC-MS analysis was used for the qualitative

> **Method CBD C.V. (%) THC C.V. (%)**  GC-FID 9.36 7.89 GC-MS Scan 27.33 17.14 GC-MS SIM 15.51 11.58

Based on the response obtained by GC-FID, the ANOVA for the quadratic regression model for each analyte, indicated that the difference was significant with a low probability value to a level of 0.05 (for the CBD pmodel = 0.0003 < α and the THC pmodel = 0.0105 < α). In that way, the models generated by the equations 2 and 3 explained satisfactorily the response in terms of the considered factors (time (t), number of extractions (n)), n being the most

Figure 3a shows the generated response surface to optimize the extraction conditions of THC, in it is observed that the response increased with the extraction time and the number of extractions. On the other hand, Figure 3b shows that the standard error associated with

CBD area= 9050 + 169\*t + 1944\*n (2)

THC area= 31518 - 2200\*t + 10177\*n + 123\*t2 - 1008\*n2 - 35\*t\*n (3)

Table 1. Coefficient of variation of the response surface for CBD and THC.

**3. Optimization of the cannabinoids analysis conditions**

mid-twentieth century by the popular classes to relieve the pain associated with arthritis. It was common that the local police take a jar of alcohol and added seized marijuana to produce a medicinal poultice. Then the habit of smoking marijuana as a pain medication was spread, but this was a practice of alternative medicine without scientific studies and without institutional control (Fernandez-Alonso et al, 2007).

Currently the forensic study over confiscated samples of *C. sativa* L., is based on the qualitative determination of THC, present in extracts obtained from plant samples, without performing an quantitative level analysis of that component and some other cannabinoids present in seized samples. As a result of the above at present there is no systematic information to determine the content of THC present in plant samples of *C. sativa* illicitly traded in Colombia, there is just an essential information for monitoring the potential levels of THC present in plant samples who smoke marijuana consumers.

The Dirección Nacional de Estupefacientes (DNE) of Colombian government has reported about the results achieved in Colombia in the fight against drug trafficking (2002, 2007). Florian et al. reported higher content of THC in vegetable samples of Colombian cannabis (2009).

This paper shows the GC as an important tool in the analysis of forensic samples, specifically in the determination of cannabinoids. Leaf samples of *C. sativa* L. in four regions of Colombia were analyzed by GC. The analyzed region were (i) Northern region (NR) at Magdalena department, (ii) Southern region (SR) at Cauca department, (iii) Eastern region (ER) at Meta department and (iv) Central region (CR) at Caldas department, see Figure 2. The analysis revealed differences in the cannabinoid content of the samples under study according to their origin region.

Fig. 2. Geographic origin of the *C. sativa* L. samples.

### **3. Optimization of the cannabinoids analysis conditions**

198 Gas Chromatography – Biochemicals, Narcotics and Essential Oils

mid-twentieth century by the popular classes to relieve the pain associated with arthritis. It was common that the local police take a jar of alcohol and added seized marijuana to produce a medicinal poultice. Then the habit of smoking marijuana as a pain medication was spread, but this was a practice of alternative medicine without scientific studies and

Currently the forensic study over confiscated samples of *C. sativa* L., is based on the qualitative determination of THC, present in extracts obtained from plant samples, without performing an quantitative level analysis of that component and some other cannabinoids present in seized samples. As a result of the above at present there is no systematic information to determine the content of THC present in plant samples of *C. sativa* illicitly traded in Colombia, there is just an essential information for monitoring the potential levels

The Dirección Nacional de Estupefacientes (DNE) of Colombian government has reported about the results achieved in Colombia in the fight against drug trafficking (2002, 2007). Florian et al. reported higher content of THC in vegetable samples of Colombian cannabis

This paper shows the GC as an important tool in the analysis of forensic samples, specifically in the determination of cannabinoids. Leaf samples of *C. sativa* L. in four regions of Colombia were analyzed by GC. The analyzed region were (i) Northern region (NR) at Magdalena department, (ii) Southern region (SR) at Cauca department, (iii) Eastern region (ER) at Meta department and (iv) Central region (CR) at Caldas department, see Figure 2. The analysis revealed differences in the cannabinoid content of the samples under study

without institutional control (Fernandez-Alonso et al, 2007).

of THC present in plant samples who smoke marijuana consumers.

(2009).

according to their origin region.

Fig. 2. Geographic origin of the *C. sativa* L. samples.

To optimize the analytic method cannabis leaves from NR were used. The first objective was the optimization of the extraction method and the selection of the analysis method. Then, the sample quantity and quantification method were defined. After, the analytic method was validated.

### **3.1 Optimization of the extraction method and the selection of the analysis method**

In order to obtain extracts of cannabinoids from plant material samples, De Meijer et al. method was taken as a based because of its quickness, efficiency and reproducibility, this method use extraction with ethylalcohol (EtOH), ultrasound and centrifugation at 4000 rpm (2003). The extraction time (t= 5, 10 and 15 min) and the number of extractions with EtOH (n= 1, 3 and 5) were considered as factors; for each extraction 750 μL were used and graduated to 5.00 mL. Using a 32 factorial model, cannabis leaves were subjected to each one of the extraction conditions, in triplicate, and at the central point (t= 10 min, n= 3) there were done five triplicates. The extracts obtained were analyzed by GC with different forms of detection: GC-FID, GC-MS scan mode and GC-MS Selected Ion Monitoring (SIM) mode. In all cases the response variable was the chromatographic area corresponding to each of the cannabinoids detected.

By comparing the coefficients of variation (C.V.) of the response surfaces for each analyte, based on the detection methods considered, we observed that the detection by GC-FID showed the lowest variation, see Table 1. Therefore, it was decided to use this method for quantitative analysis of cannabinoids, the GC-MS analysis was used for the qualitative identification of cannabinoids in the extracts.

Table 1. Coefficient of variation of the response surface for CBD and THC.

Based on the response obtained by GC-FID, the ANOVA for the quadratic regression model for each analyte, indicated that the difference was significant with a low probability value to a level of 0.05 (for the CBD pmodel = 0.0003 < α and the THC pmodel = 0.0105 < α). In that way, the models generated by the equations 2 and 3 explained satisfactorily the response in terms of the considered factors (time (t), number of extractions (n)), n being the most significant factor for the models.

Figure 3a shows the generated response surface to optimize the extraction conditions of THC, in it is observed that the response increased with the extraction time and the number of extractions. On the other hand, Figure 3b shows that the standard error associated with the analysis model, increased in the extreme values.

$$\text{CBD area} = 9050 + 169^{\circ}\text{t} + 1944^{\circ}\text{n} \tag{2}$$

$$\text{THC area} = \text{31518 - 2200\*t + 10177\*n + 123\*t2 - 1008\*n2 - 35\*t\*n} \tag{3}$$

Gas Chromatography in Forensic Chemistry:

response.

standard method DPH was added to the vegetal sample.

the internal standard method, Figures 4a and 4c (R2CBD = 0.9843; R2

response for CBD and THC, as is evidenced in Figures 4b and 4d.

Cannabinoids Content in Marijuana Leaves (*Cannabis sativa* L.) from Colombia 201

of experiments DPH at 100 ppm was used as internal and surrogate standards. In the method of internal standard DPH was added to the extract obtained, while in the surrogate

The results were analyzed by ANOVA, evaluating for each of the quantifying methods the confidence interval and the response factor for each of the cannabinoids depending on the

The linear fit showed that there was a greater linearity in the results obtained by the method of surrogate standard, Figures 4b and 4d (R2CBD = 0.9934; R2THC = 0.9941) compared against

the surrogate standard method for both CBD and THC showed less dispersion in the

From this it could be concluded that the method of surrogate standard allows more accurate answers because it offers a lower confidence interval. Regarding the amount of sample to be extracted, it was found that using 100 mg of vegetal material minimized the error in the

a) CBD internal standard method, b) CBD surrogate standard method, c) THC internal standard

Fig. 4. Linear fit for the chromatographic response depending on the quantity method of

method, d) THC surrogate standard method.

sample

THC = 0.8562). In addition,

amount of sample, the results obtained for CBD and THC are presented in Figure 4.

(a) Optimizing the extraction of THC, (b) Standard error associated with the THC RSA.

To maximize the response of each analyte with the lowest associated error, the model suggested ten solutions, which found that the optimum conditions for extraction suggested by the quadratic model based on response time (t) and the number of extractions (n) for the analysis of CBD and THC by GC-FID were three extractions with ethanol for a period of 7.5 minutes, see Table 2. These conditions suggested by the model were experimentally verified for ensuring that a fourth extraction does not provides betters detectable levels of CBD and THC.

Table 2. Solutions suggested by the model for the CBD and THC extractions.

### **3.2 Sample quantity and quantification method**

Once the method of analysis, time and number of extractions were defined, we proceeded to determine the mass of sample and the quantification method that would be used. For this there was prepared by triplicate, two extract sets: i) internal standard and ii) surrogate standard, using in each case 25.0, 50.0, 100.0 and 200.0 mg of vegetal material. For this series

(a) Optimizing the extraction of THC, (b) Standard error associated with the THC RSA.

To maximize the response of each analyte with the lowest associated error, the model suggested ten solutions, which found that the optimum conditions for extraction suggested by the quadratic model based on response time (t) and the number of extractions (n) for the analysis of CBD and THC by GC-FID were three extractions with ethanol for a period of 7.5 minutes, see Table 2. These conditions suggested by the model were experimentally verified for ensuring that a fourth extraction does not provides betters detectable levels of CBD and

**Solutions CBD THC**

Table 2. Solutions suggested by the model for the CBD and THC extractions.

Once the method of analysis, time and number of extractions were defined, we proceeded to determine the mass of sample and the quantification method that would be used. For this there was prepared by triplicate, two extract sets: i) internal standard and ii) surrogate standard, using in each case 25.0, 50.0, 100.0 and 200.0 mg of vegetal material. For this series

**3.2 Sample quantity and quantification method**

1 7.53 2.83 6.42 4.16 2 6.21 1.71 7.48 3.01 3 13.45 1.76 9.25 2.99 4 9.83 1.99 12.68 2.18 5 12.29 3.52 14.94 1.93 6 10.05 3.46 8.03 4.41 7 5.50 2.90 5.60 1.91 8 12.83 3.24 7.26 4.27 9 11.11 1.08 8.43 3.26 10 5.55 1.71 9.38 1.03

**t n t n**

Fig. 3. Response surface analysis (RSA)

THC.

of experiments DPH at 100 ppm was used as internal and surrogate standards. In the method of internal standard DPH was added to the extract obtained, while in the surrogate standard method DPH was added to the vegetal sample.

The results were analyzed by ANOVA, evaluating for each of the quantifying methods the confidence interval and the response factor for each of the cannabinoids depending on the amount of sample, the results obtained for CBD and THC are presented in Figure 4.

The linear fit showed that there was a greater linearity in the results obtained by the method of surrogate standard, Figures 4b and 4d (R2CBD = 0.9934; R2THC = 0.9941) compared against the internal standard method, Figures 4a and 4c (R2 CBD = 0.9843; R2 THC = 0.8562). In addition, the surrogate standard method for both CBD and THC showed less dispersion in the response.

From this it could be concluded that the method of surrogate standard allows more accurate answers because it offers a lower confidence interval. Regarding the amount of sample to be extracted, it was found that using 100 mg of vegetal material minimized the error in the response for CBD and THC, as is evidenced in Figures 4b and 4d.

a) CBD internal standard method, b) CBD surrogate standard method, c) THC internal standard method, d) THC surrogate standard method.

Fig. 4. Linear fit for the chromatographic response depending on the quantity method of sample

Gas Chromatography in Forensic Chemistry:

has good accuracy for each of the analytes.

**(m) SDm Intercept**

does not exceeded the tabulated value, as summarized in Table 6.

**Slope**

using DPH as surrogate standard.

THC y CBN.

Cannabinoids Content in Marijuana Leaves (*Cannabis sativa* L.) from Colombia 203

with a concentration of 100.15 ppm of DPH. With linear regression the curves were

For each one of the cannabinoids the slopes or the intercepts does not differ significantly, so the equations of the curves for CBD, THC and CBN were calculated from average values in Table 5. To eliminate the possibility of systematic errors the slopes and correlation factors were compare with those obtained by forcing the calibration curve through the origin, in both cases we obtain values of slope and correlation factor without significant differences. To determine the accuracy or the dispersion of measurements around their average value a series of ten measurements in intermediate precision conditions were made (varying the scientist) on solutions of cannabinoids of interest with DPH within the linear ranges established. For the three analytes considered, at all levels, data from the relative standard deviation are lower than those predicted by the Horwitz equation (PRSD (R) = 2C-0.15), indicating that the dispersions obtained experimentally are within acceptable ranges for each concentration. Furthermore the values obtained for HORRAT coefficients vary from 0.26 to 0.68 for CBD, between 0.29 to 1.08 for THC and from 0.32 to 0.72 for CBN, for the three cannabinoids these ratios are within the recommended range for intra-laboratory trials; values between 0.3 and 1.3. With this it can be concluded that the method of analysis

**(yb) SDb Error y**

High Medium Low

**CBD** 0.7480 0.0040 -0.1080 0.0075 0.0318 0.9981 0.1722 **THC** 0.6000 0.0050 -0.0246 0.0081 0.0376 0.9954 4.2504 **CBN** 1.0990 0.0053 -0.0577 0.0098 0.0422 0.9984 0.5899

For the three cannabinoids considered when applying the t-student test, comparing the data obtained with the tabulated critical value for nine degrees of freedom at a confidence level of 95%, we can say that the accuracy is acceptable because the three levels of concentration

**CBD** texp 1.973 2.046 2.184

**THC** texp 1.304 2.198 1.723

**CBN** texp 1.113 1.773 0.392

Moreover, the recoveries percentages and the relative deviation for the analytes, the low, medium and high, are within the ranges permitted for a nominal component that is greater

Table 6. Comparison t-student values for assessment of accuracy for the analysis of CBD,

t tab 95% 2.262 2.262 2.262

t tab 95% 2.262 2.262 2.262

t tab 95% 2.262 2.262 2.262

Table 5. Parameters for the linear calibration curves estimation for CBD, THC and CBN

Analyte **Level**

**SDxy R Error Xo**

**SDxo**

evaluated to obtain the correlation coefficient, the slope and the intercept.

Based on these arguments it was determined that the amount of sample to be used was of 100 mg and for quantification of cannabinoids by GC-FID was convenient to use the method of surrogate standard.

### **3.3 Validation of analytic method**

Regarding the validation of CBD, THC and CBN determination method by GC-FID: (i) it was determined the repeatability of the retention times of the analytes and the standard, (ii) there was calculated the chromatographic parameters (iii) the linearity was established for each analyte, (iv) precision, (v) accuracy, (vi), Limit of Detection (LOD) and Limit of Quantification (LOQ).

Retention times (tr) of DPH (compound used as surrogate standard) and the cannabinoids CBD, THC and CBN, showed an excellent repeatability, the tr of these had C.V. less than 1%, see Table 3.

Table 3. Characterization of the retention time for DPH, CBD, THC and CBN injected into the mixture.

Table 4 shows the chromatographic parameters of the cannabinoids by GC-FID. The relative tr (trr) were calculated regarding on the tr of THC.

The CBN has a response factor (RF) higher compared against the CBD and THC, compounds that exhibit relatively similar RF. Resolution (Rs) values obtained are higher than the acceptance criterion of 1.5, so the method can be considered as selective for the analytes considered. The CBN is the compound most congenial with the stationary phase and therefore it is more retained in this (K= 4.25) and the DPH has a lower retention (K= 1.04), being the compound with less affinity with the stationary phase. The highest number of theoretical plates (N) required for the separation correspond to CBN, followed in order by CBD, THC and DPH.

Table 4. Validation of chromatographic parameters for DPH, CBD, THC y CBN.

To assess the linearity in the quantification of cannabinoids of interest, ten calibration curves in the linear range were prepared (approximately 15 to 300 ppm), each point on the line

Based on these arguments it was determined that the amount of sample to be used was of 100 mg and for quantification of cannabinoids by GC-FID was convenient to use the method

Regarding the validation of CBD, THC and CBN determination method by GC-FID: (i) it was determined the repeatability of the retention times of the analytes and the standard, (ii) there was calculated the chromatographic parameters (iii) the linearity was established for each analyte, (iv) precision, (v) accuracy, (vi), Limit of Detection (LOD) and Limit of

Retention times (tr) of DPH (compound used as surrogate standard) and the cannabinoids CBD, THC and CBN, showed an excellent repeatability, the tr of these had C.V. less than

**Compound Replicas of tr (min) Avg SD C.V.**

Table 3. Characterization of the retention time for DPH, CBD, THC and CBN injected into

Table 4 shows the chromatographic parameters of the cannabinoids by GC-FID. The relative

The CBN has a response factor (RF) higher compared against the CBD and THC, compounds that exhibit relatively similar RF. Resolution (Rs) values obtained are higher than the acceptance criterion of 1.5, so the method can be considered as selective for the analytes considered. The CBN is the compound most congenial with the stationary phase and therefore it is more retained in this (K= 4.25) and the DPH has a lower retention (K= 1.04), being the compound with less affinity with the stationary phase. The highest number of theoretical plates (N) required for the separation correspond to CBN, followed in order by

**DPH** 2.04 543.6 28810.0 0.112 0.44 14.06 1.54 1.04 5290.8 **CBD** 3.92 576.2 11019.0 0.156 0.85 3.89 2.60 2.92 10111.4 **THC** 4.61 347.1 12178.9 0.197 1.00 3.35 3.30 3.61 8755.4 **CBN** 5.25 897.8 29055.0 0.184 1.14 4.25 13009.2

To assess the linearity in the quantification of cannabinoids of interest, ten calibration curves in the linear range were prepared (approximately 15 to 300 ppm), each point on the line

Table 4. Validation of chromatographic parameters for DPH, CBD, THC y CBN.

**DPH** 2.04 2.02 2.04 2.05 2.03 2.04 2.04 0.010 0.507 **CBD** 3.93 3.91 3.90 3.95 3.91 3.93 3.92 0.018 0.468 **THC** 4.60 4.59 4.62 4.60 4.61 4.63 4.61 0.015 0.319 **CBN** 5.25 5.25 5.23 5.24 5.26 5.25 5.25 0.010 0.197

**1 2 3 4 5 6 (%)**

**(s) trr Rs K N**

of surrogate standard.

Quantification (LOQ).

1%, see Table 3.

the mixture.

CBD, THC and DPH.

**3.3 Validation of analytic method**

tr (trr) were calculated regarding on the tr of THC.

**Compound tr Area RF Area/g Wb**

with a concentration of 100.15 ppm of DPH. With linear regression the curves were evaluated to obtain the correlation coefficient, the slope and the intercept.

For each one of the cannabinoids the slopes or the intercepts does not differ significantly, so the equations of the curves for CBD, THC and CBN were calculated from average values in Table 5. To eliminate the possibility of systematic errors the slopes and correlation factors were compare with those obtained by forcing the calibration curve through the origin, in both cases we obtain values of slope and correlation factor without significant differences.

To determine the accuracy or the dispersion of measurements around their average value a series of ten measurements in intermediate precision conditions were made (varying the scientist) on solutions of cannabinoids of interest with DPH within the linear ranges established. For the three analytes considered, at all levels, data from the relative standard deviation are lower than those predicted by the Horwitz equation (PRSD (R) = 2C-0.15), indicating that the dispersions obtained experimentally are within acceptable ranges for each concentration. Furthermore the values obtained for HORRAT coefficients vary from 0.26 to 0.68 for CBD, between 0.29 to 1.08 for THC and from 0.32 to 0.72 for CBN, for the three cannabinoids these ratios are within the recommended range for intra-laboratory trials; values between 0.3 and 1.3. With this it can be concluded that the method of analysis has good accuracy for each of the analytes.

Table 5. Parameters for the linear calibration curves estimation for CBD, THC and CBN using DPH as surrogate standard.

For the three cannabinoids considered when applying the t-student test, comparing the data obtained with the tabulated critical value for nine degrees of freedom at a confidence level of 95%, we can say that the accuracy is acceptable because the three levels of concentration does not exceeded the tabulated value, as summarized in Table 6.

Table 6. Comparison t-student values for assessment of accuracy for the analysis of CBD, THC y CBN.

Moreover, the recoveries percentages and the relative deviation for the analytes, the low, medium and high, are within the ranges permitted for a nominal component that is greater

Gas Chromatography in Forensic Chemistry:

SR, c) ER, d) CR.

Cannabinoids Content in Marijuana Leaves (*Cannabis sativa* L.) from Colombia 205

intermediate cannabinoid (0.83-1.94% 1.36% avg), see Figure 5. In the Table 8 cannabinoid

Fig. 5. Cannabinoids content in samples collected in different areas of Colombia a) NR, b)

**NR SR ER CR Cannabinoid Avg**. SD C.V. **Avg**. SD C.V. **Avg**. SD C.V. **Avg**. SD C.V. CBD **0.91** 0.550 60.6 **2.52** 1.441 57.2 **1.86** 1.166 62.8 **0.02** 0.020 108.9 THC **2.81** 0.792 28.2 **10.98** 3.075 28.0 **15.74** 1.340 8.5 **1.87** 0.574 30.7 CBN **0.05** 0.005 10.3 **0.11** 0.040 36.2 **0.68** 0.080 11.8 **1.36** 0.366 27.0 **Phenotype 4.14** 2.233 53.9 **7.41** 7.035 94.9 **11.82** 5.504 46.5 **414.52** 354.087 85.4

Table 8. Cannabinoids content in samples collected in different areas of Colombia.

content averages and phenotype averages of each studied region are shown.

than 10% in a recovery interval from 98 to 102%.These two tests allow us to conclude that the method has the accuracy required for low, medium and high ranges , for the analysis of these analytes, without evidence of systematic errors.

For each analyte, the linear range, the calibration curve, the LOD and the LOQ are presented in Table 7. The detection limits have very similar values for the three cannabinoids, however the lower sensitivity for the detection of THC is due because this compound has a higher limit of quantification in relation to the CBD and CBN.

Regarding this part of the work we can conclude: (i) the optimal conditions for extraction suggested by the quadratic model based on response time (t) and the number of extractions (n) for the extraction of CBD and THC from a Cannabis plant sample; (ii) the method for quantification of CBD and THC, using DPH as a surrogate standard, showed an excellent repeatability (coefficient of variation for the retention time of all substances was less than 1%); (iii) in addition, linear behavior was observed for the quantification of CBD, THC and CBN using DPH as standard in the following ranges: CBD between 19.6 to 315.2 ppm, THC between 13.2 to 287.6 ppm and for CBN 16.4 to 313.7 ppm.

Table 7. Detection and quantification limits for CBD, THC y CBN.

The results experimentally obtained allow to conclude that the analysis method for CBD, THC and CBN had a very good accuracy and the required exactitude at different concentration levels, with no systematic error evidence.

Otherwise, the detection limits using DPH as surrogate standard were of 1,02 ng for CBD, 1,06 ng for THC and 1,06 ng for CBN. The quantification limits were of 1,68 ng for CBD, 2,44 ng for THC y 1,76 ng for CBN.

### **4. Profile analysis of cannabinoids in samples collected in four areas of illicit cultivation of cannabis**

In order to determine the profile of cannabinoids in samples from four different regions of Colombia, were sampled four regions, in each region thirteen samples were taken, each was analyzed by triplicate. The areas sampled were: (i) Northern region (NR), (ii) Southern region (SR) , (iii) Eastern region (ER) and (iv) Central region (CR), see Figure 2.

In all samples of the four regions of the cannabinoid THC was the majority (NR 1.89-4.00% average 2.81%, SR 6.72-15.48% avg 10.98%, ER 13.37-17.63% avg 15.74%, CR 1.11-3.17% avg 1.87%). In regions NR, SR and ER the minor cannabinoid was CBN (NR 0.04-0.06% avg 0.05%, SR 0.05-0.17% avg 0.11%, ER 0.56-0.80% 0.68% avg), with the CBD as the intermediate (NR 0.29-2.40% avg 0.91%, SR 0.60-4.86% avg 2.52%, ER 0.92-4.14% 1.86% avg). Unlike the three previous regions, in CR CBD was minority (0.01-0.06% avg 0.02%) and CBN was the

than 10% in a recovery interval from 98 to 102%.These two tests allow us to conclude that the method has the accuracy required for low, medium and high ranges , for the analysis of

For each analyte, the linear range, the calibration curve, the LOD and the LOQ are presented in Table 7. The detection limits have very similar values for the three cannabinoids, however the lower sensitivity for the detection of THC is due because this compound has a

Regarding this part of the work we can conclude: (i) the optimal conditions for extraction suggested by the quadratic model based on response time (t) and the number of extractions (n) for the extraction of CBD and THC from a Cannabis plant sample; (ii) the method for quantification of CBD and THC, using DPH as a surrogate standard, showed an excellent repeatability (coefficient of variation for the retention time of all substances was less than 1%); (iii) in addition, linear behavior was observed for the quantification of CBD, THC and CBN using DPH as standard in the following ranges: CBD between 19.6 to 315.2 ppm, THC

Parameter CBD THC CBN Lineal Range ppm 19.6 – 315.2 13.2 – 287.6 16.4 – 313.7 Number of levels 7 7 7

Calibration curve y=0.7480x-0.1080 y=0.6000x-0.0246 y=1.0990x-0.0577 LOD (ng) 1.02 1.06 1.06 LOQ (ng) 1.68 2.44 1.76

The results experimentally obtained allow to conclude that the analysis method for CBD, THC and CBN had a very good accuracy and the required exactitude at different

Otherwise, the detection limits using DPH as surrogate standard were of 1,02 ng for CBD, 1,06 ng for THC and 1,06 ng for CBN. The quantification limits were of 1,68 ng for CBD, 2,44

**4. Profile analysis of cannabinoids in samples collected in four areas of illicit**

In order to determine the profile of cannabinoids in samples from four different regions of Colombia, were sampled four regions, in each region thirteen samples were taken, each was analyzed by triplicate. The areas sampled were: (i) Northern region (NR), (ii) Southern

In all samples of the four regions of the cannabinoid THC was the majority (NR 1.89-4.00% average 2.81%, SR 6.72-15.48% avg 10.98%, ER 13.37-17.63% avg 15.74%, CR 1.11-3.17% avg 1.87%). In regions NR, SR and ER the minor cannabinoid was CBN (NR 0.04-0.06% avg 0.05%, SR 0.05-0.17% avg 0.11%, ER 0.56-0.80% 0.68% avg), with the CBD as the intermediate (NR 0.29-2.40% avg 0.91%, SR 0.60-4.86% avg 2.52%, ER 0.92-4.14% 1.86% avg). Unlike the three previous regions, in CR CBD was minority (0.01-0.06% avg 0.02%) and CBN was the

region (SR) , (iii) Eastern region (ER) and (iv) Central region (CR), see Figure 2.

these analytes, without evidence of systematic errors.

higher limit of quantification in relation to the CBD and CBN.

between 13.2 to 287.6 ppm and for CBN 16.4 to 313.7 ppm.

Table 7. Detection and quantification limits for CBD, THC y CBN.

concentration levels, with no systematic error evidence.

ng for THC y 1,76 ng for CBN.

**cultivation of cannabis**

intermediate cannabinoid (0.83-1.94% 1.36% avg), see Figure 5. In the Table 8 cannabinoid content averages and phenotype averages of each studied region are shown.

Fig. 5. Cannabinoids content in samples collected in different areas of Colombia a) NR, b) SR, c) ER, d) CR.

Table 8. Cannabinoids content in samples collected in different areas of Colombia.

Gas Chromatography in Forensic Chemistry:

this relationship is shown in Figure 7.

Fig. 7. THC vs THC/CBN diagram for the four analyzed regions.

ionization detector GC-FID, validating the respective analytic method.

source of the region of origin of the samples analyzed.

**5. Conclusions**

By the way the ratio THC vs THC / CBN is obtain this could be an appropriate tool to discriminate between the studied samples. SR was the most dispersed group, CR was the less dispersed group, these samples had antagonistic behavior. NR and ER had antagonistic behavior too, these were the medium dispersed groups. Through this relationship we obtain four sample distinct groups, it could be the most appropriate criteria and discriminate

The present study analyzed the cannabinoids content in samples of *Cannabis sativa* L. cultivated illicitly in Colombia. The physicochemical conditions were optimized for the extraction and quantification of cannabidiol (CBD), Δ9-tetrahidrocannabinol (THC) and cannabinol (CBN) starting from a vegetable sample using gas chromatography with flame

Cannabinoids Content in Marijuana Leaves (*Cannabis sativa* L.) from Colombia 207

this could have a greater discriminatory power as the criterion of origin of the samples under study. Through this relationship we obtain four distinct groups being the most appropriate criteria and discriminate source for the region of origin of the analyzed samples,

With respect to THC, the substance which presents psychoactive effects and potentially can generate dependence in consumers, samples from ER and SR showed the highest content (15.74% and 10.98%, respectively), much higher than those reported for THC in samples from Colombia (Baker et al., 1980). The NR (2.81%) and CR (1.87%) showed lower values of THC.

The high content of THC in samples ER and SR regions could indicate the use of improved varieties of cannabis, whereas worldwide are certified varieties without genetic manipulation with less than 7% THC (Montero 2008), which comes great concern about the higher drug potential effects among consumers (Licata et al., 2005).

Phenotype values calculated according to Waller for each group of samples were found within a given range for different regions, see Figure 6. These results are consistent with data published by Turner et al. (1979), who noted that the variability in the content of cannabinoids in plant samples difficult phenotypic classification based on the result of a single analysis, and additionally Mechtler et al. (2004) found that THC may have an erratic and in some plants with hemp can be outside of the characteristic values for the population farmed.

Fig. 6. Variation ranges of the phenotype for the analyzed samples of North region (NR), South region (SR), East region (ER) and Central region (CR).

The results of the minimum and maximum phenotype values calculated according to Waller relationship, show that the ranges obtained overlap for three of the considered regions, this is NR (1.72-7.68), SR (1.39-23.08) and ER (3.56-19.54). As a criterion for differentiating the only group whose phenotype was different correspond to samples collected in the CR (40.09- 1094.50), see Figure 6.

Given the above, the Waller relationship is not useful for defining geographical origin of the samples under study. In contrast, when considering the relationship THC vs THC/CBN, this could have a greater discriminatory power as the criterion of origin of the samples under study. Through this relationship we obtain four distinct groups being the most appropriate criteria and discriminate source for the region of origin of the analyzed samples, this relationship is shown in Figure 7.

Fig. 7. THC vs THC/CBN diagram for the four analyzed regions.

By the way the ratio THC vs THC / CBN is obtain this could be an appropriate tool to discriminate between the studied samples. SR was the most dispersed group, CR was the less dispersed group, these samples had antagonistic behavior. NR and ER had antagonistic behavior too, these were the medium dispersed groups. Through this relationship we obtain four sample distinct groups, it could be the most appropriate criteria and discriminate source of the region of origin of the samples analyzed.

### **5. Conclusions**

206 Gas Chromatography – Biochemicals, Narcotics and Essential Oils

With respect to THC, the substance which presents psychoactive effects and potentially can generate dependence in consumers, samples from ER and SR showed the highest content (15.74% and 10.98%, respectively), much higher than those reported for THC in samples from Colombia (Baker et al., 1980). The NR (2.81%) and CR (1.87%) showed lower values of

The high content of THC in samples ER and SR regions could indicate the use of improved varieties of cannabis, whereas worldwide are certified varieties without genetic manipulation with less than 7% THC (Montero 2008), which comes great concern about the

Phenotype values calculated according to Waller for each group of samples were found within a given range for different regions, see Figure 6. These results are consistent with data published by Turner et al. (1979), who noted that the variability in the content of cannabinoids in plant samples difficult phenotypic classification based on the result of a single analysis, and additionally Mechtler et al. (2004) found that THC may have an erratic and in some plants with hemp can be outside of the characteristic values for the population

Fig. 6. Variation ranges of the phenotype for the analyzed samples of North region (NR),

The results of the minimum and maximum phenotype values calculated according to Waller relationship, show that the ranges obtained overlap for three of the considered regions, this is NR (1.72-7.68), SR (1.39-23.08) and ER (3.56-19.54). As a criterion for differentiating the only group whose phenotype was different correspond to samples

Given the above, the Waller relationship is not useful for defining geographical origin of the samples under study. In contrast, when considering the relationship THC vs THC/CBN,

South region (SR), East region (ER) and Central region (CR).

collected in the CR (40.09- 1094.50), see Figure 6.

higher drug potential effects among consumers (Licata et al., 2005).

THC.

farmed.

The present study analyzed the cannabinoids content in samples of *Cannabis sativa* L. cultivated illicitly in Colombia. The physicochemical conditions were optimized for the extraction and quantification of cannabidiol (CBD), Δ9-tetrahidrocannabinol (THC) and cannabinol (CBN) starting from a vegetable sample using gas chromatography with flame ionization detector GC-FID, validating the respective analytic method.

Gas Chromatography in Forensic Chemistry:

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Additionally, variation in the contents of CBD, THC and CBN in the thirteen samples collected in each region implies that the value of the Waller phenotype for each group of samples was not found around a value but becomes an interval for each source. The numerical value of this phenotype, by itself, did not serve as a criterion for differentiation on the geographical origin of the samples, due to overlapping ranges for three of the regions concerned, this is North region (NR), SR and ER. The most appropriate criteria and discriminate source for the region of origin of the four groups of samples analyzed was plotting the ratio THC vs THC / CBN, as this allows to distinguish four distinct groups.

### **6. Acknowledgment**

Grateful acknowledgements are expressed to the Dirección de Investigación sede Bogotá (DIB) of the Universidad Nacional de Colombia for their financial support (DIB project No. 8003170) and the Departamento Administrativo de Seguridad (DAS) and the Fiscalía General de la Nación for their technique support.

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335-346.

Bogotá-Colombia.

de Justicia. Bogotá-Colombia.

General de la Nación for their technique support.

**Part 3**

**Essential Oils**

Turner, C.; Elsohly, M.; Cheng, P. & Lewis, G. (1979). Constituents of Cannabis sativa L. XIV. Intrinsic problems in classifying Cannabis based on a single cannabinoid analysis. *Journal of Natural Products,* Vol. 42, pp. 317-319.

**Part 3**

**Essential Oils**

210 Gas Chromatography – Biochemicals, Narcotics and Essential Oils

Turner, C.; Elsohly, M.; Cheng, P. & Lewis, G. (1979). Constituents of Cannabis sativa L.

analysis. *Journal of Natural Products,* Vol. 42, pp. 317-319.

XIV. Intrinsic problems in classifying Cannabis based on a single cannabinoid

**10**

*Morocco*

**Chemical Composition, Antioxidant and**

**Antimicrobial Activities of Essential Oil of**

*Warionia saharae* **from Oases of Morocco**

The *Warionia saharae* which belongs to the important composite's family is an endemic species of North Africa, characterized by a discerning odour (Bonnet and Maurry, 1889). *Warionia saharae* was reported for the first time in the Oranais Sahara (Beni ounif in Algeria) by Dr.Warrion as a shrub of 1 to 3 m of height. The thick trunk, is covered of a gray peel, structural of very wavy terminal leaf bouquets, and of capitulate of yellow flowers (Photos 1 and 2). The picking of stems leafed of this bush, clear a very heady and spicy odour; the latex that flows out of injuries of the peel, glue to hands in a very tenacious way (Lebrun, 1979).

In Morocco, *Warionia saharae* is growing wild in various regions (Benabid and Fennane, 1994). The habitat is between schistose rocks (Watillon et al., 1988). *Warionia saharae* is known in Morocco by the Berber vernacular names of "âfessas" and "Tazart n-îfiss". In the Moroccan traditional medicine, the leaves of the plant are used to treat inflammatory diseases, such as rheumatoid arthritis, and for gastrointestinal disorders (Bellakhdar, 1997), inflammation of the womb, colds, Jaundice and cardiac pains (El Rhaffari et Zaid, 2002).

This work aims to study and characterise a new bioactive natural products from medicinal plants of Moroccan oases. Information concerning in vitro antioxidant, antimicrobial

The aerial part of *Warionia saharae* was collected in Er-rachidia (Morocco), during the flowering period (April/June, 2009). A duplicate specimen is held at the FST Er-rachidia. The dried plant material is stored in the laboratory at room temperature (25°C) and in the

activities of the essential oil from the *Warionia saharae* has not been reported earlier.

**1. Introduction**

**2. Materials and methods**

shade before the extraction.

**2.1 Plant material**

K. Sellam1, M. Ramchoun2, C. Alem2, Farid Khallouki2

*Faculty of the Sciences and Technology, Er-Rachidia Morocco,*

*Faculty of the Sciences and Technology, Er-Rachidia Morocco,*

B. El Moualij2 and L. El Rhaffari1\*

*1Environment and Health Research Team (EHRT),*

*2Reseach Team in Natural Product Biochemistry,*

## **Chemical Composition, Antioxidant and Antimicrobial Activities of Essential Oil of**  *Warionia saharae* **from Oases of Morocco**

K. Sellam1, M. Ramchoun2, C. Alem2, Farid Khallouki2 B. El Moualij2 and L. El Rhaffari1\* *1Environment and Health Research Team (EHRT), Faculty of the Sciences and Technology, Er-Rachidia Morocco, 2Reseach Team in Natural Product Biochemistry, Faculty of the Sciences and Technology, Er-Rachidia Morocco, Morocco*

### **1. Introduction**

The *Warionia saharae* which belongs to the important composite's family is an endemic species of North Africa, characterized by a discerning odour (Bonnet and Maurry, 1889). *Warionia saharae* was reported for the first time in the Oranais Sahara (Beni ounif in Algeria) by Dr.Warrion as a shrub of 1 to 3 m of height. The thick trunk, is covered of a gray peel, structural of very wavy terminal leaf bouquets, and of capitulate of yellow flowers (Photos 1 and 2). The picking of stems leafed of this bush, clear a very heady and spicy odour; the latex that flows out of injuries of the peel, glue to hands in a very tenacious way (Lebrun, 1979).

In Morocco, *Warionia saharae* is growing wild in various regions (Benabid and Fennane, 1994). The habitat is between schistose rocks (Watillon et al., 1988). *Warionia saharae* is known in Morocco by the Berber vernacular names of "âfessas" and "Tazart n-îfiss". In the Moroccan traditional medicine, the leaves of the plant are used to treat inflammatory diseases, such as rheumatoid arthritis, and for gastrointestinal disorders (Bellakhdar, 1997), inflammation of the womb, colds, Jaundice and cardiac pains (El Rhaffari et Zaid, 2002).

This work aims to study and characterise a new bioactive natural products from medicinal plants of Moroccan oases. Information concerning in vitro antioxidant, antimicrobial activities of the essential oil from the *Warionia saharae* has not been reported earlier.

### **2. Materials and methods**

### **2.1 Plant material**

The aerial part of *Warionia saharae* was collected in Er-rachidia (Morocco), during the flowering period (April/June, 2009). A duplicate specimen is held at the FST Er-rachidia. The dried plant material is stored in the laboratory at room temperature (25°C) and in the shade before the extraction.

Chemical Composition, Antioxidant and Antimicrobial

**2.3 Essential oil analysis**

**2.4 Antimicrobial activity**

method(Anonymous, 1995).

**2.5 Antioxidant activity**

2001; Tepe et al., 2004).

1996; Nilsson et al., 2005).

**2.2 Steam distillation apparatus and procedure**

sodium sulfate and stored at 4°C in the dark before analysis.

sample components was performed in the EI mode (70 eV).

the phytopathogenic fungi Penicillium sp. and *Candida albicans*.

the disc diffusion method (Anonymous, 1995).

assay was an average of three experiments.

preventing visible growth (May et al., 2000; Burt, 2004).

Activities of Essential Oil of *Warionia saharae* from Oases of Morocco 215

The extraction of essential oil of the aerial part of *Warionia saharae* was conducted by steam distillation in a Clevenger apparatus. The essential oil obtained was dried under anhydrous

Analyses of volatile compounds were run on a Trace GC Ultra gas chromatography equipped with a VB-5 capillary column (Methylpolysiloxane with 5% phenyl; 25 m x 0.2 mm; film thickness 0.2µm) was directly coupled to the mass spectrometer (Polaris Q. MS). Helium (1 mL/min) was used as carrier gas. The program was 2 min isothermal at 40 °C, then the temperature increased by 4 °C/min to 180 °C and 2 min isothermal at 180 °C. The injection port temperature was 250 °C and that of the detector was 280 °C. Ionization of

Kovat's retention indices were calculated using cochromatographed standards hydrocarbons. The individual compounds were identified by MS and their identity was confirmed by comparing their retention indices relatives to C8–C32 n-alkanes and by comparing their mass spectra and retention times with those of authentic samples or with

The antimicrobial activity was evaluated by paper disc diffusion and dilution methods against three selected Gram-positive and Gram-negative species: *Staphylococcus aureus* ATCC 29213, *Escherichia coli* ATCC 35218, *Pseudomonas aeruginosa* ATCC 27853, and against

The qualitative antimicrobial essay of the volatile fraction of W. saharae was carried out by

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of tested volatile fractions were determined using the Mueller Hinton broth (MHB) dilution

All samples were tested in triplicate. The MIC was defined as the lowest concentration

The antioxidant activity was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing ability (FRAP) and β-carotene bleaching method systems. Data collected for each

The method is based on the radical scavenging activity of the antioxidant (Mensor et al.,

The FRAP assay was performed as described by Benzie and Nilsson (Benzie and Strain,

The β -carotene method was carried out according to Shahidi technique (Shahidi et al., 2001).

data already available in the NIST library and literature (Adams, 1989 et 2001).

Photo 1. *Warionia saharae* (Plant) (Taken y El Rhaffari L.)

Photo 2. *Warionia saharae* (Flower) (Taken y El Rhaffari L.)

### **2.2 Steam distillation apparatus and procedure**

The extraction of essential oil of the aerial part of *Warionia saharae* was conducted by steam distillation in a Clevenger apparatus. The essential oil obtained was dried under anhydrous sodium sulfate and stored at 4°C in the dark before analysis.

### **2.3 Essential oil analysis**

214 Gas Chromatography – Biochemicals, Narcotics and Essential Oils

Photo 1. *Warionia saharae* (Plant) (Taken y El Rhaffari L.)

Photo 2. *Warionia saharae* (Flower) (Taken y El Rhaffari L.)

Analyses of volatile compounds were run on a Trace GC Ultra gas chromatography equipped with a VB-5 capillary column (Methylpolysiloxane with 5% phenyl; 25 m x 0.2 mm; film thickness 0.2µm) was directly coupled to the mass spectrometer (Polaris Q. MS). Helium (1 mL/min) was used as carrier gas. The program was 2 min isothermal at 40 °C, then the temperature increased by 4 °C/min to 180 °C and 2 min isothermal at 180 °C. The injection port temperature was 250 °C and that of the detector was 280 °C. Ionization of sample components was performed in the EI mode (70 eV).

Kovat's retention indices were calculated using cochromatographed standards hydrocarbons. The individual compounds were identified by MS and their identity was confirmed by comparing their retention indices relatives to C8–C32 n-alkanes and by comparing their mass spectra and retention times with those of authentic samples or with data already available in the NIST library and literature (Adams, 1989 et 2001).

### **2.4 Antimicrobial activity**

The antimicrobial activity was evaluated by paper disc diffusion and dilution methods against three selected Gram-positive and Gram-negative species: *Staphylococcus aureus* ATCC 29213, *Escherichia coli* ATCC 35218, *Pseudomonas aeruginosa* ATCC 27853, and against the phytopathogenic fungi Penicillium sp. and *Candida albicans*.

The qualitative antimicrobial essay of the volatile fraction of W. saharae was carried out by the disc diffusion method (Anonymous, 1995).

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of tested volatile fractions were determined using the Mueller Hinton broth (MHB) dilution method(Anonymous, 1995).

All samples were tested in triplicate. The MIC was defined as the lowest concentration preventing visible growth (May et al., 2000; Burt, 2004).

### **2.5 Antioxidant activity**

The antioxidant activity was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing ability (FRAP) and β-carotene bleaching method systems. Data collected for each assay was an average of three experiments.

The method is based on the radical scavenging activity of the antioxidant (Mensor et al., 2001; Tepe et al., 2004).

The FRAP assay was performed as described by Benzie and Nilsson (Benzie and Strain, 1996; Nilsson et al., 2005).

The β -carotene method was carried out according to Shahidi technique (Shahidi et al., 2001).

Chemical Composition, Antioxidant and Antimicrobial

fragrant odor of the oil (Figure 1 and Table 1).

6.63

**3.2 Antimicrobial activity**

RT: 0.00 - 45.02 SM: 7G

microorganism source

(Table 2).

0

20

40

60

Relative Abundance

80

100

E. coli P. aeruginosa S. aureus Candida albicans Penicillium sp.

experiments.

antibiotics.

**Nd,** not determined.

Nerolidol (17.95%), Linalool (16.79%), 1,8 cineole (6.12%).

12.78

10.68

Activities of Essential Oil of *Warionia saharae* from Oases of Morocco 217

The major components of *Warionia saharae* essential oils were β-Eudesmol (23.74%), Trans-

The high content of oxygenated identified compounds might explain the characteristic and

15.50 33.25

30.76

28.73

Fig. 1. Chromatogram *Warionia saharae* volatile fractions run on VB-5 capillary column.

18.13

0 5 10 15 20 25 30 35 40 45 Time (min)

22.56

/disc

**<sup>b</sup>** standards antibiotic; A10 : Ampicillin (Amp), N10 : Nystatin (Nys).

Table 2. Antimicrobial activity of *Warionia saharae* essential oil.

ATCC25922 ATCC27853 ATCC25923 BTCE BTCE

**BTCE,** Blood Transfusion Center Errachidia (Morocco).

Antimicrobial activities of *Warionia saharae* essential oil were evaluated by a paper disc diffusion method against bacterial strains including Gram+ and Gram-, and fungal strains

> 10µg /disc

**<sup>a</sup>** Diameter of the zone of inhibition (mm) including disk diameter of 6mm – average of three

**MIC,** minimum inhibitory concentration; values given as mg/ml for the essential oils and as µg/ml for

Inhibition (mm)a

15µg

Essential oil (EO) STAb(µg/disk)

5µg EO Amp Nys

16 18 14 Nd Nd Nd Nd Nd 22 18

38.34

42.28

NL: 1.67E7 TIC F: MS F309UATR S08GC1

/disc A10 N10

MIC (µg/ml)

> 25 5 10 Nd Nd

Nd Nd Nd 5 10

### **2.6 Statistical analysis**

For antioxidant assays the results were expressed as value±error using statistical analysis formulas referring to the value of p < 0.05 of confidence interval for the Student's t-test law.

### **3. Results and discussion**

### **3.1 Chemical composition of the essential oil**

The essential oil was extracted by the hydrodistillation of the dried leaves of *Warionia saharae* from Errachidia region (Morocco), were analyzed by GC-MS.

The essential oil yields, calculated on a dry weight basis, was about 1.1%(w/w). The identified combinations in essential oil, retention time (RT) and quantitative percentage of the compounds are presented in table 1. A total of 32 compounds, amounting 92.5% of the oils, were identified.

Total 92.5 a Compounds listed in order of elution from a DB-5 column. b Retention time (as minutes).

Table 1. Composition of essential oil of *Warionia saharae* (%).

The major components of *Warionia saharae* essential oils were β-Eudesmol (23.74%), Trans-Nerolidol (17.95%), Linalool (16.79%), 1,8 cineole (6.12%).

The high content of oxygenated identified compounds might explain the characteristic and fragrant odor of the oil (Figure 1 and Table 1).

Fig. 1. Chromatogram *Warionia saharae* volatile fractions run on VB-5 capillary column.

### **3.2 Antimicrobial activity**

216 Gas Chromatography – Biochemicals, Narcotics and Essential Oils

For antioxidant assays the results were expressed as value±error using statistical analysis formulas referring to the value of p < 0.05 of confidence interval for the Student's t-test law.

The essential oil was extracted by the hydrodistillation of the dried leaves of *Warionia saharae*

The essential oil yields, calculated on a dry weight basis, was about 1.1%(w/w). The identified combinations in essential oil, retention time (RT) and quantitative percentage of the compounds are presented in table 1. A total of 32 compounds, amounting 92.5% of the

**Peak number RTb Compounda Composition (%)**  1 6.63 α-Thujene 0.9 2 6.75 α-Pinene 0.6 3 10.19 Camphene 0.99 4 10.68 Sabinene 2.22 5 11.79 α-Phellandrene 1.50 6 12.24 α-Carene 0.60 7 12.54 p-Cymene 3.77 8 12.77 1,8-Cineole 6.12 9 13.80 3-Carene 0.77 10 14.88 α-Terpinene 0.41 11 15.49 Linalool 16.79 12 18.12 Terpinen-4-ol 3.40 13 18.60 α-Fenchol 2.59 14 18.86 Ethyl 8-fluorooctanoate 0.55 15 20.84 Geraniol 1.14 16 22.56 Carvacrol 1.34 17 23.92 Ethyl 3-phenylpropionate 0.85 18 23.99 Ocimenyl acetate 0.81 19 28.51 Tridecan-1-ol 0.55 20 28.73 Longipinane 2.05 21 29.45 Allo-Aromadendrene 0.44 22 30.20 α-Farnesene 0.51 23 30.75 Trans nerolidol 17.95 24 31.19 Caryophyllene oxide 1.91 25 33.24 β-Eudesmol 23.74

Total 92.5 a Compounds listed in order of elution from a DB-5 column. b Retention time (as minutes).

Table 1. Composition of essential oil of *Warionia saharae* (%).

**2.6 Statistical analysis**

oils, were identified.

**3. Results and discussion**

**3.1 Chemical composition of the essential oil**

from Errachidia region (Morocco), were analyzed by GC-MS.

Antimicrobial activities of *Warionia saharae* essential oil were evaluated by a paper disc diffusion method against bacterial strains including Gram+ and Gram-, and fungal strains (Table 2).

**<sup>a</sup>** Diameter of the zone of inhibition (mm) including disk diameter of 6mm – average of three experiments.

**<sup>b</sup>** standards antibiotic; A10 : Ampicillin (Amp), N10 : Nystatin (Nys).

**MIC,** minimum inhibitory concentration; values given as mg/ml for the essential oils and as µg/ml for antibiotics.

**BTCE,** Blood Transfusion Center Errachidia (Morocco). **Nd,** not determined.

Table 2. Antimicrobial activity of *Warionia saharae* essential oil.

Chemical Composition, Antioxidant and Antimicrobial

Press, San Diego.

Sc. Congrès de Paris.

**5. References**

Activities of Essential Oil of *Warionia saharae* from Oases of Morocco 219

Adams, R.P., 1989. Identification of Essential Oil by Ion Trap Mass Spectroscopy. Academic

Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatography/

Anonymous: National Committee for Clinical Laboratory, 1995. Standards methods for

Bellakhdar, J. (1997). La pharmacopée marocaine traditionnelle, Médecine arabe ancienne et

Benabid, A. and Fennane, M. (1994). Connaissance sur la végétation du Maroc: phytogéographie, phytosociologie et séries de végétations. Lazaroa 14: 21-97. Benzie, I.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as ameasure of

Bonnet and Maurry, P. (1889). Etude sur le *Warionia saharae*. Benth & coss. Assoc. Fr. Avanc.

Brehm-Stecher B F and Johnson E A. (2003). Sensitization of Staphylococcus aureus and

El Rhaffari L. et Zaid A. 2002 Pratique de la phytothérapie dans le sud-est du Maroc

Hicheri F, Ben Jannet H, Cheriaa J, Jegham S, & Mighri Z. (2003) Antibacterial activities of a

Lattaoui N, Tantaoui-Elaraki A. (1994). Individual and combined antibacterial activity of

Lebrun, J.P. (1979). Eléments pour un Atlas des plantes vasculaires de l'Afrique sèche, 2: 11-

May J, Chan CH, King A, Williams L, & French GL. (200). Time-kill studies of tea tree oils on

Mensor, L.L., Menezes, F.S., Leitão, G.G., Reis, A.S., Dos Santos, T.C., Coube, C.S., & Leitao,

Nilsson, J., Pillai, D., Önning, G., Persson, C., Nilsson, A., & Akesson, B., 2005. Comparison

in extracts of fruit and vegetables. Mol. Nutrit. Food Res. 49, 239–246.

and apritone. Antimicrobial Agents Chemotherapy 47(10): 3357–3360. Burt S. (2004). Essential oils: their antibacterial properties and potential applications in

Escherichia coli to antibiotics by the sesquiterpenoids nerolidol, farnesol, bisabolol

(Tafilalet). Un savoir empirique pour une pharmacopée rénovée. Actes du 4'ème congrès Européen d'Ethnopharmacologie: origine des pharmacopées traditionnelles et élaboration des pharmacopées savantes, Metz, 11-13 mai 2000.

few prepared derivatives of oleanolic acid and of other natural triterpenic

the main components of three thyme essential oils. Revista Italiana Eppos 13: 13-

S.G., 2001. Screening of Brazilian plant extracts for antioxidant activity by the use of

of the 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and ferric reducing antioxidant power (FRAP) methods to asses the total antioxidant capacity

"Antioxidant Power": The FRAP Assay. Anal. Biochem. 239, 70–76.

dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Wayne

QUADrupole Mass Spectroscopy. Allured, Illinois.

Pa., vol. 15, p. 15 (approved standards no. M7-A3).

foods-a review. Int J Food Microbiol. 94:223–53.

Publiés par le CRD Montpellier, 2002, pp 295-304.

clinical isolates. J Antimicrob Chemother.45:639–43.

DPPH free radical method. Phytotherapy Res. 15, 127–130.

compounds. C R Chim .6:473–83.

12. J.E.M.V.P.T., Bot 6.

19.

savoirs populaires, Ibis press, p. 208.

The comparison between in vitro bacteriostatic activity of essential oil of *Warionia saharae*, and the inhibition zone formed by standard antibiotic disc (Ampicillin (A10) and Nystatin (N10)) showed that the essential oils were more active against the microorganisms tested. These results suggest that the volatile oil of *Warionia saharae* would probably be a good therapeutic agent against these bacteria and fungus.

The bacteriostatic properties of the oil are suspected to be associated with the high Eudesmol and Nerolidol content, which has been tested previously and was found to have a significant antibiotic activity (Brehm-Stecher and Johnson, 2003; Lattaoui and Tantaoui-Elaraki, 1994).

### **3.3 Antioxidant activity**

The study of the antioxidant activities showed that the essential oil have a higher antioxidant capacity relative to the antioxidant of reference BHT and BHA (94.2 ± 0.4%).

In contrast to β-carotene/linoleic acid system, essential oil showed a moderate activity in this system (65.2 ± 0.4%).

The ferric reducing ability of plasma (FRAP) assay was used for assessing "antioxidant power" of *Warionia saharae* essential oil. The antioxidant power of the leaves oil samples were compared with BHA and BHT as reference antioxidants.

The results reported here can be considered as the first information on the antimicrobial and antioxidant properties of *Warionia saharae*. Further studies are needed to evaluate the in vivo potential of these oils in animal models.

### **4. Conclusion**

This work interested *Warionia saharae* from Er-rachidia region (south eastern Morocco). It showed that the essential oil yield is about 1.1% (w/w) in dried leaves.

The essential oils is analysed by GC-MS. Thirty two compounds were identified, they are amounting 92.5% of the oils. The major components of essential oils were ß-Eudesmol (23.74%), Trans-Nerolidol (17.95%), Linalool (16.79%), 1,8 cineole (6.12%), p-Cymene (3.77%) and terpinen 4-ol (3.40%).

Warionia oil exhibited a significant antimicrobial activity against *Staphylococcus aureus, Candida albicans and Bacillus cereus.* In each case the activity of essential oil was higher than those of the standard antibiotic.

The essential oil showed a relatively high radical scavenging ability and antioxidant activity determined by 1,1-diphenyl-1-picrylhydrazyl (DPPH) assay, ferric reducing (FRAP) assay and ß -carotene bleaching test.

The GC and GC-MS analysis was useful in order to identify the components partially involved in antimicrobial and antioxidant activities of the essential oils obtained from *Warionia saharae* leaves.

Therefore, *Warionia saharae* essential oil could be a source of pharmaceutical materials required for the preparation of new therapeutic and antimicrobial agents. This is the object of our future investigations.

### **5. References**

218 Gas Chromatography – Biochemicals, Narcotics and Essential Oils

The comparison between in vitro bacteriostatic activity of essential oil of *Warionia saharae*, and the inhibition zone formed by standard antibiotic disc (Ampicillin (A10) and Nystatin (N10)) showed that the essential oils were more active against the microorganisms tested. These results suggest that the volatile oil of *Warionia saharae* would probably be a good

The bacteriostatic properties of the oil are suspected to be associated with the high Eudesmol and Nerolidol content, which has been tested previously and was found to have a significant antibiotic activity (Brehm-Stecher and Johnson, 2003; Lattaoui and Tantaoui-

The study of the antioxidant activities showed that the essential oil have a higher antioxidant capacity relative to the antioxidant of reference BHT and BHA (94.2 ± 0.4%).

In contrast to β-carotene/linoleic acid system, essential oil showed a moderate activity in

The ferric reducing ability of plasma (FRAP) assay was used for assessing "antioxidant power" of *Warionia saharae* essential oil. The antioxidant power of the leaves oil samples

The results reported here can be considered as the first information on the antimicrobial and antioxidant properties of *Warionia saharae*. Further studies are needed to evaluate the in vivo

This work interested *Warionia saharae* from Er-rachidia region (south eastern Morocco). It

The essential oils is analysed by GC-MS. Thirty two compounds were identified, they are amounting 92.5% of the oils. The major components of essential oils were ß-Eudesmol (23.74%), Trans-Nerolidol (17.95%), Linalool (16.79%), 1,8 cineole (6.12%), p-Cymene (3.77%)

Warionia oil exhibited a significant antimicrobial activity against *Staphylococcus aureus, Candida albicans and Bacillus cereus.* In each case the activity of essential oil was higher than

The essential oil showed a relatively high radical scavenging ability and antioxidant activity determined by 1,1-diphenyl-1-picrylhydrazyl (DPPH) assay, ferric reducing (FRAP) assay

The GC and GC-MS analysis was useful in order to identify the components partially involved in antimicrobial and antioxidant activities of the essential oils obtained from

Therefore, *Warionia saharae* essential oil could be a source of pharmaceutical materials required for the preparation of new therapeutic and antimicrobial agents. This is the object

therapeutic agent against these bacteria and fungus.

were compared with BHA and BHT as reference antioxidants.

showed that the essential oil yield is about 1.1% (w/w) in dried leaves.

Elaraki, 1994).

**3.3 Antioxidant activity**

this system (65.2 ± 0.4%).

**4. Conclusion**

and terpinen 4-ol (3.40%).

those of the standard antibiotic.

and ß -carotene bleaching test.

*Warionia saharae* leaves.

of our future investigations.

potential of these oils in animal models.

**11**

*Brazil*

**Development and Validation of**

Fabíola B. Carneiro1,\*, Irinaldo D. Júnior2, Pablo Q. Lopes1 and Rui O. Macêdo2

*Universidade Federal da Paraíba - UFPB,João Pessoa – PB,*

*- Cidade Universitária, Recife – PE,*

**Analytical Methodology and Evaluation**

**of the Impact of Culture Conditions and**

*Plectranthus amboinicus* **(Lour) Spreng**

**in the Production of Essential Oil of**

**Collection Associated with the Seasonality**

*1Depto. de Ciências Farmacêuticas, Universidade Federal de Pernambuco - UFPE,*

*2Laboratórios Unificados de Desenvolvimento e Ensaios de Medicamentos – LUDEM –*

The species of Plectranthus (Lamiaceae) are used in folk medicine in many parts of the world (Hedge, 1992). The genus occurs in four continents: Africa, America, Oceania and Asia and phytochemical studies reported that abietane diterpenes and triterpenoids are the most common metabolites in the genus (Albuquerque, 2000). The β-caryophyllene, a constituent of essential oil of Plectranthus amboinicus, according to Haslam (1996), can be used in traditional medicine as a remedy for the treatment of various organic illnesses. The caryophyllene showed the following properties: antiedêmico (Shimizu, 1990), fagorrepelente (Keeler et al., 1991), antiinflammatory (Shimizu, 1990), antitumor (Zheng et al., 1992), bactericidal (Kang et al., 1992), insetífugo (Jacobson et al., 1990) and spasmolytic (Duke, 1992). Some of these activities have been given to its oxide derivative (Shimizu, 1990, Zheng et al., 1992). The registration of herbal medicines (Anvisa, 2004) establishes quality control of raw material is a prerequisite for obtaining essential herbal with reproducibility of action. The development of an analytical method, the adaptation method known or the implementation of the evaluation process involves estimating its efficiency in the laboratory routine. Particular method is considered valid if its characteristics are in accordance with the prerequisites established (Brito et al, 2003). Validation is essential to determine whether methods developed are completely suitable for their intended purposes, in order to obtain reliable results that can be satisfactorily interpreted. In this way, it allows the knowledge of the limitations and reliability of the measurements made in the analysis. The main objective

**1. Introduction**

 \*

Corresponding Author

### **Development and Validation of Analytical Methodology and Evaluation of the Impact of Culture Conditions and Collection Associated with the Seasonality in the Production of Essential Oil of**  *Plectranthus amboinicus* **(Lour) Spreng**

Fabíola B. Carneiro1,\*, Irinaldo D. Júnior2,

Pablo Q. Lopes1 and Rui O. Macêdo2 *1Depto. de Ciências Farmacêuticas, Universidade Federal de Pernambuco - UFPE, - Cidade Universitária, Recife – PE, 2Laboratórios Unificados de Desenvolvimento e Ensaios de Medicamentos – LUDEM – Universidade Federal da Paraíba - UFPB,João Pessoa – PB, Brazil*

### **1. Introduction**

220 Gas Chromatography – Biochemicals, Narcotics and Essential Oils

Shahidi F, Chavan UD, Naczk M & Amarowicz R (2001). Nutrient distribution and phenolic

Tepe, B., Daferera, D., Sokmen, M., Polissiou, M., & Sokmen, A., 2004. In vitro antimicrobial

Watillon, C., Gaspar, T., Hofinger, M. & Ramaut, J.L. (1988). La micropropagation de

Food Chem. 49: 926-933.

*Zohary*. J. Agric. Food Chem. 52, 1132–1137.

*Warionia saharae*.Benth & Coss. Al Biruniya. 4 (1): 35-38.

antioxidants in air-classified fractions of beach pea (*Lathyrus maritimus* L.). J. Agric.

and antioxidant activities of the essential oils and various extracts of *Thymus eigii*

The species of Plectranthus (Lamiaceae) are used in folk medicine in many parts of the world (Hedge, 1992). The genus occurs in four continents: Africa, America, Oceania and Asia and phytochemical studies reported that abietane diterpenes and triterpenoids are the most common metabolites in the genus (Albuquerque, 2000). The β-caryophyllene, a constituent of essential oil of Plectranthus amboinicus, according to Haslam (1996), can be used in traditional medicine as a remedy for the treatment of various organic illnesses. The caryophyllene showed the following properties: antiedêmico (Shimizu, 1990), fagorrepelente (Keeler et al., 1991), antiinflammatory (Shimizu, 1990), antitumor (Zheng et al., 1992), bactericidal (Kang et al., 1992), insetífugo (Jacobson et al., 1990) and spasmolytic (Duke, 1992). Some of these activities have been given to its oxide derivative (Shimizu, 1990, Zheng et al., 1992). The registration of herbal medicines (Anvisa, 2004) establishes quality control of raw material is a prerequisite for obtaining essential herbal with reproducibility of action. The development of an analytical method, the adaptation method known or the implementation of the evaluation process involves estimating its efficiency in the laboratory routine. Particular method is considered valid if its characteristics are in accordance with the prerequisites established (Brito et al, 2003). Validation is essential to determine whether methods developed are completely suitable for their intended purposes, in order to obtain reliable results that can be satisfactorily interpreted. In this way, it allows the knowledge of the limitations and reliability of the measurements made in the analysis. The main objective

<sup>\*</sup> Corresponding Author

Development and Validation of Analytical Methodology and Evaluation

**2.1.2 Quantification of β-caryophyllene**

1.0 μl. Analysis time of 30 minutes.

diluted with 25 ml of hexane.

interferences.

**2.1.3 Preparation of standard solution**

**2.1.4 Preparation of sample solution**

thus set the time of 7 hours for the extraction of essential oils.

**2.1.5 Procedure for validation of analytical methodology**

of the Impact of Culture Conditions and Collection Associated with the Seasonality... 223

Quantification studies were performed on a Shimadzu Gas Chromatograph (model GC-17A) equipped with a capillary column DB-1 dimethylpolysiloxane (30 mx 0.25 mm, 0.25 micron) and flame ionization detector (FID) was used for analysis of samples. The carrier gas was N2 with a flow of 1.3 ml / min, split 1:5, injector temperature 260 ° C, detector temperature 280 ° C, initial column temperature equal to 60 º C heated at a rate of 8 ° C / min up to 280 º C for 10 minutes remaining at this temperature. The injection volume was

The standard solution of 0.180 g / ml was prepared by diluting 0.20 ml of the chemical reference (β-caryophyllene) in 20 ml of hexane and an aliquot of 0.5 ml of this solution was

The essential oils obtained from fresh plant Plectranthus amboinicus (Lour) Spreng were analyzed in order to observe the presence of markers (β-caryophyllene) and possible

The plants were collected at 7 am, 12h and 16h and submitted separately to hydrodistillation in Clevenger apparatus, according to the parameters of the Brazilian Pharmacopoeia Fourth Edition, using approximately 60 g of plant material. The extraction time was determined by quantifying the hexane fractions obtained by clavenger at intervals of 1, 2, 3, 4, 5, 6 and 7 hours and analyzed by gas chromatography with flame ionization detector (GC / FID) for so after 7 hours of analysis was no longer observed the presence of volatile components, and

The analytical validation protocol applied in the study was based on the recommendations of Resolution 899 RE, the National Agency of Sanitary Surveillance for raw materials, establishing the following parameters: The specificity of the method was demonstrated by injecting the standard solution, sample solution and the solvent (hexane). The injections were performed to demonstrate the absence of interference that may promote the elution peak of the chemical reference (β-caryophyllene). The linearity of the curve area versus concentration of β-caryophyllene (standard) was obtained in the following concentration range 0.050 mg / mL to 0.450 mg / mL. The linear curve was evaluated by linear regression analysis by the least squares of the midpoints of three (3) calibration curves authentic. Studies of repeatability and intermediate precision were performed using six replicas of dilutions of the standard solution. The repeatability was evaluated on the same day, for each sample, and intermediate precision was performed on three consecutive days, also for each sample. The studies followed the proposed method and the data were expressed as relative standard deviation (rsd). The repeatability and intermediate precision of the standard solution were analyzed at concentrations of β-caryophyllene equivalent to 0.180 mg / ml, respectively. The accuracy of the method was performed through studies of recovery of β-caryophyllene from the parent plant through the addition of a known amount of standard β-caryophyllene to extract

of the validation of analytical methodologies is to ensure that the method is accurate, specific, reproducible and durable within the range specified for the substance under examination, thus ensuring its credibility in routine use (USP 1994). One of the difficulties to develop herbal medicines that have these characteristics of reproducibility, batch to batch, the therapeutic action is the variability in the content of major constituents, due to the effects of seasonality in the cultivation of plants. Temporal and spatial variations in the total content as well as the relative proportions of secondary metabolites in plants occur at different levels (seasonal and daily, intraplantar, inter-and intraspecific) and, despite the existence of a genetic control, the expression may undergo changes resulting from interaction of biochemical processes, physiological, ecological and evolutionary (Lindroth et al. 1987; Hartmann, 1996). In fact, secondary metabolites represent an interface between chemistry and the environment surrounding the plants (Kutchan, 2001). The constituents of essential oil are biosynthesized mainly in glandular trichomes of leaves and floral cups (Lawrence, 1992) and depend, in addition to genetic factors, also of physiological and environmental factors (Davis et al., 2004). Whereas the amount of constituents present in plants vary considerably depending on external factors, including temperature, irrigation, solar irradiation, soil nutrients, time of collection, plant age, among others, it is necessary a detailed study of these features aiming at the quality of the raw plant, ensuring product quality and clinical effectiveness of herbal medicine. Our objectives are the development and validation of analytical methodology for the determination of β-caryophyllene in essential oil extracted from Plectranthus amboinicus (Lour) Spreng and the relationship between some variables that may be related to variations in the amount of essential oil of the species Plectranthus amboinicus (Lour.) Spreng., Lamiaceae, grown. We evaluated the influences of the following variables: incidence solar, irrigation, time of collection, stages of plant development and different types of fertilization. For this was monitored quantitatively by gas chromatography, the essential oil β-caryophyllene.

### **2. Materials and methods**

### **2.1 Development and validation of analytical method**

The chemical used reference (β-caryophyllene) was obtained from the manufacturer Sigma-Aldrich (USA). The fresh plant Plectranthus amboinicus (Lour) Spreng was collected in an experimental plot of the Laboratory Rabelo. The quantitative and qualitative studies of essential oils obtained from Plectranthus amboinicus (Lour) Spreng were performed on two types of detection techniques

### **2.1.1 Identification of volatile constituents of Plectranthus amboinicus (Lour) Spreng**

The analysis of identification of volatiles were performed by gas chromatography coupled to a mass spectrometer. Was performed on a Shimadzu GC/MS-QP5050A system equipped with a capillary column DB-5 5 phenyl methylpolysiloxane (30 mx 0.25 mm, 0.10 micron). The carrier gas used was helium with a flow of 1.6 ml / min, split 1:200, injector temperature 260 ° C, initial column temperature equal to 60 º C heated at a rate of 10 ° C / min up to 280 º C. The injection volume was 1.0 μl. Time analysis 30 minutes. The mass spectrometer was operated in SCAN mode with scan range of 50-400 with an electron impact (70 eV) and the detector temperature equal to 280 º C.

### **2.1.2 Quantification of β-caryophyllene**

222 Gas Chromatography – Biochemicals, Narcotics and Essential Oils

of the validation of analytical methodologies is to ensure that the method is accurate, specific, reproducible and durable within the range specified for the substance under examination, thus ensuring its credibility in routine use (USP 1994). One of the difficulties to develop herbal medicines that have these characteristics of reproducibility, batch to batch, the therapeutic action is the variability in the content of major constituents, due to the effects of seasonality in the cultivation of plants. Temporal and spatial variations in the total content as well as the relative proportions of secondary metabolites in plants occur at different levels (seasonal and daily, intraplantar, inter-and intraspecific) and, despite the existence of a genetic control, the expression may undergo changes resulting from interaction of biochemical processes, physiological, ecological and evolutionary (Lindroth et al. 1987; Hartmann, 1996). In fact, secondary metabolites represent an interface between chemistry and the environment surrounding the plants (Kutchan, 2001). The constituents of essential oil are biosynthesized mainly in glandular trichomes of leaves and floral cups (Lawrence, 1992) and depend, in addition to genetic factors, also of physiological and environmental factors (Davis et al., 2004). Whereas the amount of constituents present in plants vary considerably depending on external factors, including temperature, irrigation, solar irradiation, soil nutrients, time of collection, plant age, among others, it is necessary a detailed study of these features aiming at the quality of the raw plant, ensuring product quality and clinical effectiveness of herbal medicine. Our objectives are the development and validation of analytical methodology for the determination of β-caryophyllene in essential oil extracted from Plectranthus amboinicus (Lour) Spreng and the relationship between some variables that may be related to variations in the amount of essential oil of the species Plectranthus amboinicus (Lour.) Spreng., Lamiaceae, grown. We evaluated the influences of the following variables: incidence solar, irrigation, time of collection, stages of plant development and different types of fertilization. For this was monitored quantitatively by gas chromatography, the

The chemical used reference (β-caryophyllene) was obtained from the manufacturer Sigma-Aldrich (USA). The fresh plant Plectranthus amboinicus (Lour) Spreng was collected in an experimental plot of the Laboratory Rabelo. The quantitative and qualitative studies of essential oils obtained from Plectranthus amboinicus (Lour) Spreng were performed on two

**2.1.1 Identification of volatile constituents of Plectranthus amboinicus (Lour) Spreng**  The analysis of identification of volatiles were performed by gas chromatography coupled to a mass spectrometer. Was performed on a Shimadzu GC/MS-QP5050A system equipped with a capillary column DB-5 5 phenyl methylpolysiloxane (30 mx 0.25 mm, 0.10 micron). The carrier gas used was helium with a flow of 1.6 ml / min, split 1:200, injector temperature 260 ° C, initial column temperature equal to 60 º C heated at a rate of 10 ° C / min up to 280 º C. The injection volume was 1.0 μl. Time analysis 30 minutes. The mass spectrometer was operated in SCAN mode with scan range of 50-400 with an electron

essential oil β-caryophyllene.

**2. Materials and methods**

types of detection techniques

**2.1 Development and validation of analytical method**

impact (70 eV) and the detector temperature equal to 280 º C.

Quantification studies were performed on a Shimadzu Gas Chromatograph (model GC-17A) equipped with a capillary column DB-1 dimethylpolysiloxane (30 mx 0.25 mm, 0.25 micron) and flame ionization detector (FID) was used for analysis of samples. The carrier gas was N2 with a flow of 1.3 ml / min, split 1:5, injector temperature 260 ° C, detector temperature 280 ° C, initial column temperature equal to 60 º C heated at a rate of 8 ° C / min up to 280 º C for 10 minutes remaining at this temperature. The injection volume was 1.0 μl. Analysis time of 30 minutes.

### **2.1.3 Preparation of standard solution**

The standard solution of 0.180 g / ml was prepared by diluting 0.20 ml of the chemical reference (β-caryophyllene) in 20 ml of hexane and an aliquot of 0.5 ml of this solution was diluted with 25 ml of hexane.

### **2.1.4 Preparation of sample solution**

The essential oils obtained from fresh plant Plectranthus amboinicus (Lour) Spreng were analyzed in order to observe the presence of markers (β-caryophyllene) and possible interferences.

The plants were collected at 7 am, 12h and 16h and submitted separately to hydrodistillation in Clevenger apparatus, according to the parameters of the Brazilian Pharmacopoeia Fourth Edition, using approximately 60 g of plant material. The extraction time was determined by quantifying the hexane fractions obtained by clavenger at intervals of 1, 2, 3, 4, 5, 6 and 7 hours and analyzed by gas chromatography with flame ionization detector (GC / FID) for so after 7 hours of analysis was no longer observed the presence of volatile components, and thus set the time of 7 hours for the extraction of essential oils.

### **2.1.5 Procedure for validation of analytical methodology**

The analytical validation protocol applied in the study was based on the recommendations of Resolution 899 RE, the National Agency of Sanitary Surveillance for raw materials, establishing the following parameters: The specificity of the method was demonstrated by injecting the standard solution, sample solution and the solvent (hexane). The injections were performed to demonstrate the absence of interference that may promote the elution peak of the chemical reference (β-caryophyllene). The linearity of the curve area versus concentration of β-caryophyllene (standard) was obtained in the following concentration range 0.050 mg / mL to 0.450 mg / mL. The linear curve was evaluated by linear regression analysis by the least squares of the midpoints of three (3) calibration curves authentic. Studies of repeatability and intermediate precision were performed using six replicas of dilutions of the standard solution. The repeatability was evaluated on the same day, for each sample, and intermediate precision was performed on three consecutive days, also for each sample. The studies followed the proposed method and the data were expressed as relative standard deviation (rsd). The repeatability and intermediate precision of the standard solution were analyzed at concentrations of β-caryophyllene equivalent to 0.180 mg / ml, respectively. The accuracy of the method was performed through studies of recovery of β-caryophyllene from the parent plant through the addition of a known amount of standard β-caryophyllene to extract

Development and Validation of Analytical Methodology and Evaluation

distributed in groups of three, according to Table 2.

**Sun \*Shadow Organic**

Table 2. Experimental groups.

**2.2.3 Collection of plant material**

homogeneous sample, at 7, 12 and 16

**Vegetative cycle 1** 12 a

**Vegetative cycle 2** 11 a

Table 3. Colection period.

extraction of essential oils.

23/02/07

22/06/07

**2.2.4 Essential oil extraction and determination of extraction time**

of the Impact of Culture Conditions and Collection Associated with the Seasonality... 225

P. amboinicus were grown in experimental plots during the months of December 2006 to September 2007. The substrate was organic fertilizer (manure), a mineral fertilizer [NPK (nitrogen, phosphorus and potassium)] and two mineral fertilizer (NPK with limestone) under different treatments. In each experimental plot eight seedlings were grown,

**fertilizer NPK NPK c/**

**Bed 2** X X X

**Bed 4** X X X **Bed 6** X X X **Bed 7** X X X

It was regarded as the start date of the experiment 12 December 2006, samples were collected in two vegetative cycles DAP (days after planting or plant age) according to the dates of Table 3. In each treatment were collected aerial parts of six plants to obtain a

> 12 a 23/03/07

> 16 a 27/07/07

The collected material was submitted separately to hydrodistillation in Clevenger apparatus, according to the parameters of the Brazilian Pharmacopoeia Fourth Edition, using approximately 60 g of plant material. The extraction time was determined by quantifying the hexane fractions obtained by Clevenger at intervals of 1 h and analyzed by gas chromatography with flame ionization detector (GC / FID) for both after 7 h of analysis was no longer observed the presence of volatile components, and thus set the time of 7 h for

**DAYS AFTER PLANTING (dap) 60 90 120 150**

> 16 a 2 7/04/07

13 a 24/08/07

**Bed 1** X X X

**Bed 3** X X X

\* Shadow = Covered with shade 50% \*\* Irrigation alternating = Three times a week.

**limestone**

**Daily irrigation (DI)**

**\*\*Alternating irrigation (AI)**

> 14 a 25/05/07

> 10 a 21/09/07

**2.2.2 Local cultivation and preparation of the substrate: Experimental groups**

amboinicus Plectranthus (Lour) Spreng (Brazil, 2003) being expressed from the results found (mean concentration experimental) divided by value added (theoretical concentration) multiplied by 100, and the average recovery obtained from the analysis in triplicate. The limits of detection and quantification were obtained with data from the calibration curve of the chemical reference and mathematically calculated using the equations below:

Equation 1. Calculation for determining the limits of detection (LOD) and quantification (LQ).

where:

S = standard deviation of the intercept of the three standard curves;

I = average slope of the three curves.

Were performed on the strength variations related to different temperatures and flows evaluating the impact of these changes in the areas obtained. The studies were performed with dilutions of standard solution with concentration level of 0.180 mg/ml. Analyses were prepared in triplicate and the data evaluated using the relative standard deviation (RSD).

### **2.2 Study of seasonality**

### **2.2.1 Plant material**

The plant material [amboinicus Plectranthus (Lour.) Spreng. Lamiaceae] was grown in experimental plots on the premises of the Laboratory Rabelo, in the municipality of Cabedelo that is inserted into the drive and geoenvironmental coastal tableland has an average altitude from 50 to 100 m. In general, the soils are deep and low natural fertility. The climate is wet tropical with dry summer. The rainy season begins in the fall starting in February and ending in October, as rainfall amounts shown in Table 1. A sample of the plant material was identified, herborized and incorporated into the collection of the Agricultural Research Institute of Pernambuco. Proof: MB Costa e Silva, excicata No. 024/2006.

\* Source: EMATER. \*\* Source: Solar energy laboratory UFPB.

Table 1. Precipitation of rain (rainfall) and temperature.

### **2.2.2 Local cultivation and preparation of the substrate: Experimental groups**

P. amboinicus were grown in experimental plots during the months of December 2006 to September 2007. The substrate was organic fertilizer (manure), a mineral fertilizer [NPK (nitrogen, phosphorus and potassium)] and two mineral fertilizer (NPK with limestone) under different treatments. In each experimental plot eight seedlings were grown, distributed in groups of three, according to Table 2.

\* Shadow = Covered with shade 50% \*\* Irrigation alternating = Three times a week.

Table 2. Experimental groups.

224 Gas Chromatography – Biochemicals, Narcotics and Essential Oils

amboinicus Plectranthus (Lour) Spreng (Brazil, 2003) being expressed from the results found (mean concentration experimental) divided by value added (theoretical concentration) multiplied by 100, and the average recovery obtained from the analysis in triplicate. The limits of detection and quantification were obtained with data from the calibration curve of the

Equation 1. Calculation for determining the limits of detection (LOD) and quantification (LQ).

Were performed on the strength variations related to different temperatures and flows evaluating the impact of these changes in the areas obtained. The studies were performed with dilutions of standard solution with concentration level of 0.180 mg/ml. Analyses were prepared in triplicate and the data evaluated using the relative standard

The plant material [amboinicus Plectranthus (Lour.) Spreng. Lamiaceae] was grown in experimental plots on the premises of the Laboratory Rabelo, in the municipality of Cabedelo that is inserted into the drive and geoenvironmental coastal tableland has an average altitude from 50 to 100 m. In general, the soils are deep and low natural fertility. The climate is wet tropical with dry summer. The rainy season begins in the fall starting in February and ending in October, as rainfall amounts shown in Table 1. A sample of the plant material was identified, herborized and incorporated into the collection of the Agricultural Research Institute of Pernambuco. Proof: MB Costa e Silva,

**MONTHS \*PRECIPITATION (mm) \*\*TEMPERATURE (ºC)**

**December/2006** 42,2 **29,8 January/2007** 31,7 **30,0 February/2007** 162,1 **30,0 March/2007** 194,0 **29,9 April/2007** 262,7 **29,5 May/2007** 224,7 **28,8 June/2007** 616,9 **27,4 July/2007** 127,8 **27,3 August/2007** 203,1 **27,0 September/2007** 201,2 **28,0**

\* Source: EMATER. \*\* Source: Solar energy laboratory UFPB. Table 1. Precipitation of rain (rainfall) and temperature.

chemical reference and mathematically calculated using the equations below:

S = standard deviation of the intercept of the three standard curves;

I = average slope of the three curves.

where:

deviation (RSD).

**2.2 Study of seasonality**

**2.2.1 Plant material**

excicata No. 024/2006.

### **2.2.3 Collection of plant material**

It was regarded as the start date of the experiment 12 December 2006, samples were collected in two vegetative cycles DAP (days after planting or plant age) according to the dates of Table 3. In each treatment were collected aerial parts of six plants to obtain a homogeneous sample, at 7, 12 and 16

Table 3. Colection period.

### **2.2.4 Essential oil extraction and determination of extraction time**

The collected material was submitted separately to hydrodistillation in Clevenger apparatus, according to the parameters of the Brazilian Pharmacopoeia Fourth Edition, using approximately 60 g of plant material. The extraction time was determined by quantifying the hexane fractions obtained by Clevenger at intervals of 1 h and analyzed by gas chromatography with flame ionization detector (GC / FID) for both after 7 h of analysis was no longer observed the presence of volatile components, and thus set the time of 7 h for extraction of essential oils.

Development and Validation of Analytical Methodology and Evaluation

relative peak area (Y) and the standard concentration (X).

\*Relative standard deviation

the values shown in Table 6.

\*N=3 \*\*Relative standard deviation

of -caryophyllene.

**Parâmetros**

of the Impact of Culture Conditions and Collection Associated with the Seasonality... 227

Fig. 1. Equation of the linear calibration of -caryophyllene (y = a + bx), which shows the

shown in Table 5, and expressed as mean (Xm), relative standard deviation (RSD).

**Repeatability Área of the peaks of the sampls Concentration**

Table 5. Test of repeatability (precision intra-run) with the average values of peak areas.

Médias\* 0,1759 0,1751 0,1737 *Dpr\*\** 0,65 1,19 1,31

Table 6. Assessment of intermediate precision expressed on different days at concentrations

Tests of intermediate precision (inter-run precision) were performed on different days and

**Conc. of -caryophyllene in essential oils of Plectranthus amboinicus ( g / ml)**  Day 03 Day 02 Day 03

1 70803 0,1803 2 70768 0,1802 3 71438 0,1819 4 71260 0,1814 5 70577 0,1897 6 70093 0,1785 Média 70823,2 0,1804 dpr (%)\* 0,68 0,67

The repeatability (intra-run precision) was evaluated from injections in six times as Brazil (2003) where the concentration used was 0.180 mg / ml, representing the midpoint of the range specified by the procedure (0.050 mg / mL to 0.450 mg / mL), whose results are

**(g/ml)**

### **2.2.5 Analysis of essential oil**

### **<IP_ADDRESS> Identification of essential oils**

The analyses of identification of volatiles were performed by gas chromatography coupled to a mass spectrometer. Was performed on a Shimadzu GC/MS-QP5050A system equipped with a capillary column DB-1 dimethylpolysiloxane (30 mx 0.25 mm, 0.25 micron). The carrier gas used was helium with a flow of 1.6 mL/min, split 1:200, injector temperature 260 ° C, and initial column temperature equal to 60 º C heated at a rate of 10 ° C/min up to 280 º C. The injection volume was 1.0 μl. The mass spectrometer was operated in SCAN mode with scan range of 50 to 400 with an electron impact (70 eV) and the detector temperature equal to 280 º C.

### **<IP_ADDRESS> Quantification of β-caryophyllene**

Quantification studies were performed on a Shimadzu Gas Chromatograph (model GC-17A) equipped with a capillary column DB-1 dimethylpolysiloxane (30 m x 0.25 mm, 0.25 micron) and flame ionization detector (FID) was used for analysis of samples. The carrier gas was N2 with a flow of 1.3 mL/min, split 1:5, injector temperature 260 ° C, detector temperature 280 ° C, initial column temperature equal to 60 º C heated at a rate of 8 ° C / min remaining up to 280 º C for 10 min at this temperature. The injection volume was 1.0 mL.

### **3. Results and e discussion**

### **3.1 Validation**

### **Validation testing**

The solutions of the samples and blank (placebo) were analyzed by the proposed method. No interference of other constituents was observed in time corresponding to the peak marker β-caryophyllene. The data area and retention time of peak marker showed no coeluting peaks, indicating that the method is specific. The identification of peaks was based on comparison of retention time of standard and sample, and comparing their mass spectra obtained under the same conditions and with the mass spectrum of the instrument library (Wiley, 6th Edition for Class-5000 1999, 229,119 spectra). After optimization of analytical conditions, the parameters selectivity, linearity, precision, accuracy, limits of detection and quantification and robustness were evaluated. Linearity was tested with a diluted solution in hexane, concentration of 0.050 mg / mL to 0.450 mg / mL of β-caryophyllene, performing three replicates authentic as described in Table 4. The results shown graphically in Figure 1 showed linearity in the track, where the correlation coefficient should not be less than 0.99.

\*Average, \*\* Coefficient of variation, \*\*\*Relative standard deviation

Table 4. Linearity of the method from a dilute solution in hexane their respective areas, medium and three replicates authentic.

Fig. 1. Equation of the linear calibration of -caryophyllene (y = a + bx), which shows the relative peak area (Y) and the standard concentration (X).

The repeatability (intra-run precision) was evaluated from injections in six times as Brazil (2003) where the concentration used was 0.180 mg / ml, representing the midpoint of the range specified by the procedure (0.050 mg / mL to 0.450 mg / mL), whose results are shown in Table 5, and expressed as mean (Xm), relative standard deviation (RSD).

\*Relative standard deviation

226 Gas Chromatography – Biochemicals, Narcotics and Essential Oils

The analyses of identification of volatiles were performed by gas chromatography coupled to a mass spectrometer. Was performed on a Shimadzu GC/MS-QP5050A system equipped with a capillary column DB-1 dimethylpolysiloxane (30 mx 0.25 mm, 0.25 micron). The carrier gas used was helium with a flow of 1.6 mL/min, split 1:200, injector temperature 260 ° C, and initial column temperature equal to 60 º C heated at a rate of 10 ° C/min up to 280 º C. The injection volume was 1.0 μl. The mass spectrometer was operated in SCAN mode with scan range of 50 to 400 with an electron impact (70 eV) and the detector temperature

Quantification studies were performed on a Shimadzu Gas Chromatograph (model GC-17A) equipped with a capillary column DB-1 dimethylpolysiloxane (30 m x 0.25 mm, 0.25 micron) and flame ionization detector (FID) was used for analysis of samples. The carrier gas was N2 with a flow of 1.3 mL/min, split 1:5, injector temperature 260 ° C, detector temperature 280 ° C, initial column temperature equal to 60 º C heated at a rate of 8 ° C / min remaining up to 280 º

The solutions of the samples and blank (placebo) were analyzed by the proposed method. No interference of other constituents was observed in time corresponding to the peak marker β-caryophyllene. The data area and retention time of peak marker showed no coeluting peaks, indicating that the method is specific. The identification of peaks was based on comparison of retention time of standard and sample, and comparing their mass spectra obtained under the same conditions and with the mass spectrum of the instrument library (Wiley, 6th Edition for Class-5000 1999, 229,119 spectra). After optimization of analytical conditions, the parameters selectivity, linearity, precision, accuracy, limits of detection and quantification and robustness were evaluated. Linearity was tested with a diluted solution in hexane, concentration of 0.050 mg / mL to 0.450 mg / mL of β-caryophyllene, performing three replicates authentic as described in Table 4. The results shown graphically in Figure 1 showed linearity in the track, where the correlation coefficient should not be less than 0.99.

**Conc. de -caryophyllene (µg/ml) Avarege\* ± dp\*\* dpr(%)\*\*\***

Table 4. Linearity of the method from a dilute solution in hexane their respective areas,

0,45 201926,6 ± 2736,0 1,35 0,23 98561,0 ±1169,5 1,19 0,18 71092,6 ±1415,3 1,99 0,09 41221,3 ±797,1 1,93 0,05 18300,6 ±1812,6 9,90

**2.2.5 Analysis of essential oil**

equal to 280 º C.

**3.1 Validation Validation testing**

**<IP_ADDRESS> Identification of essential oils**

**<IP_ADDRESS> Quantification of β-caryophyllene**

**3. Results and e discussion**

C for 10 min at this temperature. The injection volume was 1.0 mL.

\*Average, \*\* Coefficient of variation, \*\*\*Relative standard deviation

medium and three replicates authentic.

Table 5. Test of repeatability (precision intra-run) with the average values of peak areas.

Tests of intermediate precision (inter-run precision) were performed on different days and the values shown in Table 6.

\*N=3 \*\*Relative standard deviation

Table 6. Assessment of intermediate precision expressed on different days at concentrations of -caryophyllene.

Development and Validation of Analytical Methodology and Evaluation

**3.1.1 Performance equipment**

**<IP_ADDRESS> System Suitability**

\*\* Standard deviation

**3.2 Seasonality**

**3.2.1 Vegetative cycle 1**

of the Impact of Culture Conditions and Collection Associated with the Seasonality... 229

Validation using the software Class -5000 (SHIMADZU, Japan) were calculated area and retention time according to the USP-24. The standard deviation (SD) of replicate injections of standard solution was calculated for the peak areas of the marker. The system suitability data are shown in Table 9 and indicate an acceptable level with a standard deviation less than 2%.

**Injections Area Retention time**  78807 9,786 75724 9,404 77134 9,579 76887 9,548 78583 9,759 **Media** 77427 9,615 **dpr (%)\*** 1,65 1,64

In the period from February to May, corresponding to a growth cycle according to Figure 2, one can deduce that the highest concentration of β-caryophyllene as a function of

Fig. 2. Study of the seasonality of β-caryophyllene (Vegetative Cycle 1). Concentration

(mg / mL) x time of collection X DAP (days after planting).

Table 9. Suitability of the chromatographic system for the -caryophyllene.

Intermediate precision expresses the effects of variations between different conditions. The value of standard deviation below 5% indicates that the method has an acceptable level of accuracy.