Comparison of Volatile Constituents Present in Commercial and Lab-Distilled Frankincense (Boswellia carteri) Essential Oils for Authentication

A comparative analysis of the chemical constituents present in twenty-one commercial and two lab-distilled frankincense (Boswellia carteri) essential oils was carried out using gas chromatography-mass spectrometry (GC-MS) and chiral gas chromatography-mass spectrometry (CGC-MS) for authentication. Out of the twenty-one commercial samples, six were adulterated with synthetic limonene, three were contaminated with synthetic octyl acetate, three were adulterated with castor oil, and two samples each were contaminated with frankincense resin and Boswellia occulta species, respectively, and one was contaminated with the Boswellia serrata species. Additionally, one sample was contaminated with phthalates as well as a cheap essential oil with similar compositions. Furthermore, one sample was adulterated with copaiba resin and frankincense resin in combination with synthetic octyl acetate. Additionally, one was contaminated with Boswellia serrata species, which was further adulterated with castor oil and frankincense resin. To the best of our knowledge, this is the first report to compare the enantiomeric distribution of chiral terpenoids present in commercial frankincense essential oil with lab-distilled frankincense oil for authentication. The CGC-MS analysis showed the presence of a total of eight chiral terpenoids in lab-distilled frankincense essential oils, which can be used as chemical fingerprints for the authentication of frankincense essential oil.


Introduction
Frankincense oil, commonly known as the king of essential oil, is extracted by the hydro-distillation or steam distillation of aromatic oleo-gum resin produced by the trees of the Boswellia genus. There are around twenty species of Boswellia distributed across Africa, Arabia, and South Asia. The oleo-gum resin has been used in traditional medicine, cultural, and religious ceremonies since ancient times, but now, the essential oil derived from these resins has become popular in perfumery and aromatherapy [1]. Among the various species of Boswellia, only a few are traded in a significant amount: B. sacra, B. carteri, B. frereana, B. papyrifera, and B. serrata. The highest quality frankincense resins are produced by B. carteri trees [2].
The essential oil of frankincense can be distinguished based on its chemical composition. However, various factors such as climate, geographical location, and harvesting period may also affect the chemical compositions of frankincense essential oils [3]. B. carteri resins, native to Somaliland and Somalia, are rich in α-pinene and limonene while Omani B. sacra species are dominated by α-pinene [4,5]. Similarly, B. frereana grown in northern Somalia are rich in α-pinene, α-thujene, and p-cymene [4], while B. serrata samples originating from India are rich in α-thujene [6]. In addition, B. papyrifera originating from Ethiopia and Sudan shows notably different chemical profiles compared to the others as octyl acetate is the predominant compound present [7].
The essential oil market has grown rapidly in recent years and is a billion-dollar industry due to its reputed health benefits and application in several industries [8]. Therefore, it is a huge challenge for different stakeholders to maintain the quality of essential oils. Different types of adulteration can be observed in the commercial essential oil market. The addition of carrier oils or cooking oils is the most common way of adulterating essential oils [9]. However, more recently, clever ways of adulteration such as the addition of petrochemicalderived synthetic compounds have increased, for example, the addition of synthetic methyl salicylate in wintergreen or birch essential oil, linalool or linalyl acetate to bergamot or lavender oil, and (E)-cinnamaldehyde to cinnamon oil [10][11][12]. Similarly, the addition of natural oils with similar compositions is another innovative way of adulteration such as the addition of sweet orange oil to grapefruit oil and wintergreen oil to birch oil. Additionally, adding a natural isolate is a creative way of conducting adulteration (e.g., linalool extracted from ho leaf oil added to lavender or bergamot oil) [11]. Finally, creating an essential oil by combining similar natural oils is a sophisticated and shrewd way of doing an adulteration (e.g., lavender and lemon oil have a similar makeup to bergamot oil and can be used to make bergamot oil (https://www.aromaticplant.org/post/bergamot-adulteration, (accessed on 15 June 2022). Figure 1 presents the different degrees of adulteration in essential oils.
others as octyl acetate is the predominant compound present [7].
The essential oil market has grown rapidly in recent years and is a bil industry due to its reputed health benefits and application in several ind Therefore, it is a huge challenge for different stakeholders to maintain the qu sential oils. Different types of adulteration can be observed in the commercial e market. The addition of carrier oils or cooking oils is the most common way ating essential oils [9]. However, more recently, clever ways of adulteration s addition of petrochemical-derived synthetic compounds have increased, for ex addition of synthetic methyl salicylate in wintergreen or birch essential oil, linalyl acetate to bergamot or lavender oil, and (E)-cinnamaldehyde to cinnam 12]. Similarly, the addition of natural oils with similar compositions is another way of adulteration such as the addition of sweet orange oil to grapefruit oi tergreen oil to birch oil. Additionally, adding a natural isolate is a creative w ducting adulteration (e.g., linalool extracted from ho lea f oil added to laven gamot oil) [11]. Finally, creating an essential oil by combining similar natur sophisticated and shrewd way of doing an adulteration (e.g., lavender and have a similar makeup to bergamot oil and can be used to make ber (https://www.aromaticplant.org/post/bergamot-adulteration, (accessed on 15 Figure 1 presents the different degrees of adulteration in essential oils. The first and second degrees of adulteration can be detected through ma analysis using GC-MS and 14 C radiocarbon activity [12,13] whereas third to fif of adulteration are much more complicated to detect as they can easily pass carbon activity tests due to the use of natural oil or their isolates. Therefore, we cast an eye over the enantiomeric distributions of different chiral terpenoids pr  The first and second degrees of adulteration can be detected through marker-based analysis using GC-MS and 14 C radiocarbon activity [12,13] whereas third to fifth degrees of adulteration are much more complicated to detect as they can easily pass 14 C radiocarbon activity tests due to the use of natural oil or their isolates. Therefore, we needed to cast an eye over the enantiomeric distributions of different chiral terpenoids present in oil for their purity authentication and species identification [5,14,15]. Thus, chiral gas chromatographymass spectrometry should always be the choice of a researcher to detect adulterations in essential oils. In, this research, we analyzed the composition of twenty-one commercial frankincense essential oils and compared them with lab-distilled Boswellia carteri essential oil using gas chromatography-mass spectrometry (GC-MS) and chiral GC-MS.

GC-MS Analysis of Commercial and Lab-Distilled Frankincense Essential Oil
The results of the GC-MS analysis of the commercial (F1-F21) and lab-distilled (F22 and F23) frankincense essential oils are presented in the Supplementary File (Table S1), and Table 1 shows the selected constituents. These frankincense essential oils were particularly rich in monoterpene hydrocarbons.
α-Thujene in the commercial samples ranged from 0.4% to 11.6%. Surprisingly, the F3 and F12 samples contained 39.4% and 52.9%, respectively, which was similar to the Boswellia serrata species, as previously reported [6]. In the commercial samples, α-pinene ranged from 24.1% to 46.4%, while F3 and F12 showed significantly lower concentrations of αpinene, 19.1% and 10.6%, respectively, due to the contamination with B. serrata as described above. Furthermore, major compounds such as δ-3-carene, limonene, and β-caryophyllene were notably different in samples F3 and F12 compared to the lab distilled samples. Instead, other major compounds such as sabinene, β-pinene, myrcene, and p-cymene were in ratios comparable to the lab-distilled F22 and F23.
Ethyl isopropyl phthalate (0.1%) was detected in sample F18, which is generally regarded as a contaminant from plastics [16]. Samples F11, F12, F19, and F20 contained ricinelaidic acid lactone, indicating the addition of castor oil to the frankincense oil [17]. In sample F15, copalic acid was also detected in a trace amount, which indicates the addition of copaiba resin to the frankincense oil [18]. Additionally, the F2, F4, F12, and F17 samples showed the presence of the methyl commate isomer and methyl commate B, which indicates the addition of frankincense resin to frankincense essential oil [19]. Samples F4-F11 along with F19 and F20 contained relatively high percentages of limonene, which might be due to the addition of limonene from petrochemical sources.
Most of the commercial samples, except for F3, F4, F6, F11, F12, and F18, contained either octyl methyl ether or decyl methyl ether, or both, which are generally regarded as contamination from Boswellia occulta species, as previously described [1, 20,21]. Samples that were not contaminated by B. occulta were adulterated in other ways: F3 and F12 were samples of B. serrata, F18 contained some other cheap oil, F11 was adulterated using castor oil, F4 was contaminated with frankincense resin, and in F6, synthetic limonene may have been added. Additionally, the F1, F2, F14, F15, and F21 samples contained remarkably high percentages of octyl acetate, which might be due to the addition of synthetic octyl acetate to B. carteri essential oils [22]. Finally only two samples, F2 and F7, contained both the biomarker compounds incensole and serratol of B. carteri, which were present in the pure lab-distilled samples.
Due to different therapeutic benefits, consumers are using frankincense essential oil in skin care [23] and aromatherapy [3]. Frankincense oil also shows impressive antiinflammatory and anticancer properties [24,25], but the present study showed that the commercial essential oil market is full of adulterated oils. Therefore, consumers are vulnerable to safety issues of synthetic compounds present in adulterated frankincense oil, as oils are non-compliant with natural labels [26]. Ethyl isopropyl phthalate - "-" indicates not detected and "t" indicates trace (≤0.05%).

Enantiomeric Distribution Analysis of Commercial and Lab-Distilled Frankincense Essential Oils
The results of the CGC-MS analysis of the commercial (F1-F21) and lab-distilled (F22 and F23) frankincense essential oils are presented in Table 2. The enantiomeric distributions of chiral terpenoids were used in a previous study to differentiate the species of Boswellia [5]. In this study, we used chiral-GC-MS as a tool for the authentication of commercial frankincense essential oil. To the best of our knowledge, this is the first report on the enantiomeric distribution analysis of chiral terpenoids that was used for the authentication of commercial frankincense essential oils. In both the commercial (F1-F21) and lab-distilled (F22 and F23) frankincense oils, eight chiral terpenoids were detected, except in sample F12, where only seven were detected. The enantiomeric distribution analysis of the lab-distilled samples F22 and F23 showed absolute levorotatory α-thujene and nearly racemic α-pinene and camphene. Additionally, most of the chiral terpenoids were dominated by the levorotatory forms, namely sabinene, β-pinene, limonene, terpinen-4-ol, and α-terpineol.
First, in the F2, F14, and F21 samples, significant changes in the enantiomeric ratios of limonene were observed, while in F1 and F15, both limonene and α-terpineol were changed. Additionally, the GC-MS results of these samples showed a high percentage of octyl acetate. In F3 and F12, the enantiomeric distributions of all chiral terpenoids changed drastically, which is supported by the GC-MS results of these samples with a high percentage of α-thujene from B. serrata [6].
Samples F4 and F17 were adulterated with frankincense resin as the GC-MS analysis showed and chiral GC-MS also showed the change in the enantiomeric distributions of β-pinene. From this, we can say that the addition of frankincense resin changes the enantiomeric distributions of β-pinene. Additionally, in the F5-F10 samples, the enantiomeric ratios of limonene changed, in particular, (+)-limonene increased. The GC-MS results of these samples showed an increase in the percentage of limonene compared to those that were lab-distilled. Additionally, in F7, only the enantiomeric ratios of limonene changed while in F10, along with limonene, the enantiomeric ratios of camphene changed and in F5, F6, F8, and F9, the enantiomeric ratios of α-pinene, camphene, and limonene in all three samples changed. From this, it seems that with an increase in the addition of limonene from external sources, the enantiomeric ratios of all chiral terpenes will be altered.
Interestingly, in F11, F19, and F20, the enantiomeric ratios of α-pinene, camphene, and limonene were all changed and the GC-MS results of these samples indicate that they were adulterated with castor oil, along with a high limonene percentage. Furthermore, in F18, the enantiomeric ratios of chiral terpenoids were drastically changed and this was also supported by the GC-MS results, showing an unusual increase in the percentage of some minor compounds that might be due to the addition of some cheap oils. On the other hand, in F13 and F16, although the enantiomeric ratios of all chiral terpenoids were within the ±10 range compared to the lab-distilled samples, the GC-MS results showed that they were contaminated with B. occulta. Thus, with the incidental contamination of B. carteri by B. occulta species, the enantiomeric ratio of chiral terpenoids did not change significantly.
Thus, from the GC-MS and chiral GC-MS analyses, all samples of frankincense essential oil purchased from the market with the claim of pure Boswellia carteri were either adulterated or contaminated with other species such as Boswellia occulta, and Boswellia serrata.