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Determination of enantiomeric distribution of terpenes for quality assessment of Australian tea tree oil Noel W. Davies, Tony Larkman, Philip John Marriott, and Ikhlas A Khan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01803 • Publication Date (Web): 15 May 2016 Downloaded from http://pubs.acs.org on May 29, 2016
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Journal of Agricultural and Food Chemistry
Determination of Enantiomeric Distribution of Terpenes for Quality Assessment of Australian Tea Tree Oil Noel W. Davies a*, Tony Larkman b, Philip J. Marriott c, Ikhlas A. Khan d
a.
Central Science Laboratory, University of Tasmania, Churchill Avenue, Sandy Bay Hobart, TAS 7005, Australia
b.
Australian Tea Tree Industry Association (ATTIA Ltd.), PO Box 903, Casino, NSW 2470, Australia
c.
Australian Centre for Research on Separation Science, School of Chemistry, Monash University, Wellington Road, 3800 Victoria, Australia
d.
National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA
*
Corresponding author : email:
[email protected], phone 613 62262157, fax 613 62262494
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Journal of Agricultural and Food Chemistry
Abstract 1
A number of papers have appeared in recent years proposing the use of enantiomeric
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ratios of key monoterpenes in Australian tea tree oil (TTO) for detection of adulterated oils.
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There are however a range of reported values, even from exactly the same suite of
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authentic oils, and we address here probable reasons for these differences, and stress the
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importance of establishing reference ratios within each laboratory based on oils of known
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provenance. Any biological variation in the ratio for the key terpene terpinen-4-ol has been
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demonstrated to be effectively unmeasurable, as the standard deviation on multiple
8
measurements of the same oil is of the same order as that of multiple authentic oils.
Keywords: 9 10
Tea tree oil authenticity, Melaleuca alternifolia, enantiomeric ratios, terpinen-4-ol, αterpineol, interlaboratory study
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Journal of Agricultural and Food Chemistry
Correspondence 11
Australian tea tree oil (TTO) steam distilled from Melaleuca alternifolia is widely used as
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a topical antiseptic, anti-bacterial, anti-fungal, anti-inflammatory and anti-viral agent and is
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also widely formulated into many cosmetic and personal care products in an industry now
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worth in the order of AU$ 29 million per year in Australia. In 2015 more than 800 tonnes
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were produced (T.Larkman, personal communication). There have been increasing problems
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of adulteration of genuine oils, and oils of other species being substituted,
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existence of an International Standard
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appeared over the last 20 years in which the chiral ratios of specific terpenes are explored
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as potential indicators of oil quality. 3-7 Two very recent articles (Wong et al., 8 Wang et al. 9)
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based on one-dimensional gas chromatography in three independent laboratories (Monash
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University and the University of Tasmania, both in Australia, and the University of
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Mississippi) have measured these ratios for key terpenes as indicators of adulteration using
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the same set of samples provided by the Australian Tea Tree Association Ltd (ATTIA Ltd).
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This sample set included 57 genuine Melaleuca alternifolia oils of known Australian
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provenance, covering seven different growing seasons and five different geographical
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regions ranging from latitudes 17oS to 34oS, as well as including a range of 43 commercial
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oils from North America, Europe, Asia, Australia and South Africa. While the concept of
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enantioselective analysis has been employed widely in the field of essential oil quality, it has
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yet to be applied to tea tree oil by any regulatory authority. This article is intended to
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provide guidance in developing and maintaining an environment where data can be
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generated with good repeatability and acceptable accuracy, and to comment on the reasons
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for the different reported values from the same suite of oils.
2
1
despite the
for many years now. A number of articles have
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In these two recent studies, as with previous studies, there are noticeable differences in
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the measured chiral ratios between and, to a lesser extent, within them where more than
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one laboratory is used. Table 1 shows the measured ranges of the percentage of the major
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(+) enantiomer for terpinen-4-ol and α-terpineol from these 57 genuine tea tree oils of
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known provenance, as well as ± 3 standard deviations around the mean values. In all cases
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measured terpinen-4-ol values fell with the 3 standard deviation limit. While the standard
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deviations within each laboratory were extremely small across the 57 samples (relative
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standard deviations of 0.6% or less for terpinen-4-ol and 1.9% or less for α-terpineol), the
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difference in mean values both within and between these two studies has implications for
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the setting of valid benchmark values for the enantiomeric composition of the target
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compounds, despite the fact that each compound clearly has an absolute enantiomeric
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ratio.
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We believe that differences in the measured enantiomeric ratios of compounds in the
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same samples can be caused by three main factors. Firstly, when there is incomplete
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baseline resolution of enantiomers that otherwise have symmetrical peak shapes, as can
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occur readily for terpinen-4-ol, there is a very small overestimation of the larger peak – this
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effect can be observed between the University of Tasmania and Monash University data for
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terpinen-4-ol, in which the respective average values were 69.8% and 68.5% respectively for
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the (+)-terpinen-4-ol enantiomer. While these are very close, this ~1% difference was
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consistently observed across all samples and the resulting mean values were highly
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significantly different (p
