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40609 03/2022
 Editor’s Note

Welcome to the Essential E-book

JENNA RIMENSNYDER
Managing Editor
[email protected]

2022 ESSENTIALS E-BOOK

CONTENTS

A
n emphasis on well-being has nearly consumed consumer demand
4 Editor’s Note due to the global pandemic. Wellness is a trend that is likely to
By Jenna Rimensnyder
continue to as frontrunner in a variety of markets including, but
5 Allergenic Fragrance Testing— not limited to, food and beverage, personal care, cosmetics and
New Certified Reference Materials aromatherapy. Essential oils play a key role in these markets and has in turn
Markus Obkircher, Head of Reference Materials R&D,
Hanspeter Sprecher, R&D Senior Scientist, been consistently growing at a swift rate.
Leonard Sidisky, Gas Separations R&D Manager, The global essential oils market is expected to grow from $9.62 billion
Kathleen H. Kiefer, Principal Scientist,
in 2021 to $18.25 billion in 2028, according to Fortune Business Insightsa,
Matthias Nold, Global Product Manager
Reference Materials growing at a CAGR of 9.57% during the 2021-2028 period.
This book comprises handpicked columns by Perfumer & Flavorist+
6 Essential Oils’ Identity Crisis
guest columnists digging into the authenticity, traceability and analysis of
By Wladyslaw S. Brud and Alastair Hitchen
essential oils.
16 HPTLC Fingerprinting for Essential Oil
We hope this e-book, sponsored by Millipore Sigma, gives F&F experts
Authenticity
By Monika Baeumle, Matthias Nold, Ilona Trettin, and insight into the technical nuances of the rapidly growing essential oils
Melanie Broszat market. We hope you enjoy.
20 Application of Gas-Liquid Chromatography to
the Analysis of Essential Oils Best,
By M. J. Milchard, N. C. Dacosta, R. Esdale, L. Gates,
P. Maccormick, P. Merle, G. Moran, N. Moss, D. A.
Moyler, N. Owen, N. Roach, A. Sherlock, T.D. Smith,
B. Starr, J. Webb and R. Wood

41 Genetic Traceability of the Bitter Almond J. Rimensnyder

Essential Oil Supply Chain
By Nelly Dubrulle, Benjamin Marteaux, Nicole
Giraud, Claire Delbecque and Pierre-Philippe Garry

awww.fortunebusinessinsights.com/industry

4 Editor’s Note 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

 Technical Article

Allergenic Fragrance Testing—New Certified

Reference Materials
Calibration mixes for the new IFRA method now available
Markus Obkircher, Head of Reference Materials R&D, Hanspeter Sprecher, R&D Senior Scientist,
Leonard Sidisky, Gas Separations R&D Manager, Kathleen H. Kiefer, Principal Scientist,
Matthias Nold, Global Product Manager Reference Materials

This workflow typically starts with a series of preliminary investigations

such as hygroscopy and volatility of the substances. Further
determinations ensure the compatibility of each analyte with the
internal standard used for the qNMR measurements.

Since reliable weighing values are mandatory, having a direct influence

on the result, they are performed in a metrological way. Using a
micro-balance in a specific, dedicated setup is the key to success
because less sensitive equipment will lead to higher uncertainty
contributions deriving from the weighing procedure. This is of the
The International Fragrance Association (IFRA) published a new method utmost importance for these components as they often have relatively
for the testing of 57 potentially allergenic fragrance compounds in low boiling points. In the qNMR measurements, a value for the mass
response to an opinion by the European Scientific Committee on fractions (g/g) was obtained and direct traceability to the SI unit was
Consumer Safety (SCCS), which also proposed to correspondingly achieved through the use of acknowledged NIST primary reference
amend EC regulation No. 1223/2009. materials. In addition, early insight into the homogeneity and stability
of the components could be gained. If signals of interest of different
This new analytical method has been developed by the IFRA Analytical isomers could not be completely separated in NMR, the ratio was
Working Group to identify and to quantify the 57 fragrance substances determined by Gas Chromatography (GC) measurement under ISO/IEC
and their relevant isomers at a concentration higher than 0.002% 17025 accreditation.
(20 mg/kg) in ready to inject fragrance materials and oils using
Gas Chromatography with FID (GC-FID) or with Mass Spectrometry The raw materials were then gravimetrically dissolved and diluted
(GC-MS) detection. This new method will allow the screening of in methyl tert-butyl ether following the ISO 17034 accreditation
complex fragrance mixtures for the presence of these chemically workflow. Amber glass ampoules were then filled with the resulting
defined allergens. bulk solutions and the process subsequently controlled by GC-MS
homogeneity testing.
In this article we will discuss the introduction of new certified reference
materials and their analysis by GC using an ionic liquid column as The final ampoules were thoroughly investigated to guarantee
an alternative with unique selectivity and stability as compared to stability throughout the entire shelf life of the product at the storage
traditional wax/PEG phases. temperature, as well as during transport to the customer at a
potentially higher temperature. In order to be fully compliant with
ISO 17034, Long-Term Stability studies (LTS) as well as Accelerated
New Certified Reference Material Stability Tests (AST) were performed by GC-MS.
(CRM) Mixtures The results from stability testing, from the conducted homogeneity
experiments and from the qNMR measurements themselves all
We are proud to introduce two ready-to-use certified reference material contribute to the overall uncertainty budget that is included individually
mixtures suitable for use as calibrants for the above mentioned new for each component in the certificate. In addition to the certified
IFRA method. The individual substances of these two mixes consist of values of the concentrations and their respective overall expanded
a range of compound classes (predominantly of alcohol and carbonyl uncertainties in g/kg and g/L (through a certified density measurement
components), which were all individually thoroughly characterized and at 20 °C), the certificate contains the lot number, an expiry date,
their content determined by quantitative nuclear magnetic resonance intended use, accreditation stamps and signatures of the producing and
(qNMR) spectroscopy. qNMR is a relative primary method, as the releasing chemists.
signal intensity is proportional to the number of protons contributing
to the resonance. It is therefore possible to directly compare the signal To guarantee a suitable solution for the customer, the mixtures were
intensities of an analyte via an internal reference standard to a NIST intensively tested in a series of interlaboratory comparison studies
SRM being a different compound. The individual qNMR certifications before the launch.
were all performed following the well-established qualification
workflow developed in Buchs, Switzerland, according to the ISO/IEC For more details and a GC-FID/GC-MS application please visit:
17025 accreditation. https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/
marketing/global/documents/426/487/analytix-reporter-2018-
3-ms.pdf

MilliporeSigma is the U.S. and Canada Life Science business of Merck KGaA, Darmstadt, Germany.
© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. MilliporeSigma, the vibrant M and Supelco are
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Detailed information on trademarks is available via publicly accessible resources.
40620 04/2022
 ESSENTIAL OILS’
IDENTITY CRISIS
How data problems can create challenges for industry.

Essential oils qualified as natural complex substances and, due to their composition and
diversity, are one of the most difficult ingredient groups used in industries such as flavors,
fragrances, cosmetics, pharmaceuticals, aromatherapy and many others.

Reproduction in English or any other language of all or part of this article is strictly prohibited. © 2022 Allured Business Media.

6 Fragrance
6 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com
www.PerfumerFlavorist.com
 BY WLADYSLAW S. BRUD and ALASTAIR HITCHEN,
International Federation of Essential Oils and
Aroma Trade, London
P roper identification of essential oils
remains a challenge even as demand
for natural ingredients, particularly
essential oils, continues to grow.
Therefore, the relationship between
industry factors and research on
essential oils are the subject of this article.

Market Drivers
One can go to any supermarket from New York
to Mumbai or Tokyo and see the same international
Vol. 46 • March 2021
brands of soft drinks, bars of chocolate, chewing
gum, potato chips, tomato ketchup, cereals and
toothpaste. Every duty-free shop in every airport sells
the same top-class perfumes. Every hotel bar around
the world will sell you the same international brand
of gin, whiskey, vodka, brandy or beer.
For these reasons, the global flavor and fragrance
market was valued by different sources at $22-28
billion in 2018. The industry is changing quickly
with new companies entering the market, especially
in Asia, South America and South Africa. That said,
the top compounders of flavors and fragrances
include Givaudan, Firmenich, International Flavors
& Fragrances, Symrise, Mane, Robertet, Kerry, T.
Hasegawa, and Takasago.
According to Global Newswire Inc.a, the flavor
and fragrance market will grow to $35-plus billion in
2024. This, however, can change significantly due to
numerous markets, economies, agricultural condi-
tions, climate and legislative factors.
To achieve conformity of flavor, the major
companies purchase commercial essential oils so
that regardless of the season (good or bad) they can
replicate target flavors and fragrances. The volumes
required today are huge and therefore not possible to
acquire from a single source. This diversity of sourc-
ing ensures continuity of manufacturing in the event
of a disaster.
Sometimes this system fails. When cyclones hit
Madagascar in 2002, the price of vanilla beans went
from $25 per kilo to more than $600 per kilo. Only
the high-end ice cream manufacturers and chocolate

awww.globenewswire.com

7
Essential Oils’ Identity Crisis

companies were able to afford to keep pure vanilla This factor is also crucial for the industry use of
extract in their products. essential oils.
This will be exacerbated by growing demand for
more/only natural (botanically sourced, organic,
vegan, etc.) raw materials such as essential oils Key Application Areas
in food and beverage and cosmetics and related There are two main areas of industrial uses of
products, as well as certain household chemicals. essential oils: flavor and fragrance compounds.
This will force producers to find new resources of These are applied to several primary product
natural materials. categories: food and beverage flavors (for odor and
On the other hand, climate change, devastation taste), fragrances (for odor), household chemicals
of natural resources, and the economic aspects of (for odor), pharmacy (for biochemical activity and
growing essential oil-bearing plants create limits taste/odor), cosmetics (for odor and bioactivity) and
both in supply and price. An additional problem is aromatherapy (for bioactivity and odor).
legislation, which, based on protection of human In the flavor industry, top applications include
safety, eliminates or forces limits in the contents of beverages (comprising almost 30% of the market),
essential oils in market products. dairy, savory, bakery, confectionery, meat, snacks,
Whatever the growing demand for essential oils oral hygiene and pharmaceuticals.
will be, the main factors of basic importance for Meanwhile, in the pharmacy category, a
manufacturers of products containing the essential growing number of essential oils are used not
oils will be always the same: quality and reproduc- only as flavors but as auxiliary or main bioactive
ibility, properties, and safety. ingredients of medicaments.
However, essential oils are categorized as natural Essential oils are used in fragrance compositions
complex substances and, due their composition in major product categories such as household
and diversity, are one of the most challenging chemicals, cosmetics, perfumes and related
ingredient groups used in industries such as flavors, products, and a wide range of products in which
fragrances, cosmetics, pharmaceuticals, aroma- an original fragrance is covered or improved.
therapy and many others. Sales of essential oils
used in consumer products are affected, as noted
above, by market conditions, quality, reproduc-
ibility, properties and safety, as well as the position
of manufacturers and, importantly, legislation.
Proper identification is also an issue. Therefore, the
relationship between industry factors and research
on essential oils are the subject of this article.

The Essential Oil Value Chain

The essential oil production and trade industry
consists of:
• Plantations: Ranging from a family owned piece
of ground to hundreds of hectares and growing
sites.
• Distilleries: Ranging from small field distilleries
to professional computerized factories operating
with hundreds of tons of raw materials.
• Processing and trade: Users and producers of
essential oils often create processing enterprises,
usually related to traders who collect the oils from
different sources and, by professional blending,
sometimes perform additional rectification or
other physical processes to adjust the products to
industry standards.

Swiss scientist Carl von Linné created a binominal system of essential oil classification
Essential oils research should consider all which, in many cases, is used to this day with its signature “L.” following the binominal
above factors for precise identification of products. name of the plant.

8 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

Oregano, marjoram and thyme (pictured) appear in numerous chemotypes, which often are very different.

Cosmetics and perfumes contain essential oils as distillation, after separation of the aqueous phase—if
part of their fragrance and, with development of any—by physical process.”
cosmeceuticals, as their bioactive ingredients. ISO Standard 4720c, “Essential Oils—
Household chemicals comprise an almost infinite
Nomenclature,” contains a list of essential oils,
number of articles, including washing products,
including the Latin names of plants and families,
cleaning materials, disinfectants, air conditioners,
English or French common names, reference to the
car fresheners, paints, bathroom and toilet care, and
oil standard (if available), and part of plant from
insect repellents.
which oil is obtained. Unfortunately, it covers only
a minority of the hundreds of oils and their chemo-
ISO Standards for Essential Oils types which are on the market.
The above definitions and nomenclature are
One of the most important points in essential oil reviewed and updated but remain insufficient for
research and publications is the correct definition both industry and research needs. The importance
of investigated materials, considering biodiversity, of standardization of the oils, especially consider-
agro technology, methods of processing, source and, ing expanding legislation, was known already in
of course, methods of examination of the oils and the middle of the 20th century when ISO and the
their properties. American Essential Oil Association started the
According to ISO Standard 9235b, “Aromatic standardization.
Natural Raw Materials—Vocabulary” essential
oils are:
“Products obtained from natural raw materials Historical Naming Complexities
of plant origin, by steam distillation, by mechanical The first trial of binomial classification of plants
process from the epicarp of citrus fruits, or by dry was published by the Swiss scientist Casper Bauhin

bISO Standard 9235; www.iso.org/standard/51017.html cISO Standard 4720, www.iso.org/standard/69688.html

9
Essential Oils’ Identity Crisis

(1560-1624), followed by few others, including Polynesia—Santalum insulare; and Hawaii—

the famous classification of organisms, “Systema Santalum ellipticum, S. paniculatum and four
Naturae” by Swiss scientist Carl von Linné (Latin: others of minor importance.
Carolus Linnaeus 1707-1778). Von Linné created a The odor, properties, market prices and industrial
binominal system of classification, which in many application of these oils differ significantly. But
cases is used to this day with its signature “L.” fol- on the retail market—especially in aromatherapy,
lowing the binominal name of the plant. cosmetics and related products—only an educated
The Linnaeus system was used for more than consumer will discover that, per the appropri-
century, a period when researchers were developing ate INCI information (if available), what is called
the investigation of plants and their products, includ- sandalwood oil on a product label is not necessar-
ing essential oils. This system shows that plants were ily the oil one expected to buy—Santalum album.
considered as one group with similar or identical This can happen also in some research work. The
contents and properties, though in reality many basic composition of some select sandalwood oils is
were very different. Thus, although we continue to depicted in T-1.2
use the original binominal system, the number of When we consider well-known and -used plants
names with the same first part and different second such as oregano, marjoram and thyme, we enter
is growing almost daily. a jungle of names, contents and chemotypes. The
And herein starts the first problem we face both in family Lamiaceae comprises more than 200 genus
industry and research. and 7,000 species, including 50 species in the
Origanum genus and 350 species in the Thymus
genus. Oregano, marjoram and thyme appear in
The Nomenclature Jungle numerous chemotypes, which often are very differ-
A common and well-known essential oil, san- ent. Surprisingly, different genus may have similar
dalwood, is found in ISO 4720 under item 158, contents, like wild marjoram (Origanum marjorana
sandalwood—Santalum album L., and is obtained ct. linalool), containing more than 65% linalool,
from wood, per ISO Standard 3518. Under item 159 and thyme (Thymus vulgaris ct. linalool), containing
of ISO 4720 we will find “Australian sandalwood— more than 75% linalool. This phenomenon is shared
Santalum spicatum (R.Br.) A. DC, syn. Eucarya across a large portion of essential oil-bearing plants.
spicata (R.Br.) Sprag et Summ,” obtained from Furthermore, there are differences in composi-
heartwood, per ISO Standard 22769. Both belong tions among specimens of the same plants according
to family Santalaceae. But if we investigate market to location of the plantation, weather, agriculture,
offers, we will also find: harvesting and processing. Therefore, ISO standards
for individual oils present all data—physicochemical
• Northern Australian sandalwood oil—Santalum
parameters and contents of ingredients as minimum-
lanceolatum R.Br.
maximum. Otherwise, it would be impossible to
• Fiji sandalwood oil, or brown sandalwood—
use the standards for identification of the oils.
Santalum yasi Seem.
Furthermore, as mentioned before, ISO standards
• New Caledonian sandalwood oil, or Pacific
cover only part of the essential oils used in the
sandalwood, or Vanuatu sandalwood—Santalum
industry.
austrocaledonicum Viell.
Therefore, we approach the most important
There are several other members of the conclusion concerning any investigation of essential
sandalwood family in the Pacific: Papua New oils: Any research work on quality, properties and
Guinea—Santalum macgregorii F. Muell.; any other essential oil data must contain detailed
Fiji, Tonga and Niue—Santalum yasi; French (minimum GC-MS) analyses and identification of its

T-1. Basic composition of selected sandalwood oils2

Type Santalum a-santalol (%) b-santalol (%) (Z )-nuciferol (%) (Z )-lanceol (%)
lanceolatum (Queensland) 4.48 1.7 10.4 22.8
lanceolatum (Cape York) 39 16 nd nd
album 41-55 41-55 8.26 4.54
spicatum 15-25 5-20 3.19 1.17
yasi 37-39 26-28 nd nd
austrocaledonicum 38-48 16-24 nd 8-14

10 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

contents, source, and all data available on produc-
tion and processing.

Market Significance of Essential Oil

Identification To achieve conformity
Accurate identification of essential oils is
required to support industry’s demand for quality
of flavor, the major
and reproducibility, properties (specifications and
composition), and safety.
companies purchase
commercial essential oils
1. Quality and reproducibility
Fragrance and flavor should be the identical in all so that regardless of the
deliveries because there is no way to change them
without changing the end product. Chanel No. 5 season (good or bad)
and 4711 Echte Kölnisch Wasser, for instance, have
reportedly smelled the same since their creation in they can replicate target
1921 and 1725, respectively. These are not provable
claims, but consumers believe in them. Therefore,
flavors and fragrances.
they will be very sensitive to any changes of the odor
they know from daily use.
As a result of this phenomenon, industry will
expect flavor and fragrance suppliers to deliver
reproducible products at the same price, whatever
the difficulties are with raw materials on the market Although odor and taste are very important and
(supply, quality, source, price). often crucial for success or failure of cosmetics,
household chemicals and similar products, exact
reproducibility of essential oil content and quality
2. Properties
is indispensable when those materials are used as
This factor is particularly important for essential
bioactive ingredients in pharmaceuticals, aroma-
oils used in cosmetics, pharmaceuticals and aroma-
therapy and cosmetics.
therapy as bioactive ingredients. Specifications and
3. Quality and properties are the most important
compositions should be strictly controlled.
factors in all research used for elaboration of
safety standards and legislation. Mistakes or
3. Safety inaccuracy here create problems, which some-
A growing number of national and international times require years and large financial sums to
regulations and internal industry standards (GRAS, be clarified.
IFRA, IOFI, FMA, EFEO, etc.) have introduced
additional problems with stable odor and taste due
to changing limits of ingredient contents or prohibi- Examples of Data Problems
tion of thew use of certain materials. A few examples hereafter will illustrate problems
with the correct use of data and their interpretation.
Most important is the proper nomenclature used
The Need for Sound Research for essential oils, which should identify without any
All of these challenges first and foremost require doubts what was investigated. ISO standard GLC
high-quality scientific research on the essential oils. with MS identification must be an integral part of
any research work on essential oils. The methodol-
1. Problems with raw materials require studies in ogy of examination of data and correct conclusions
agriculture on quality and yield of essential oils, should generate useful literature.
new plants or their varieties, plant care, methods
of processing and production, blending, testing Example 1: Turpentine
both organoleptic and instrumental, storage,
A group of Spanish authors published a paper,
transportation, etc.
“Allergic contact hobby dermatitis from turpen-
2. Investigation of properties is one of most
tine,” beginning with the following description
important areas of research on essential oils.

11
Essential Oils’ Identity Crisis

“Turpentine is an oleoresin obtained from various

species of pine.”5
Considering that error in fact, the authors clearly
had no idea what they were investigating, although
somewhere in the paper they refer to a volatile oil Whatever the growing
obtained by resin distillation. The process in fact is
steam distillation, which produces an essential oil of
demand for essential oils
turpentine, sometimes called “balsamic turpentine”
to differentiate from other turpentine products.
will be, the main factors
Furthermore, the research and conclusions of basic importance for
were based on examination of just one person—an
amateur painter who, as can be concluded from the manufacturers of products
containing the essential oils
paper, used a common solvent, sulfate turpentine
obtained as side product in paper production. This
substance can be a sensitizer due to impurities.
There is also turpentine produced by destructive will be always the same:
distillation of pine wood.
Without any clarification of what was really
quality and reproducibility,
used, the authors conclude: “Contact allergy to oil
of turpentine was reported to have become rare in
properties, and safety.
Europe but over the last few years, increased rates
of turpentine sensitization have been reported.” No
other data to support this conclusion are presented.
According to IFRA Standards, concentration The author did not understand the original paper
of turpentine in fragrances is safe up to 0.6% and from Gothenburg, and of course was not aware of
products of oxidation are dangerous. In folk medi- the difference between linalyl acetate and products
cine, dissolved balsamic turpentine is used for chest of its oxidation. However, it shall be emphasized
massage in case of cold. that web pages are more often studied by law
regulators than original scientific papers. The title
Example 2: Linalyl Acetate of the piece alone could be enough for an unwise
In 2015 a paper, “Air-oxidized linalyl acetate—an regulatory decision.
emerging fragrance allergen?” was published by
authors from University of Gothenburg.6 Very well Example 3: Vetiveryl Acetate
documented research, with clear methodology, led to In 2009, IFRA published its Standard for veti-
the conclusion that, “The frequency of positive reac- veryl acetate (in 2015 it was renamed as acetylated
tions to oxidized linalyl acetate is comparable to that vetiver oil). In June 2013, a dossier was submitted
of previously studied oxidized fragrance terpenes. to the EU’s Scientific Committee on Consumer
Oxidized linalyl acetate could thus be a common Safety (SCCS).
fragrance contact allergen … and its hydroperoxides On the basis of that dossier, SCCWS elabo-
were classified as moderate sensitizers.” rated and adopted “Opinion on Vetiveryl Acetate
It should be mentioned that oxidation of linalyl (Fragrance Ingredient).” The SCCS adopted this
acetate requires long direct exposure to air. It is opinion at its eighth plenary meeting on December
common knowledge that products of oxidation of 16, 2014. The opinion was based on 99 references
several terpenes are sensitizing and act as allergens. and analysis of 14 different properties of the product.
On the basis of that publication, A. McDougall The SCCS' overall comment on mutagenicity/
published an article, “Study questions potential genotoxicity was as follows: “Based on the submitted
fragrance allergen not currently declared in studies, the mutagenic/genotoxic of vetiveryl acetate
cosmetics,” with the following conclusion: “Linalyl cannot be evaluated as only partial and insuf-
acetate, a fragrance chemical that is one of the ficient information on the composition of vetiveryl
main constituents of the essential oil of laven- acetate on the market is reported (see 3.1.4) and as
der, may be an emerging fragrance allergen and the composition of the test substances used in the
should appear in the declaration of ingredients for submitted mutagenic/genotoxic studies is unknown
cosmetics, according to a study by the University to the SCCS.”
of Gothenburg, which suggests that it can cause The conclusion was: “On the basis of the inad-
allergic eczema.”7 equate data provided, a reliable safety assessment

12 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

cannot be performed whether vetiveryl acetate on the The report continued: “The following sub-
market is safe for use in cosmetics at the concentra- stances were included, and shall be identified on
tion limits proposed by the IFRA. However, due to the product label (as INCI ingredients): amyl cin-
major concern of genotoxicity the SCCS considers namal, amylcinnamyl alcohol, anisyl alcohol,
vetiveryl acetate unsafe as a cosmetic ingredient.” benzyl alcohol, benzyl benzoate, benzyl cinnamate,
The following trade names and abbreviations benzyl salicylate, 2-(4-tert-butylbenzyl)-propio-
were used in references for the product: vetiveryl naldehyde, cinnamal, cinnamyl alcohol, citral,
acetate, vetiver acetate, vetivert acetate, vetyvenyl citronellol, coumarin, eugenol, farnesol, geraniol,
acetate, vetiverol acetate, vetyveryl acetate, hexyl cinnamylaldehyde, hydroxy-citronellal,
Vetiveria zizanioides, ext., acetylated, acetyver, and hydroxy-methylpentylcyclohexene carboxalde-
vetiveryl acetate. hyde, isoeugenol, d-limonene, linalool, methyl
There was not enough data and the SCCS had heptin carbonate, 3-methyl-4-(2,6,6-tri-methyl-2-
no idea what was evaluated. Nevertheless, the final cyclohexen-l-yl)-3-buten-2-one, oak moss extract,
negative conclusions were reached. treemoss extract.”
It shall be noted that although there is no doubtful
Example 4: Allergenic Reactions evidence of allergic properties of above chemi-
In 2003 the “Directive 2003/15/EC of the cals, and any chemical is likely to cause allergenic
European Parliament and of the Council of 27 reaction—i.e., can be a “potential allergen”—this
February 2003 amending Council Directive 76/768/ regulation created problems for manufacturers of
EEC on the laws of the Member States relating many products. This is especially true for those
to cosmetic products” was publishedd. One of the products that are small in size and lack space for
important points based on previous opinions (see a label for listing ingredients, especially those that
below) concerned so-called “potential allergens.” appear in natural raw materials, e.g., essential oils.
Per the publication: “A number of substances The meaning of “pragmatic administrative decision”
have been identified by the SCCNFP (The Scientific in relation to safe concentrations will remain a secret
Committee on Cosmetic Products and Non-Food of the science applied by SCCS.
Products Intended for Consumers) as likely to cause
allergenic reactions and it will be necessary to Scientific Research Needs
restrict their use and/or impose certain conditions
The provided examples emphasize how impor-
concerning them. Information should be provided to
tant quality research is. The following areas are of
consumers about their presence in cosmetic prod-
most import:
ucts. At the time there were insufficient scientific
data to allow for the determination of dose/response 1. Agriculture: New plants in the wild and planted
relationships and/or thresholds for these allergens. to extend the range of essential oils plants and
Nevertheless, in a pragmatic administrative decision their natural modifications (location, cultivation,
the limits of 0.01% and 0.001% were set, for rinse-off harvesting methods, time etc.).
and leave-on products, respectively.” 2. Processing: From the harvest through storage,
drying, distillation, equipment and technology,
dSCCNFP/0017/98;
and transportation used to achieve the highest
published 30.09.1999, adopted by SCCS** 1459/11 of
13.12.2011 yield and quality at the lowest cost.

13
Essential Oils’ Identity Crisis

3. Quality evaluation: New and improved methods 2. Analysis of Investigated Sample: Methods and
of analysis of final products, as well as produc- instruments used for analysis and detail condi-
tion process, and instrumental and organoleptic tions (especially for instrumental analysis),
testing of product composition and compatibility olfactometric evaluation methods, sample condi-
with approved samples. tion, packing and time from sample collection to
4. Properties: This is the most difficult but opening for analysis.
extremely important area of research that should 3. Intended Aims of Investigation: A)
prove essential oil composition and activity of its Improvement of agriculture to achieve better
ingredients. This is especially valid for essential yield or quality, more efficient processing and
oils used as bioactive agents in pharmaceuticals, of essential oil including modification (if any),
aromatherapy and cosmetics. production technology. B) Fragrance and
5. Safety: For humans, animals, environment, water flavor properties with detailed descriptions of
etc. Crucial for potential influence on legislation. evaluation methods applied. C) Properties of
investigated product intended for use as bioac-
All research areas mentioned above can detect tive ingredients, including standards obligatory
much more than a few decades ago due to progress for ingredients to be used in pharmaceuticals,
in the equipment that laboratories are using. The biomaterials used for tests (e.g., bacteria) should
cost of a growing variety of more complicated and be standardized and available for other laborato-
accurate analytical equipment has come down ries. D) Safety of use for humans, environment,
significantly. Therefore, these capabilities are more etc., including all standards either established by
affordable to most companies within our industry, official regulations or approved by appropriate
not just the top 10. industry organizations.
This progress allows industry researchers to move
from PPM via PPB to PPT. There are also instru- In conclusion any publication on scientific
ments which allow researchers to not only separate research of essential oils must be:
ingredients but quantify their content and identify • Complete and precise
their chemical structure. • Reliable
Several other parameters can be analyzed to give • Fully documented with results and references
detailed descriptions of essential oils, their sources, • Possible to repeat in any other research center
nature, possible adulteration, etc. This has brought
its own difficulties because we are finding undesir-
able components that we don’t want but which have Author Contact
probably always been present in certain essential Wladyslaw S. Brud, International Federation of
oils. On the other hand, with that information in Essential Oils and Aroma Trade; [email protected].
hand, problems of safety of use of the essential oils
can be examined with complete knowledge of the
chemical content of the product. References
1. P. Rovesti, In search of perfumes lost, Venetia 1980

Conclusion: Research & Publication 2. B. Lawrence, Essential Oils 1988-1991 Wheaton,1993 180-182

Conditions 3. W.S. Brud in K.H.C. Baser, G. Buchbauer, “Handbook of

Essential Oils Science, Technology and Applications” CRC
To improve the essential oil industry’s data, the Press, Boca Raton, 2020, p.1033
following research and publication conditions must
4. Ibid. p.1035
be observed.
5. L. Barchino-Ortiz , et al,. Allergol Immunopathol (Madr).
1. Exact Description of Investigated Product:
2008 Mar-Apr;36(2):117-9 https://www.ncbi.nlm.nih.gov/
Essential oil, processed essential oil (rectified, pubmed/18479665
modified, x-less, etc.), fraction of essential oil,
ingredient or mixture. This shall include: botani- 6. L. Hagvall, V. Berglund, J. B.Christensson, Contact Dermatitis,
72 (4), 2015 p. 216-223
cal identity of plant raw material (especially if
new product is analyzed), source location, time 7. A. McDougall, Cosmeticsdesign-Europe; www.cosmeticsdesign-
and method of harvesting, processing before europe.com/Article/2015/05/28/Study-questions-potential-
distillation, distillation or expression technology fragrance-allergen-not-currently-declared-in-cosmetics

including size of charge, time, water source, instal-

lation with method of product collection, handling
and storage, packing and sample collection.

14 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

With the two new ready-to-use Certified Reference Materials, fragrance
manufacturers and testing labs are being provided with reliable, high
quality standard calibration solutions suitable for the new IFRA method.
This is a major advancement in improving the safety of personal care
products and allowing manufacturers to retain their customer's trust.

Featured Products
Description Qty. Cat. No.
Fragrance Allergen Standard A1 2 mL/5 mL 89131
Fragrance Allergen Standard A2 2 mL/5 mL 16558
SLB®-IL60i, 30 m x 0.25 mm I.D., 0.20 µm 1 ea 29832-U

Fragrance Allergen Mixes A1 and A2 with Composition.

The nominal concentration for most components is 2000 mg/kg (exceptions
are marked with *). The lot specific certified values are given in the certificate
provided with the products. For both mixes, 2 mL and 5 mL packages are
available (amber glass ampoules).

Fragrance Allergen Mix A1 Fragrance Allergen Mix A2

Cat. No.: 89131 Cat. No.: 16558
Qty.: 1 mL, 5 mL Qty.: 1 mL, 5 mL
Solvent: Methyl-tert-butyl Isoeugenol (E+Z) Solvent: Methyl-tert-butyl Dimethylbenzylcarbinyl acetate
ether (MTBE) ether (MTBE) (DMBCA)
α-Amylcinnamyl alcohol R(+)-Limonene 3-Propylidene phthalide Eugenyl acetate
trans-Anethole Linalool α-Acetyl Cedrene Galaxolide 1
Anise alcohol Menthol Isoeugenyl Acetate Galaxolide 2
Benzyl alcohol α-Pinene α-Amylcinnamaldehyde Geranyl acetate
β-Caryophyllene β-Pinene Amyl salicylate Hexadecanolactone /
Cinnamyl alcohol α-Santalol Dihydroambrettolide

Citronellol β-Santalol Benzaldehyde α-Hexylcinnamaldehyde

Ebanol 1 Sclareol Benzyl benzoate Hydroxycitronellal

Ebanol 2 α-Terpinene Benzyl cinnamate Hydroxyisohexyl 3-cyclohexene

carboxaldehyde - major
Eugenol Terpineol, mainly α
Benzyl salicylate Hydroxyisohexyl 3-cyclohexene
trans,trans-Farnesol Trimethyl-benzenepropanol carboxaldehyde - minor*
Geraniol Butylphenyl methylpropional α-Isomethylionone
Camphor Linalyl acetate
Carvone Methyl salicylate
Cinnamaldehyde Methyl-2-octynoate
Neral Salicylaldehyde
Geranial Terpinolene
Coumarin α-Tetramethylacetyloctahy-
dronaphthalene (ISO E® α)*
β-Damascenone β-Tetramethylacetyloctahy-
(Rose Ketone-4) dronaphthalene (ISO E® β)
α-Damascone γ-Tetramethylacetyloctahy-
dronaphthalene (ISO E® γ)*
ß-Damascone "E" Vanillin
δ-Damascone (Rose Ketone-3)
*Compounds deviate from the nominal concentration of 2000 mg/kg

MilliporeSigma is the U.S. and Canada Life Science business of Merck KGaA, Darmstadt, Germany.
© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. MilliporeSigma, the vibrant M and Supelco are
trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners.
Detailed information on trademarks is available via publicly accessible resources.

40651 04/2022
 Understanding this analytical method and its role in analyzing botanicals.

BY MONIKA BAEUMLE, product manager TLC, MilliporeSigma; MATTHIAS NOLD, product manager reference materials
MilliporeSigma; ILONA TRETTIN, application specialist, CAMAG; and MELANIE BROSZAT, scientific business development
manager, CAMAG
Vol. 46 • September 2021

Sponsored by:

With the growing popularity of essential oils, the likelihood of adulteration by the addition of cheaper oils or oil constituents increases.

Reproduction in English or any other language of all or part of this article is strictly prohibited. © 2022 Allured Business Media.

16 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

 he use of essential oils is showing no
signs of slowing down, with an
expected growth at a compound
annual growth rate (CAGR) of 7.5%
from 2020 to 2027 in the global
marketa. Consumer demand is
driving the use in food and beverage,
personal care, cosmetics and aromatherapy.
Because some essential oils have been known
to have anti-inflammatory and antiviral properties,
there is a long history of using them as a natural
home remedy for a range of ailments. Such remedies
have caused added interest in the use of essential oils
to help combat the symptoms of COVID-19.1
With the growing popularity of essential oils, the
likelihood of adulteration by the addition of cheaper
oils or oil constituents increases to maximize profits.
Since there are many possible ways adulteration can
occur, it can be a challenge to analyze these products. Some essential oils have been known to have anti-inflammatory and antiviral properties.
An efficient method for the detection of such adultera-
tion is the use of chromatographic fingerprinting
methods, in particular, high-performance thin-layer
chromatography (HPTLC).
Create with Confidence
Defining HPTLC Essential Oils: Field to Formulation
HPTLC is the most advanced form of thin-layer
chromatography (TLC), a well-known method We set the standard when it comes to safe
for the analysis of botanicals and other complex and compliant ingredients. We partner with
samples. The main differentiators between the trusted growers and processors
two are the silica particle size in the layer (10-12 to bring you the highest quality
microns for TLC and 5-6 microns for HPTLC) essential oils. Our ingredients
and the layer thickness of the plate (250 microns go beyond the industry norm
for TLC; below 200 microns for HPTLC). These by providing both all quality and
differences allow a separation in shorter times by regulatory documentation online.
having a shorter migration distance and increased The ingredients are verified natural,
separation efficiencies. The increased separation follow the guidelines designated by IFRA (International Fragrance
efficiency is crucial when it comes to fingerprinting Association) and meet the EU Regulation 1223/2009. Reference
analysis. A quick identity test can be done by TLC, Materials for Allergenic Fragrances Listed in the New IFRA Method.
but comprehensive fingerprinting or profiling tests We exclusively offer two ready-to-use certified reference material
are more reliable by HPTLC. mixtures suitable for use as calibrants with the new IFRA method to
HPTLC is a powerful, yet simple, cost-effective test 57 potentially allergenic fragrances. The new method allows the
tool for testing the identity, purity and strength screening of complex fragrance mixtures for the presence of these
(content) of botanicals, as well as for identifying chemically defined allergens by GC-MS.
adulteration during quality control. With the publica- We also have a number of new individual reference materials
tion of general chapters by the U.S. Pharmacopoeia as neat compounds for some of the allergenic fragrances contained
(USP <203>) and European Pharmacopoeia (Ph. Eur. in the mixes. These reference materials have been certified by
2.8.25), HPTLC has officially come into existence as quantitative NMR (qNMR) and are traceable to NIST SRMs. The
a highly standardized and, therefore, reproducible certificate contains information about traceability, homogeneity and
analytical technique. stability and the values are given with calculated uncertainties.
The use of high-performance plates, suitable More information about our standards can be found at:
instruments and software, a standardized meth- www.sigmaaldrich.com/products/analytical-chemistry/
odology and validated methods ensures reliable reference-materials
results that are fully compliant with current Good Learn more about all we have to offer when formulating new
Manufacturing Practice (cGMP). scents at: SigmaAldrich.com/flavors-fragrances
awww.grandviewresearch.com/industry-analysis/essential-oils-market

17
HPTLC Fingerprinting for Essential Oil Authenticity

F-1. Lemon oil, UV 254 nm prior to F-2. Lemon oil, UV 366 nm after F-3. Lemon oil, white light after
derivatization derivatization derivatization

F-4. Pine needle oil, UV 366 nm after derivatization F-5. Pine needle oil, white light after derivatization

Detecting Adulteration variety of detection methods (UV, numerous chemical

derivatizations and MS) increases the chance to detect
HPTLC is a fingerprinting method that allows con-
adulteration (please see figures F-1 to F-5).
venient visual comparison of multiple samples, even
To perform the work done in this article, the
if they originate from different plates (and different
HPTLC plates, TLC accessories and solvents and
laboratories worldwide). Reference images (HPTLC
suitable reference materials for common chemical
fingerprints of botanical reference materials or other
markers used in essential oils authenticity testing
references) can be used to qualify data and pass or fail
were provided by MilliporeSigma. Fingerprints for
samples based on similarity or difference.
more essential oils can be found in the Analytix
In this study, fingerprints for a range of essential
Reporter: Issue 7 at sigmaaldrich.com/analytix.
oils were obtained with the standard HPTLC method
for identification (submitted to Ph. Eur. for evalua-
References
tion) by comparison of RF values, colors of reference
1. Asif, M., Saleem, M., Saadullah, M., Yaseen, H. S., & Al
substances and matching zones in the oils.
Zarzour, R. (2020). COVID-19 and therapy with essential oils
While not all potential components of essential oils having antiviral, anti-inflammatory and immunomodulatory
are detectable with this method (e.g., camphene, men- properties. Inflammopharmacology, 28(5), 1153–1161.
thofuran, a-pinene, b-pinene or g-terpinene), the broad www.doi.org/10.1007/s10787-020-00744-0

18 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

 Essential Oils
Transparency is essential to all formulations. That's why we have
partnered with key growers and manufacturers to bring you essential
oils you can trust. We are proud to partner with Essential Oils and Herbs
(Bulgaria), Simone Gatto (Sicily) and Dutch Essentials BV (Netherlands)
to bring you high quality essential oils. To see the full list of essential oils
visit: sigmaaldrich.com/essential-oils

Product
FEMA Product Name Number Availability
2657 Mandarin Oil, product of Simone Gatto W265722 North America Only
2657 Mandarin Oil, colorless, product of Simone W265799 North America Only
Gatto
2153 Bergamot terpenes, product of Simone Gatto W215377 North America Only
2153 Bergamot Oil, product of Simone Gatto W215355 North America Only
2153 Bergamot Oil, colorless, product of Simone W215311 North America Only
Gatto
4848 Lemon terpenes, natural, product of Simone W484815 North America Only
Gatto
2625 Folded Lemon Oil, Verdelli, product of Simone W262513 North America Only
Gatto
2625 Distilled Lemon Oil, product of Simone Gatto W262577 North America Only
2823 Bitter Orange Oil, product of Simone Gatto W282301 North America Only
2823 Sweet Blonde Orange Oil, product of Simone W282111 North America Only
Gatto
2821 Sweet Blood Orange Oil, product of Simone W282155 North America Only
Gatto
2112 Melissa Oil, product of Essential Oils & Herbs W211233 Global – All Regions
Ltd
2622 Bulgarian Lavender Oil, product of Essential W262255 Global – All Regions
Oils & Herbs Ltd
2989 Rose Oil, Bulgarian, product of Essential W298944 Global – All Regions
Oils & Herbs Ltd
2275 Roman Chamomile Oil, product of Essential W227511 Global – All Regions
Oils & Herbs Ltd
2466 Eucalyptus Oil, 80% product of Dutch W246680 Global – All Regions
Essentials
2466 Eucalyptus Oil, 65%, product of Dutch W246665 Global – All Regions
Essentials

MilliporeSigma is the U.S. and Canada Life Science business of Merck KGaA, Darmstadt, Germany.
© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. MilliporeSigma, the vibrant M and Sigma-Aldrich are
trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed
information on trademarks is available via publicly accessible resources.

40621 04/2022
 Fingerprint chromatograms of esters-containing essential oils: Chamomile
English.
Vol. 46 • August 2021, September 2021, October 2021

BY M. J. MILCHARD, N. C. DACOSTA, R. ESDALE, L. GATES, P. MACCORMICK, P. MERLE, G. MORAN, N. MOSS,

D. A. MOYLER, N. OWEN, N. ROACH, A. SHERLOCK, T.D. SMITH, B. STARR, J. WEBB and R. WOOD

Gas Liquid Chromatography (GLC) fingerprint chromatograms of seven authentic essential oils were obtained in a collaborative study.

Reproduction in English or any other language of all or part of this article is strictly prohibited. © 2021 Allured Business Media.

20
20 Fragrance 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com
G
as Liquid Chromatography Institute of Science and Technology) WebBook of
(GLC) fingerprint retention indices.3
chromatograms of seven This method has since been used by the
authentic essential ISO TC (International Standards Organization
oils were obtained in a Technical Committee) 54 and used in this series of
collaborative study using the RSC publications.
recommended procedure
given in Part XVII of this
series and are presented here (this paper represents Results
Part XXIV, the cedarwood oils were Part XXIII).1,2 The results for each essential oil are presented
The samples examined were chamomile English in the form of an annotated chromatogram and
(also known as chamomile Roman), bergamot FCF a table of identified components with their RRI
(furocoumarin-free), clary sage, spike lavender, (relative retention indices) and area percentage
neroli and two oils of petitgrain, bigarade and concentrations. The numbers in brackets in the
Paraguay. These were selected in consultation component list refer to peaks in the corresponding
with the UK essential oil trade through the BEOA chromatogram(F-1). The published chromato-
(British Essential Oil Association). The expert grams are representative of those obtained by
working group gratefully acknowledges the staff individual expert working group members. Only
time and samples of essential oils provided by the those results obtained on columns that had g-pack
companies involved in this study. values within the accepted range were used for the
This paper explores six other esters including ber- calculation of relative retention index and compo-
gamont, clary sage, spike lavender, neroli, petitgrain nent concentration.1 The tables also contain other
bigarade and petitgrain Paraguay. published results, where available, from ISO, EFFA
(European Flavour and Fragrance Association),
AFNOR (Association Française de Normalisation)
Experimental and Schmidt for comparison.4 Where a positive
These NCS (natural complex substances) were identification could not be made, the chemical
subjected to GLC analysis in one long auto-sampler class is given as follows: STHC (Sesquiterpene
run over a weekend, starting and finishing with Hydrocarbon); STOH (Sesquiterpene Alcohol).
an NC (Standard GLC Fingerprinting Calibration In all of the chromatograms, the x axis is the time
Mixture) polarity calibration mix (aliphatic hydro- in minutes and the y axis is the detector response
carbon based), as described in the RSC (Royal in counts.
Society of Chemistry) fingerprinting
paper in The Analyst.1 The assumption
was made that if the calibration was
the same at the start and finish of the
run, all of the samples in between were
correctly run.
Basic chromatographic condi-
tions used by the group were a 25-60
meter length columns of about 0.25
mm diameter and 0.25-micron film
thickness of a non-polar stationary
phase. Temperature program used was
50-240oC at 4oC/minute and no initial
hold time. Any inert carrier gas phase
was used and flame ionization detec-
tion with supporting data provided by
the use of a polarity calibrated wax
phase column and mass spectrometry.
The injected sample size and dilution
was in accordance with individual
laboratories instrumentation. This is
the detailed level of GLC conditions
that is also listed in the NIST (National
The flowering herb has a small chrysanthemum-like flower.

21
Application of Gas-Liquid Chromatography to the Analysis of Essential Oils

RRI of Early Eluting Components run. This effect was not apparent on monoterpene
hydrocarbon constituents after RRI of 950, i.e.,
During the compilation of GLC results from b-pinene, sabinene and myrcene.
various laboratories, it was noted that some of the The reported indices are from the laboratories
laboratories’ NC calibrations consistently gave low that reported linearity between the C8 and C10.
values for early eluting, non-polar constituents of
essential oils on non-polar columns, when MS
(mass spectrometry) had confirmed the correct Procedure to Confirm the Identification
identification, e.g., a-pinene RRI 920 or 932 on
methyl silicone columns.
of Constituents Seen in GC-MS Analyses
The NC column polarity calibration mixture uses The confidence in the identification of constitu-
C8 H18 to C24 H50 even number carbon hydrocar- ents in complex samples is critical to achieving
bons and assumes linearity between the C8 and C10. reliable results that are acceptable to third parties
When retested with a calibration mixture of C5, C6, and publications.
C7, C8, C9, C10 hydrocarbons, a-pinene was found There are a number of key steps which, if applied
to have an RRI of 932. in a stepwise manner, can significantly improve the
This value corresponded to the median value reliability of the identification of constituents in a
given in the NIST database monograph collated GC-MS analysis.
information for hundreds of separate laboratories’
measurements of a-pinene CAS 80-56-8. 1. Application of Retention Times or Retention Indices
It is believed that this effect of low values could be a). This refers to the calibration of the GC condi-
linked to the injector dead volume, giving rise to this tions being applied to the analysis. The retention time
non linearity at the start of the temperature program (RT) is a unique factor dependant on the GC column

F-1 GLC of chamomile oil English

Current Chromatogram(s)
counts
7
80000

13 & 14

70000 16

60000

50000

10
40000
2 12

30000

11

20000

1
17
4 56 9
10000 3 15
18

5 7.5 10 12.5 15 17.5 20 22.5 25 27.5 min

22 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

and the method parameters
being used. It is always advisable
to develop and then set a given
method, whereby the retention
times of known constituents are
well characterized and, therefore,
unknown components can be
more reliably estimated.
b). The use of retention
indices is more widespread than
retention times, and indices
are widely available for many
constituents and GC phases, as
well as being increasingly given
in MS Library entries as well.3
This relies on the calibration of
the GC column phase (typically
non-polar or a polar wax phase)
using an ascending serial group
of compounds (alkanes or alkane
esters) which have a systematic
and defined retention behavior.
Once calibrated, all other con- Chamomile oil is used as a traditional medicine and in herbal infusions.
stituents have an index relative to
the nearest calibration neighbor(s) and can therefore d). Any figure below 500 is considered to
be referenced to those in terms of its own retention not be a match and should be discarded for
index. This is one parameter used to characterize a reporting purposes.
potential unknown constituent.
3. Combined Searching
2. MS Library Matching a). This relies on the use of the calibrated RT/RRI
The quality, or match factor, of that constituent’s process and linking that to the MS Library entry for
mass spectrum is assessed by comparing it to a each constituent.
reference spectrum in either a commercial library b). Each constituent is either run under the
or, ideally, the spectrum of that constituent in a user precise GC conditions and the RT/RRI generated for
generated library. The quality of the match can be that material or, alternatively, a commercial library
further defined by the following criteria. can be used which already has those index entries as
a). A match factor of >850: This indicates a good part of each library entry.a
match with the library spectrum; the closer to 999 c). Only those MS Library entries found within
this becomes, the increasing confidence one can have a defined RT/RRI window are considered as being
with the proposed candidate. appropriate for searching against; this reduces the
b). A match factor of >750 but <850: This potential for incorrect identification being reported.
indicates that the match is relatively strong, but
that caution should be taken with taking this as
being 100% the proposed candidate. This may be Conclusions
due to other constituents providing some spec- Overall, confirming a constituent’s identity should
tral interference with the mass spectrum under be a combination of both the closeness of the RT/
consideration. Other supporting evidence should RRI record on the GC phase and the quality of the
be provided to improve confidence in the identifi- MS Library match. Clearly, a review of the quality of
cation (RT; RRI, etc.). both the separation and the mass spectrum should
c). A match factor of <750: Typically not consid- be undertaken to assess how clean both are. Are they
ered as indicating the library and constituent spectra subject to co-elution with another constituent? Is
are the same. If there is additional evidence to the peak at very low concentration, therefore having
support this identification (RT or RRI, etc.), then this poor spectral quality (too few ions), and would a
should only be reported as a tentative identification
at best and used with warning.
achemdata.nist.gov/dokuwiki/doku.php?id=chemdata:start#libraries

23
Application of Gas-Liquid Chromatography to the Analysis of Essential Oils

T-1. Composition of Chamomile oil English

RRI Constituent Area % AFNOR Schmidt
829 3-methylpentanol 0.1 0.2
900 isobutyl isobutyrate 0.9 2-9 1.5-7.1
911 (E)-2-butenyl butyrate 0.2
912 2-methylallyl isobutyrate 0.1
926 isobutyl methacrylate [1] 1.4 0.5-3 0.9-2.8
932 a-pinene [2] 5.2 2.2-11.5
937 methylallyl methacrylate [3] 1.0 1.7
946 camphene 0.7 0.4-1.4
960 3-methylpentyl acetate 0.2 0.9
972 b-pinene 0.6 0.3-1.1
978 n-pentyl acrylate 0.5 0.2-2.1
986 isobutyl -2-methylbutyrate 0.7 0.6-1.7
996 isoamyl isobutyrate 0.3 2.5-5 0.6-2.4
1000 2-methylbutyl isobutyrate [4] 1.3 0.5-4.7
1010 p-cymene 0.5 0.05-0.2
1020 isoamyl methacrylate [5] 1.1
1021 1,8-cineole 0.1 0.1
1023 2-methylbutyl methacrylate [6] 1.0 0.5-1.5 0.7-1.5
1025 d,l-limonene 0.4 0.05-0.1
1033 isobutyl angelate [7] 13.7 30-45 11.2-34.4
1039 3-methyl-2-butenyl isobutyrate 0.3 0.2
1050 methallyl angelate [8] 10.5 6-10 6.8-8
1063 3-methyl-2-butenyl methacrylate 0.3 0.3
1069 butyl angelate 0.6 0.07-0.7
1084 isoamyl-2-methyl butyrate 0.2 0.3-0.8
1087 isoamyl isovalerate 0.5
1099 3-methylpentyl isobutyrate [9] 1.4 0.6-1.4
1124 (E)-pinocarveol [10] 7.2 2-7 1.7-5.2
1130 3-methylpentyl methacrylate [11] 3.9 0-1.8
1131 isoamyl angelate [12] 6.4 12-22 2.2-22.3
1138 pinocarvone [13]* 5.0 1.3-6 2.3-5.6
1139 2-methyl butyl angelate [14]* 9.0 2-9.7
1142 ester 0.5
1148 borneol 0.7 0.1-0.3
1149 isopinocamphone 0.6
1164 myrtenal 0.5 0.5-0.8
1166 a-terpineol 0.2 0.03
1172 angelyl angelate [15] 1.2 0.9-1.4
1174 myrtenol 0.6 0.4
1182 ester 0.1
1188 2-methylbutyl ester 0.5
1215 2-hydroxy-2-methyl-3-butenyl angelate 0.2 0.3-0.4
1240 3-methyl pentyl angelate [16] 13.3 3-7 0.4-12.8
1259 (Z)-3-hexenyl angelate 0.1 0.2
1374 a-copaene 0.1 <0.5
1411 b-caryophyllene 0.2 0.5
1461 ar-curcumene 0.1
1472 germacrene D [17] 1.9 0.9-1.3
1476 b-selinene 0.2
1495 (E,E)-a-farnesene [18] 1.0 0-0.2
1511 d-cadinene 0.1 <0.5
1551 myrtenal angelate 0.1 trace
1585 hexahydrofarnesyl acetone 0.2
Total: 97.7%
*peaks 13 and 14 separated on a wax column

24 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

repeat injection of the sample
at higher concentration if
poor sensitivity, or more dilute
if the peak is overloaded on
the GC phase, give a more
reliable result?
Improving the quality of
peak identification by these
simple routines should be
an aim of all laboratories
undertaking analysis of
these complex samples. It
is not difficult in itself, but
will require some rigor and
robustness within the whole
methodology used, from
ensuring the GC phase used
is performing to an accept-
able standard, the mass
spectrometer is correctly cali-
brated for mass accuracy and
ionization profile and that
the simple rules for accepting
library matches against either
internal (recommended) or
external (also recommended
but with more care) MS The oil has a small use in fruit flavors where its unusual esters add complexity in alcoholic beverages.
Libraries is followed.

unusual esters add complexity in alcoholic beverages.

Chamomile Oil English (Roman) It is applied in oral care products, skin cosmetics and
The flowering herb Anthemis nobilis L., syn- fruity floral fragrances, where its diffusive odor means
onyms Chamaemelum nobile L. All., Ormenis that it only needs to be used in small amounts, more
nobilis L. J.Gay ex Coss. & Germ. is part of the often to add complexity in fine fragrances.
Asteraceae family and has a small chrysanthemum- The oil tested was from a single consignment
like flower. CITES (Convention on International made by a grower and distiller in England and is
Trade in Endangered Species) lists this oil as characteristic of this complex oil(T-1). AFNOR stan-
being from a plant of least concern. dard 75-253 applies for this oil.
It is cultivated and steam distilled from semi- This paper is dedicated to the memory of the late
dried flowers in England, Egypt, Tunisia, Italy David Moyler for his valuable contribution to the
and Germany. Oils with different constituents are work of the Royal Society of Chemistry’s Analytical
produced from the other chamomile varieties, Methods Committee Expert Working Group on
e.g., blue German or Hungarian chamomile Essential Oils over the last 40 years.
Matricaria chamomilla and the Moroccan chamo-
mile Ormenis multicaulis; these oils and are also Bergamot
available commercially. The small, fruiting citrus tree Citrus bergamia
English chamomile is a clear, pale blue (unlike Risso, synonym Citrus aurantium subsp. ber-
the dark blue Matricaria) mobile oil with an herbal gamia [Risso & Poit.] Wight & Arn. ex Engl. is of
odor, fruity and tea leaf-like character, which derives the Rutaceae family and mainly cultivated in the
from a wide range of esters, giving an ester content Calabria coastal region of southern Italy, with some
of about 80%. production also in Brazil and the Ivory Coast. Like
Chamomile oil is used as a traditional medicine many commercial citrus trees, they are usually
and in herbal infusions, but there was shown not to grafted onto the hardier, more frost-resistant bitter
be sufficient evidence of clinical benefits and so its use orange root stock. The fruits produced in the winter
is not supported by the European Medicines Agency. season are the size of oranges, the shape of lemons
The oil has a small use in fruit flavors where its and picked while the fruit is still a dark green color.

25
Application of Gas-Liquid Chromatography to the Analysis of Essential Oils

This mobile oil is extracted from the peel of Bergamot Oil, Furocoumarin Free (F-2)
the whole fruit using pelatrice extractors, is dark
There are several proprietary methods for
green in color and has a fresh, sweet, fruity, floral
removing the furocoumarins. Most involve vacuum
odor. This zest rasping method keeps the peel oil
distillation, but some care is needed as the furocou-
separated from the acidic juice with which it might
marins can sublime if very high vacuum conditions
react. Unlike other citrus oils, bergamot oil has a
are used. This group has published a paper on the
high level of linalol and linalyl acetate, as well as
determination of bergaptene in bergamot oil by
the ubiquitous dextro-limonene that is found in
HPLC (High Performance Liquid Chromatography)
citrus peel oils.6
with UV detection, including the method for the
It is used as a flavoring for consumer products
preparation of a standard.8 The collaborative study
like earl grey tea, liqueurs, confectionery, tobacco,
followed work by the Japanese National Institute of
baked goods, desserts and chewing gum.
Hygienic studies on bergamot oil in cosmetics.9
This expressed oil has an ISO standard 3520:1998
IFRA have developed and collaboratively tested
and is only used for flavorings, as it contains natu-
a comprehensive method for the determination of
rally occurring skin photosensitizing furocoumarins
six furocoumarin marker constituents in simple
(about 3000 ppm) that are subject to a limiting
essential oils, totalling 5 mg/kg (5 ppm) maximum
low level of 5 ppm in the IFRA (International
to be present in leave-on consumer products and
Fragrance Association) standard for fragrance uses.
50 mg/kg (50 ppm) maximum in wash-off products.
The analysis of the cold pressed oil is described in
This method is published by IFRA on their website
detail in many publications and only reported in
and has a quantification limit of 10 mg/L, stated as
this paper’s T-2 for comparison purposes with the
not being a suitable method for the quantification of
FCF grade, to illustrate the compositional changes
mixtures of essential oils, compounded fragrances
that occur during the process that removes the skin
or consumer products like cosmetics.10 It is note-
photosensitizers.7
worthy that the Swiss cosmetic regulation of May 1,
2017, set a furocoumarin
limit of 1 mg/kg (1 ppm)
for all cosmetic products
that can be exposed to
sunlight.
The typical guide
for FCF oil grade is a
content of <5 ppm total
furocoumarins and the
oils commercially avail-
able are well below this
level. It should only be
the waxes, carotenoids
and furocoumarins,
e.g., bergaptene, ber-
gamottin, bergaptol,
citropten, 5-geranyloxy-
7-methoxycoumarin etc,
that are removed without
any appreciable thermal
degradation of the linalyl
acetate content level of
the cold pressed oil. Any
appreciable processing
thermal degradation is
indicated by raised levels
of p-cymene formed by
the breakdown and lower
content of g-terpinene.
The tested sample indeed
The bergamot fruits produced in the winter season are the size of oranges, the shape of lemons and picked while the
fruit is still a dark green color.
shows a slightly higher

26 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

but acceptable level of p-cymene, compared to the Clary Sage Oil (F-3)
level found in the cold pressed oil.11
This non-skin photosensitizing grade of bergamot The flowering herb Salvia sclarea L. is part of
oil is traditionally extensively used in fine fragrances, the Lamiaceae family and is cultivated in France,
classic eau de colognes and cosmetic creams. Bulgaria, Russia and Morocco. It is solvent extracted
The FCF oil tested here is from a single consign- to make a concrete and absolute, as well as a steam
ment from an Italian manufacturer and the results distilled oil. It is related to, but of very different
meet this criteria. composition to, Dalmatian sage, S. officinalis L., and

T-2. Composition of bergamot oil, FCF

RRI Constituent Area % ISO 3520* Schmidt*

924 a-thujene 0.2 0.08-0.4
932 a-pinene 0.8 0.6-1.7
946 camphene trace 0.02-1.1
959 sabinene 0.8 0.03-1.3
963 b-pinene [1] 5.8 5.5-9.5 4.8-9.1
978 b-myrcene [2] 1.0 0.7-1.8
995 a-phellandrene trace 0.01-0.07
1007 a-terpinene trace 0.06-0.2
1014 p-cymene** [3] 1.3 0.1-0.7
1021 b-phellandrene trace 0-0.6
1023 limonene [4] 43.1 30-45 31.4-45.1
1025 (Z)- b-ocimene trace 0.02-0.3
1035 (E )- b-ocimene 0.2 0.03-0.6
1046 g-terpinene [5] 7.0 6-10 5.1-8.5
1075 terpinolene 0.3 0.1-0.4
1082 linalol [6] 11.3 3-15 4.7-16.7
1134 citronellal trace 0-0.1
1175 a-terpineol trace 0.04-0.5
1185 decanal trace 0.02-0.3
1196 octyl acetate trace 0.01-0.3
1213 nerol trace 0.02-0.2
1217 neral 0.2 0.05-0.3
1237 geraniol trace trace-0.1
1240 linalyl acetate [7] 26.4 22-36 20.3-35.5
1247 geranial 0.2 0.25-0.5 0.2-0.6
1330 terpinyl acetate 0.1 0.01-0.3
1340 neryl acetate 0.3 0.1-0.7
1358 geranyl acetate 0.2 0.04-0.6
1411 b-caryophyllene 0.2 0.05-0.9
1433 (Z)- b-farnesene trace 0.01-0.4
1434 (E )- a-bergamotene 0.2 0.02-1.2
1448 (E )- b-farnesene trace trace-0.07
1497 (E,E)- a-farnesene trace trace-0.08
1498 b-bisabolene 0.2 0.3-0.55 0.01-0.5
Trace <0.01 %
Total: 99.8%
*The ranges reported by ISO and Schmidt were for cold expressed oils obtained before the removal of furocoumarins.4
**Note the slightly raised para-cymene level in the FCF grade of oil which has been caused by minimal thermal degradation of principally g-terpinene.

27
Application of Gas-Liquid Chromatography to the Analysis of Essential Oils

F-2 GLC of bergamot oil, FCF

Spanish sage, S. lavandulifolia Vahl. Steam distilla- constituents, dependent on the method of steam
tion of clary sage takes place principally in Russia, distillation from either herb dried in the field and
but an oil can also be made as a fraction from the distilled months later, or by immediate hydro-distil-
solvent extracted concrete after removal of the lation after harvesting.
sclareol that is used for the chemical manufacture of The oil tested is from a single consignment of
macrocyclic amber and musk perfumery ingredients. Russian origin and the results conform to the COE
The oil has traditionally been used in pharmacy, and AFNOR monograph for fresh herb oil, as can
medicines and aromatherapy; the name clary is said be seen from its higher linalol level, compared to
to be derived from “clear eye.” It is also used as a the standard for oil distilled classically from dried
flavoring for foods, liqueurs and tobacco. Fragrance herb (T-3).
uses are in skin care and oral cosmetics, as well as in
perfumes. Clary sage oil is a clear, mobile liquid with
a floral herbaceous odor whose major constituents Spike Lavender Oil (F-4)
are linalyl acetate and linalol. The flowering herb Lavandula latifolia Medik.,
There are monographs by AFNOR and COE part of the Lamiaceae family, is cultivated in Spain
(Council of Europe) 415. In the AFNOR NF-T 75-255 and it is the flowering tops that are steam distilled at
monograph, there are two ranges for the major origin to produce oil. It is related to, but different in

28 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

composition from, true lavender L. angustifolia Mill. Spike lavender is a pale yellow mobile oil with a
and the lavandin hybrids. The lavandin hybrid oils, fresh, floral and more camphoraceous odor than the
abrialis, grosso and super have already been part of other Lavandulas; characteristics that are given by its
a fingerprint GLC study in a previous publication by higher levels of 1,8-cineole and camphor.
this group.12 It has limited flavoring applications but is widely

F-3 GLC of clary sage oil

29
Application of Gas-Liquid Chromatography to the Analysis of Essential Oils

T-3. Composition of clary sage oil

AFNOR 75-255 AFNOR 75-255

RRI Constituent Area % Schmidt
classic en vert
932 a-pinene trace 0.01-0.3
946 camphene trace 0.02-0.2
968 sabinene trace 0.01-0.2
972 b-pinene trace 0.02-0.3
978 b-myrcene 0.7 0.2-1.5
1010 p-cymene trace 0.02-0.06
1017 b-phellandrene trace 0.08-0.2
1022 1,8-cineole trace trace-0.08
1023 limonene 0.3 0.1-0.6
1028 (Z)- b-ocimene 0.3 0.2-4.8
(E)-b-ocimene 0.5 0.3-1.3
1060 (Z)-linalol oxide furan trace 0-0.3
1074 (E)-linalol oxide furan trace trace-0.5
1079 terpinolene trace 0.09-0.2
1083 linalol [1] 23.2 6.5-13.5 13-24 18.6-31
1123 camphor trace trace-0.04
1155 borneol trace 0.04-0.06
1166 terpinen-4-ol trace 0.02-0.1
1175 a-terpineol [2] 2.9 trace-1.2 1-5 0.6-5.1
1199 linalyl formate trace trace-0.3
1206 nerol 0.3 0.06-1.3
1233 geraniol [3] 1.2 0.2-3.6
1241 linalyl acetate [4] 62.8 62-78 56-70.5 31.2-64.2
1270 bornyl acetate trace trace-0.07
1284 geranyl formate trace 0.05-0.8
1337 a-terpinyl acetate trace 0.02-0.2
1343 neryl acetate [5] 1.3 0.3-1.4
1358 geranyl acetate [6] 2.4 0.5-4
1371 a-copaene 0.2 0.2-7.8
1378 b-elemene 0.1 trace-0.4
1385 b-bourbonene trace 0.1-0.5
1411 b-caryophyllene [7] 2.3 0.6-4.8
1448 (E )-b-farnesene trace 0.01-0.3
1451 a-humulene trace 0.02-0.2
1470 germacrene D 0.7 1.5-12 1.2-7.5 0.4-5.4
1485 bicyclo-germacrene trace 0.2-4.8
1497 (E,E)-a-farnesene trace trace-0.3
1515 d-cadinene trace 0.1-0.5
1568 spathulenol trace 0.2-1
1569 caryophyllene oxide trace 0.1-0.7
1868 sclareol oxide trace trace-0.2
2179 sclareol 0.5 0.4-2.6 0.4-2.6 0.08-1
Trace <0.01 %
total: 99.7%

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T-4. Composition of spike lavender oil

RRI Constituent Area % ISO 4719 Schmidt

857 hexan-1-ol 0.1 trace-0.1
932 a-pinene [1] 1.6 0.6-3.6
944 a-fenchene trace 0.02-1
946 camphene 0.5 0.09-0.8
964 1-octen-3-ol trace 0.04-2.6
967 sabinene 0.6 0.02-0.8
967 hexyl acetate trace 0.01-1
968 3-octanone trace trace-0.4
972 b-pinene [2] 2.0 0.4-2.6
978 b-myrcene 0.6 0.05-0.9
1007 a-terpinene trace trace-0.2
1014 p-cymene 0.2 0.05-1.1
1022 1,8-cineole [3] 26.6 16-39 14.2-31.2
1023 Limonene trace 0.5-3 0.2-3
1028 (Z)-b-ocimene 0.3 0.09-1
1038 (E)-b-ocimene trace 0.08-0.2
1046 g-terpinene 0.2 0.2-0.3
1050 (E)-sabinene hydrate 0.1 trace-0.8
1055 (Z)-sabinene hydrate 0.1 trace-0.3
1060 (Z)-linalol oxide, furan trace trace-0.3
1069 (E)-linalol oxide, furan 0.1 0.01-0.4
1075 Terpinolene 0.2 0.09-0.4
1084 linalol [4] 43.8 34-50 24.9-42.3
1116 camphor [5] 12.2 8-16 8.7-35.1
1126 (E)-pinocarveol 0.1 0.05-0.5
1135 hexyl isobutyrate trace 0.05-0.3
1142 (Z)-linalol oxide, pyran 0.4
1144 borneol [6] 1.2 0.07-1.3
1152 lavandulol 0.1 trace-0.9
1157 terpinen-4-ol 0.5 0.4-0.8
1166 Myrtenal 0.1 0.2-0.3
1168 a-terpineol [7] 1.1 0.2-2 0.08-1.4
1173 hexyl butyrate 0.1 0.07-0.1
1220 nerol 0.2 trace-0.3
1224 hexyl 2-methylbutyrate 0.1 trace-0.1
1238 geraniol 0.1 trace-1
1235 bornyl formate trace trace-0.2
1235 linalyl acetate 0.3 0-1.6 0.6-0.9
1241 geranyl acetate trace 0.06-0.2
1262 bornyl acetate trace trace-0.3
1270 hexyl valerate trace trace-0.2
1270 lavandulyl acetate trace 0.09-0.7

31
Application of Gas-Liquid Chromatography to the Analysis of Essential Oils

F-4 GLC of spike lavender oil

T-4. Composition of Spike Lavender oil (Cont.)

RRI Constituent Area % ISO 4719 Schmidt

1273 hexyl tiglate trace 0.09-0.4
1310 neryl acetate trace 1.1-1.3
1343 b-bourbonene 0.2 trace-0.2
1383 STHC 0.1
1397 STHC 0.1
1411 b-caryophyllene [8] 1.6 0.09-1.9
1428 (E)- a-bergamotene 0.1 0.1-0.2
1444 b-farnesene 0.5 0.2-4.3
1451 a-humulene trace 0.1-0.2
1471 germacrene D 0.7 0.05-0.4
1493 STHC 0.3 0.1-1.6
1498 STHC 0.2 trace-0.1
1502 b-bisabolene 0.2 0.2-0.4
1529 (E)-a-bisabolene [9] 1.9 0.4-2.5 0.1-1.5
1562 caryophyllene oxide 0.2 trace-1.7
1617 STOH 0.1
Trace <0.01 %
total: 99.7%

32 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

Neroli oil is used in prepared foods and can also be solvent extracted to make an ingredient known as orange flower water absolute.

used in cosmetics, soaps and detergents, technical The oil steam distilled from the leaves is called
fragrances like room sprays and disinfectants, as petitgrain bigarade oil. A similar leaf oil of not-so-
well as in veterinary medicines and aromatherapy. fine-odor quality is made in South America and
The oil tested is from a single consignment of known as petitgrain Paraguay oil.
Spanish origin and the results conform to the ISO All of these oils are widely used in the manu-
standard. ISO standard 4719:2112 applies (T-4). facturing of perfumery products, but neroli oil is
substantially more expensive due to its lower yield.
Bitter Orange
The bitter orange tree, Citrus aurantium L.,
syn. C. amara Link, syn. C. bigaradia Loisel, syn. C. Neroli oil (F-5)
vulgaris Risso, is part of the Rutaceae family and Neroli oil is cultivated and steam distilled prin-
grows notably in Spain and North Africa. Besides cipally in Tunisia, Morocco and Egypt. The oil is
the fruit’s traditional use in marmalade, its various a pale-yellow mobile oil with a sweet, fresh, bitter,
plant parts are extracted in many ways to produce floral odor and its principle constituents are linalyl
a large range of ingredients, such as expressed acetate, linalol and indole.13
bitter orange oil, terpeneless oil and oleoresin from It is traditionally used in the best eau de
the fruit pericarp, five different extracts of the colognes and fine fragrances with some limited (due
orange blossom and five different extracts from the to its high cost) flavoring applications in confec-
leaves. Neroli oil is steam distilled from the flowers tionery, beverages, ice cream, baked goods, chewing
(blossom) and, as the water from this distillation gum and aromatherapy.
has flavor, it is used as such in prepared foods and The oil tested is from a single consignment of
can also be solvent extracted to make an ingredient Tunisian origin and meets the criteria of the ISO stan-
known as orange flower water absolute. dard, COE and Ph. Eur (European Pharmacopoeia)

33
Application of Gas-Liquid Chromatography to the Analysis of Essential Oils

monographs.14 ISO standard 3517:2012 and COE 136, This oil is also a clear, pale-yellow mobile oil, but
Ph. Eur monographs apply (T-5). with a slightly harsher, fresh, floral odor, the main
constituents also being linalyl acetate and linalol.
Paraguay petitgrain oil finds use in soaps and
Petitgrain bigarade oil detergents; more functional fragrance products
This oil is a clear, pale yellow mobile oil with a than the bigarade and neroli oils are used for.
fresh, floral odor, the main constituents being linalyl The oil tested was from a single consignment
acetate and linalol.15 Often, this oil is used as a more of Paraguayan origin and meets the criteria of the
cost-effective alternative to neroli oil in many of its ISO standard. ISO standard 3064:2015 applies. For
uses besides fine fragrances. GLC profile and composition, please see F-6 and
The oil tested was from a single consignment of T-6 where it is compared to petitgrain bigarade oil
Tunisian origin and meets the criteria of the ISO together with both of their ISO standard constitu-
standard. ISO standard 8901:2003 applies. ent data ranges.

References
Petitgrain oil Paraguay
1. Analytical Methods Committee (1997). Application of Gas–
The EFFA REACH (Registration Evaluation Liquid Chromatography to the Analysis of Essential Oils
Authorisation and Restriction of Chemicals) NCS Part XVII. Fingerprinting of Essential Oils by Temperature-
working group statistically treated the analysis programmed Gas–Liquid Chromatography Using Capillary
of 211 commercially traded oils from 1999-2008, Columns with Non-polar Stationary Phases. The Analyst,
122(10), 1167-1174.
and this group’s results are consistent with those
obtained by EFFA.16

F-5 GLC of neroli oil

Current Chromatogram(s)
counts
5

80000

70000

60000

50000

40000 3

30000

20000 9

6
4
10000
8 11 12
2 13
10
7

5 10 15 20 25 30 min

34 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

T-5. Composition of neroli oil

RRI Constituent Area % ISO 3517 Schmidt

924 a-thujene trace 0.01-0.04
932 a-pinene 0.5 0-2 0.3-1.2
946 camphene trace 0.03-0.1
965 benzylnitrile 0.1 0.1-0.5
967 sabinene 0.8 0-3 0.5-1.4
972 b-pinene [1] 8.0 2-17 3.8-16.9
983 b-myrcene [2] 1.4 1-4 1-2.6
998 a-phellandrene trace 0.01-0.09
1005 d-3-carene trace 0.03-0.2
1007 a-terpinene trace 0.02-0.2
1010 p-cymene 0.1 0.02-0.1
1021 b-phellandrene trace 0.09-0.3
1023 limonene [3] 15.0 7-18 10.1-20.3
1028 (Z)-b-ocimene 0.4 0.4-1.8
1035 (E)-b-ocimene [4] 3.7 3-9 3.5-7.9
1055 g-terpinene 0.2 0.09-0.3
1069 (Z)-linalol oxide, furan 0.1 0.1-0.4
1076 (E)-linalol oxide, furan 0.2 0.02-0.3
1079 terpinolene Trace 0.3-0.5
1082 linalol [5] 43.0 26-55 31.9-57.7
1158 terpinen-4-ol 0.3 0.2-0.5
1168 a-terpineol [6] 4.5 2-8 3.2-7.6
1206 nerol [7] 1.0 0.5-2 0.8-1.5
1216 neral trace trace-0.2
1231 geraniol [8] 2.8 1-5 2-3.6
1238 linalyl acetate [9] 7.6 1.5-20 1.4-15.1
1251 geranial 0.1 0.03-0.1
1266 indole 0.1 0-0.5 0.08-0.2
1302 methyl anthranilate 0.1 0-1 0.01-0.4
1330 a-terpineol acetate 0.2 0.01-0.2
1340 neryl acetate [10] 1.4 0-7 0.8-2
1358 geranyl acetate [11] 2.8 1-5 1.7-3.7
1398 b-elemene 0.1 0.09-0.2
1412 b-caryophyllene 0.5 0.4-1.2
1444 a-humulene 0.1 0.04-0.1
1536 (Z)-nerolidol trace trace-0.2
1544 (E)-nerolidol [12] 2.6 0.5-5 1.7-4.9
1696 (E,E)-farnesol [13] 2.1 0.5-4 0.3-3.5
Trace <0.01 %
Total: 99.8%

35
Application of Gas-Liquid Chromatography to the Analysis of Essential Oils

T-6. Composition of petitgrain bigarade and Paraguay oils

RRI Constituents Bigarade Area % Bigarade ISO 8901 Paraguay Area % Paraguay ISO 3064
924 a-thujene Trace trace
932 a-pinene 0.1 0.1
962 sabinene 0.3 0.2 0.1-0.5
973 b-pinene [1] 1.4 1.3 0.5-2
983 b-myrcene [2] 1.6 2.1 1.3-3.0
995 a-phellandrene trace trace
1002 d-3-carene 0.7 0.3
1014 p-cymene trace trace
1018 limonene [3] 2.6 1-6 1.1 0.7-3.5
1024 (Z)-b-ocimene 0.5 0.7 0.7-1.5
1035 (E)-b-ocimene [4] 1.6 1-4 1.9 0.5-3.5
1048 g-terpinene trace trace
1060 (Z)-linalol oxide, furan trace trace
1074 (E)-linalol oxide, furan trace trace
1075 terpinolene 0.3 0.4
1083 linalol [5] 26.5 10-32 24.4 15-30
1134 citronellal trace trace
1141 (Z)-linalol oxide, pyran trace trace
1145 (E)-linalol oxide, pyran trace trace
1157 terpinen-4-ol 0.1 0.1
1168 a-terpineol [6] 4.4 1-7 6.1 3.0-7.0
1206 nerol [7] 1.0 1.2 0.5-2
1211 neral 0.3 trace
1233 geraniol [8] 2.4 1-4 3.0 2-4.5
1241 linalyl acetate [9] 49.6 40-72 49.4 40-60
1247 geranial trace trace
1333 a-terpineol acetate trace trace
1340 neryl acetate [10] 1.9 2.4 1-3
1358 geranyl acetate [11] 3.6 1.5-5.5 4.1 2-5
1411 b-caryophyllene 0.6 0.8 0.3-1.5
1486 bicyclo-germacrene trace 0.1
1544 (E)-nerolidol trace trace
1569 caryophyllene oxide trace trace
Trace <0.01 %
Total: 99.5% 99.7%

2. Analytical Methods Committee (2019), Application of Gas– 3. NIST Office of Data and Informatics. (2017). NIST Chemistry
Liquid Chromatography to the Analysis of Essential Oils Part WebBook, SRD 69. Retrieved from https://webbook.nist.gov/
XXIII. Fingerprint GLC of selected Texas, Virginia, China, Atlas chemistry
and Himalaya cedarwood oils intended for REACH registration
4. deGroot, A. C., & Schmidt, E. (2016). Essential oils: Contact
Perfumer & Flavorist, 44, November 31-42
allergy and chemical composition. Boca Raton: CRC Press

36 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

 F-6 GLC of petitgrain bigarade oil

Current Chromatogram(s)
counts
9

175000

150000

125000

100000

75000

50000

6
25000 11
3
12 4 8 10
7

5 10 15 20 25 30 min

6. Dugo, G. & Bonaccorsi, I (2014) In Citrus bergamia Boca XIX. Fingerprints of 12 essential oils Perfumer & Flavorist, 29,
Raton CRC press July/August 28-36

7. Dugo, P. & Russo, M. (2011) Chapter 8 Oxygen Heterocyclic 13. Lawrence, B. M. (1976-2017). Essential oils. Carol Stream, IL:
components of Citrus essential oils in Citrus oils (pp. 405-444) Allured Publishing Corporation
edit Dugo, G. & Mondello, L Boca Raton CRC press
14. Peyron, L.(2002) Chapter 9 Production of Neroli and Petitgrain
8. Analytical Methods Committee (1987) Application of High–
oils. In The Genus Citrus (pp 148-152) edit Dugo, G.&
Performance Liquid Chromatography to the Analysis of
DiGiacomo, A. Boca Raton CRC Press
Essential Oils Part I Determination of Bergaptene in Oils of
Bergamot The Analyst, 112, 195-98, 15. Dugo, G., Cotroneo, A.& Bonaccorsi, I. (2011) Chapter 5
Composition of Petitgrain oils. In Citrus oils (pp 253-332) edit
9. Suzuki H, Nakamura K, Iwaida M. (1979) J, Soc. Cosmet.
Dugo, G. & Mondello, L. Boca Raton CRC Press
Chem. 30, 393-400

10. International Fragrance Association website (2019) www. 16. EFFA REACH NCS working group (2008) private
ifraorg.org communication

11. Adams, R. P. (2009). Identification of essential oil components

by gas chromatography/quadrupole mass spectroscopy. Carol Address for correspondence:
Stream: Allured Publ.
The Secretary, Analytical Methods Committee,
12. Analytical Methods Committee (2004) Application of Gas– Analytical Division, The Royal Society of Chemistry,
Liquid Chromatography to the Analysis of Essential Oils Part
Piccadilly, London W1J 0BA, United Kingdom.

37
White Paper

Essential Oils: What to Watch for

in these Complex Natural Extracts
Luke Grocholl, Regulatory Affairs Expert, Sigma-Aldrich® Flavors & Fragrances

A Dangerous Outbreak, Suspicious

Claims, and Regulatory Response
In late June 2014, a worker in a cotton factory in
Sudan was hospitalized with severe diarrhea and
dehydration. The patient died within a week and was
unfortunately the first victim of an unknown disease.
The subsequent outbreak killed over 150 people and
sickened almost 300, some with severe symptoms that
included internal, and even external bleeding such as
bleeding through the tear ducts. It would be months
until the virus responsible for this disease was identified
and named. The world had experienced its first Ebola
outbreak. This horrible disease would help shape global
health policies, such as travel bans and screenings, that
would eventually have an impact during the COVID-19
pandemic. Ebola would also lead the FDA to some of What is an Essential Oil?
the first policies and strong enforcement on essential Essential oils are the mixtures of aroma chemicals
oil therapeutic claims. and related compounds such as terpenes and
When the first Ebola cases were detected outside of vegetable oils extracted or physically isolated
sub-Saharan Africa in 2014, there was significant public directly from botanicals. They may not be oily,
concern. A US-base aromatherapy company began but often have a viscous oily-like appearance.
marketing some of their products with claims they They could even be waxy solids or resins. IOFI
could be used to treat Ebola as well as other conditions (the International Organization of the Flavor
such as cancer, infections, and brain injuries. Prompted Industry, iofi.org) and ISO (the International
by concerns over fake cures and false claims. The FDA Organization for Standardization, iso.org) both
issued several warning letters and began responding offer similar definitions of essential oils and
aggressively to firms that made therapeutic claims related complex, aromatic, botanically derived
without the proper evidential support and regulatory ingredients.1,2 The IOFI and ISO documents
filings. Much of the regulatory enforcement of essential define many types of botanically derived,
oils involves health claims, but there is still confusion aromatic mixtures distinguished primarily by
over how to define and market essential oils for all extraction, isolation, and/or purification methods.
applications. For the sake of this article, I will not differentiate
between such forms as gums, waxes, resins, and
essential oils but will use the term essential oil as
a generic term for all direct botanical extracts.

MilliporeSigma is the U.S. and Canada

Life Science business of Merck KGaA,
Darmstadt, Germany.
Manufacturing Methods Confirming Quality
The most common method for extracting essential Addition of top or base notes to oils may be fully
oils from botanicals is steam distillation, but they can legitimate as long as the oil is properly labeled to
be obtained through solvent extraction or even by indicate such additions. However, oils may also be
expression using physical presses. Some essential intentionally adulterated to save costs. This type
oils can be obtained through other highly specialized of intentional adulteration for economic purposes
methods, such as slow-folding methods for extracting includes diluting the oil with other cheaper oils, or even
citrus oils known as sfumatura (from the Italian for synthetic mineral oil. Fortunately, careful analytical
softening or blurring indicating the soft, slow method) testing can often detect adulteration. Essential oils
or digestion, diffusion, or immersion of flowers to have distinct “fingerprints” detected by different
obtain pomades. The extracts and distillates may be chromatographic tests. Gas chromatography (GC)
isolated ready-to-use or may require further purification can separate and characterize each component of
ranging from simple decanting to fractional distillation. an oil and when coupled with mass spectroscopy
Regardless of the manufacturing method, the end- (GCMS), analytical chemists have a powerful tool
product is a complex mixture of aroma chemicals and to identify potential adulterants in essential oils.
related compounds. Some simple methods, such as viscosity or specific
gravity analysis can also identify adulteration such
Natural Declarations and Verification as dilution. Naturalness verification by carbon-14
ratio (14C) will indicate if an oil has been diluted with
Since these methods all involve physical separation synthetic materials. Sensory testing can also be used
of the aroma chemicals from their botanical sources, by experienced flavorists and perfumers who can
essential oils meet regulatory and other definitions of identify off-notes that may indicate poor quality or
natural flavors. Essential oils, when used as flavoring, adulterated oils. A strong reliable supply chain and
can be labeled as a natural flavor in the US, EU, and a good relationship with the suppliers is one of the
most other global regions, but some caution should best methods to help address the risk of intentionally
be taken when evaluating the flavoring. Similarly, adulterated essential oils. Regardless of the controls in
essential oils inherently meet the definition of “natural place, buyers should always be skeptical of essential
raw material” in ISO 9235, often used for cosmetic or oils offered below market price.
fragrance products so long as there are no synthetic
additives in the essential oil. Processing and purification
Watch for Allergens
methods for isolating essential oils sometimes result
in the loss of some aroma chemicals. Oils may As with all flavors and fragrances, allergens can be
therefore sometimes be top-noted or base-noted by a concern for essential oils. Undeclared allergens
the addition of aroma chemicals. Care should be taken continue to be one of the primary reasons for food
when evaluating oils to determine if they have had recalls and fragrance allergens are of particular concern
any modifications or additions. Such material may be for essential oils. IFRA (the International Fragrance
synthetic flavors which then disqualifies natural claims Association, ifrafragrance.org) has identified hundreds
associated with the essential oil. of products of concern in cosmetic and household
products. The EU has a similarly extensive list in its
Natural verification of essential oils can be difficult.
regulations on cosmetics.3 Some essential oils are
Since they are complex mixtures, it is often difficult
specifically identified as skin irritants, whereas others
to analytically identify a single component that could
could contain components on the EU and/or IFRA
be added. Furthering the complexity are expected
prohibited or restricted lists. For example, juniper oil
variations of the composition of the natural oil itself.
(Juniperus sabina) is prohibited for use in cosmetics in
Botanical crop quality is subject to regional and
the EU and citral, a constituent of lemon grass oil as
seasonal variations which may impact the composition
well as lemon oil, basil oil, and related oils is a known
of the oil. Extraction, purification, and storage methods
skin allergen in the EU. Therefore, juniper oil should
also impact essential oil quality and composition.
not be used in cosmetic applications, and where citral-
containing oils are used, care should be taken to ensure
the citral levels are below the thresholds of concern
provided by the regulations.

2
When the oils are used as food flavoring, there is Summary
considerably less concern from potential allergens. The
US FDA, the EU, and many other regulatory bodies Essential oils are a wonderful addition to any perfumer
recognize that highly refined oils are free or nearly free or flavorist’s toolbox. These complex mixtures
of the proteins that could cause food allergies. Highly provide flavor and odor sensations not realized by
refined oils are therefore exempted from allergen simple aromatic chemicals. However, a thorough
labeling requirements. Nevertheless, it is recommended understanding of the potential risks from adulterated
that flavor producers disclose to their customers or mislabeled essential oils is important. The complex
any essential oils derived from major food allergens. nature of these ingredients may make it easier for bad
Food companies often require a full list of products players to try and pass off inferior or even dangerous
they receive that could be derived from a major food ingredients as high-quality essential oils. Extreme
allergen. care must also be taken before promoting beneficial
claims from essential oils. Only registered drug
products can make therapeutic claims and regulatory
Therapeutic Claims agencies monitor false claims related to therapies
In addition to flavor and fragrance applications, and treatments. Knowing the risks associated with
essential oils are sometimes promoted for therapeutic essential oils allows formulators to use these important
uses. Regulatory enforcement agencies are consistent ingredients to develop new and rich formulations for a
that any products with therapeutic claims are drugs and demanding marketplace.
must meet all the stringent requirements associated 1. OFI GPS 2015 – Definitions of Forms
with manufacturing, registering, and marketing of a 2. ISO 9235 Aromatic Natural Raw Materials – Vocabulary
drug. Therapeutic claims are defined as any claim the
3. Regulation (EC) No 1223/2009 of the European Parliament and of
product would treat a disease or affect the function the Council of 30 November 2009 on cosmetic products
of the body. Claims such as digestive relief, reduction
of muscle soreness, or treating depression are all
therapeutic claims. Proper wording of any claim and
evidence for those claims is important. For example,
a claim that an essential oil will help a person sleep
is a therapeutic claim, but statements promoting the
conditions important for sleep may be acceptable. Any
claims promoting specific health benefits or treatment
of health conditions should be carefully reviewed, and
it is recommended that any questions on such claims
be brought to regulatory authorities to understand their
position.

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trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed 41225
information on trademarks is available via publicly accessible resources. 04/2022
 Examining barcoding and metabarcoding analyses to authenticate and
trace raw materials.

BY NELLY DUBRULLE, BENJAMIN MARTEAUX and, NICOLE GIRAUD, DNA Gensee; and CLAIRE DELBECQUE
and PIERRE-PHILIPPE GARRY, Bontoux SAS
Vol. 46 • April 2021

Almond trees in M’Gouna, Morocco; all photos courtesy of Elise Leclerc, Julie Paumier and Claire Delbecque of Bontoux SAS.

41
 B
itter almonds and apricot almonds traceability of plants across the process chain is
used in the production of the difficult or even impossible.
two corresponding bitter almond To improve these analyses, plant genetic research-
essential oils can be authenticated ers uncovered the specific genetic signatures of plant
via DNA analyses to ensure purity species.6 The increase of genomic resources has
and traceability at every step of the thereby enabled the development of specific genetic
supply chain process. methods such as barcoding and metabarcoding.
DNA barcoding and metabarcoding technologies
use a fragment of the genome of a plant as a genetic
Essential F&F Ingredients marker for the identification and discrimination
Natural essential oils of Prunus species, com- of plant species. Thanks to the barcoding method,
monly called bitter almond essential oils, are famous plant genetic profiles can be detected, and thanks
in the flavor and fragrance industry for their sweet, to metabarcoding, one can sequence multiple DNA
powerful and typical almond aroma with a pleas- markers at the same time, allowing for plant compo-
ant cherry note. In the fragrance industry, they sition analyses of mixed samples.7
are increasingly used in fine fragrance in alcoholic Thus, carrying out genetic analysis is an essen-
perfumes. In the flavor industry, they are widely and tial way to obtain plant compositions for botanical
traditionally used in carbonated beverages, alcoholic authentication and traceability. These methods have
beverages, baking and confections.1 Natural bitter already shown their effectiveness in ecology, the
almond essential oils can be produced by steam environment, feeding livestock and paleontology.8-11
distillation of different natural sources containing
glycoside amygdalin: ripe kernels of bitter almonds
(Prunus amygdalus, synonym of Prunus dulcis),
apricots (Prunus armeniaca), peaches (Prunus
persica), or other kernels containing amygdalin such
as cherries (Cerasus species) and plums (Prunus
domestica).1,4 The smell and aroma of these essential
oils can differ from one to another.
Mainly composed of natural benzaldehyde (97-
99.5%), these essential oils are far more expensive
than synthetic benzaldehyde due to low yields in a
multi-step process of production.1,2 Therefore, these
essential oils have always suffered from adulteration,
which can now be better controlled thanks to chemi-
cal analyses described in different studies, enabling
differentiation of synthetic benzaldehydes from
natural and genuine ones.3,4

Botanical Authentication Processes

Botanical authentication and traceability from Almond oil cake.
raw materials to products are becoming essential for
consumers and the cosmetics, flavor and fragrance,
and nutraceutical industries.
Today, quality controls of these raw materials
and products are made by morphological, or
chemical analyses.5 When using botanical identifi-
cations based on morphological aspects, it is easy
to confuse a species with another one. Moreover,
contamination of plants can be missed. However, it
is mandatory to perform these tests to release a new
product on the market.
For plant authentication, chemical analysis has
advantages: it is affordable and most of the time
efficient. The main disadvantage is that chemistry
fails to identify some plant species. Moreover, Apricot almond and shells.

Reproduction in English or any other language of all or part of this article is strictly prohibited. © 2022 Allured Business Media.

42 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

Production in Morocco Two different qualities of essential oils were devel-
oped: one starting from apricots kernels and the
The Bontoux Company used to produce bitter
other starting from bitter almonds.
almond essential oil in Provence. In 2012, a part-
The processes carried out in Morocco (F-1) are
nership between Phytotagante and Bontoux was
similar to the ones previously described:1
initiated to share technologies and transfer this
production to Morocco, where almond fruits were 1. Separation of almonds and kernel shells:
traditionally produced for the local market. The crushing of kernels followed by sifting and then
production was developed in ARD Guisser’s facilities. almond sorting

F-1 Processes of production of the two qualities of natural bitter almond essential oil, one from almond the
other from apricot kernels

43
Genetic Traceability of the Bitter Almond Essential Oil Supply Chain

2. Separation of vegetal oil and almond fat-free oil • Sample 2: Whole apricot almonds from apricot
cakes: via several steps of pressing kernels: smaller almonds with a brown,
3. Steam distillation of almond cakes to produce yellowish color
the corresponding essential oil • Sample 3: Leaves of a tree whose almonds have a
bitter taste
• Sample 4: Leaves of a tree whose almonds have a
Genetic Traceability: from Almonds to sweet taste
Oil Cakes and Vegetal Oils
The aim of the present study is to offer full trace- Processed products:
ability of the raw material from the almonds, the • Sample 5: Dried oil cake of bitter almonds: big flat
vegetal oils and the oil cakes before distillation of the pieces with a brown, reddish color
essential oil. • Sample 6: Dried oil cake of apricot almonds: big flat
pieces with a brown, yellowish color
• Sample 7: Vegetal oil of bitter almond
Material & Methods • Sample 8: Vegetal oil of apricot almond
• Sample 9: Essential oil produced from
a. Samples studied and morphological description
bitter almonds
All the plant samples studied came from
M’Gouna, Morocco in the country’s Ouarzazate
region. The processed products came from ARD b. DNA barcoding & metabarcoding technologies
Guisser in the Casablanca region. DNA Gensee has developed protocols for DNA
barcoding and metabarcoding technologies. Several
Plant materials: steps are necessary to access the DNA sequences from
• Sample 1: Whole bitter almonds: large, with a samples (extraction, amplification, sequencing and
brown, reddish color bioinformatic analysis).

F-2 Samples studied presented in their flow chart of production

44 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

Barcoding sequencing was then performed using a next-gener-
Extraction and PCR (polymerase chain reac- ation sequencer (NGS) (MiSeq, Illumina Inc.). The
tion) amplification of DNA are carried out using sequencing results were analyzed using Geneious
both universal and specific primers and a well- software and Genbank.15-17
defined protocol).14 Before sequencing, the DNA
concentration could be measured spectroscopi-
cally (SimpliNano 4285 V2.0.0, GE Health Care).
Results & Discussions
DNA sequencing was then performed using Sanger The Prunus genus contains more than 200
method (SeqStudio Genetic Analyzer, Applied species. Among them, one can find bitter almonds,
Biosystems). The sequencing results were analyzed Prunus amygdalus, and apricot almonds, Prunus
using Geneious software) and Genbank).15-17 armeniaca, the two species of interest of this study.

Metabarcoding Barcoding genetic identification

Extraction and PCR amplification of DNA To confirm that the species used to produce the
are carried out using specific primers for the oils are bitter almonds and apricot almonds, we per-
Rosaceae family and a well-defined protocol.14 DNA formed a barcoding analysis on oil cakes (samples

T-1. Results of barcoding analyses of oil cakes and leaves

Combined results of
Samples Names Marker 1 Marker 2 Marker 3 Marker 4 the four markers (the
sample is assigned to)
Leaves of a tree
100% Prunus 100% Prunus 100% Prunus 100% Prunus
3 whose almonds Prunus amygdalus
amygdalus amygdalus amygdalus amygdalus
have a bitter taste
Leaves of a tree
100% Prunus 100% Prunus 100% Prunus 100% Prunus
4 whose almonds Prunus amygdalus
amygdalus amygdalus amygdalus amygdalus
have a sweet taste
100% Prunus 100% Prunus
Dried oil cake of 100% Prunus 100% Prunus
5 (Including P. amygdalus Prunus amygdalus
bitter almonds amygdalus amygdalus
amygdalus ) Sequence A

100% Prunus 100% Prunus

100% Prunus 100% Prunus
Sequence B Including P.
Including P. Including P.
Including P. armeniaca et
armeniaca, armeniaca,
armeniaca, sibirica
Dried oil cake of salicina, mume, andersonii,
6 cerasifera, Excluding P. Prunus armeniaca
apricot almonds angustifolia japonica, sibirica
mandshurica cerasifera,
Excluding P. Excluding P.
Excluding P. salicina,
andersonii, et P. mume et P.
sibirica et P. mume,
japonica mandshurica
angustifolia andersonii

T-2. Major and minor sequences found in all the analyzed samples

Sample 1 Sample 5 Sample 7 Sample 9

Major sequence (>90% of Genus Prunus Genus Prunus Genus Prunus

No amplified DNA
the total read number) Sequence A Sequence A Sequence A

Sample 1 Sample 5 Sample 7

Major sequence (>90% of Genus Prunus Genus Prunus Genus Prunus

the total read number) Sequence B Sequence B Sequence B

Minor sequence Prunus mume

45
Genetic Traceability of the Bitter Almond Essential Oil Supply Chain

5 and 6). We also analyzed and compared leaves of certified between samples made with the plant
almond trees (samples 3 and 4). species used for the production.
As shown in T-1, DNA was extracted from the Interestingly, but not surprisingly, no DNA is
oil cakes and leaves of almond trees. Four genetic found in the essential oil (F-3). Indeed, the distilla-
markers, chosen accordingly to the bioinfor- tion process of essential oils does not allow for the
matic study on available data, were amplified and DNA to be steam-driven.
sequenced. The results indicate that the samples
3 and 4 (leaves) both belong to Prunus amygdalus
(synonym of Prunus dulcis), and samples 5 and 6 (oil Conclusion
cakes) respectively belong to Prunus armeniaca and Apricot, bitter and sweet almond products have
Prunus amygdalus. often suffered from adulteration. Chemical analyses
These results confirm that the authentication enable the differentiation of synthetic benzaldehydes
can be performed on oil cakes with long markers from natural and genuine ones. The fragrances and
(400-1300 pb) and that the two species of interest flavor industries are now looking for complete trace-
correspond to their vernacular names. Interestingly, ability of natural products to authenticate the whole
the transformation process from almonds to oil supply chain of their high-value products.
cakes does not impair the DNA quality and quantity, As shown in this study, barcoding and metabar-
allowing one to analyze this type of product with a coding analyses are powerful tools that allow us to
barcoding approach. It also confirms that bitter and identify and trace plant DNA from crude raw mate-
sweet almonds belong to the same species and are rial to a product. The use of both approaches allows
probably two different varieties of this species. researchers to authenticate the raw materials and to
trace them in vegetal oils and oil cakes.
The Bontoux company is currently working on
Metabarcoding profiles and traceabil- chemical analyses using new technologies to com-
ity analyses plete the traceability on essential oils that cannot be
Metabarcoding using next generation sequencing done by DNA analyses. The final aim is to authenti-
(NGS) is a very powerful tool for authenticating and cate benzaldehyde from apricot and from almond,
detecting plant contamination in any type of sample, while differentiating between the two as well as all
including plant mixtures, plants powders, extracts other sources of benzaldehyde.
and oils.
Regarding the species of interest, Bontoux used
Acknowledgments
metabarcoding to authenticate and trace in the
The authors would like to thank Moustapha
raw material, almonds, in the oil cakes and in the
Chahboun from ARD Guisser and Jamal Chahboun
vegetal oils.
from Phytotagante for their long-term partnership in
The analysis results of these samples show the
the whole production of essential oils in Morocco.
presence of several genetic sequences, different
They would also like to thank Dr. Florian Philippe
for the two species (Prunus amygdalus and Prunus
for his help and comments on this article.
armeniaca; T-2 and F-3).
Address correspondence to Nicole Giraud, DNA
For bitter almonds, a sequence belonging to the
Gensee, 17 rue du Lac St André, 73370 Le Bourget
Prunus genus is found in the almonds, the oil cake,
du Lac, France; [email protected]. For
and the vegetal oil. The taxonomic level of these
correspondence to Bontoux: 583 Route du Col de
results can’t be pushed further because several
Peyruergue, Quartier Aguzon, 26170 Saint-Auban-
species of this genus have the same sequence for
sur-l’Ouvèze, France; [email protected].
this marker. Nevertheless, this sequence is strictly
identical to a genetic reference of Prunus amygdalus.
Interestingly, no other plant species were detected in Bibliography
all samples. This proves that no plant contamination
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Perfumer & Flavorist, 16, 17–24 (1991).
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analyzed samples we found a sequence assigned 2. Clark G. S., An aroma profile: benzaldehyde, Perfumer &
to Prunus, which is strictly identical to the Prunus Flavorist, 20, 53–60 (1995).

armeniaca genetic reference. A sequence assigned to 3. Remaud G., Debon A. A., Martin Y., Martin G. G., Martin G.
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often called Chinese plum—is found at trace levels Application of the SNIF-NMR Method to Benzaldehyde, J.
Agric. Food Chem., 45, 4042–4048 (1997).
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These results show that traceability can be

46 2022 Essentials Ebook | www.sigmaaldrich.com www.PerfumerFlavorist.com

 F-3 Images of capillary electrophoresis obtained after amplification of Samples 1, 2, 5, 6, 7, 8 and 9; except
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of plant material, Methods Mol. Biol. Clifton NJ, 862, 23–33 Sturrock S., Buxton S., Cooper A., Markowitz S., Duran C.,
(2012). Thierer T., Ashton B., Meintjes P., Drummond A., Geneious
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