SEPTEMBER 2018
Tea Tree Oil Laboratory Guidance Document
By Stefan Gafner, PhDa* and Ashley Dowellb aAmerican
Botanical Council, PO Box 144345, Austin, TX
78714 bSouthern Cross
University, Military Road, East Lismore, NSW 2480, Australia *Corresponding author: email
Citation
(JAMA style): Gafner S, Dowell A. Tea tree oil laboratory guidance document.
Austin, TX: ABC-AHP-NCNPR Botanical Adulterants Prevention Program. 2018.
Keywords: Adulteration, Eucalyptus globulus, eucalyptus oil, Melaleuca
alternifolia, Melaleuca
linariifolia, tea tree oil, white camphor oil CONTENTS
1.
Purpose
Tea tree oil (TTO) is the
essential oil of tea tree (Melaleuca
alternifolia or M. linariifolia, Myrtaceae).
Adulteration of TTO has become more apparent in recent years. Adulteration occurs
with single essential oil components (e.g., sabinene from pine oil), waste
products derived from other essential oils such as pine (Pinus spp., Pinaceae), eucalyptus (Eucalyptus globulus and other Eucalyptus
spp., Myrtaceae), and camphor (Cinnamomum
camphora, Lauraceae) oils, or with essential oils from other Melaleuca species and the closely
related genus Leptospermum. This Laboratory
Guidance Document presents a review of the various analytical technologies used
to differentiate between authentic tea tree oil and essential oils containing
adulterating materials. This document can be used in conjunction with the Tea
Tree Oil Botanical Adulterants Bulletin published by the ABC-AHP-NCNPR
Botanical Adulterants Prevention Program in 2017.1
2.
Scope
The various analytical
methods were reviewed with the specific purpose of identifying strengths and
limitations of the existing methods for differentiating tea tree oil from its
potentially adulterating materials. Less emphasis will be given to authenticate
whole, cut, or powdered tea tree leaves and distinguish them from potential
confounding materials by macroscopic, microscopic and genetic analysis. Analysts
can use this review to help guide the appropriate choice of techniques for qualitative
purposes. The suggestion of a specific method for testing TTO materials in
their particular matrix in this Laboratory
Guidance Document does not reduce or remove the responsibility of laboratory
personnel to demonstrate adequate method performance in their own laboratories
using accepted protocols outlined in the United States Food and Drug
Administration’s Good Manufacturing Practices (GMPs) rule (21 CFR Part 111) and
those published by AOAC International, International Organization for
Standardization (ISO), World Health Organization (WHO), and International
Conference on Harmonisation (ICH).
3.
Common and Scientific Names
3.1
Common name: Tea tree
Note:
According to the American Herbal Products Association’s Herbs of Commerce, 2nd ed.,2 the
standardized common name of M.
alternifolia is tea tree. Melaleuca
linariifolia, although rarely used for TTO production, is another accepted
source material for TTO according to the ISO,3 but
is not listed as such source in the second edition of Herbs of Commerce.
3.2
Other common names for Melaleuca
alternifolia
English: paperbark
tree, narrow-leaved paperbark4-6 Chinese: Hùshēng yè bái qiān céng (互生叶白千层)7
French: Mélaleuca (arbre
à thé),8 tea tree, théier Australien9,10
German: Teebaum,8 australischer Teebaum
Italian: Melaleuca,8 tea tree, albero del tè
Spanish: Árbol de té,10 Melaleuca alternifolia8
International Nomenclature of Cosmetic Ingredients (INCI):
Melaleuca alternifolia (tea tree) leaf
oil
China INCI: 互生叶白千层(Melaleuca alternifolia)叶油
3.3 Latin binomial:
Melaleuca
alternifolia (Maiden & Betche) Cheel
3.4 Synonyms: Melaleuca linariifolia var. alternifolia Maiden &
Betche.
3.5
Botanical family: Myrtaceae
4.
Botanical Description and Geographical Range
Melaleuca
alternifolia is an evergreen tree native to Australia, where it
is endemic to the East coastal littoral of continental Australia from
Maryborough in the north to Port Macquarie in the south and west to the Great
Dividing Range. The native habitat of M. alternifolia is low-lying, swampy, sub-tropical, coastal ground.4-6 Botanical descriptions have been
published by a number of sources.6,11-13 Melaleuca
alternifolia has been introduced and cultivated in China, Indonesia, Kenya,
Madagascar, Malaysia, South Africa, Tanzania, Thailand, United States, and
Zimbabwe.5
Melaleuca
linariifolia is a tree or shrub growing up to 10 m (32.8
ft.). It has a more limited distribution range, being endemic to the Australian
states of Queensland and New South Wales, where it is mostly found in coastal
areas. It grows in swampy shrub land or open forest, low shrubby or dry
sclerophyll forests, eucalyptus woodlands, and on sandy and sandstone soils.11
5.
Adulterants and Confounding Materials
Table
1.
Scientific Names, Family, and Common Names
of Known Tea Tree Oil Adulterants*
Speciesa
|
Synonym(s)a
|
Family
|
Common
nameb
|
Other
common namesc
|
Cinnamomum
camphora (L.)
J.Presl.
|
C. camphora f. linaloolifera (Y.Fujita) Sugim. C. camphora f. parvifolia Miq. C. camphora var. cyclophylla Nakai C. camphora var. glaucescens (A.Braun) Meisn. C. camphora var. hosyo (Hatus.) J.C. Liao C. camphora var. linaloolifera Y.Fujita C. camphora var. rotundifolia Makino
|
Lauraceae
|
Camphor
|
Camphor-laurel, Japanese camphor tree
|
Eucalyptus globulus Labill
|
E. gigantea Dehnh E. glauca A. Cunn. Ex DC. E. globulosus St.-Lag. E. maidenii subsp. globulus (Labill.) J.B. Kirkp. E. perfoliata Desf.
|
Lauraceae
|
Eucalyptus
|
Blue gum, southern blue gum, Tasmanian blue gum
|
Melaleuca cajuputi Maton & Sm. ex R. Powell
|
M. saligna (J.F.Gmel.) Reinw. ex Blume M. trinervis Buch.-Ham. Myrtus saligna J.F.Gmel. Pimentus saligna (J.F. Gmel.)
|
Myrtaceae
|
Cajuput
|
Cajeput, cajuput-tree, paperbark tea tree, swamp tea tree
|
Melaleuca
leucadendra Maton & Sm. ex R. Powell
|
Cajuputi leucadendron (L.) A. Lyons Leptospermum leucodendron (L.) J.R. Forst. & G. Forst. Char. Meladendron leucocladum St.-Lag. Melaleuca amboinensis Gand. M. leucadendra var. angusta C.Rivière M. leucadendra var. cunninghamii F.M.Bailey M. leucadendra var. lancifolia F.M.Bailey M. leucadendra var. mimosoides (A.Cunn. ex Schauer)
Cheelin A.J.Ewart & O.B.Davies, M. mimosoides A.Cunn. ex Schauerin
W.G.Walpers, Repert. M. rigida Roxb. Metrosideros coriacea K.D.Koenig & Simsin
R.A. Myrtus alba Noronha Myrtus leucadendra L. Myrtus saligna Burm.f.
|
Myrtaceae
|
Cajuput
|
Paper bark tree, river tea tree, swamp tea tree, weeping tea tree,
weeping paper bark, white tea tree, white wood
|
Melaleuca quinquenervia (Cav.) S.T. Blake
|
M. quinquenervia var. albida Cheel. M. quinquenervia var. angustifolia L.f. M. quinquenervia var. coriacea (Poir.) Cheel. M. maidenii R.T. Baker M. smithii R.T. Baker Metrosideros quinquenervia Cav.
|
Myrtaceae
|
Broadleaf paperbark
|
Broadleaf teatree, coastal teatree, five-vein paperbark, paperbark
teatree
|
Pinus massoniana Lamb.
|
P.
massoniana (Lamb.)
Opiz P. argyi Lemée & H.Lév. P.
canaliculata
Miq. P. cavaleriei Lemée & H.Lév. P. crassicorticea Y.C.Zhong & K.X.Huang P. nepalensis J.Forbes P. sinensis D.Don
|
Pinaceae
|
Masson pine
|
Chinese red pine, southern red pine
|
Pinus pinaster Aiton
|
P. lemoniana Benth. P. nigrescens Ten. P. syrtica Thore
|
Pinaceae
|
Maritime pine
|
Cluster pine, pinaster pine
|
Pinus roxburghii Sarg.
|
|
Pinaceae
|
Chir pine
|
Long-leaf Indian pine
|
aThe Plant
List and the Kew Medicinal Plant Names Services database.14,15 A
comprehensive list of synonyms can be accessed through both websites. bHerbs of Commerce, 2nd
ed.2 cHerbs of Commerce, 2nd
ed.,2 and the USDA GRIN database.16
Hagers
Handbuch der Pharmazeutischen Praxis lists additional Melaleuca species as potential sources
of essential oil, i.e., M. decora, M. dissitiflora, M. quinquenervia, and M.
viridiflora.13 Melaleuca viridiflora, which is the source for niaouli oil, is also
listed in Herbs of Commerce.2 Melaleuca ericifolia essential oil is a relatively limited boutique
production and is sold at a premium price compared to TTO; so, it is not a
likely adulterant. Melaleuca dissitiflora
is indicated as another source for TTO in the European Pharmacopoeia,3 but is no longer
permissible according to the latest ISO guidelines. However, there is currently
no evidence that adulteration with these Melaleuca
species is an issue in the marketplace.
Sections 6-10 of this
document discuss macroscopic, microscopic, organoleptic, genetic, and phytochemical
authentication methods for M.
alternifolia. A comparison among the various chemical methods is presented
in Table 2 at the end of section 11.
6.
Identification and Distinction using Macroanatomical Characteristics
Botanical descriptions of tea tree leaves have been
published in a number of papers and books.11-13,17 Criteria to distinguish M. alternifolia from other Melaleuca species (M. leucadendra; M.
quinquenervia; M. cajuputi, subsp. cajuputi, M. cajuputi, subsp. platyphylla;
M. armillaris, and M.
ericifolia) have been published
by Barbosa et al.18 For obvious reasons,
macroscopic analysis is not applicable to TTO.
7.
Identification and Distinction using Microanatomical Characteristics
Two
references with details on microanatomical features of M. alternifolia leaves have been retrieved.17,18 Shah et al. also published microscopic characteristics of M. leucodendra.19 Images of cross-sections of leaves and petioles to
distinguish tea tree leaves from those of other Melaleuca spp. are provided in the publication by Barbosa et al.18 Based on the available information, M. alternifolia is readily distinguished
from E. globulus using botanical
microscopy, e.g., according to the drawings provided by Eschrich.20 As with macroscopic analysis, microscopy
is not applicable to TTO authentication
8.
Organoleptic Identification
Prior to the development of
modern chemical analysis, the assessment of aroma was the primary means to
authenticate the essential oils. The odor evaluation is still part of most
routine tests in quality control laboratories. The odor of tea tree oil is
described as myristic in the WHO
monograph.21 It is also characterized
as having a spicy, fresh and camphor-like aroma with a dry hay-like undertone.
(C. Beaumont [Doterra] email to S. Gafner, June 15, 2018). While experts in
organoleptic assessment of tea tree oil will be able to distinguish authentic
TTO from other essential oils, and from the various TTO chemotypes, some of the
subtler ways of adulteration may be missed. Therefore, the organoleptic
evaluation is not suitable as a stand-alone method for TTO authentication, and
has to be combined with an appropriate chemical method for an unambiguous
determination of the identity.
9.
Genetic Identification and Distinction
A few authors have looked into differences among
nucleotide sequences of various gene regions for Melaleuca spp. and closely related species to determine
phylogenetic relationships. Ladiges et al. and Brown et al. used the nuclear
ribosomal 5S and internal transcribed
spacer (ITS) regions to distinguish
among Melaleuca, Callistemon, and related genera, but
did not include any M. alternifolia
samples.22,23 Edwards et al. used the chloroplast gene region NADH dehydrogenase F (ndhF) in addition to morphologic
criteria to establish a phylogenetic relationship within the Melaleuceae tribe,
and found that the ndhF region is
better resolved than the ITS region.24 As such, the ndhF
genetic region may be suitable to distinguish among the various Melaleuca species and species from
closely related genera, but data on successful authentication of commercial M. alternifolia materials by genetic
means are lacking.
Comments: As
outlined by the studies above, the use of genetic techniques is considered to
be a suitable means for authentication of crude M. alternifolia materials such as leaves or twigs. The use of
genetic technologies to determine the authenticity of essential oils is not
appropriate because essential oils are generally devoid of DNA.25 This is due to the
lack of solubility of DNA in highly lipophilic materials such as essential
oils, and the low volatility of DNA (since the production of these oils using
steam-distillation, DNA would have to be volatile to be present in the
essential oil).
10.
Physicochemical Tests
Several monographs include
specifications for the density, optical rotation, refractive index, and/or
miscibility of TTO in ethanol.3,9,26 While these simple tests are helpful as
a screening test for TTO adulteration, they must be used in combination with a
chemical analysis to rule out adulteration with some of the materials mentioned
in section 5.
11.
Chemical Identification and Distinction
A large number of analytical
methods has been published for identifying TTO based on its chemistry. These
methods are cited in the Laboratory Methods section below (Section 11.2). Distinction
based on the phytochemical profile requires detailed knowledge of the
constituents of TTO, its chemotypes, and its adulterants. The important
components in TTO and its adulterating species are listed below. When
distinction is based on chromatographic or spectral patterns, identification of
specific constituents may not be necessary.
11.1
Chemistry of Melaleuca alternifolia, Melaleuca linariifolia, Melaleuca dissitiflora and potential adulterants
Melaleuca alternifolia: The
main compounds in tea tree oil are mono- and sesquiterpenes. Most often,
authors have suggested three distinct chemotypes of M. alternifolia oil, dominated by 1,8-cineol, terpinolene, or (+)/(-)-terpinen-4-ol,
respectively, although classification of up to seven chemotypes has been
proposed.27-31 The TTO on the market is made from plants
of the terpinen-4-ol chemotype.† Besides 30-48% terpinen-4-ol, the
oil of this chemotype contains 10-28% γ-terpinene, 5-13% α-terpinene, <0.01%-15%
of 1,8-cineole, 0.5-8% p-cymene, 1-6%
(+)/(-)-α-pinene, and 1.5-5% of terpinolene. Minor compounds include
aromadendrene, δ-cadinene, ledene, limonene, and sabinene.9
Figure
1: Major monoterpenes in tea tree oil
Note: The absolute
configuration is often not indicated in the published literature. In these
cases, the compounds are collectively referred to as terpinen-4-ol, α-pinene,
and α-terpineol, respectively.
The 1,8-cineole type M.
alternifolia oil contains between 36-71% of 1,8-cineole, 6-22% of terpinen-4-ol,
and 12-14% of α-pinene. Contents of 1,8-cineole, terpinolene, and terpinen-4-ol
vary between 17-34%, 10-57%, and 1-20%, respectively, in the terpinolene
chemotype.27 Of particular
interest for the authentication of TTO are the enantiomeric ratios of (+)-terpinen-4-ol
and (-)-terpinen-4-ol, as well as (+)-α-terpineol and (-)-α-terpineol. Ratios
ranged between 63.3-69.8/36.7-30.2 for (+)-terpinen-4-ol/(-)-terpinen-4-ol and
between 74.2-79.5/25.8-20.5 for (+)-α-terpineol/(-)-α-terpineol in authentic TTO.32 A phytochemical screening suggests that
flavonoids, triterpenes, and tannins are also present in tea tree leaves.33 An ellagic acid
derivative, 3,3′-di-O-methylellagic
acid-4-O-glucoside, has been reported
by Shah et al., but discrepancies among the NMR data, the alleged structure, and
the structure drawing (3,5,3′,5′-tetrahydroxy-4,4′-dimethoxydiphenic acid-5-O-xylopyranoside) cast a doubt about the
veracity of these findings.34
Melaleuca linariifolia: There
are two main chemotypes described based on differences in the composition of essential
oil obtained from the leaves and branchlets of this species: the 1,8-cineole
and the terpinen-4-ol chemotypes.28 The oil of the
terpinen-4-ol type is very similar to essential oil of the same chemotype from M. alternifolia. Melaleuca linariifolia oil can be distinguished from M. alternifolia oil by its higher
concentrations of t-sabinene hydrate
and by the ratio of α-pinene to α-thujene.35 No information on compounds
other than the essential oil could be retrieved.
Melaleuca dissitiflora:
Essential oil of the terpinen-4-ol chemotype of M. dissitiflora is also accepted as “tea tree oil” by the European Pharmacopoeia, and was listed
in older ISO standards as a source of TTO.26,36 However, the most
recent ISO standard does not include M.
dissitiflora as an acceptable source, partly because the species is not
important in commerce.9 There are two chemotypes of M. dissitiflora, distinguished by the concentrations of 1,8-cineole
(63-66% and 2-7%, respectively). The 1,8-cineole chemotype also contains 5-7%
limonene, 1-2% of α-pinene, 3-4% of terpinolene, and 1-7% of terpinen-4-ol.37 Williams and Lusunzi
later analyzed the leaf oils of 30 M.
dissitiflora trees from the Alice Springs region, which were mainly of the
terpinene-4-ol (low 1,8-cineole) type but noticed a few oils with intermediate
levels of 1,8-cineole.38 The composition of
the oil made from the terpinen-4-ol chemotype is similar to TTO of M. alternifolia, but has generally higher
(0.7-7.6%) sabinene concentrations.39 A recent publication
suggested that methyl eugenol‡ levels are also substantially higher
in M. dissitiflora than in M. alternifolia or M. linariifolia. The
data were limited to one M. dissitiflora
product, though, and need to be confirmed with a larger sample size.40
Cinnamomum camphora: The
essential oil obtained from the wood, leaves, or twigs of the camphor tree
shows substantial differences in the composition depending on the subspecies,
varieties, chemotype, and plant part.41,42 As an example to
illustrate the point, Zhu et al. analyzed three chemotypes from China, with
chemotype I containing 50.0% of 1,8-cineole, 14.4% of α-terpineol, 6.9%
β-pinene, 3.1% bornyl acetate, and 0.3% camphor; chemotype II is made up of
81.8% borneol, 3.0% camphor, 2.8% of α-pinene, and 1.6% of 1,8-cineole;
chemotype III contains 57.7% isonerolidol, 3.6% of α-terpineol, 2.3% linalool,
and 0.3% camphor.42
The crude essential oils obtained for commercial use are
rich in crystalline camphor, which is obtained in pure form after filter
pressing.42 The remaining
essential oil is rectified by fractional distillation, yielding a camphor-rich
fraction, and several fractions low in camphor. The fraction with the lowest
boiling point is known as white camphor oil, while higher boiling fractions are
separated into brown (sometimes also termed yellow, red, or black camphor oil
depending on the safrole content providing its color) and blue camphor oil, the
latter containing mainly sesquiterpenes.41-43 White camphor oil, which is the product
that is described as a TTO adulterant, contains mainly monoterpenes, e.g., 1,8-cineole,
α-pinene, α-terpineol, camphor, camphene, furfural, limonene, β-pinene, and
safrole.44,45 Lumpkin et al. suggest
that α-terpinene and sabinene are also among the main compounds in white
camphor oil.46 Quantitative data
report the contents of 1,8-cineole, α-pinene, and camphor at 46%, 22% and 21%,
respectively.47 Unpublished results
from close to 100 samples of white camphor oil give the following ranges of the
eight major compounds: 30-40% of 1,8-cineole, 14-30% limonene, 2.3-14.4% of α-pinene,
4.2-10.0% p-cymene, 0.5-9.0% of γ-terpinene, 1.3-8.4% sabinene, 1.8-7.5%
myrcene, and 0.9-5.2% β-pinene. In all these samples, the camphor level was
below 1.5%. (E. Schmidt [University of Vienna] email communication, December 9,
2017) The high contents of 1,8-cineole and α-pinene can be used to distinguish
white camphor oil from TTO.
Eucalyptus globulus: Among
the numerous Eucalyptus spp., E. globulus is the main source of
eucalyptus oil, since it is grown for the wood and pulp industry and the leaves
are used for the essential oil production.48 The leaf contains
1.2-3.0% essential oil, with 65-80% of 1,8-cineole as the main component, and α-pinene,
α-terpineol, aromadendrene, β-pinene, glubulol, limonene, and t-pinocarveol as minor constituents.49-51 Commercial eucalyptus oils are most
often rectified. In the rectification process, the crude essential oil is
treated with an alkaline substance and subjected to fractional distillation to
remove a majority of mono- and sesquiterpenes, leading to a product that
contains higher amounts in 1,8-cineole.49 In order to comply
with the European Pharmacopoeia
monograph on eucalyptus oil, the content of 1,8-cineole has to be greater than
70%, with other components in the following ranges: α-pinene: 0.05-10.0%;
β-pinene: 0.05-1.5%; sabinene: not more than 0.3%; α-phellandrene: 0.05-1.5%;
limonene: 0.05-15.0%; camphor: not more than 0.1%.52 The US National
Formulary (NF) standard requires the oil to contain not less than 70.0% and not
more than 95.0% of 1,8-cineole.53 The standard
published by the International Organization for Standardization (ISO 3065:2011)
demands an even higher content of 80-85% of 1,8-cineole.54 Besides the
essential oil, eucalyptus leaves contain ellagitannins, proanthocyanidins,
flavonoids, triterpenes, and formylated phloroglucinol derivatives.50 The latter are
characteristic for the genus Eucalyptus.
Besides E. globulus, the European Pharmacopoeia, the National Formulary, as well as
the Personal Care Products Council’s International Nomenclature of Cosmetic
Ingredients allows essential oils made from the leaves of E. polybractea and E. smithii to be sold as eucalyptus oils, as long as these oils
comply with the composition outlined in the monograph.52 Similarly, the ISO
standard allows E. radiata ssp. radiata, E. smithii, E.
plenissima, E. dives and other 1,8-cineole-rich eucalyptus species as
sources of eucalyptus oil.54 Chemically, the
large amount of 1,8-cineole can be used to distinguish eucalyptus oil from TTO.
It is not clear to what extent commercial eucalyptus oil
is used to adulterate TTO, but reports suggest that the waste oil products
obtained during the rectification process may represent a more important source
of adulterants. Publications detailing the composition of these waste oils
could not be retrieved, but since fractional distillation is used for
rectification, some of the compounds found in TTO (e.g., α-pinene, α-terpineol,
limonene) may be obtained in high amounts among the purified fractions and used
to dilute authentic TTO.
Melaleuca cajuputi: There
are three morphologically distinct subspecies, M. cajuputi subsp. cajuputi, subsp. cumingiana, and
subsp. platyphylla. The chemical composition of essential oils derived
from each of the subspecies is markedly different. Cajuput oil, which can also
be obtained from M. leucadendra (see
below) is also a medicinally used oil, e.g., as an ingredient in topical
products to treat sore muscles or as a topical decongestant. The commercial
cajuput oil is primarily made using leaves and branchlets of the subspecies cajuputi, of which there exist various
chemotypes based on the concentrations of 1,8-cineol, which is present between 3-60%.39,55 The sesquiterpene
alcohols globulol (trace–9%), viridiflorol (trace–16%) and spathulenol
(trace–30%) are present in rather variable concentrations. Minor compounds in
cajuput oil are α-pinene, α-terpineol, limonene, β-caryophyllene, humulene, and
viridiflorene.55,56 However, cajuput
oils from Myanmar, Thailand, Vietnam, or Indonesia may have an altogether
different composition.57-59 Overall, the essential oil of M. cajuputi can be distinguished from TTO
by the larger relative concentration of 1,8-cineole, and lower levels of terpinen-4-ol,
γ-terpinene , and α-terpinene.60
Melaleuca leucadendra: M. leucadendra oil has two distinct
chemotypes; chemotype I contains 10-45% of 1,8-cineole as the main component,
and 5-22% of p-cymene, 4-19% of α-pinene, 3-6% limonene, and 6-9% of α-terpineol.37,61 Chemotype II, also
called the aromatic ether chemotype, is dominated by methyl eugenol (95-97%) or
methyl isoeugenol (74-88%). Minor terpenes in the oil of chemotype II include t-β-ocimene, and calamene.39 Chemotype I is
distinguished from TTO by the higher contents of 1,8-cineole, while chemotype
II differs by the large amounts of methyl eugenol or methyl isoeugenol.37
Melaleuca quinquenervia:
According to Hager’s Handbuch der
Pharmazeutischen Praxis, the essential oil of broadleaf paperbark (also
called cajuput oil or niaouli oil depending on the author, although the latter
should be derived from M. viridiflora)2,39 from Madagascar can be
separated into four chemotypes: chemotype I has been reported to contain 37% of
1,8-cineole, 24% viridiflorol, 9.3% of 8, 9.3% of viridiflorene, 5.8% of 7 and
5.8% of α-thujene. Chemotype II contains 22.8% of 1,8-cineole, 20%
viridiflorol, 4.8% of α-pinene, and 4.8% α-thujene. Chemoype III is
characterized by high amounts (47.8%) of viridiflorol, followed by
β-caryophyllene (8.5%), 1,8-cineole(8.2%), and ledol (4.4%). Finally, chemotype
IV consists predominately of E-nerolidiol
(86.7%), with lesser amounts of β-caryophyllene (3.5%), and 1,8-cineole
(1.1%).39 Two additional
chemotypes have been described from Australia and Papua New Guinea. Chemotype V,
commonly known as Nerolina, is comprised of E-nerolidol
(74–95%) and linalool (14–30%)
and is found along the east coast of Australia. Chemotype VI contains predominantly
1,8-cineole (10–75%)
or viridiflorol (13–66%), with α-terpineol (0.5–14%)
and β-caryophyllene (0.5–28%)
occurring at lower concentrations. Trees yielding essential oil of this
chemotype are found from Sydney along the eastern coast of Australia and north
to Papua New Guinea and New Caledonia.62 Melaleuca quinquenervia oil can be distinguished from TTO by high
amounts of either 1,8-cineole, viridiflorol, or E-nerolidol.
Pinus spp: A number of pine
species are used for essential oil production.63 Pine oil can be
obtained by steam distillation of the needles, young shoots, and young branches
with shoots and needles (pine needle oil) or of the wood chips of the heartwood
and roots (pine oil). Important sources of pine needle oil are P. mugo, P. palustris, and P.
sylvestris.64,65 While a number of pine species are used
to produce essential oil from the wood, the largest volumes of turpentine oil are
obtained from P. massoniana, P. pinaster, and P. roxburghii.66 (E. Schmidt [University
of Vienna] email communication, December 9, 2017)
Due to the high cost of pine needle oil, only the use of
pine resin oil makes economic sense as an adulterant of TTO. Many turpentine
oils can be used as adulterants, primarily as a source of α-pinene and
β-pinene. These two compounds may be used directly to dilute TTO or may serve as
starting materials for the semi-synthesis of additional monoterpenes.
The turpentine oil of Masson pine is dominated by α-pinene
(84.6%) and β-pinene (9.6%), with lower concentrations of limonene (1.7%) and
longifoliene (0.4%).67 Maritime pine
contains 63-65% of (-)-α-pinene, 18-27% (-)-β-pinene, ca. 8% limonene, and
traces of terpinolene, camphene, and myrcene,63,66 while the turpentine
oil from Chir pine is dominated by 3-carene, which makes it a less likely
source as adulterant.63,66
Tea tree oil and pine oils can be differentiated by the
altogether different composition, with pine resin oils dominated by α-pinene
and/or β-pinene, or 3-carene, while TTO contains substantially higher amounts
of terpinen-4-ol, γ-terpinene and α-terpinene.
11.2
Laboratory methods
Note: Unless otherwise
noted, all methods summarized below are based on chemical analysis of the
essential oil. Analytical tests evaluating tea tree leaf extracts are beyond
the scope of this document.
11.2.1
HPTLC
Methods from the following sources were evaluated in this
review: the European Pharmacopoeia
(EP 7.0),26 the British Pharmacopoeia (BP),68 and the
High-Performance Thin Layer Chromatography (HPTLC) Association.69
Comments: The
conditions described in the EP26 and BP68 are the same, and differ
from those described by the HPTLC Association69 in that the EP/BP
method uses a mobile phase of higher polarity and lists 1,8-cineole, terpinen-4-ol,
and α-terpineol as reference compounds rather than the two compounds (1,8-cineole
and nerolidol) suggested by the HPTLC Association. Both methods detail
appropriate conditions to separate the essential oil constituents of TTO.
Developed to detect adulteration of niaouli (Melaleuca viridiflora) essential oil, the HPTLC Association’s
method provides suitable separation for M.
alternifolia leaf oil, and enables its distinction from cajuput oil,
eucalyptus oil, kanuka (Kunzea ericoides,
Myrtaceae) oil, manuka (Leptospermum scoparium,
Myrtaceae) oil, and neroli (Citrus
aurantium var. amara, Rutaceae).69
Since the method proposed by
the HPTLC Association (Figure 2) has documented its suitability to detect
adulteration with a variety of potential TTO adulterants based on an evaluation
of the fingerprints, it is suitable for the routine identity testing in a
quality control laboratory. However, dilution of tea tree oil with essential
oil fractions obtained from the waste stream of a number of essential oils may
be difficult to detect, and may warrant the quantitative determination of the
individual tea tree oil compounds and the enantiomeric ratios of terpinen-4-ol
and α-terpineol.
Figure
2: HPTLC analysis of commercial tea tree and authentic Melaleuca spp. essential oils Lane 1: Isoeugenol and
isoeugenyl acetate (with increasing Rf); Lane 2: α-terpineol, terpinen-4-ol,
and 1,8-cineol (with increasing Rf); Lanes 3-9: commercial tea tree oils; Lanes
10-12: Melaleuca alternifolia oils;
Lanes 13-15: Melaleuca linariifolia
oils; Lanes 16-18: Melaleuca quinquenervia
oils. Conditions as specified by the HPTLC Association.69 Detection after
derivatization with anisaldehyde reagent. Top: white light; bottom: UV at 366
nm. Image provided by Camag AG; Switzerland.
11.2.2
Infrared, mid-infrared, and near infrared spectroscopy
Four infrared-based authentication methods were evaluated
in this review (Tankeu et al.70, and Gallart-Mateu et al.71).
Figure 3: Fourier transform-infrared (FT-IR) spectra of tea tree oil
samples in the range between 4,000 and 600 cm-1 Image provided by Prof. Miguel de la Guardia (University of Valencia,
Valencia, Spain) Figure 4: Near infrared (NIR) spectra of tea tree oil samples in the
range between 14,000 and 4,000 cm-1
Image provided by Prof. Miguel de la Guardia (University of Valencia, Valencia,
Spain)
Comments: Sixty-four
TTO samples were evaluated using near-infrared (NIR), or mid-infrared (MIR), and
compared to results obtained by gas-chromatographic (GC) methods. The
concentrations of seven major essential oil components (1,8-cineole,
terpinolene, terpinen-4-ol,
γ-terpinene, α-terpinene, α-terpineol, and limonene) were calculated using a
partial least square regression analysis based on quantitative GC data.70 Based on the published results, the quantitative
models constructed for the infrared data provides a fairly good correlation
with the results from the GC measurements. Partial least square (PLS) regression models were constructed based on
MIR versus GC-MS and NIR versus GC-MS results, with coefficients of
determination ranging from 0.76 – 0.97 for MIR, and 0.75 - 0.95 for NIR. In
general, the coefficients of determination were higher for the models
constructed with MIR data compared with NIR data. Prediction of quantitative
levels was better with compounds at higher concentrations than those, e.g.,
limonene, that are in the low single percentage range.70 In the second study,71 a set of 267 samples
was used to build a chemometric model for FT-IR and NIR (Figures 3 and 4) authentication
of tea tree oil. The models were built using a partial least square analysis.
After optimization of the models, the overall accuracy of FT-IR was 87% (153
correctly assigned samples out of 175 that were tested by FTIR), and 98% for
NIR (3 misidentified samples out of 125 analyzed by NIR).
The FT-IR, MIR and NIR
methods provide a fast, easy and affordable approach to get a good idea about
the TTO quality. Unusual amounts of any of the seven target compounds can be
used to detect adulteration, and the chemometric model based on the NIR method
by Gallart-Mateu et al. provided good accuracy.71 While highly
sophisticated types of adulteration may be difficult to detect with this method,
it has a lot of promise as a method in routine quality control laboratories.
Samples that are close to the discriminant threshold (the limit that separates
the authentic from the adulterated samples) in this NIR model can be verified
using a GC method (see section 11.2.3). For its use in quality control, further
method validation is needed, and system suitability parameters must be
established.
11.2.3
Gas chromatography
Methods described in the following literature were
evaluated in this review: EP 7.0,26 ISO 4730:2017,9 Leach
et al.,72 Brophy
et al.,73 Gallart-Mateu
et al.,74 Wong
et al.,75,76 Wang et al.,77 Sciarrone et al.,78 Shellie
et al.,79 Padalia et al.,80 and
Southwell et al.81 Specific comments on
strengths and weaknesses of each of the methods are listed in Appendix 1, Table
3.
Figure
5: GC-FID chromatogram of authentic tea tree oil, terpinen-4-ol type Conditions
according to Southwell and Russell.31
Comments:
Gas chromatography (Figure 5) has been the method of
choice to analyze TTO for decades. Sample preparation consists of a dilution of
the analyte in acetone, ethanol, or hexane. For routine analysis, the validated
methods published by the European
Pharmacopoeia26 or ISO 4730:20179 represent attractive
choices. Parameters for composition ranges of a number of TTO constituents have
been tightened in the ISO 4730:2017 (versus the 2004 version) to better reflect
the quality of TTO being produced currently, and to mitigate the trading of adulterated
oils.
Adulteration of TTO can occur with addition of terpinen-4-ol,
a molecule which can be readily synthesized. In evaluation of TTO quality, the
enantiomeric ratio of terpinen-4-ol provides a clear indication of the
authenticity. The enantiomeric ratio of chiral terpenoids is genetically
predefined and serves as a useful signature for essential oil identification. The
application of chiral GC methods to measure enantiomers of terpinen-4-ol helps
to detect the addition of this compound from synthetic or natural non-TTO
sources.
Multi-dimensional GC analysis
is an interesting approach, in particular in a research setting, but is
impractical for commercial use due to costs (combination of two columns) and
time savings since acceptable peak separation can be achieved by conventional
or chiral chromatography.
Table 2. Comparison among
the different chemical methods to authenticate tea tree oil
Method
|
Pro
|
Contra
|
HPTLC
|
Quick Basic systems affordable
|
No statistics High-end equipment
expensive Dilution with essential
oil fractions from other materials may be difficult to detect Need for standard
compounds
|
GC-FID
|
Standard equipment in many
laboratories Basic systems affordable Detection of adulteration
possible using a fingerprint and concentration ranges
|
Mainly quantitative method Dilution with essential
oil fractions from other materials difficult to detect Need for standard
compounds
|
GC-FID (chiral)
|
Detection of adulteration
possible based on enantiomeric ratios of (+)/(-)-terpinen-4-ol and (+)/(-)-α- pinene
|
Mainly quantitative method Higher costs compared to
conventional GC-FID Need for standard
compounds
|
GC-MS
|
Qualitative and
quantitative State-of-the-art
statistical evaluation possible
|
Equipment expensive Dilution with essential
oil fractions from other materials difficult to detect Need for standard
compounds
|
MIR/NIR
|
Quick Affordable State-of-the-art
statistical evaluation possible
|
Mostly qualitative Accuracy and precision for
low-concentration compounds insufficient Dilution with essential
oil fractions from other materials difficult to detect Need to build-up reference
library
|
12.
Conclusion
Identification of TTO can be
achieved by a range of analytical techniques. In practice, gas chromatography
combined with physical measurements of optical rotation, refractive index,
density and miscibility in ethanol, provide a robust identification of TTO.
While HPTLC and NIR represent good screening methods, more sophisticated types
of adulteration will require the use of chiral GC to establish TTO authenticity
with confidence.
* Note: The list of known
adulterants is based on published data, e.g., those listed in the Botanical
Adulterants Bulletin on TTO.1 The adulterating materials
may not be the essential oil of the species listed in Table 1, but materials
enriched in desirable terpenes obtained from the waste stream after
rectification of camphor, eucalyptus, and pine essential oils. Materials
obtained by fractional distillation from species not listed in Table 1 or pure
compounds made by chemical synthesis, e.g., terpinen-4-ol or α-terpineol, may
also be used to dilute TTO without the knowledge of the buyer. † Tea
Tree oil was traditionally obtained from bush cuts, where leaf and twig are
removed manually by machete from wild stands. Bush cut oil is typically
from older leaf which naturally has higher p-cymene
(as high as 10%) contents, and variable amounts (sometimes below 35%) of
terpinen-4-ol. It is also collected from a more genetically diverse
mixture of plants, and so it may contain more material from the higher
1,8-cineole types. The manufacture of bush cut oil is not competitive with
broad-acre production from tea tree cultivation, due to manual harvesting, but
also because unselected wild material yields lower amounts of oil overall. ‡ In the European Union, the permissible
levels of methyl eugenol from natural sources have been restricted to 0.001% in
rinse-off products (e.g., shower gels, bar soaps) and to 0.0002% in leave-on
(creams, lotions) and oral hygiene products.
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D, Ragonese C, Carnovale C, et al. Evaluation of tea tree oil quality and
ascaridole: A deep study by means of chiral and multi heart-cuts
multidimensional gas chromatography system coupled to mass spectrometry
detection. J Chromatogr A. 2010;1217(41):6422-6427.
- Shellie
RA, Xie L-L, Marriott PJ. Retention time reproducibility in comprehensive
two-dimensional gas chromatography using cryogenic modulation: An
intralaboratory study. J Chromatogr A. 2002;968(1):161-170.
- Padalia
RC, Verma RS, Chauhan A, Goswami P, Verma SK, Darokar MP. Chemical composition
of Melaleuca linariifolia Sm. from
India: a potential source of 1,8-cineole. Ind
Crops Prod. 2015;63(Supplement C):264-268.
- Southwell
IA, Dowell A, Morrow S, Allen G, Savins D, Shepherd M. Monoterpene chiral
ratios: Chemotype diversity and interspecific commonality in Melaleuca alternifolia and M. linariifolia. Ind Crops Prod. 2017;109(Supplement C):850-856.
Appendix 1
Table 3: Comments on
the published GC methods for tea tree oil. In order to compare methods, the run
times indicate only the duration of gradient elution, since initial and final
hold times are often not indicated
Reference
|
Comments
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EP 7.026
|
This is a validated GC-FID method with
36-minute run time using a stationary phase similar to the polar column used
in the ISO standard 4730:2017.9 Authentication
is based on the ranges of 11 TTO components. The determination of
enantiomeric ratios for authentication of TTO oil is not part of the EP
monograph.
|
ISO 4730:20179
|
This ISO standard includes three
different methods using columns of low, medium and high polarity and run
times of 65 min., 20 min., and 30 min., respectively. Depending on the
conditions, the peaks of 1,8-cineole, β-phellandrene, or p-cymene are not resolved. The determination of the terpinen-4-ol
enantiomeric ratio is proposed as additional measure to ensure authenticity,
but no specific conditions to measure this ratio are given.
|
Leach et al.72
|
This GC-MS
method separates the TTO constituents in 43 minutes. The
use of a chiral column allows the determination of terpinen-4-ol, α-pinene,
and α-terpineol enantiomers. The resolution among the peaks is acceptable,
despite the fact that terpinen-4-ol enantiomers are not fully resolved.
Suitable to detect adulteration of TTOs; method validation data are lacking.
|
Brophy et al.73
|
The publication details two routine GC
methods using FID detection. The stationary phases are of low and high
polarity with run times of 21 min. and 13 min., respectively (not including
the hold time at the end, which was not detailed in the paper). The peaks of 1,8-cineole,
β-phellandrene, and limonene are not always resolved, which hampers accurate
quantification of 1,8-cineole, one of the markers of adulteration. The method
is able to detect low quality or adulterated TTOs based on low concentrations
of terpinen-4-ol and high contents of 1,8-cineole. Method validation data are
lacking.
|
Gallart-Mateu et al.74
|
This validated GC-MS method is modified
from Brophy et al.73
using a low polarity column with a run time of 17 min. Limonene and 1,8-cineole
are not resolved, but the use of selective ion monitoring may allow
quantification of these compounds. The use of a headspace injector reduces
the amount of solvent necessary for the sample preparation. Data on the
method’s ability to detect adulteration are not presented, but it is expected
to give results similar to other GC-MS fingerprinting methods.
|
Wong et al.75
|
The GC-FID method uses a chiral column
to measure the enantiomeric ratios of (+)-terpinen-4-ol/(-)-terpinen-4-ol and
(+)-α-terpineol/(-)-α-terpineol with a run time of ca. 35 min. (the initial
hold time is not detailed). The method has been developed specifically with
the goal to detect adulteration of TTO. Although the accuracy has not been
evaluated, the method provides good repeatability and results of over 50
different samples have been confirmed by tests in multiple laboratories.32 The ratios of
(+)/(-)-limonene could not be established due to overlapping with p-cymene, but this issue can be
resolved using mass spectrometric detection, providing an additional
criterion for the authenticity of TTO.
|
Wang et al.77
|
Wang and co-workers present a GC-MS
method using a chiral column to determine the enantiomeric ratios of (+)-terpinen-4-ol/(-)-terpinen-4-ol,
(+)-α-pinene/(-)-α-pinene, (+)-α-terpineol/(-)-α-terpineol, and
(+)/(-)-limonene. The run time of close to 60 min. is longer than durations
of other methods discussed here. The use of a MS detector allows determining
additional enantiomeric ratios (e.g., those of (+)-limonene/(-)-limonene, which
co-elute despite the long run time) and the statistical evaluation of the
results is ideal for a quality control laboratory. Validation data are not
provided.
|
Wong et al.76
|
This is a heart-cut bi-dimensional
GC-FID method where after a short (< 10 min.) run on a column of medium
polarity, compounds of interest are collected and released onto a second, chiral
column. The authors present two chiral separation methods, with the longer
method giving baseline separation of enantiomers in 40 minutes, thus allowing
to determine the enantiomeric ratios of (+)-terpinen-4-ol, (-)-terpinen-4-ol,
(+)-α-terpineol, (-)-α-terpineol, and (+)- and (-)-limonene as indicators of
tea tree oil authenticity without the need of a costly MS instrument. However,
it is not clear how widespread instrumentation that allows running
bi-dimensional GC is in the industry. The method has not been validated.
|
Sciarrone et al.78
|
Sciarrone et al. assessed the quality
of TTO using a chiral GC column with a FID detector, and bi-dimensional GC-MS
(using a heart-cut system) combining a non-polar and polar stationary phase
with FID and MS detection. The run times for each of the separations are long
at 75-77 min. The use of a chiral separation allows determination of the
enantiomeric ratios of (+)-terpinen-4-ol, (-)-terpinen-4-ol, (+)-α-terpineol,
(-)-α-terpineol, while the addition of the bi-dimensional GC provides more
accurate quantitative data on co-eluting peak clusters, e.g., 1,8-cineole, p-cymene, and limonene, or terpinen-4-ol
and p-cymen-8-ol. Method validation
data is limited to the repeatability of retention times, and the limits of
detection and quantification.
|
Shellie et al.79
|
This method represents another
bi-dimensional GC approach using two columns of different polarity. The
compounds elute directly from column 1 onto column 2 after being trapped
using a cryogenic modulator. Due to the improved resolution, this seems a
suitable approach for the detection of adulteration, although actual data on
the determination of TTO identity are not presented. With run times of 75
minutes in each dimension, this method is too time-consuming for a routine
assay. The method has not been validated.
|
Padalia et al.80
|
Padalia et al. describe two methods
using either FID or MS detection on columns of low polarity with a 60 min.
run time for each method. The peaks of 1,8-cineole, p-cymene, and limonene are not resolved, which prevents accurate
quantification of 1,8-cineole. No data on its ability to detect adulteration are
given, but due to the similarity with other methods, the GC-FID or GC-MS
fingerprints might be used as a criterion for authentication of TTO. Method
validation data are not presented.
|
Southwell et al.81
|
Two different GC-FID methods are described
in this paper. One method, using an intermediate polarity column, allows
distinguishing M. alternifolia and M. linariifolia chemotypes in 21 min.
based on concentrations of 1,8-cineole, terpinolene, and terpinen-4-ol.
Enantiomeric GC analysis enables verification of the ratios of (+)-terpinen-4-ol/(-)-terpinen-4-ol,
(+)-α-terpineol/(-)-α-terpineol, and (+)-limonene/(-)-limonene. The run time for the
chiral separation is 85 min. The authors point out that the chiral separation
becomes overly labor intensive if ethanol extracts of tea tree leaves are
used rather than the distilled oil due to the need for increased column
cleaning. While the combination of these two methods provide adequate data to
determine the authenticity of TTO, the 85-minute chiral separation may prove
too long for adoption in a quality control setting. Method validation data are
not given.
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