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Article

HPTLC Analysis and Chemical Composition of Selected Melaleuca Essential Oils

United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Subtropical Horticulture Research Station (SHRS), Miami, FL 33158, USA
*
Author to whom correspondence should be addressed.
Molecules 2023, 28(9), 3925; https://doi.org/10.3390/molecules28093925
Submission received: 7 April 2023 / Revised: 28 April 2023 / Accepted: 5 May 2023 / Published: 6 May 2023

Abstract

:
Tea tree oil (TTO) is a volatile essential oil obtained by distillation, mainly from the Australian native plant Melaleuca alternifolia (Maiden & Betche) Cheel (Myrtaceae). In this study, a comparative analysis of the chemical constituents of seven tea tree oils (M. alternifolia) and four other Melaleuca spp. oils (M. cajuputi, (MCa), two chemotypes of M. quinquenervia, (MNe and MNi), and M. ericifolia (MRo)) was carried out using gas chromatography–mass spectrometry (GC-MS) and high-performance thin-layer chromatography (HPTLC). Among the seven TTOs, terpinen-4-ol (37.66–44.28%), γ-terpinene (16.42–20.75%), α-terpinene (3.47–12.62%), α-terpineol (3.11–4.66%), and terpinolene (2.75–4.19%) were the most abundant compounds. On the other hand, the most abundant compounds of the other Melaleuca oils varied, such as 1,8-cineole (64.63%) in MCa oil, (E)-nerolidol (48.40%) and linalool (33.30%) in MNe oil, 1,8-cineole (52.20%) in MNi oil, and linalool (38.19%) and 1,8-cineole (27.57%) in MRo oil. HPTLC fingerprinting of Melaleuca oils enabled the discrimination of TTO oils from other Melaleuca spp. oils. Variation was observed in the profile of the Rf values among EOs. The present study shows that HPTLC is one of the best ways to identify and evaluate the quality control in authenticating TTOs, other Melaleuca EOs, or EOs from other species within the Myrtaceae.

1. Introduction

Plant essential oils (EOs), originally used in the perfume and aromatherapy market, have gained widespread acceptance in other industries. Tea tree oil (TTO), for example, an essential oil isolated from Melaleuca alternifolia (Maiden & Betche) Cheel (Myrtaceae) [1], has long been recognized as a safe and effective topical antiseptic in Australia [2,3,4,5]. During World War II, it was considered a necessary commodity for first aid kits and was also used as an insect repellent [6]. Current commercial products that contain TTO include assorted ointments, lotions, shampoos, soaps, toothpastes, and mouthwashes [2,5,7]. Additionally, many EOs have since been evaluated for their antimicrobial [8,9], insect attractant [10,11], repellent [12], or insecticidal properties [13,14,15,16,17]. Isman [18] investigated the potential effect of several EOs as attractants, repellents, and toxicants against insects and other organisms.
Since EOs consist of concentrated plant terpenoids, they have provided an ideal substrate for the development of host-based (kairomone) lures for invasive pests in Florida, including the redbay ambrosia beetle Xyleborus glabratus Eichhoff, the tea shot hole borer Euwallacea perbrevis Schedl [19], and the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), which has been a destructive pest in Europe, the Middle East, Australia, Central and South America, and Hawaii, USA [20,21,22]. Behavioral studies conducted at the USDA-ARS in Miami, Florida [23,24], have indicated that tea tree oil (TTO) has a strong short-range attractive effect on sterile male medflies in laboratory bioassays [17,25,26,27,28,29,30,31]. Therefore, it has good potential as an economical new attractant for male medflies, and the identification of the key components of TTO is important for developing lures for pest management.
In recent experiments [27], we used preparative thin-layer chromatography (prep TLC) to separate TTO into five fractions. Bioassays conducted after these separations revealed that two TTO fractions are responsible for the observed attraction in male medflies. Furthermore, TLC-based bioassays played an important role in the isolation of insect kairomones from complex mixtures such as EOs. Although it is not a new technique, TLC continues to be a valuable tool as a preparative technique for a variety of studies in both chemical and biological fields [25,32,33,34,35]. While gas chromatography–mass spectrometry (GC-MS) is exceptional in the identification of unknown chemicals, it works best for trace analysis [36]. In addition, the sample is destroyed in the process of obtaining its fingerprint fragments for proper identification. In cases where a much larger sample is needed or it is required to remain intact for further analysis, TLC proves to be a more suitable method. With the development of high-performance thin-layer chromatography (HPTLC), this separation technique has evolved from a mostly qualitative procedure into a quick and cost-effective quantification method used by the European Pharmacopoeia for the quality control of some EOs [37]. The automated sample application, and the capability of using a universal HPTLC standard mix [38], ensures accurate sample and standard amounts, and provides reproducible quantitative results.
Various Melaleuca species, and even other Myrtaceae, are often confused under the common name ‘tea tree’, (e.g., ‘‘swamp tea tree’’, M. cajuputi; ‘‘paperbark tea tree’’, “broad-leaved tea tree”, or “broad-leaved paper bark”, M. quinquenervia; “black tea tree” or “river tea tree”, M. bracteate; “lemon scented tea tree”, Leptospermum petersonii, etc.). Moreover, kanuka and manuka EOs derived from Kunzea ericoides and Leptospermum scoparium, respectively, are referred to as New Zealand TTOs [2,3,5,6,39,40].
Considering the diversity of compounds present in Melaleuca EOs, and their current and potential applications, including prospective IPM strategies, it is important to study the chemical composition and degree of variability in commercially available Melaleuca EOs. In this study, HPTLC methods were developed for the evaluation of seven TTOs and four different Melaleuca spp. oils selected mainly from open markets in the United States. Separation patterns of various brands of TTO, as well as other Melaleuca oils, were compared to ensure the presence and consistent amount of chemicals attractive for male medflies.

2. Results and Discussion

2.1. Identification of Components in Melaleuca EOs

The identification of the components from the seven M. alternifolia EOs (TTAA, TTAS, TTEG, TTFC, TTNG, TTPT, and TTSAT) and four other Melaleuca EOs (MCa, MNe, MNi, and MRo) (Table 1) was achieved on the GC-MS using a non-polar DB-5 column. One hundred thirty-eight compounds were identified in total among the eleven Melaleuca EOs, accounting for 99.49-99.97% of the total composition (Table 2).
The TTOs were characterized by a high amount of terpinen-4-ol (37.66–44.28%), followed by γ-terpinene (16.42–20.75%), α-terpinene (3.47–12.62%), α-terpineol (3.11–4.66%), terpinolene (2.75–4.19%), p-cymene (1.66–11.47%), α-pinene (1.29-2.66%), aromadendrene (1.20–1.90%), 1,8-cineole (1.17–4.79%), ledene (0.47–1.69%), and limonene (0.30–1.63%).
The GC-MS data indicated that the components of the other Melaleuca EOs varied notably from each other. The MCA oil contained 1,8-cineole (64.43%) as a principal component, followed by α-pinene (5.48), α-terpineol (5.44%), β-caryophyllene (3.82%), γ-terpinene (3.24%), α-humulene (1.81%), β-selinene (1.28%), α-selinene (1.12%), β-pinene (1.11%), and terpinolene (1.04%). The most abundant constituents identified in the MNe oil were largely (E)-nerolidol (48.40%) and linalool (33.30%), whereas 1,8-cineole (52.20%), α-pinene (9.75%), (E)-nerolidol (6.89%), viridiflorol (6.23%), α-terpineol (5.08%), limonene (2.83%), β-caryophyllene (2.66%), β-pinene (2.56%), and ledene (1.76%) were identified as the main constitutes in the MNi oil. Linalool (38.19%) and 1,8-cineole (27.57%) were the main constituents of the MRo oil.
The International Standard, ISO 4730, requires terpinen-4-ol chemotype to be present in commercial TTO production [58]. ISO standards allow terpinen-4-ol between 30 and 48%, along with γ-terpinene (10–28%), α-terpinene (5–13%), 1,8-cineole (<0.01–15%), α-terpineol (1.5–8%), p-cymene (0.5–8%), α-pinene (1–6%), and terpinolene (1.5–5%), and containing a mixture of minor terpenoids with sabinene (<0.01–3.5%), aromadendrene (<0.01–3.0%), δ-cadinene (<0.01–3.0%), ledene (viridiflorene, <0.01–3.0%), limonene (0.5–1.5%), globulol (<0.01–1.0), and viridiflorol (<0.01–1.0%) [ISO]. Our GC-MS analysis revealed that all the TTO samples fitted into the terpinen-4-ol chemotype (37.7–44.3%) (Table 2). The therapeutic use of TTO is attributed to the concentration of terpinen-4-ol and 1,8-cineole (eucalyptol), yet 1,8-cineole has been reported to cause skin and mucous membranes irritation [59,60,61]. Therefore, low concentrations of 1,8-cineole are preferred to maximize the therapeutic use of TTO, and it is critical to distinguish between TTOs and other commercially available Melaleuca oils.
M. cajuputi oil has three chemotypes. Chemotype 1 contains a high concentration (50–70%) of 1,8-cineole, while chemotype 2 contains a lower concentration of 1,8-cineole (31%), and chemotype 3 contains no 1,8-cineole. M. cajuputi subsp. cajuputi is the main source of cajuput oil, which does contain 1,8-cineole [61]. Our M. cajuputi (MCa) oil from Thailand was found to be dominated by a 1,8-cineole-rich chemotype (Table 2).
M. quinquenervia can be a source of 1,8-cineole-rich essential oil [61]. Four chemotypes were reported for M. quinquenervia [62]; the cineole chemotype (1) contains 1,8-cineole (55.0–65.0%), α-pinene (7.0–12.0%), limonene (6.0–12.0%), α-terpineol (4.0-10.0%), β-pinene (1.5–4.5%), viridiflorol (1.0–3.5%), β-caryophyllene (0.01–2.0%), and myrcene (0.01–2.0%), and is called niaouli oil; the linalool chemotype (2) contains (E)-nerolidol (61.1%), linalool (23.9%), 1,8-cineole (2.6%), α-pinene (1.9%), terpinene-4-ol (1.8%), viridiflorol (1.6%), and β-caryophyllene (1.1%), and is called nerolina oil; the nerolidol chemotype (3) contains (E)-nerolidol (75.7–92.5%), β-caryophyllene (0.5–8.7%), 1,8-cineole (0.01–6.6%), caryophyllene oxide (0.1–6.1%), α-pinene + α-thujene (0–4.5%), δ-cadinol (0–2.5%), viridiflorol (0.1–1.7%), and α-terpineol + viridiflorene (ledene) (0-1.5%), and is also called niaouli oil; and the viridiflorol chemotype (4) contains viridiflorol (40.0–45.0%), 1,8-cineole (30.0–35.0%), (E)-nerolidol (3.0–6.0%), and ledol (0.01–4.0%), and is called niaouli oil, too. Our M. quinquenervia oil (MNi) from Madagascar represents chemotype 1, and the oil (MNe) from Australia represents chemotype 2 (Table 2).
M. ericifolia is from native Australian plants and is known as the lavender tea tree or rosalina oil. The major compounds of rosalina oil were identified as linalool (35.0–55.0%), 1,8-cineole (18.0–26.0%), and α-pinene (5.0–12.0%) [61,62]. Our rosalina oil (MRo) from Australia contained a high abundance of linalool and 1,8-cineole, followed by α-pinene (Table 2). Consequently, TTOs can be distinguished from other Melaleuca oils by the high amount of terpinene-4-ol and low amounts of 1,8-cineole and linalool, and the absence of (E)-nerolidol.
TTO and other Melaleuca EOs were subjected to PCA and HCA in order to identify which constituents (detected at ≥0.5%) are different among the TTOs and the four other Melaleuca species. Principle components are reflected by eigenvalues. Table 3 shows that the seven components with eigenvalues greater than one account for 96.35% of the total variance. According to the rules of PCA, the highest eigenvalues, F1 (14.92) and F2 (8.60), were selected and then subjected to the PCA analysis. The Bartlett′s sphericity test carried out on the correlation matrix shows a calculated x2 = 1519.98, greater than the critical value x2 = 52.19 with 37 degrees of freedom (p < 0.0001), thus proving that PCA can achieve a significant reduction in the dimensionality of the original data set.
The PCA plot established according to the first two PCA axes is shown in Figure 1. Principal component 1 (F1, 39.26%) was strongly loaded for TTOs, with negative scores in MCa, MNe, MNi, and MRo. Principal component 2 (F2, 22.62%) demonstrated strong scores in MCa, MNe, MNi, and low correlations with TTOs and MRo, adding up to 61.88% of the variance in eleven Melaleuca EOs.
PCA provides a simple way to visualize similarities among different samples; a short range between the samples means a small or very little difference, and a long distance means a strong difference. Factor loadings and squared cosine (cos2) indicate the importance of components representing the individual components for a given principal component (Table 4). The cos2 similarity always tends to be 1, showing a high linear correlation relationship of the variables with the components. The highest cos2 values were in F1: γ-terpinene (0.902), terpinene-4-ol (0.900), terpinolene (0.886), δ-cadinene (0.844), trans-cadina-1(6),4-diene (0.828), bicyclogermacrene (0.798), α-gurjunene (0.798), zonarene (0.762), and α-terpinene (0.724). Similarly, the highest cos2 value for F2 was a-terpineol (0.858). When the relationship between the factor loadings and their percentage contributions to the matrix was analyzed, it could be concluded that α-thujene, α-pinene, β-pinene, 1,8-cineole, linalool, α-terpinyl acetate, α-terpineol, β-caryophyllene, α-humulene, (E)-β-farnesene, (E)-nerolidol, viridiflorol, and ledol had a negative relationship with the TTOs. Higher percentages of compounds then appear to be of interest for the selection of quality assessment of tea tree M. alternifolia EOs.
HCA classified Melaleuca EOs in two main groups (Figure 2): group I clustered samples with high contents of terpinene-4-ol, γ-terpinene, α-terpinene, and terpinolene. Although MRo did not belong to M. alternifolia (TTOs), it was grouped in cluster I due to its high levels of interfering compounds such as limonene, aromadendrene, and alloaromadendrene. The Euclidean distance between TTAA and TTPT was 1.74, and between TTAA and MRo it was 7.51. Group II clustered samples MCa, MNi, and MNe, which presented high contents of 1,8-cineole, linalool, and (E)-nerolidol, with intermediate values of α-pinene, α-terpineol, and β-caryophyllene, and lower contents of terpinene-4-ol, γ-terpinene, terpineol, and α-terpinene, indicating that the TTOs contained significantly higher concentrations of terpinen-4-ol when compared to the other Melaleuca EOs.

2.2. HPTLC Analysis of Melaleuca EOs

The less-polar components of the UHM, labeled (e) through (h) according to Do et al. [38], separated well under the selected HPTLC development conditions, as shown in Figure 3A. This indicated that the initial method involving Hex/EtOAc 90:10 (v/v) as the solvent system was appropriate for the separation of non-polar components of interest in our samples. However, the Rf of some Melaleuca oil components appear to exceed that of the highest UHM component. For future purposes, an additional component with higher Rf may need to be added to the UHM to better encompass the range of our samples.
In general, the HPTLC results complemented the findings obtained from the GC-MS analysis. The oils of M. alternifolia (TTOs) (Figure 3B), despite their wide variety of sources, exhibited a very similar separation pattern dominated by terpinen-4-ol as the primary component. When developed with Hex/EtOAc 90:10 (v/v), this main constituent of TTO appeared at an Rf value of 0.278 ± 0.067. A group of mono- and sesquiterpenes, including α-and β-pinene (Rf = 0.767 ± 0.031) and (+)-aromadendrene (Rf = 0.738 ± 0.062), was showcased as the second most prominent band, with a collective Rf of 0.747 ± 0.065. Included in this group were ledene and δ-cadinene, identified by GC-MS. This band was followed in intensity by α-terpineol (Rf = 0.147 ± 0.052) and 1,8-cineole (Rf = 0.504 ± 0.081), respectively.
In cases where oil components were merged, as observed by the overlapping or blending of colors, such components exhibited a shift in Rf values compared to those of the corresponding reference standards. This may be caused by interactions due to large amounts of components competing for the limited silica surface area. The Rf of the merged oil constituents, as well as the presence of additional constituents, was established by GC-MS.
The other four Melaleuca EOs (Figure 3C) displayed a diverse pattern compared to each other, as well as in comparison to the TTOs. 1,8-cineole could be observed as the most characteristic component in MCa (track 9) and MNi (track 11), merged with small amounts of α-terpinyl acetate (Rf = 0.539 ± 0.050) and limonene (Rf = 0.486 ± 0.047). In contrast with MCa and MNi, cineole was less prominent in MRo (track 12) and almost insignificant in MNe (track 10). No terpinyl acetate was observed in either MNe or MRo, yet limonene was still present in MRo.
The second most noticeable band of MCa was a group of mono- and sesquiterpenes merged at Rf = 0.743 ± 0.052. These were identified by GC-MS as α- and β-pinene, β-caryophyllene (Rf = 0.727 ± 0.059), α-humulene (Rf = 0.727 ± 0.062), and α-phellandrene (Rf = 0.759 ± 0.031). Also identified by GC-MS in small amounts were α- and β-selinene. Two other chemicals present at a lower intensity, yet highly identifiable on MCa, were α-terpineol and linalool (Rf = 0.243 ± 0.056).
The two most prominent bands in MNe, combined near 0.3 Rf, were identified as nerolidol (Rf = 0.279 ± 0.056) and linalool, respectively. They were followed in intensity by the mono- and sesquiterpene band near 0.75 Rf, comprised of β-caryophyllene, β-pinene, β-ocimene (Rf = 0.747 ± 0.047), and farnesene (Rf = 0.737 ± 0.060), as identified by GC-MS. Small, yet still characteristic of MNe, was geraniol at Rf = 0.124 ± 0.067. A bright-pink band at Rf = 0.432 ± 0.045 was recognized as caryophyllene oxide by isolating the component on a TLC preparation plate (Section 3.4.2) and confirmed by GC-MS analysis against its reference standard and available GC-MS libraries. Caryophyllene oxide appeared to be a byproduct of β-caryophyllene since it was present in both TLC and TIC reference standard chromatograms.
The MNi oil was characterized by a large amount of 1,8-cineole. As with MCa, this large band was mixed with a small amount of terpinyl acetate and limonene. The second-largest band was composed of mono- and sesquiterpenes, confirmed by GC-MS as α- and β-pinene, β-caryophyllene, and ledene. Other main components were nerolidol, α-terpineol, and viridiflorol (Rf = 0.213 ± 0.021).
In MRo, the signature component was linalool, with a small amount of terpinen-4-ol merging into it. Mono- and sesquiterpenes composed the second-strongest band near 0.76 Rf, followed by cineole as the third characteristic band. Constituents of the second band were recognized as α-pinene, aromadendrene, alloaromadendrene, and ledene by GC-MS. Merged into a fourth band was α-terpineol, followed by globulol a bit below, at Rf = 0.137 ± 0.022. System suitability as well as Rf values for the major components were established by SSTs (Figure 4A) and reference standards (Figure 4B), analyzed under the same HPTLC conditions.
An enhanced separation of more polar constituents in the oils (lower Rf range) was obtained when developed with Hex/EtOAc at a ratio of 80:20 (v/v). Figure 5A emphasizes an increased distance among UHM constituents. Additionally, target oil components that merged at low Rf values under the previous solvent system were now appearing mid-range and better separated.
The most significant improvement with a more polar solvent system was an increased separation of terpinen-4-ol (Rf = 0.532 ± 0.049) and α-terpineol (Rf = 0.385 ± 0.054) in TTOs (Figure 5B). With the other Melaleuca oils (Figure 5C), where the differences among samples relied more on the polar region, a greater distinction could be made among nerolidol (Rf = 0.549 ± 0.052), linalool (Rf = 0.514 ± 0.055), viridiflorol (Rf = 0.494 ± 0.027), and terpinen-4-ol (Rf = 0.532 ± 0.049), as well as between α-terpineol (Rf = 0.385 ± 0.054), globulol (Rf = 0.376 ± 0.021), and geraniol (Rf = 0.263 ± 0.052). These differences in Rf were more evident at lower concentrations, as demonstrated by the SSTs (Figure 6A) and the individual reference standards (Figure 6B) analyzed under exact HPTLC developing conditions.

3. Materials and Methods

3.1. Sample Selection and Preparation

TTOs from M. alternifolia were selected based on their previously established biological activity as a potential attractant for the male Mediterranean fruit fly [23,24,26,27]. Essential oils from other Melaleuca species were also included for the purpose of comparison (Table 1). Each sample was diluted to 20% of its original purity using methylene chloride, ACS Reagent, CAS# 75-09-2 (J.T. Baker-Avantor, Center Valley, PA, USA). If necessary, concentration and application volume were adjusted for optimum HPTLC separation.

3.2. Standard Selection and Preparation

A universal HPTLC calibration mix (UHM) (Sigma-Aldrich, St. Louis, MO, USA) was used as a reference standard for HPTLC separations. It contains eight different compounds diluted in methanol at ready-to-use concentrations [38], four of which were observed under our HPTLC conditions using a UV254 light source (Table 5).
Stock solutions of isoeugenol, CAS# 97-54-1, and isoeugenyl acetate, CAS# 93-29-8 (Sigma-Aldrich, St. Louis, MO, USA), were prepared in methylene chloride at a concentration of 100 µL/mL and 100 µg/mL, respectively. A 20 µL/mL (21.6 mg/mL; δ = 1.08 g/mL) methylene chloride dilution of isoeugenol, as well as a 20 mg/mL dilution of isoeugenyl acetate, were prepared from their corresponding stock and used as system suitability standards (SST1 and SST2).
A series of reference standards (Table 6) were obtained from Sigma-Aldrich (St. Louis, MO, USA), and were prepared and analyzed under the same conditions as the EOs to confirm the Rf values of the oil components on HPTLC.

3.3. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

Melaleuca EO samples were analyzed on an Agilent 7890B GC coupled with a 5977B mass selective detector (GC-MS) (Agilent Technologies, Santa Clara, CA, USA). A DB-5 column (30 m × 0.25 mm inner diameter with 0.25 μm film thickness) was used with an electron ionization source set at 70 eV. The temperatures of the ion source and quadrupole were 230 °C and 150 °C, respectively. The mass spectrometry transmission line was 250 °C. Injector and detector temperatures were kept at 220 °C and 230 °C, respectively. The oven temperature program was set at 60 °C for 1.3 min and increased to 246 °C at 3 °C/min. A constant helium flow of 1.3 mL/min was applied [41]. The selected mass range was m/z 35 to 450 Da and scan rate was 2.8 scans/s. Mass Hunter B.07.06 software (Agilent Technologies) was used for data acquisition and processing. One μL of diluted samples was injected into the GC–MS on splitless mode.
Linear retention indices (RIs) were calculated using the van Den Dool and Kratz [42] equation in relation to a homologous series of n-alkanes (C9–C21). Compound identification was achieved by comparison of their corresponding mass spectra and RIs to those reported in a mass spectral library developed at the USDA-ARS-SHRS laboratory with authentic compounds and with the commercial libraries MassFinder [43], Adams Library [41], Flavours and Fragrances of Natural and Synthetic Compounds 3 (FFNSC-3) [44], Wiley 12/NIST 2020 [45], and an in-house library “SHRS Essential Oil Constituents-DB-5 Column”. Retention indices were also verified with data reported in the specific literature [46,47,48,49,50,51,52,53,54] and internet sources [55,56,57]. Each oil was analyzed in triplicate. Relative percentages were directly obtained from peak total ion current (TIC) areas. All these standards were purchased from the following sources: α-pinene (CAS# 80-56-8), camphene (CAS# 79-92-5), benzaldehyde (CAS# 100-52-7), sabinene (CAS# 3387-41-5), β-pinene (CAS# 127-91-3), myrcene (CAS# 123-35-3), 6-methyl-5-hepten-2-ol (CAS# 1569-60-4), α-phellandrene (CAS# 99-792-5), δ-3-carene (CAS# 13466-78-9), 1,4-cineole (CAS# 470-67-7), α-terpinene (CAS# 99-86-5), p-cymene (CAS# 99-87-6), limonene (CAS# 5989-27-5), 1,8-cineole (CAS# 470-82-6), ocimene mixture (CAS# 13877-91-3), γ-terpinene (CAS# 99-85-4), linalool oxide (CAS# 60047-17-8), terpinolene (CAS# 586-62-9), linalool (CAS# 78-70-6), terpinen-4-ol (CAS# 20126-76-5), α-terpineol (CAS# 10482-56-1), citronellol (CAS# 106-22-9), geraniol (CAS# 106-24-1), thymol (CAS# 89-83-8), carvacrol (CAS# 499-75-2), α-terpinyl acetate (CAS# 80-26-2), eugenol (CAS# 97-53-0), geranyl acetate (CAS# 105-87-3), methyl eugenol (CAS# 93-15-2), β-caryophyllene (CAS# 87-44-5), aromadendrene (CAS# 489-39-4), α-humulene (CAS# 6753-98-6), (E)-β-farnesene (CAS# 18797-84-8), farnesene, mixture of isomers (product number W383902), alloaromadendrene (CAS# 25246-27-9), nerolidol (CAS# 7212-44-4), caryophyllene oxide (CAS# 1139-30-6), globulol (CAS# 489-41-8), viridiflorol (CAS# 552-02-3), α-bisabolol (Cas# 23089-26-2), farnesol mixture (CAS# 4602-84-0) from Sigma-Aldrich, St. Louis, MO, USA; β-phellandrene (CAS# 555-10-2) from Toronto Research Chemicals (Toronto, ON, Canada); α-copaene (CAS# 3856-25-5) and β-elemene (CAS# 515-13-9) from Fluka Chemical Co., Buchs, SG, Switzerland); and (+)-ar-curcumene (CAS# 4176-06-1) from BOC Sciences Shirley, NY, USA.

3.4. Thin-Layer Chromatography Analysis

3.4.1. Automated High-Performance Thin-Layer Chromatography (HPTLC) Analysis

Chromatography was performed using a CAMAG HPTLC system equipped with VisionCATS 3.1 software (CAMAG, Muttenz, Switzerland). Initial conditions were set following the established HPTLC/TLC protocol for essential oils [37,63]. An HPTLC Silica gel 60 F254 glass-backed plate, 20 × 10 cm (Supelco Merck KGaA, Darmstadt, Germany, operating as Millipore-Sigma in St. Louis, MO, USA), was activated by heat using a TLC Plate Heater III (CAMAG, Muttenz, Switzerland) for 10 min. at 65 °C prior to analysis. Toluene/ethyl acetate 93:7 (v/v) was used as the mobile phase. However, previous bioassays (P.E.K. unpublished data) had indicated that sterile male medflies were repelled by toluene residue, prompting the search for an alternative mobile phase. Hexane was selected due to its similar polarity to toluene.
Melaleuca EO constituents appear in different amounts and cover a relatively wide polarity range when separated by TLC. Tabanca et al. [27] used hexane/acetone 90:10 (v/v) and obtained a good separation that produced two TTO fractions attractive to sterile male medflies, yet these fractions still contained a mixture of chemicals. Further separation was necessary to identify possible individual attractants. Various ratios of hexane/ethyl acetate were then attempted in preliminary experiments and it was decided that two separate solvent combinations provided improved resolution of the fractions of interest.
For the separation of monoterpenes and other non-polar oil constituents, a solution of 45 mL hexane (Hex), Certified ACS, CAS# 92112-69-1 (Fisher Chemical, Thermo Fisher Scientific, Waltham, MA, USA), and 5 mL ethyl acetate (EtOAc), HPLC grade, ≥99.7%, CAS#141-78-6 (Sigma-Aldrich, St. Louis, MO, USA), was prepared for a ratio of 90:10 (v/v). To favor the separation of more polar compounds, 40 mL Hex and 10 mL EtOAc were mixed for an 80:20 (v/v) ratio.
An aliquot of each oil sample was dispensed into a 1.5 mL screw-cap vial, covered with TFE/SIL septum cap (J.G. Finneran Associates, Inc., Vineland, NJ, USA), and placed into an Automatic TLC Sampler (ATS4) (CAMAG, Muttenz, Switzerland). An activated silica gel plate was placed in its corresponding holder. Samples were applied as thin bands (8 mm long, 8 mm from the bottom edge of the plate) using a 25 µL Hamilton syringe with spray application needle and nozzle. Syringe and needle were automatically rinsed 5 times with methanol, ACS grade, CAS# 67-56-1 (Supelco Merck KGaA, Darmstadt, Germany, as EMD Millipore Corporation, Burlington, MA, USA), between samples.
The HPTLC plate was developed in an Automatic Developing Chamber (ADC2) (CAMAG, Muttenz, Switzerland). The chamber containing a saturation pad was saturated for 20 min with 25 mL of the selected mobile phase. To remove as much moisture as possible, the system was also activated for 10 min with a saturated magnesium chloride aqueous solution prepared from magnesium chloride hexahydrate, (MgCl2.6H2O), CAS# 7791-18-6 (Sigma-Aldrich, St. Louis, MO, USA). Development was automatically started and stopped once the solvent front reached a preset height of 85 mm. After development, the plate was allowed to dry for 1 min at room temperature in the fume hood.
A vanillin/sulfuric acid derivatizing reagent was prepared following Wagner and Bladt [63], by adding 0.4 g of vanillin Reagent Plus, 99%, CAS# 121-33-5 (Sigma-Aldrich, St. Louis, MO, USA), to 100 mL of 190 proof ethanol, USP, CAS# 64-17-5 (Decon Labs, Inc., King of Prussia, PA, USA). The ethanolic solution was kept refrigerated until use. Concentrated sulfuric acid, Certified ACS Plus, CAS# 7664-93-9 (Fisher Chemical, Thermo Fisher Scientific, Waltham, MA, USA), was added shortly before use at a proportion of 20 µL acid per mL of vanillin solution.
Automatic derivatization of the plate to generate color occurred inside a Derivatizer chamber (CAMAG, Muttenz, Switzerland) with 2 mL of vanillin/H2SO4 reagent. The derivatizing reagent was applied by spraying through a yellow nozzle at spray level 3. Colors were observed after heating the plate for 1.5 to 3.0 min at 100 °C on a CAMAG Plate Heater III, depending on color intensity. Images of the plate were taken at various stages of the process using a Visualizer 2 (CAMAG, Muttenz, Switzerland) with a 16 mm lens under RT White, UV254, and UV366 light. The retention factors (Rf) values were calculated by VisionCATS software version 3.1. Profiles and comparisons were also generated using VisionCATS.

3.4.2. Automated Preparative Thin-Layer Chromatography Analysis

Preparative TLC was used to isolate unknown bands for identification in cases where a component was not readily identified, and a standard could not be easily referenced for confirmation. An HPTLC Silica gel 60 F254 glass-backed plate, 20 × 10 cm (Supelco Merck KGaA, Darmstadt, Germany, operating as Millipore-Sigma in St. Louis, MO, US), was used to separate and collect a reasonable amount of the unknown chemical for further identification. The plate was initially activated by heating at 100 °C for 15 min on a CAMAG TLC Plate Heater III.
Sample application was conducted in a CAMAG ATS4 autosampler, where 2 µL oil was applied in 20 consecutive bands, 8 mm long each, making a solid horizontal line 8 mm from the bottom edge of the plate. Once the sample was applied, the plate was developed in a previously saturated CAMAG ADC2 chamber. A total of 35 mL mobile phase was used, 25 mL for saturation and 10 for development. Development stopped when solvent front reached 85 mm.
In the case of nerolina oil, a bright pink band at Rf = 0.451 ± 0.022 (40–45 mm from the bottom) was our target chemical. A 5 × 10 cm strip was cut out of the developed plate and was sprayed with vanillin reagent in a CAMAG Derivatizer. The bright pink band on the derivatized strip provided the measurements of the area to scrape to obtain our unknown from the remaining (non-derivatized) portion of the plate. Scraped silica containing our compound of interest was extracted with 1 mL methylene chloride and filtered through a 0.2 µm Whatman AUTOVIAL™ 5 syringeless filter (Global Life Sciences Solutions USA LLC–Cytiva, Marlborough, MA, USA) for GC-MS analysis.

3.5. Statistical Analysis

Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to TTOs and other Melaleuca EOs and their chemical constituents, using the XLSTAT 2021 (Addinsoft, New York, NY, USA) for PCA and JMP (JMP® Pro 17.0.0, SAS Institute Inc. Cary, NC, USA) for HCA. Both PCA and HCA were performed on the means of those volatile constituents higher than 0.5%; the covariance data matrix was 38 × 11 (418 data). Pearson’s correlation model was used for PCA, Euclidean distance for measure, and Ward’s method for HCA analysis.

4. Conclusions

The results of this study demonstrate that HPTLC serves as a quick and effective analytical technique for the screening of selected Melaleuca oils. Its automated steps eliminate most human error and provide better reproducibility. A wide variety of samples may be analyzed by combining the most suitable mobile and stationary phases for the target analytes. This allows for the selection of less toxic solvents, such as hexane instead of toluene, while maintaining comparable retention factors to those in the Pharmacopeia. It also provides a fast detection tool for more polar additives or contaminants that may not be detected under GC-MS conditions. Samples can be applied as a long, narrow band, allowing multiple samples to be simultaneously analyzed and a cleaner separation of individual components.
An advantage over other analytical techniques is that multiple samples and standards may be analyzed at the same time and under true identical conditions using HPTLC. Moreover, the development process is nondestructive, which allows samples to be scraped and extracted from the plate for further studies. For this purpose, a template may be created from a prior plate derivatized with color reagent.
On the other hand, there are some disadvantages to this procedure. In the case of highly volatile constituents, there is a high probability of evaporation during the process. In addition, some oil components do not react with the derivatizing reagent and therefore do not emit a visible color. While some may be seen under UV light, others may not be visible at all. Another complicating factor is that compounds found in trace amounts may fall under the detection limit of the HPTLC instrument. A more complex mixture of coeluting chemicals also represents a challenge. Two or more developments may be required for better separation of these target constituents.
New studies are currently in process, involving two-dimensional and multigradient developments to address the above-mentioned challenges. Future work prospects include the addition of a densitometry module and a TLC-MS interface to achieve a more accurate quantification and precise recovery of individual oil components for further analysis. With so many favorable features and few obstacles, this technique proved to be an efficient and reliable screening tool for the selected Melaleuca oils.

Author Contributions

Conceptualization, A.V., N.T. and P.E.K.; methodology, A.V. and N.T.; software, A.V. and N.T.; validation, A.V., N.T. and P.E.K.; formal analysis, A.V. and N.T.; investigation, A.V., N.T. and P.E.K.; data curation, A.V. and N.T; writing—original draft preparation, A.V. and N.T.; writing—review and editing, A.V., N.T. and P.E.K.; visualization, A.V.; supervision, N.T. and P.E.K.; project administration, P.E.K. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by appropriated funds from the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) (Project Number: 6038-22000-007-00D), and an appointment (AV) to the ARS Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE), an interagency agreement between the U.S. Department of Energy (DOE) and the USDA, managed under DOE contract # DE-SC0014664.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data are available from the authors upon request.

Acknowledgments

The authors are grateful to Elena Q. Schnell (USDA-ARS, Miami, FL, USA) and Wayne S. Montgomery (USDA-ARS, Miami, FL, USA) for their technical assistance with this work, and to Kevin R. Cloonan (USDA-ARS, Miami, FL, USA), Xiangbing Yang (USDA-ARS, Miami, FL, USA), and three anonymous referees for providing reviews of a previous version of this manuscript.

Conflicts of Interest

The authors declare no conflict of interest. Mention of a proprietary product does not constitute an endorsement by the USDA-ARS. USDA is an equal opportunity provider and employer.

Sample Availability

Not applicable.

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Figure 1. PCA biplot of TTO and other Melaleuca EOs based on their chemical composition. EO sample codes are listed according to the code reported in Table 1. The nomenclature of volatile compounds is listed in Table 2. Same color highlights similarity in chemical composition based on statistical analysis (See Section 3.5).
Figure 1. PCA biplot of TTO and other Melaleuca EOs based on their chemical composition. EO sample codes are listed according to the code reported in Table 1. The nomenclature of volatile compounds is listed in Table 2. Same color highlights similarity in chemical composition based on statistical analysis (See Section 3.5).
Molecules 28 03925 g001
Figure 2. Two-way dendrogram of the hierarchical cluster analysis (HCA) performed on the chemical composition of the seven tea tree (M. alternifolia) EOs and four other Melaleuca EOs. Sample codes refer to Table 1. The color box indicated the abundance of each compound. Red represents high density and blue represents low density.
Figure 2. Two-way dendrogram of the hierarchical cluster analysis (HCA) performed on the chemical composition of the seven tea tree (M. alternifolia) EOs and four other Melaleuca EOs. Sample codes refer to Table 1. The color box indicated the abundance of each compound. Red represents high density and blue represents low density.
Molecules 28 03925 g002
Figure 3. Separation of the UHM under UV254 (A) (see Table 5), tea tree oils (B), and other Melaleuca oils (C) (see Table 1) under visible light, developed with Hex/EtOAc 90:10 (v/v) on Silica gel 60 F254.
Figure 3. Separation of the UHM under UV254 (A) (see Table 5), tea tree oils (B), and other Melaleuca oils (C) (see Table 1) under visible light, developed with Hex/EtOAc 90:10 (v/v) on Silica gel 60 F254.
Molecules 28 03925 g003
Figure 4. SSTs (A) are shown under UV254 light. Reference standards (B) (see Table 6) are shown under visible light. Developed with Hex/EtOAc 90:10 (v/v) on Silica gel 60 F254.
Figure 4. SSTs (A) are shown under UV254 light. Reference standards (B) (see Table 6) are shown under visible light. Developed with Hex/EtOAc 90:10 (v/v) on Silica gel 60 F254.
Molecules 28 03925 g004
Figure 5. Separation of the UHM under UV254 (A) (see Table 5), tea tree oils (B), and other Melaleuca oils (C) (see Table 1) under visible light, developed with Hex/EtOAc 80:20 (v/v) on Silica gel 60 F254.
Figure 5. Separation of the UHM under UV254 (A) (see Table 5), tea tree oils (B), and other Melaleuca oils (C) (see Table 1) under visible light, developed with Hex/EtOAc 80:20 (v/v) on Silica gel 60 F254.
Molecules 28 03925 g005
Figure 6. SSTs (A) are shown under UV254 light. Reference standards (B) (see Table 6) are shown under visible light. Developed with Hex/EtOAc 80:20 (v/v) on Silica gel 60 F254.
Figure 6. SSTs (A) are shown under UV254 light. Reference standards (B) (see Table 6) are shown under visible light. Developed with Hex/EtOAc 80:20 (v/v) on Silica gel 60 F254.
Molecules 28 03925 g006
Table 1. Species, sample codes, and sources of oils used in this study.
Table 1. Species, sample codes, and sources of oils used in this study.
SpeciesCodeSource
M. alternifolia (Maiden & Betche) CheelTTAAAromappeal (Puritan’s Pride, Inc.), Oakdale, NY, USA
M. alternifolia (Maiden & Betche) CheelTTASApothecary Shoppe, Portland, OR, USA
M. alternifolia (Maiden & Betche) CheelTTEGEden’s Garden, San Clemente, CA, USA
M. alternifolia (Maiden & Betche) CheelTTFCFloracopeia, Grass Valley, CA, USA
M. alternifolia (Maiden & Betche) CheelTTNGNature’s Gift, Madison, TN, USA
M. alternifolia (Maiden & Betche) CheelTTPTPlant Therapy, Inc, Twin Falls, ID, USA
M. alternifolia (Maiden & Betche) CheelTTSATSAT Group, Kannauj, India
M. cajuputi PowellMCaNature’s Gift, Madison, TN, USA
M. quinquenervia (Cav.) S.T. Blake MNeNature’s Gift, Madison, TN, USA
M. quinquenervia (Cav.) S.T. BlakeMNiNature’s Gift, Madison, TN, USA
M. ericifolia Sm.MRoNature’s Gift, Madison, TN, USA
Table 2. Comparative percentage composition of the Melaleuca EOs.
Table 2. Comparative percentage composition of the Melaleuca EOs.
#* RI Exp** RILitCompoundsTTAATTASTTEGTTFCTTNGTTPTTTSATMCaMNeMNiMRo
1938930α-Thujene RI, MS0.85 ± 0.100.77 ± 0.050.76 ± 0.040.48 ± 0.020.97 ± 0.060.79 ± 0.010.30 ± 0.100.77 ± 0.100.27 ± 0.031.07 ± 0.280.17 ± 0.00
2946939α-Pinene RI, MS, Std1.89 ± 0.062.66 ± 0.032.40 ± 0.411.67 ± 0.052.48 ± 0.161.39 ± 0.021.29 ± 0.165.48 ± 0.110.57 ± 0.129.75 ± 0.135.09 ± 0.03
3954954Camphene RI, MS0.000.000.000.000.000.01 ± 0.000.01 ± 0.000.15 ± 0.020.000.04 ± 0.020.28 ± 0.05
4969960Benzaldehyde RI, MS, Std0.000.000.000.000.000.000.000.000.38 ± 0.040.09 ± 0.040.00
5970975Sabinene RI, MS, Std0.72 ± 0.030.05 ± 0.000.36 ± 0.040.45 ± 0.060.94 ± 0.030.64 ± 0.070.08 ± 0.020.18 ± 0.040.000.000.00
6989979β-pinene RI, MS, Std0.73 ± 0.010.88 ± 0.070.89 ± 0.100.97 ± 0.161.06 ± 0.020.80 ± 0.080.93 ± 0.011.11 ± 0.070.94 ± 0.082.56 ± 0.330.64 ± 0.03
7998990Myrcene RI, MS, Std0.48 ± 0.060.58 ± 0.050.63 ± 0.040.62 ± 0.120.77 ± 0.030.41 ± 0.060.63 ± 0.020.65 ± 0.030.60 ± 0.160.72 ± 0.200.61 ± 0.02
810099916-Methyl-5-hepten-2-ol RI, MS, Std0.000.000.000.000.000.000.000.000.19 ± 0.030.000.00
910101002α-Phellandrene RI, MS, Std0.14 ± 0.070.54 ± 0.020.45 ± 0.070.47 ± 0.100.53 ± 0.080.33 ± 0.100.43 ± 0.130.50 ± 0.020.09 ± 0.010.11 ± 0.030.03 ± 0.01
1010121003Pseudolimonene RI, MS0.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.000.000.000.000.00
1110181011δ-3-Carene RI, MS, Std0.000.000.000.000.000.000.000.000.000.000.20 ± 0.04
12102110141,4-Cineole RI, MS, Std0.000.000.04 ± 0.010.000.000.000.000.000.000.000.00
1310221017α-Terpinene RI, MS, Std6.14 ± 0.0312.62 ± 0.419.62 ± 0.148.35 ± 0.138.89 ± 0.377.63 ± 0.193.47 ± 0.060.22 ± 0.010.13 ± 0.030.000.17 ± 0.04
1410301024p-Cymene RI, MS, Std5.18 ± 0.551.66 ± 0.153.51 ± 0.043.59 ± 0.273.22 ± 0.283.71 ± 0.1711.47 ± 0.190.79 ± 0.110.03 ± 0.020.07 ± 0.021.21 ± 0.06
1510391029Limonene RI, MS, Std1.63 ± 0.230.30 ± 0.091.10 ± 0.071.15 ± 0.020.87 ± 0.031.10 ± 0.050.51 ± 0.190.10 ± 0.000.19 ± 0.032.83 ± 0.183.03 ± 0.05
1610391029β-Phellandrene RI, MS, Std0.10 ± 0.010.26 ± 0.010.86 ± 0.040.83 ± 0.060.03 ± 0.000.44 ± 0.040.39 ± 0.010.07 ± 0.010.01 ± 0.000.01 ± 0.000.00
17104010311,8-Cineole RI, MS, Std2.85 ± 0.381.17 ± 0.013.76 ± 0.123.28 ± 0.074.79 ± 0.143.90 ± 0.084.50 ± 0.0964.63 ± 1.191.91 ± 0.1652.20 ± 0.2827.57 ± 0.56
1810491037(Z)-β-Ocimene RI, MS, Std0.000.000.000.000.000.000.000.000.69 ± 0.100.000.04 ± 0.01
1910621050(E)-β-Ocimene RI, MS, Std0.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.000.000.74 ± 0.100.03 ± 0.010.76 ± 0.08
2010711059γ-Terpinene RI, MS, Std16.42 ± 0.8318.91 ± 0.4619.58 ± 0.1019.60 ± 0.1016.58 ± 0.4520.75 ± 0.2016.61 ± 0.413.24 ± 0.200.12 ± 0.050.57 ± 0.001.84 ± 0.01
2110811070cis-Sabinene hydrate RI, MS0.000.01 ± 0.000.14 ± 0.040.20 ± 0.090.29 ± 0.070.07 ± 0.020.09 ± 0.010.01 ± 0.010.000.000.00
2210841072cis-Linalool oxide RI, MS, Std0.000.000.30 ± 0.140.000.000.000.000.000.07 ± 0.000.000.13 ± 0.13
2310981088Terpinolene RI, MS, Std2.83 ± 0.053.61 ± 0.014.19 ± 0.043.63 ± 0.213.39 ± 0.123.16 ± 0.062.75 ± 0.041.04 ± 0.050.26 ± 0.100.45 ± 0.072.14 ± 0.02
2411071096Linalool RI, MS, Std0.07 ± 0.020.13 ± 0.010.45 ± 0.151.12 ± 0.031.04 ± 0.040.13 ± 0.060.22 ± 0.020.26 ± 0.0433.30± 0.630.47 ± 0.2938.19 ± 0.06
2511151097Hotrienol RI, MS0.000.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.000.000.12 ± 0.060.000.15 ± 0.02
2611231121cis-p-Menth-2-en-1-ol 0.14 ± 0.080.21 ± 0.060.21 ± 0.050.000.37 ± 0.080.28 ± 0.010.36 ± 0.050.000.000.000.00
2711251122trans-p-Menth-2-en-1-ol0.21 ± 0.010.18 ± 0.010.17 ± 0.020.15 ± 0.020.31 ± 0.040.22 ± 0.010.28 ± 0.040.000.000.000.00
2811681166δ-Terpinol RI, MS0.000.000.000.000.000.000.000.12 ± 0.010.000.11 ± 0.010.12 ± 0.01
2911831177Terpinene-4-ol RI, MS, Std44.28 ± 0.9038.62 ± 0.3337.66 ± 0.2440.36 ± 0.5338.42 ± 0.2240.55 ± 0.6739.57 ± 1.370.71 ± 0.030.27 ± 0.000.62 ± 0.010.79 ± 0.01
3011881182p-Cymen-8-ol RI, MS0.14 ± 0.040.05 ± 0.020.12 ± 0.000.17 ± 0.030.11 ± 0.010.10 ± 0.020.19 ± 0.100.02 ± 0.010.000.000.10 ± 0.01
3111981188α-Terpineol RI, MS, Std3.74 ± 0.053.11 ± 0.013.35 ± 0.073.61 ± 0.094.66 ± 0.033.40 ± 0.044.20 ± 0.095.44 ± 0.100.46 ± 0.015.08 ± 0.064.05 ± 0.08
3212061196cis-Piperitol RI, MS, Std0.08 ± 0.020.07 ± 0.000.07 ± 0.000.03 ± 0.000.08 ± 0.010.09 ± 0.010.13 ± 0.020.000.000.000.00
3312181208trans-Piperitol RI, MS0.14 ± 0.010.09 ± 0.010.10 ± 0.000.05 ± 0.000.08 ± 0.000.15 ± 0.000.14 ± 0.060.000.000.000.00
3412261216trans-Carveol RI, MS,0.000.000.000.000.000.000.000.000.000.000.01 ± 0.01
3512321221cis-Sabinene hydrate acetate RI, MS0.000.000.000.000.000.000.000.000.08 ± 0.000.000.00
3612361225Citronellol RI, MS, Std0.000.000.000.000.000.000.000.000.000.01 ± 0.000.02 ± 0.00
3712381229Nerol RI, MS0.000.000.000.000.000.000.000.000.01 ± 0.000.000.16 ± 0.01
3812431237Ascaridole RI, MS0.000.000.000.000.000.000.05 ± 0.030.000.000.000.00
3912451238Neral0.000.000.000.000.000.000.000.000.000.000.09 ± 0.01
4012531252Geraniol RI, MS, Std0.05 ± 0.050.000.04 ± 0.030.02 ± 0.000.03 ± 0.000.000.02 ± 0.020.04 ± 0.020.19 ± 0.020.02 ± 0.010.20 ± 0.00
41126412582-Phenyl ethyl acetate RI, MS0.000.000.000.000.000.000.000.000.000.000.08 ± 0.02
4212711267Geranial RI, MS0.000.000.000.000.000.000.000.000.000.000.12 ± 0.01
4312731269trans-Ascaridol glycol RI, MS0.12 ± 0.020.01 ± 0.000.04 ± 0.000.05 ± 0.020.02 ± 0.000.04 ± 0.010.06 ± 0.020.000.000.000.00
4412911288cis-Ascaridol glycol RI, MS0.04 ± 0.020.01 ± 0.000.02 ± 0.000.04 ± 0.010.000.01 ± 0.000.04 ± 0.010.000.000.000.00
4512951290Thymol RI, MS, Std0.000.000.000.01 ± 0.00l0.000.000.02 ± 0.010.000.000.000.00
4613061299Carvacrol RI, MS, Std0.01 ± 0.010.000.02 ± 0.010.02 ± 0.010.000.000.02 ± 0.010.000.000.000.00
4713301324Methyl geranate RI, MS0.000.000.000.000.000.000.000.000.000.000.07 ± 0.03
4813431338δ-Elemene RI, MS0.01 ± 0.000.01 ± 0.000.01 ± 0.000.000.05 ± 0.000.01 ± 0.000.02 ± 0.010.01 ± 0.000.000.000.02 ± 0.00
4913551347α-Terpinyl acetate RI, MS, Std0.000.000.000.000.000.000.000.64 ± 0.010.001.30 ± 0.020.00
5013561348α-Cubebene RI, MS0.01 ± 0.010.04 ± 0.000.03 ± 0.000.01 ± 0.000.07 ± 0.000.03 ± 0.000.06 ± 0.020.000.000.000.00
5113591359Eugenol RI, MS, Std0.000.000.000.000.000.000.000.000.01 ± 0.000.000.00
5213761375α-Ylangene RI, MS0.000.000.000.000.000.000.000.13 ± 0.020.000.000.00
5313781376Isoledene RI, MS0.06 ± 0.010.08 ± 0.010.03 ± 0.010.04 ± 0.000.06 ± 0.000.05 ± 0.010.06 ± 0.020.000.000.000.15 ± 0.02
5413801376α-Copaene RI, MS, Std0.10 ± 0.010.14 ± 0.010.10 ± 0.010.07 ± 0.000.13 ± 0.000.06 ± 0.020.13 ± 0.020.08 ± 0.010.000.03 ± 0.000.02 ± 0.01
5513821381Geranyl acetate RI, MS, Std000000000.000.000.02 ± 0.01
5613951390β-Elemene RI, MS, Std0.03 ± 0.010.01 ± 0.000.02 ± 0.000.01 ± 0.000.06 ± 0.000.01 ± 0.000.03 ± 0.010.05 ± 0.030.000.000.32 ± 0.02
5714031402α-Funebrene RI, MS, Std0.000.000.000.000.000.000.000.000.03 ± 0.010.000.00
5814041403Methyl eugenol RI, MS, Std0.000.000.01 ± 0.000.01 ± 0.010.000.000.02 ± 0.000.000.000.000.00
5914091408Isocaryophyllene RI, MS0.000.000.000.000.000.000.000.01 ± 0.010.02 ± 0.010.000.00
6014101409α-Gurjunene RI, MS0.41 ± 0.010.58 ± 0.010.36 ± 0.010.18 ± 0.060.45 ± 0.010.35 ± 0.010.43 ± 0.060.06 ± 0.030.000.07 ± 0.020.39 ± 0.01
6114121411α-Cedrene RI, MS0.000.000.000.000.000.000.000.000.06 ± 0.030.000.00
6214171416β-Maaliene RI, MS0.01 ± 0.000.02 ± 0.000.02 ± 0.000.000.01 ± 0.000.000.02 ± 0.000.000.000.000.03 ± 0.01
6314211419β-Caryophyllene RI, MS, Std0.37 ± 0.010.74 ± 0.020.29 ± 0.010.21 ± 0.010.56 ± 0.020.28 ± 0.030.40 ± 0.043.82 ± 0.122.40 ± 0.052.66 ± 0.050.16 ± 0.01
6414291425γ-Maaliene RI, MS0.06 ± 0.010.08 ± 0.000.07 ± 0.000.05 ± 0.010.06 ± 0.000.04 ± 0.000.06 ± 0.010.01 ± 0.010.000.01 ± 0.010.10 ± 0.01
6514331433β-Gurjunene RI, MS0.01 ± 0.000.01 ± 0.000.01 ± 0.000.000.01 ± 0.000.000.02 ± 0.000.05 ± 0.010.000.000.10 ± 0.00
6614351433α-Maaliene RI, MS, Std0.07 ± 0.010.09 ± 0.000.07 ± 0.010.05 ± 0.010.07 ± 0.000.05 ± 0.000.07 ± 0.020.05 ± 0.010.000.000.12 ± 0.01
6714391441Aromadendrene RI, MS, Std1.51 ± 0.021.90 ± 0.021.20 ± 0.031.25 ± 0.041.24 ± 0.011.51 ± 0.191.27 ± 0.090.65 ± 0.040.000.16 ± 0.083.38 ± 0.02
6814431443Selina-5,11-diene RI, MS0.16 ± 0.010.22 ± 0.010.14 ± 0.010.08 ± 0.040.17 ± 0.000.11 ± 0.010.15 ± 0.020.06 ± 0.010.000.000.26 ± 0.01
6914481451Amorpha-4,11-diene RI, MS0.000.000.000.000.000.000.000.000.07 ± 0.030.000.00
7014501453trans-Muurola-3.5-diene RI, MS0.11 ± 0.010.19 ± 0.010.12 ± 0.010.03 ± 0.000.31 ± 0.010.14 ± 0.010.18 ± 0.030.02 ± 0.010.000.01 ± 0.000.06 ± 0.01
7114531454α-Humulene RI, MS, Std0.08 ± 0.000.12 ± 0.000.07 ± 0.000.05 ± 0.000.15 ± 0.020.07 ± 0.010.09 ± 0.011.81 ± 0.080.40 ± 0.010.40 ± 0.030.03 ± 0.00
7214571456(E)-β-Farnesene RI, MS, Std0.000.000.000.000.000.000.000.001.93 ± 0.400.12 ± 0.050.00
7314591460Alloaromadendrene RI, MS, Std0.67 ± 0.010.96 ± 0.010.53 ± 0.020.47 ± 0.020.62 ± 0.010.54 ± 0.010.62 ± 0.050.36 ± 0.020.000.51 ± 0.051.41 ± 0.02
7414641466α-Acoradiene RI, MS0.000.000.000.000.000.000.000.000.01 ± 0.000.000.00
7514671470β-Acoradiene RI, MS0.000.000.000.000.000.000.000.000.03 ± 0.010.000.00
761471147510-epi-β-Acoradiene RI, MS0.000.000.000.000.000.000.000.000.02 ± 0.010.000.00
7714731476trans-Cadina-1(6),4-diene RI, MS0.36 ± 0.010.55 ± 0.020.36 ± 0.010.20 ± 0.010.45 ± 0.020.42 ± 0.020.45 ± 0.050.000.02 ± 0.010.000.00
7814741477γ-Gurjunene RI, MS0.000.000.000.000.000.000.000.000.000.000.35 ± 0.01
7914761479γ-Muurolene RI, MS0.04 ± 0.000.05 ± 0.000.04 ± 0.000.03 ± 0.010.04 ± 0.000.03 ± 0.000.05 ± 0.010.45 ± 0.030.000.07 ± 0.010.08 ± 0.01
8014801480ar-Curcumene RI, MS, Std0.000.000.000.000.000.000.000.000.13 ± 0.040.000.00
8114811482γ-Curcumene RI, MS0.000.000.000.000.000.000.000.000.09 ± 0.020.000.00
8214831484α-Amorphene RI, MS0.000.000.000.000.000.000.000.30 ± 0.010.000.000.00
8314841485Germacrene D RI, MS0.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.000.000.18 ± 0.020.00
8414861490β-Selinene RI, MS0.09 ± 0.010.11 ± 0.010.10 ± 0.010.09 ± 0.010.06 ± 0.000.05 ± 0.010.08 ± 0.021.28 ± 0.050.000.20 ± 0.010.23 ± 0.01
8514881490Alloaromadendr-9-ene RI, MS0.11 ± 0.010.14 ± 0.010.07 ± 0.040.08 ± 0.010.11 ± 0.000.07 ± 0.010.11 ± 0.020.000.000.18 ± 0.000.29 ± 0.01
8614911492δ-Selinene RI, MS0.000.000.000.000.000.000.000.35 ± 0.010.000.000.00
8714921493cis-β-Guaiene RI, MS0.19 ± 0.010.28 ± 0.010.18 ± 0.010.11 ± 0.010.31 ± 0.010.16 ± 0.010.21 ± 0.030.11 ± 0.010.000.01 ± 0.010.16 ± 0.01
8814941496Ledene RI, MS1.14 ± 0.011.62 ± 0.020.86 ± 0.030.47 ± 0.031.06 ± 0.011.38 ± 0.051.69 ± 0.050.19 ± 0.030.16 ± 0.061.76 ± 0.170.98 ± 0.02
8914951498α-Selinene RI, MS0.000.000.000.000.000.000.001.12 ± 0.080.000.000.00
9014961499(Z,E)-α-Farnesene RI, MS0.000.000.000.000.000.000.000.000.64 ± 0.140.000.00
9114971500Bicyclogermacrene RI, MS0.91 ± 0.010.96 ± 0.060.67 ± 0.020.31 ± 0.010.72 ± 0.020.91 ± 0.040.60 ± 0.030.000.000.02 ± 0.010.62 ± 0.02
9214981500α-Muurolene RI, MS0.17 ± 0.010.20 ± 0.000.14 ± 0.000.11 ± 0.010.22 ± 0.000.16 ± 0.010.20 ± 0.020.06 ± 0.010.000.09 ± 0.010.03 ± 0.00
9315011501Epizonarene RI, MS0.02 ± 0.000.03 ± 0.000.02 ± 0.000.01 ± 0.000.03 ± 0.000.01 ± 0.000.02 ± 0.000.19 ± 0.010.000.000.00
9415031502trans-β-Guaiene RI, MS0.000.000.000.000.000.000.000.000.09 ± 0.070.000.00
9515051505(Z)-α-Bisabolene RI, MS0.000.000.000.000.000.000.000.000.23 ± 0.030.000.00
9615061505(E,E)-α-Farnesene RI, MS, Std0.000.000.000.000.000.000.000.001.67 ± 0.380.11 ± 0.040.00
9715081512δ-Amorphene RI, MS0.000.000.000.000.000.000.000.000.000.000.19 ± 0.01
9815121513γ-Cadinene RI, MS0.01 ± 0.000.02 ± 0.000.02 ± 0.000.01 ± 0.000.02 ± 0.000.01 ± 0.000.03 ± 0.000.07 ± 0.010.000.25 ± 0.020.06 ± 0.01
9915141515(Z)-γ-Bisabolene RI, MS0.000.000.000.000.000.000.000.000.21 ± 0.050.000.00
100151715227-epi-α-Selinene RI, MS0.000.000.000.000.000.000.000.02 ± 0.010.000.000.06 ± 0.01
10115181522trans-Calamene RI, MS0.07 ± 0.060.01 ± 0.000.01 ± 0.000.01 ± 0.000.000.01 ± 0.000.000.000.000.000.00
10215211523δ-Cadinene RI, MS1.35 ± 0.011.71 ± 0.021.22 ± 0.041.06 ± 0.041.32 ± 0.021.78 ± 0.141.79 ± 0.090.23 ± 0.030.13 ± 0.040.35 ± 0.010.17 ± 0.03
10315271529Zonarene RI, MS0.75 ± 0.020.58 ± 0.040.33 ± 0.020.30 ± 0.010.37 ± 0.020.54 ± 0.040.48 ± 0.060.04 ± 0.010.04 ± 0.010.000.03 ± 0.02
10415291531(E)-γ-Bisabolene RI, MS0.000.000.000.000.000.000.000.000.19 ± 0.050.000.00
10515311534trans-Cadina-1,4-diene0.22 ± 0.010.28 ± 0.010.19 ± 0.010.14 ± 0.010.29 ± 0.010.24 ± 0.010.27 ± 0.020.000.000.000.00
10615351538α-Cadinene RI, MS0.000.000.000.000.000.000.000.06 ± 0.030.000.000.01 ± 0.00
10715401545α-Calacorene RI, MS0.01 ± 0.000.01 ± 0.000.02 ± 0.000.02 ± 0.010.000.01 ± 0.000.04 ± 0.030.000.000.000.03 ± 0.03
10815421546Selina-3,7(11)-diene RI, MS0.000.000.01 ± 0.000.000.000.01 ± 0.000.000.16 ± 0.020.000.000.00
10915481547(E)-α-Bisabolene RI, MS0.000.000.000.000.000.000.000.000.41 ± 0.110.000.00
11015591561Germacrene B RI, MS0.000.000.000.000.000.000.000.11 ± 0.010.000.000.00
11115601563(E)-Nerolidol RI, MS, Std0.000.000.000.000.000.000.000.0048.40 ± 1.216.89 ± 0.440.00
11215621567Maaliol RI, MS0.06 ± 0.010.06 ± 0.010.06 ± 0.010.13 ± 0.010.03 ± 0.000.01 ± 0.010.01 ± 0.000.000.000.000.00
11315631568Palustrol RI, MS0.07 ± 0.010.07 ± 0.010.09 ± 0.010.19 ± 0.020.04 ± 0.000.02 ± 0.010.08 ± 0.020.01 ± 0.000.000.000.09 ± 0.01
11415721578Spathulenol RI, MS0.12 ± 0.020.07 ± 0.010.07 ± 0.010.14 ± 0.010.05 ± 0.000.03 ± 0.010.10 ± 0.050.01 ± 0.000.000.000.25 ± 0.01
11515781583Caryophyllene oxide RI, MS, Std0.000.000.000.000.000.000.000.01 ± 0.000.20 ± 0.050.000.00
11615851590Globulol RI, MS, Std0.40 ± 0.020.43 ± 0.020.35 ± 0.020.94 ± 0.040.25 ± 0.010.22 ± 0.010.23 ± 0.020.38 ± 0.030.31 ± 0.040.35 ± 0.010.59 ± 0.02
11715861592Viridiflorol RI, MS, Std0.19 ± 0.010.17 ± 0.010.18 ± 0.010.41 ± 0.020.10 ± 0.000.11 ± 0.010.16 ± 0.030.40 ± 0.020.07 ± 0.016.23 ± 0.170.15 ± 0.01
11815881595Cubeban-11-ol RI, MS0.15 ± 0.010.16 ± 0.010.13 ± 0.010.35 ± 0.010.07 ± 0.010.08 ± 0.010.12 ± 0.020.000.000.000.12 ± 0.01
11915941600Rosiflorol RI, MS0.11 ± 0.010.12 ± 0.010.12 ± 0.010.29 ± 0.010.06 ± 0.000.04 ± 0.010.10 ± 0.020.000.000.000.10 ± 0.01
12015971600Guaiol RI, MS0.000.000.000.000.000.000.000.03 ± 0.010.01 ± 0.000.09 ± 0.010.11 ± 0.01
12115991602Ledol RI, MS0.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.01 ± 0.000.03 ± 0.020.01 ± 0.000.05 ± 0.010.01 ± 0.000.72 ± 0.020.03 ± 0.00
122160916075-epi-7-epi-α-Eudesmol RI, MS0.11 ± 0.010.13 ± 0.010.12 ± 0.010.30 ± 0.020.04 ± 0.000.04 ± 0.010.08 ± 0.020.000.000.000.14 ± 0.01
12316111608Humulene epoxide II RI, MS0.000.000.000.000.000.000.000.02 ± 0.010.02 ± 0.000.02 ± 0.010.00
124161916191,10-di-epi-Cubenol RI, MS0.20 ± 0.010.22 ± 0.010.19 ± 0.010.40 ± 0.020.14 ± 0.000.14 ± 0.010.22 ± 0.040.000.000.02 ± 0.010.00
1251626162310-epi-γ-Eudesmol RI, MS0.000.000.000.000.000.000.000.02 ± 0.010.000.000.00
126163016281-epi-Cubenol RI, MS0.10 ± 0.010.10 ± 0.010.10 ± 0.010.17 ± 0.010.07 ± 0.000.06 ± 0.010.15 ± 0.010.000.000.06 ± 0.030.01 ± 0.00
12716321631Muurola-4,10(14)-dien-1β-ol RI, MS0.000.000.000.000.000.000.000.03 ± 0.000.02 ± 0.000.000.00
12816331632γ-Eudesmol RI, MS0.000.000.000.000.000.000.000.15 ± 0.010.000.000.00
12916391640T-Cadinol RI, MS0.000.000.000.000.000.000.000.01 ± 0.000.000.000.00
13016431646α-Muurolol RI, MS0.06 ± 0.020.03 ± 0.010.03 ± 0.010.07 ± 0.000.02 ± 0.000.01 ± 0.000.03 ± 0.010.000.000.03 ± 0.000.02 ± 0.00
13116451646Cubenol RI, MS0.000.01 ± 0.000.000.02 ± 0.010.000.000.02 ± 0.000.000.000.06 ± 0.010.00
13216491650β-Eudesmol RI, MS0.000.000.000.000.000.000.000.10 ± 0.010.000.000.00
13316521653α-Eudesmol RI, MS0.000.000.000.000.000.000.000.18 ± 0.010.000.01 ± 0.000.00
13416681671Bulnesol RI, MS0.000.000.000.000.000.000.000.01 ± 0.000.000.01 ± 0.000.00
13516791684epi-α-Bisabolol RI, MS0.000.000.000.000.000.000.000.000.02 ± 0.000.000.00
13616811685α-Bisabolol RI, MS, Std0.000.000.000.000.000.000.000.000.03 ± 0.010.000.00
13717101715(E,Z)-Farnesol RI, MS, Std0.000.000.000.000.000.000.000.000.01 ± 0.010.000.00
13817201723(Z,E)-Farnesol RI, MS, Std0.000.000.000.000.000.000.000.000.01 ± 0.010.000.00
Total99.70 ± 0.0799.85 ± 0.0499.65 ± 0.0099.84 ± 0.0399.87 ± 0.0199.97 ± 0.0299.73 ± 0.2699.49 ± 0.3799.72 ± 0.1199.79 ± 0.1399.53 ± 0.05
* RIexp: Retention indices (RIs) calculated from the current study; ** RIlit: RI from the literature [41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57]. Identification method: RI: retention index; MS: computer matching of the mass spectra libraries and comparison with the literature data; Std: standards compounds were purchased. TTO and samples and their corresponding major components are represented in blue. Other Melaleuca oils and their respective major components are highlighted in gray.
Table 3. Eigenvalues and percentage of variability and cumulative.
Table 3. Eigenvalues and percentage of variability and cumulative.
F1F2F3F4F5F6F7
Eigenvalue14.928.604.803.851.971.311.17
Variability (%)39.2622.6212.6210.135.203.453.07
Cumulative (%)39.2661.8874.5084.6389.8393.2796.35
Table 4. Factor loadings, contributions (%), and squared cosine (cos2) values. The positive important contributions are highlighted in blue, and the negative important contributions are highlighted in pink. The highest cos2 values are highlighted in green.
Table 4. Factor loadings, contributions (%), and squared cosine (cos2) values. The positive important contributions are highlighted in blue, and the negative important contributions are highlighted in pink. The highest cos2 values are highlighted in green.
Factor Loadings and
Contributions (%)
Squared Cosines
(cos2)
#CompoundsF1F2F1F2
1α−Thujene0.138 (0.129)−0.649 (4.945)0.0190.425
2α−Pinene−0.546 (2.024)−0.759 (6.695)0.3020.575
5Sabinene0.563 (2.191)−0.055 (0.042)0.3270.004
6β-Pinene−0.517 (1.799)−0.601 (4.208)0.2680.362
7Myrcene−0.341 (0.824)−0.317 (1.311)0.1230.113
9α-Phellandrene0.525 (1.832)−0.237 (0.681)0.2730.059
13α-Terpinene0.853 (4.851)0.033 (0.010)0.7240.001
14p-Cymene0.621 (2.579)0.033 (0.011)0.3850.001
15Limonene−0.157 (0.163)−0.357 (1.474)0.0240.127
16β-Phellandrene0.539 (1.963)0.052 (0.036)0.2930.003
171,8-Cineole−0.685 (3.144)−0.667 (5.139)0.4690.442
18(Z)-β-Ocimene−0.561 (2.106)0.794 (7.323)0.3140.629
19(E)-β-Ocimene−0.527 (1.866)0.628 (4.594)0.2780.395
20γ-Terpinene0.947 (6.044)0.013 (0.002)0.9020.000
23Terpinolene0.943 (5.940)−0.001 (0.000)0.8860.000
24Linalool−0.502 (1.693)0.605 (4.263)0.2530.366
29Terpinene-4-ol0.948 (6.034)0.047 (0.024)0.9000.002
31α-Terpineol0.074 (0.037)−0.923 (9.987)0.0060.858
49α-Terpinyl acetate−0.637 (2.725)−0.708 (5.793)0.4070.498
60α-Gurjunene0.862 (4.918)−0.023 (0.008)0.7340.001
63β-Caryophyllene−0.833 (4.665)−0.247 (0.703)0.6960.060
67Aromadendrene0.483 (1.556)0.015 (0.003)0.2320.000
71α-Humulene−0.586 (2.293)−0.338 (1.318)0.3420.113
72(E)-β-Farnesene−0.582 (2.268)0.753 (6.598)0.3380.567
73Alloaromadendrene0.373 (0.904)−0.237 (0.654)0.1350.056
77trans-Cadina-1(6),4-diene0.911 (5.546)0.050 (0.026)0.8280.002
84β-Selinene−0.434 (1.261)−0.469 (2.534)0.1880.218
88Ledene0.431 (1.232)−0.422 (2.043)0.1840.176
89α-Selinene−0.407 (1.106)−0.369 (1.568)0.1650.135
90(Z,E)-α-Farnesene−0.549 (2.022)0.786 (7.179)0.3020.617
91Bicyclogermacrene0.894 (5.351)0.067 (0.053)0.7980.005
96(E,E)-α-Farnesene −0.584 (2.283)0.751 (6.563)0.3410.564
102δ-Cadinene0.917 (5.655)−0.024 (0.005)0.8440.000
103Zonarene0.873 (5.105)0.073 (0.065)0.7620.006
111(E)-Nerolidol−0.620 (2.580)0.707 (5.811)0.3850.499
116Globulol0.016 (0.001)0.005 (0.000)0.0000.000
117Viridiflorol−0.486 (1.593)−0.602 (4.188)0.2380.360
121Ledol−0.506 (1.718)−0.599 (4.143)0.2560.356
Table 5. List of UHM * components used in this study.
Table 5. List of UHM * components used in this study.
LabelNameCAS#
(e) Phthalamide85-41–6
(f) 9-Hydroxyfluorene1689-64-1
(g) Thioxanthen-9-one492-22-8
(h) 2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl) phenol3147-75-9
* UHM components visible under the established analytical conditions.
Table 6. System suitability standards (SSTs) and reference standards for Melaleuca EOs.
Table 6. System suitability standards (SSTs) and reference standards for Melaleuca EOs.
TrackNameCAS#TrackNameCAS#
1Isoeugenol97-54-1111,8-Cineole470-82-6
2Isoeugenyl acetate93-29-812(S)-(−)-Limonene5989-54-8
3(1R)-(+)-α-Pinene7785-70-813(−)-Caryophyllene oxide1139-30-6
4α-Phellandrene99-83-214Nerolidol7212-44-4
5Ocimene mix13877-91-315(−)-Terpinen-4-ol20126-76-5
6(+)-Aromadendrene489-39-416(−)-Linalool1126-91-0
7Farnesene mix502-61-417Viridiflorol0552-02-03
8α-Humulene6753-98-618(−)-α-Terpineol10482-56-1
9β-Caryophyllene87-44-519(−)-Globulol489-41-8
10α-Terpinyl acetate80-26-220Geraniol106-24-1
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Vázquez, A.; Tabanca, N.; Kendra, P.E. HPTLC Analysis and Chemical Composition of Selected Melaleuca Essential Oils. Molecules 2023, 28, 3925. https://doi.org/10.3390/molecules28093925

AMA Style

Vázquez A, Tabanca N, Kendra PE. HPTLC Analysis and Chemical Composition of Selected Melaleuca Essential Oils. Molecules. 2023; 28(9):3925. https://doi.org/10.3390/molecules28093925

Chicago/Turabian Style

Vázquez, Aimé, Nurhayat Tabanca, and Paul E. Kendra. 2023. "HPTLC Analysis and Chemical Composition of Selected Melaleuca Essential Oils" Molecules 28, no. 9: 3925. https://doi.org/10.3390/molecules28093925

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