Introduction
Essential oil is a concentrated hydrophobic liquid and volatile chemical compounds from the essence of the plants’ fragrance. Distillation is generally used for extracting oil from plants. Extracted oils have the characteristic fragrance of the plant in which they are derived. One of the characteristics of essential oils is that they usually evaporate completely so they are volatile as well as dissolve only in oils and non-polar solvents like lipophobic compounds. Peppermint oil is used more in pharmacy, medicine, and aromatherapy. Lavender is one of the most popular herbs in the world. In addition to being a herbal medicine used in traditional medicine, its oil is antibacterial and a natural antibiotic. This experiment conducted to identify the separated components of peppermint oil and lavender oil and to identify specific ions in the spectra that will be used for quantitative analysis. In this experiment, The gas chromatography (GC) technique was used for separating and analyzing non-polar compounds in a mixture that is reasonably volatile and sufficiently thermally stable.
GC-Mass spectrometry (GC-MS) still dominates the analysis of volatile and semivolatile molecules and remains the platform of choice in many metabolic phenotyping studies, but this method is not capable of directly analyzing non-volatile, polar and thermally sensitive compounds and these limits the applications of this method.
Using of full scan mode of GC-MS, qualitative analysis of compounds in lavender and peppermint can be identified by matching the full mass spectrum of unknown peaks with a mass spectral library or a chemical database. Because of the low sensitivity and selectivity of the full scan mode, we used the different scan modes to improve quantitative analysis, including Selected Ion Monitoring (SIM) and GC-MS-MS that offer the greatest sensitivity and specificity. SIM method programs the mass spectrometer to monitor only certain ionic fragments at certain retention times for the target compounds. GC-MS-MS indicates that the instrument is capable of separating and fragmenting a favorite molecular weight, and the resulting fragments can then be analyzed using mass spectrometry.
One of the most important advantages of these two methods is the high selectivity and sensitivity in the minimum analysis time.
Materials and Methods
| Materials and Instrument | |
| 1 | Varian 320MS Triple Quadrupole Mass Spectrophotometer |
| 2 | Varian CP3800 (Bruker) Gas chromatography |
| 3 | Linalool, Sigma-Aldrich, Inc, MO, USA |
| 4 | Lavender essential oil, Xenex Laboratories Inc, BC, Canada, |
| 5 | Peppermint Oil, Aura Cacia c/o Frontier Co-op, IA, USA |
| 6 | (–)-trans-Caryophullene, Sigma-Aldrich, Inc, MO, USA |
Method
The method of this experiment is divided into two segments, where the first one is the quality analysis of the Peppermint and Lavander essential oils using GC-MS, and the second one is the determination of the concentration of Caryiphyllene and Linalool in oils by GC-MS-MS and GC-SIM.
Method of the first experiment:
Samples of Lavender essential and peppermint oils were diluted 1:100 in absolute ethanol. Using the Varian Software automated injection was performed and the Chromatogram of the solutions has been measured and recorded.
Method of the Second Experiment:
The samples examined are Lavender, Peppermint oil diluted 1:100 in absolute alcohol, 4 dilutions of Linalool, and Caryophyllene in the concentration of 1:100, 1:200, 1:400 and 1:800 was prepared. Using the Varian Software automated injection was performed for each of the solutions and the Chromatogram of the solutions has been measured and recorded.
Results:
Table 1. The retention time for the identified compounds of Peppermint and Lavender oils by GC–MS in full scan mode.
| Retention Time (min) | ||
| Peppermint | Lavender | |
| Caryophyllene | 16.725 | 16.720 |
| Linalool | 11.822 | 11.822 |
| Pulegone | 14.121 | 14.121 |
| Ledol | 18.915 | 18.915 |
| Menthyl acetate | 14.597 | 14.597 |
| Mint Furanone | 17.713 | 17.713 |
| Limonene | 10.459 | 8.088 |
| menthol | 13.153 | 13.153 |
| Menthofurane | ——- | 17.713 |
| Eucalyptol | 10.503 | 10.518 |
| 1,2-Dihydroinabol | 12.355 | ——- |
| Acetophene | 8.863 | ——- |
| Heptasiloxane | 24.832 | ——- |
| 21.151 | ——- | |
Qualitative analysis involves the identification of a component by means of peak data on the chromatogram. Since the retention time is a specific property of a component, it may be used as a tool to identify the component by comparing the retention time of the peak of the analyte in the sample with the chemical databases (NIST). In this experiment, as reported in table 1, by comparing the retention time of various peaks of Peppermint and Lavender oils in chromatogram with NIST, we were able to identify several compounds in these oils. In the next step, we performed quantitative measurement of Caryophyllene and Linalool in oils.
Figure 1. Standard curve fitted to GC-MS-MS peak area for calculating the unknown concentration of Caryophyllene in oils by comparing the unknown to a set of standard samples of known Caryophyllene concentration. (Q1=69).
Using a set of Caryophyllene standard solutions with known concentration, we created the Caryophyllene standard curve (Figure 1) to calculate the approximate concentration of Caryophyllene in Lavender and Peppermint oils by GC-MS-MS mode.
The linearity of the response interpolating over the concentration range (0.01-0.00125 μg/ml) and linear regression analysis determined the correlation coefficient (R²) as 0.9873 (Figure 1). The equation from the standard curve was used to determine the Caryophyllene content of Lavender and Peppermint oils by interpolating in curves or by placing the peak area of unknown concentration of Caryophyllene in the resulted standard equation (eq. 1: y=3E+08x). The results for unknown Caryophyllene concentration in oils are shown in table 2.
Table 2. The relationship between peak area and caryophyllene concentration in sample and oil by GC-MS-MS.
| Peak Area | Caryophyllene concentration in the sample (μg/ml) | Caryophyllene concentration in oil (μg/ml) | |
| Peppermint oil | 497255 | 0.00166 | 0.166 |
| Lavender oil | 456756 | 0.00152 | 0.152 |
According to equation 1, the calculation is as follows:
y = 3E+08x à 497255 = 3E+08x à x=0.00166
Since we initially diluted the solution 100 times with alcohol, multiplied the obtained concentration of Caryophyllene to100.
0.00166 × 100=0.166
The amount of caryophyllene 0.166 μg/ml in lavender and 0.152 μg/ml in Peppermint was obtained.
Figure 2. Standard curve to GC-MS-MS peak area for calculating the unknown concentration of Linalool in oils by comparing the unknown to a set of standard samples of known Linalool concentration. (Q1=198, Q3=50).
Similar to figure 1, here with the standard solutions of with known concentration of Linalool, we determined the linear standard curve by GC-MS-MS at the correlation coefficient (R²) as 0.9903 (Figure 2). The approximate concentration of Linalool in Lavender and Peppermint oils is determined by interpolating in curves or by placing peak area of unknown concentration of Linalool in the resulted standard equation (eq.2: y = 1E+08x). The results for unknown Linalool concentration in oils are shown in table 3.
Table 3. The relationship between peak area and Linalool concentration in sample and oil by GC-MS-MS.
| Peak Area | Linalool concentration in sample (μg/ml) | Linalool concentration in oil (μg/ml) | |
| Peppermint | 101374 | 0.0010 | 0.10 |
| Lavender | 2.967E+5 | 0.00297 | 0.297 |
According to the equation 2, the calculation is as follows:
y = 1E+08x à 101374 = 1E+08x à x=0.001
Since samples examined (Lavender, Peppermint oil) was diluted 1:100 in alcohol we have to multiply the obtained concentration of linalool to100.
0.0010 × 100=0.10
The amount of Linalool 0.1 μg/ml in lavender and 0.297 μg/ml in Peppermint was obtained.
Figure 3. Standard curve to GC-MS-SIM peak area corresponding to m/z 69 (determined mass for the fragments we monitored) for calculating the unknown concentration of Caryophyllene in oils by comparing the unknown to a set of standard samples of known Caryophyllene concentration. (Q1=69).
Here, we determined the linear standard curve using the GC-MS with SIM mode with the standard solutions with the known concentration of Caryophyllene at the correlation coefficient (R²) as 0.9915 (Figure 3). By interpolating in curves or by placing peak area of unknown concentration of Caryophyllene in the resulted standard equation (eq.3: y = 9E+09x), we were able to determine the approximate concentration of Caryophyllene in Lavender and Peppermint oils. The results for unknown Caryophyllene concentration in oils are shown in table 4.
| Peak Area | Caryophyllene concentration in the sample (μg/ml) | Caryophyllene concentration in oil ( μg/ml) | |
| Peppermint | 1.503e+7 | 0.00167 | 0.167 |
| Lavender | 1.249e+6 | 0.00139 | 0.139 |
Table 4. The relationship between peak area and Caryophyllene concentration in sample and oil by GC-MS-SIM
According to the equation 3, the calculation is as follows:
y = 9E+09x à 1.503E+7 = 9E+09x à x=0.00167
Since we initially diluted the solution 100 times with alcohol, multiplied the obtained concentration of Caryophyllene to 100.
0.00167 × 100=0.167
The amount of caryophyllene 0.167 μg/ml in lavender and 0.139 μg/ml in Peppermint was obtained.
Figure 4. Standard curve to GC-MS-SIM peak area corresponding to m/z 76.9 (determined mass for the fragments we monitored) for calculating the unknown concentration of Linalool in oils by comparing the unknown to a set of standard samples of known Linalool concentration. (Q1=198, Q3=50).
Also, with the standard solutions with the known concentrations of Linalool, we determined the curve by GC-MS with SIM mode at the correlation coefficient (R²) as 0.9915 (Figure 3). The approximate concentration of Linalool in Lavender and Peppermint oils determined by interpolating in curves or by placing peak area of unknown concentration of Linalool in the resulted standard equation (eq.4: y=1E+09x). The results for unknown Linalool concentration in oils are shown in table 5.
Table 5. The relationship between peak area and Linalool concentration in sample and oil by GC-MS-SIM
| Peak Area | Linalool concentration in sample (μg/ml) | Linalool concentration in oil (μg/ml) | |
| Peppermint | 807460 | 0.00081 | 0.081 |
| Lavender | 6.464E+6 | 0.00646 | 0.646 |
Since samples examined (Lavender, Peppermint oil) was diluted 1:100 in alcohol, we have to multiply the obtained concentration of linalool to100.
According to the equation 4, the calculation is as follows:
y = 1E+09x à 807460 =1E+09x à x=0.00081
Since we initially diluted the solution 100 times with alcohol, multiplied the obtained concentration of linalool to 100.
0.00081 × 100=0.081
The amount of Linalool 0.081 μg/ml in lavender and 0.646 μg/ml in Peppermint was obtained.
Discussion:
As shown in table 1, we were able to identify different compounds (such as Caryophyllene, Linalool and etc.) in peppermint oil and lavender oil based on matching the retention time of the peaks with NIST. Around 11 of the total number of components in each sample were reliably characterized.
A similar result has been found by Shellie et al (2002) which identified the component of Limonene, Pulegone, Menthol, caryophyllene, Linalool, etc. using the GC-MS method. The compounds were identified according to Beigia et al (2018) for the Peppermint essential oils by GC-MS includes Limonene, Pulegone, Menthol, caryophyllene, Linalool, and Menthofuran. In the experiment, these components also were identified in Lavender and Peppermint oils.
For several causes, the assignment of some components in each oil could not be performed. The main cause that these oils components were not characterized is that sufficiently accurate mass spectra could not be achieved for the components. Many of the components which remain uncharacterized eluted from GC, are those poorly resolved in the solvent. In this case, correction of background is loose and hence spectral quality is degraded. Identification of special components was difficult in the case where the retention time of a minor components’ was very similar to a component of more prominent. According to Beigia et al (2018), different drying methods of peppermint leaves like increasing drying temperature decreases the essential oil content. For the quantitative analysis of two compounds in Lavender and Peppermint essential oils, standard curves are also reported in figures 1 to 4. Quantitation was performed using a triple quadrupole mass spectrometer. Although GC–MS full screen is sometimes used to obtain quantitative sample information, its response factors for various analysts often differs significantly and for multi-component samples cannot always provide accurate quantitative results.
SIM allows the mass spectrometer to detect particular compounds with very high sensitivity. In SIM mode, the device is adjust to collect data at masses of interest (69 for Caryophyllene and 76.9 for Linalool), instead of stepping the mass filter over a wide range of masses. Since the mass spectrometer gathers data at only the masses of interest, it responds only to compounds of the selected mass fragments. In essence, the device is focused on only the interest compounds. Also, because only a few masses are detected, much more time for looking at these masses may be consumed, with the associated increase sensitivity. SIM also allows the gathering of more points across a chromatographic peak, thus increasing the precision and accuracy of quantitative results.
Beigia et al (2018) performed quantitative analysis of the peppermint component by GC-MS, but Shellie et al (2002) to get better results, used GC with flame ionization detection (FID) for Semi-quantitative analysis. However, the sensitivity of GC in different scan modes available within the Triple Quad Mass Spectrometer that used in this experiment is greater than the two previous methods and gives more accurate results.
For the quantitative analysis of caryophyllene and linalool in oils, we used the standard curve. A standard curve is a graph relating a measured quantity (peak area) to concentration of the substance of interest in “known” samples. By interpolating in curves the approximate concentration of Linalool in Lavender and Peppermint oils determined
Figure 5. Standard curve for sensitivity comparison of MS-MS and SIM modes for Caryophyllene measurement.
Figure 6. Standard curve for sensitivity comparison of MS-MS and SIM modes for Linalool measurement.
Both MS-MS and SIM methods were used as a quantitative measurement, but to compare two methods, the standard curves of MS-MS and SIM merged together for linalool and caryophyllene in figures 5 and 6. As it is clear from these figures, the sensitivity in the SIM method is greater than MS-MS method. The sensitivity of the method is a measure of its ability to discriminate between small differences in analyte concentration. So, the higher the slope of standard curves, the higher the sensitivity of method for that particular component.
Conclusion
The essential oil industry uses reliable methods for characterizing essential oils to ensure product quality. For essential oils to be used as food products, or as pharmaceutical products reliable characterization methods are mainly important. Using GC-MS identification of the components in the samples investigated was greatly simplified. This study has shown that GC-MS analysis discloses a clearer indication of the true molecular complexity of essential oils and used for qualitative analysis of essential oils but for quantitative analysis, we used GC-MS with MS and SIM modes. The GC–MS-MS and GC–MS-SIM provide improved sensitivity and selectivity with a drastic reduction of the background signal and a high capability of confirmation. With the optimisation of some parameters such as oven temperature ramp rate and column film thickness and flow rate, an increase in assay efficiency can be achieved.
Here are some the chemistry writing services we provide:
– Chemistry assignment writing services
– Chemistry Essay Writing Service
– Chemistry Dissertation Writing Service
