Detection of fraudulent practices in commercially available cinnamon : analyses by HS-GC-MS, EDXRF, FT-Raman spectroscopy, TGA and DNA-techniques.
Date: 2025-09-24
Institutions: Behr, Marc, Garlant, Linda, Ghidotti, Michele, Jimenez Pena, Manuel, Joint Research Centre, Papoci, Sergej, Pellegrin, Clement, Pietretti, Danilo, de la Calle Guntiñas, Maria Beatriz
Subjects: aromatic plant, consumer protection, food fraud, market, report, sampling, spectrometry

ISSN 1831-9424 

Detection of fraudulent practices in 
commercially available cinnamon 

Analyses by HS-GC-MS, 
EDXRF, FT-Raman spectroscopy, TGA and DNA-techniques 

Behr, M., Boix Sanfeliu, A., Chatzipanagis, K., de la Calle 
Guntiñas, M.B., Garlant, L., Ghidotti, M., Jimenez Pena, 
M., Papoci, S., Pellegrin, C., Pietretti, D. 

2025 

 EUR 40356 

 
 
  
This  document  is  a  publication  by  the  Joint  Research  Centre  (JRC),  the  European  Commission’s  science  and  knowledge 
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Address: Retieseweg 111, B-2440 Geel 
Email: lourdes.alvarellos@ec.europa.eu  
Tel. +32(0)14-571 316 

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JRC142672 

EUR 40356 

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ISBN 978-92-68-28691-3   ISSN 1831-9424   doi:10.2760/5195608  

KJ-01-25-338-EN-N 

Luxembourg: Publications Office of the European Union, 2025  

© European Union, 2025  

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How to cite this report: Behr, M., Box Sanfeliu, A., Chatzipanagis, K., de la Calle Guntiñas, M.B., Garlant, L. et al., Detection of fraudulent practices 
in commercially available cinnamon - Analyses by HS-GC-MS, EDXRF, FT-Raman spectroscopy, TGA and DNA-techniques, Publications Office of 
the European Union, Luxembourg, 2025, https://data.europa.eu/doi/10.2760/5195608, JRC142672. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Contents 

Abstract ..............................................................................................................................................................................................................................................................1 
1 
Introduction ............................................................................................................................................................................................................................................3 
2  Test materials and methods ...................................................................................................................................................................................................5 
2.1 Test materials ......................................................................................................................................................................................................................................5 
2.2 Analytical methods used ............................................................................................................................................................................................................7 
3  Results and discussion on the findings regarding cinnamon integrity ...............................................................................................9 
4  Conclusions ......................................................................................................................................................................................................................................... 14 
5  Annexes .................................................................................................................................................................................................................................................. 15 
5.1 Annex 1: Test Samples .............................................................................................................................................................................................................. 15 
5.2 Annex 2: X-ray fluorescence based method .......................................................................................................................................................... 19 
5.3 Annex 3: Thermogravimetric analysis (TGA) .......................................................................................................................................................... 23 
5.4 Annex 4: Vibrational spectroscopy based method ............................................................................................................................................ 24 
Raman screening method using benchtop instrument ........................................................................................................................ 24 
5.5 Annex 5: Gas chromatography-based method .................................................................................................................................................... 33 
5.6 Annex 6: Feasibility study for the identification of adulterants by DNA-based methods .............................................. 35 
Quantification of pepper and clove in admixed samples ................................................................................................................... 35 
Extension to other adulterants in commercial cinnamon samples ........................................................................................... 35 
6  References ........................................................................................................................................................................................................................................... 40 
List of abbreviations and definitions ..................................................................................................................................................................................... 43 
List of figures ............................................................................................................................................................................................................................................ 44 
List of tables ............................................................................................................................................................................................................................................... 45 

 
 
 
 
 
 
   
Abstract  

Upon request of DG SANTE, the JRC carried out a study to address the subject of fraud in the cinnamon market 
within  the  EU.  Two  screening  methods  based  on  Energy  Dispersive  x-Ray  Fluorescence  and  FT-Raman 
spectroscopy,  respectively,  were  developed  and  in-house  validated  to  detect  fraudulent  practises  in  the 
cinnamon sector, in particular substitution of Ceylon cinnamon by cassia. 

A method based on Head Space-Gas Chromatography-Mass Spectrometry, was developed and validated in-
house as a quantitative confirmatory method for the results obtained by the two screening methods. If needed, 
after proper validation via an inter-laboratory comparison, the method could be used by control laboratories to 
detect samples suspect of fraud.  

A method based on DNA analyses, was developed and in-house validated to detect the fraudulent substitution 
of cinnamon by clove and/or pepper, following a specific request made by DG SANTE. 

The four methods were used to study a set of more than hundred commercially available cinnamon samples. 
The total ash content in those materials was determined by Thermo-gravimetric Analyses. 

The outcome of the study is presented and discussed in this report. Summarising, a high rate of suspicious 
samples was found, in particular among Ceylon cinnamon, the most expensive cinnamon species on the market.  

1 

 
 
 
 
Authors 

Name 

BEHR Marc 

BOIX SANFELIU Ana 

CHATZIPANAGIS Konstantinos 

Contribution 

E, R 

C, E, R 

E, R 

DE LA CALLE GUNTINAS Maria Beatriz 

C, E, R 

GARLANT Linda 

GHIDOTTI Michele 

JIMENEZ PENA Manuel 

PAPOCI Sergej 

PELLEGRIN Clement 

PIETRETTI Danilo 

C: conception, E: experiments, R: reporting 

E, R 

E, R 

E, R 

E, R 

E, R 

E, R 

2 

 
 
1 

Introduction  

Cinnamon is a spice originated from the genus Cinnamomum, it belongs to the Lauraceae family and includes 
about 250 species, many of them aromatic and flavouring, spread over South East Asia, China and Australia,. 
The  most  commercially  valued  cinnamon  is  the  dried  inner  bark  of  the  tree  Cinnamomum  verum  (syn.  C. 
zeylanicum Blume) native to Sri Lanka, main producer in the world, and considered the highest quality cinnamon.  
Cinnamomum verum is also produced in small quantities in Madagascar, Seychelles and India. It is pale in colour 
and has a mildly sweet flavour. Cassia is a similar, stronger flavoured spice of lower quality, native to Myanmar 
(Burma).  Cassia  has  several  species:  Chinese  cassia  (C.  cassia  syn.  C.  aromatica)  from  China  and  Vietnam, 
Vietnamese cassia (C. loureiroi), Indonesian cassia (C. burmannii) from Sumatra and Java, and Indian cassia (C. 
tamala) from the north-eastern India and Myanmar (Baruah and Nath, 2004; Thomas and Kuruvilla, 2012).  For 
clarity,  this  report  refers  to  C.  zeylanicum  (or  C.  verum)  as  Ceylon  cinnamon,  while  Cassia  cinnamon 
encompasses all other species, including C. aromaticum, C. loureiroi, C. burmannii and C. tamala. 

Cinnamon has been used for medicinal and culinary purposes, and it has been cultivated at least since 3000 
BC (Dasanayake et al., 2016, Wang et al., 2023). The trade of cinnamon from South Asia has been documented 
as far back as 2000 BC. Cinnamon is an important ingredient in a growing number of new products (e.g. dairy 
products, beverages) (CBI, 2023); the popularity of some of its medicinal applications, is growing in popularity 
throughout the world (Pages-Rebull et al, 2024). Currently, global production is dominated by China, Vietnam, 
Indonesia, Sri Lanka, and Madagascar, which together account for more than 99% of the world's production 
(238,105 tons in 2023), (FAO, 2025).  China and Vietnam produce about 89% of the world's cinnamon (FAO, 
2025). In 2023, cinnamon was the fifth most imported spice in the European Union, after ginger, paprika, pepper 
and turmeric, with 13,300 tons imported, mostly from Vietnam (Eurostat, 2025).  

There are four major grades of Ceylon cinnamon, namely Alba, Continental, Mexican and Hamburg, based on 
quill diameter. Among these, Alba with a 6mm quill is the most expensive (Ceylon Golden Cinnamon, 2022)  

Cinnamon marketed in the EU must comply with several regulations (Regulation (EC) No 178/2002; Regulation 
(EU) No 1169/2011) and precisely Commission Regulations (EU) No 2023/915 and (EC) No 1333/2008) which 
respectively set a maximum level of 2.0 mg lead /kg and 150 mg of sulphur dioxide-sulphites /kg in cinnamon. 
Additionally, Regulation (EC) No 1334/2008 sets maximum levels of coumarin ranging from 5 mg/kg in desserts 
to  50  mg/kg  in  traditional  and/or  seasonal  bakery  products  labelled  as  containing  cinnamon.  However,  no 
specific provisions apply to coumarin naturally present in cinnamon. Coumarin is a liver toxic compound with a 
Tolerable Daily Intake (TDI) of 0.1mg/kg bodyweight (EFSA, 2008; ANSES, 2021) that can be found in Cassia 
cinnamon. 

Cinnamon demand is expected to grow the coming years, with a global compound annual growth rate (CAGR) 
of  6.4-12.4%,  with  Europe  following  the  same  trend  (KBV,  2024;  Fact,  2024).  The  increase  in  demand  has 
already triggered an increase in the price of cinnamon in Europe (CBI, 2023). The significant price difference 
between Ceylon and Cassia cinnamon, the former having approximately twice the price of the later, creates a 
motivation for adulteration or enhancement with additives, in addition to the general risk of fraudulent practices 
associated with spices, which are among the most vulnerable food commodities (Velazquez et al, 2023). These 
fraudulent practices may include the substitution (partial or complete) of Ceylon cinnamon by Cassia cinnamon, 
particularly in ground cinnamon products, or adulteration of either Ceylon or Cassia cinnamon with parts of the 
cinnamon  plant  other  than  the  bark  (e.g.,  root,  leaves,  flowers,  seeds),  or  with  a  different  plant.  Moreover, 
cinnamon products are vulnerable to non-compliance with health-related regulations, including contamination 
with lead, as highlighted by multiple recent alerts from both the FDA (FDA, 2023 and FDA, 2024) and the RASFF 
(RASFF,  2024)  highlighting  the  need  for  rigorous  quality  control  measures.  To  address  such  concerns,  the 
International Organization for Standardization (ISO) has established standards for cinnamon, specifically for 
Ceylon cinnamon (ISO 6539:2014) and Cassia cinnamon (ISO 6538:1997), which provide guidelines for quality 
control and testing to minimize the risk of contamination and ensure compliance with regulatory and quality 
requirements.  

3 

 
 
 
The JRC-DG SANTE Administrative Agreement had two primary objectives regarding cinnamon: (1) to further 
develop and refine existing analytical methods, as well as explore innovative analytical approaches, and (2) to 
analyse a representative set of commercially available cinnamon samples. The goal of this analysis was to 
provide a snapshot of the current challenges and issues associated with cinnamon in the European market, 
thereby informing future regulatory and quality control efforts. It is important to note that, due to the limited 
number of samples analysed compared to an EU-Coordinated Control Plan (EU-CCP), this commercial sample 
analysis cannot be as comprehensive but rather provides a preliminary overview of the potential problems that 
may exist. 

report  provides  first  an 

This 
to  detect 
adulteration/substitution in cinnamon, in line with the specific deliverables associated with each method and 
then  presents  the  findings  of  applying  these  methods  to  a  selection  of  commercially  available  cinnamon 
products.  

the  analytical  methods  used 

in-depth  description  of 

4 

 
 
2  Test materials and methods 

2.1  Test materials 

One hundred and four commercially available cinnamon samples were purchased in Austria, Belgium, Bulgaria, 
France (including some in Martinique), Germany, Greece, Italy, Malta, Serbia, Slovakia, Spain, Sri Lanka and UK. 
Samples were bought at supermarkets of different chains and small retailers, to avoid the risk of all the samples 
belonging  to  the  same  batch/original  producer.  Six  samples  were  sold  as  bulk  and  the  remaining  were  pre-
packaged  and  branded  as  registered  trademarks.  Fourteen  samples  were  purchased  on-line,  all  of  them 
commercialised by registered trademarks.  

According to the information provided on the labels, 44 samples were C. verum referred to as Ceylon cinnamon 
in this work, 4 C. burmannii, 1 C. burmannii/cassia/loureiroi, 1 C. aromaticum and 11 cassia cinnamon; for the 
remaining 43 no information about botanical variety was provided. Regarding the origin of the cinnamon, 22 
were from Sri Lanka, 6 from Madagascar, 7 from Vietnam, 2 from India, 3 from Indonesia, 2 were a mixture of 
cinnamon from Sri Lanka and Indonesia and 1 indicated Tropics. The remaining samples either indicated “outside 
EU”, or 4 “non-EU”, or no information about the origin was provided.  

Six samples from Madagascar were included in the study. Ceylon cinnamon from Madagascar seems to have a 
special chemical composition when compared with that of other origins, as reflected by the higher total ash 
contents accepted by the ISO standard (7.5 % for Ceylon cinnamon from Madagascar, while for the remaining 
geographical origins the limit is 5 %). For this reason, test materials labelled as coming from Madagascar were 
analysed but they were not further evaluated, those samples are referred to in figures and in the Annex 1 as 
not evaluated (NE). 

Seventy samples were ground (according to the labels 27 C. verum, 3 C. burmanii, 1 C. burmannii/cassia/loureiroi, 
1  C.  aromaticum,  and  9  were  labelled  as  cassia;  no  information  on  botanical  variety  was  provided  for  the 
remaining  29).  The  remaining  34  samples  were  commercialised  as  bark  (17  C.  verum  and  2  cassia,  no 
information on botanical variety was provided for the remaining 15). 

A summary of the previous information is provided in Figure 1. All available information about the cinnamon 
samples included in this study is summarised in Annex 1. Figure 1 and Annex 1 also indicates if the sample was 
produced with organic or conventional agriculture, referred to in Annex 1 as BIO and NON BIO, respectively, to 
reflect  the  labelling  nomenclature  in  many  samples.  This  information  is  recorded  because  the  elemental 
composition of food can be different for the same food commodity depending if the product is organic or not 
(Fiamegos et al, 2021), what could influence the outcome of the screening method based on the use of X-Ray 
Fluorescence. 

All test samples were kept at room temperature and in sealed bags to prevent absorption of humidity. For the 
cinnamon samples purchased in sticks, milling was applied to produce powder for testing. 

5 

 
 
 
 
 
Figure 1: Summary of information about the cinnamon test materials included in this study. 

Source: JRC 

6 

 
 
 
 
2.2  Analytical methods used 

All test materials included in the study, were analysed with four different techniques, energy dispersive x-ray 
fluorescence (EDXRF), FT-Raman spectroscopy, head space-gas chromatography-mass spectrometry (HS-GC-
MS), and thermo gravimetric analysis (TGA). Test items sold ground were also analysed by DNA-based methods. 

EDXRF and Raman were used as targeted and untargeted screening methods, respectively. A previous study 
carried out by the JRC (Ghidotti et al., 2023) showed that in general, the elemental content of cassia samples 
is lower than that of Ceylon cinnamon, and for this reason, the elemental profile determined by EDXRF can be 
used to detect substitution of Ceylon cinnamon by cassia. Raman can be used to detect the same substitution 
because the content of cinnamaldehyde, main compound in cinnamon oil, is higher in cassia than in Ceylon 
cinnamon. Information on the in-house validation of the EDXRF and Raman methods is provided in Annex 2 and 
3, respectively. The results obtained with both screening methods were combined with multivariate analyses 
and  modelling  techniques,  to  identify  Ceylon  cinnamon  suspicious  of  having  been  substituted  by  cassia 
cinnamon or by other species/materials. 

HS-GC-MS  was  used  to  confirm  the  information  obtained  by  the  screening  methods.  For  that  purpose,  14 
compounds  (pinene,  limonene,  benzyl  alcohol,  camphor,  hydrocinnamaldehyde,  anethole,  cinnamaldehyde, 
eugenol,  caryophyllene,  coumarin,  cinnamyl  acetate,  eugenol  acetate,  caryophyllene  oxide,  benzyl  benzoate) 
were selected based on a previous study carried out by the JRC (Ghidotti et al., 2023). The selected compounds 
would  allow  not  only  the  detection  of  substitution  of  Ceylon  cinnamon  with  cassia,  but  also  to  detect  the 
presence of clove and/or pepper if present, as well as the substitution of bark with other parts of the cinnamon 
plant  (leaves,  flowers,  roots,  seeds,  etc),  based  on  the  difference  in  relative  abundances  of  the  selected 
compounds (Rao et al, 2014), for instance eugenol is the most abundant volatile compound in Ceylon cinnamon 
leaves, 70 to 95% in comparison with the 5 to 10% in bark, camphor is the most abundant volatile in the roots 
of  the  plant.  Cinnamyl  acetate,  is  the  predominant  compound  in  fruit,  flowers,  and  fruit  stalks  of  Ceylon 
cinnamon (Jayaprakasha et al, 2011). Ceylon cinnamon can be identified by the presence of eugenol and benzyl 
benzoate, both absent in cassia, while coumarin and δ-cadinene, present in cassia, are either absent or present 
in small amounts in Ceylon cinnamon. δ-cadinene was not added to the list of compounds measured by HS-CG-
MS due to its very high cost. Figure 2 shows the box and whisker plot for eugenol, benzyl benzoate and coumarin 
in Ceylon cinnamon and cassia. 

Figure 2: Box and whisker plot for eugenol, benzyl benzoate and coumarin in Ceylon cinnamon and cassia test 
materials according to their label. 

Source: JRC 

7 

 
 
 
 
 
 
Source: JRC 

Eugenol acetate and anethole could be used to detect the adulteration of cinnamon with clove and pepper, 
because they are present in high amounts in those two spices, respectively.  

Information about the HS-GC-MS method and its in-house validation can be found in Annex 4.  

TGA was used to determine the total ash content in all test materials, to detect samples that do not fulfil the 
quality requirements of ISO 6539 and ISO 6538. Test materials that do not fulfil those requirements are not 
necessarily suspect of adulteration but lack the required quality to be compliant with international standards. 
The description of the TGA method is provided in Annex 5.  

DNA  methods,  namely  New  Generation  Sequencing  (NGS)  and  Quantitative  real-time  PCR  (qPCR),  were 
specifically used to detect the presence of clove and pepper in ground cinnamon. Two  qPCR methods were in-
house developed and validated to quantify clove and black pepper as known adulterants of cinnamon (Castro 
et al., 2023; Gopu et al., 2008). DNA-based methods provided also information about the presence of unusually 
high  content  of  species  different  than  cinnamon,  or  the  presence  of  a  large  amount  of  different  species, 
although this information can only be considered as indicative and further developments and validation studies 
are needed before it can be considered conclusive. Information about the DNA-based methods is given in Annex 
6.  

Principal  Component  Analyses  (PCA),  a  non-supervised  modelling  technique  that  reduces  the  number  of 
dimensions in large datasets into a reduced number of principal components that retain most of the original 
information, and Partial Least Square-Discriminant Analyses (PLS-DA), a supervised modelling technique that 
maximises the differences between two categories, were used to observe in Ceylon cinnamon and cassia test 
materials form separated clusters, and to visually detect outliers. 

8 

 
 
 
 
 
 
3  Results and discussion on the findings regarding cinnamon integrity 

The main objectives of the JRC-DG SANTE Administrative Arrangement in relation to cinnamon integrity were: 

• 

• 

• 

• 

To propose two screening methods based on FT-Raman spectroscopy and EDXRF to detect substitution 
of Ceylon cinnamon with cassia, 

To  develop  and  validate  in-house  HS-GC-MS  method  to  confirm  the  results  of  the  two  screening 
methods, proposing substances that can be used as markers to detect cinnamon adulteration, 

To propose HS-GC-MS and molecular biology-based methods to detect substitution of cinnamon by 
pepper and/or clove, 

To evaluate by TGA the compliance of commercially available cinnamon with ISO standards regarding 
the total content of ash. 

Both screening methods, Raman and EDXRF were able to detect four test materials labelled as Ceylon cinnamon 
that had been substituted totally or partially by cassia. The four samples were projected among cassia samples 
in the two PCA score plots created using the two techniques, respectively. A fifth sample without any indication 
on the label about its botanical variety, but labelled as produced in Sri Lanka, was also identified by the two 
screening methods, as cassia. The label claims of having been produced in Sri Lanka, could be considered as an 
intention to deceit the consumer. The five cases of suspicion of substitution of Ceylon cinnamon by cassia were 
confirmed  by  HS-GC-MS,  based  on  the  content  of  the  mentioned  samples  in  eugenol,  benzyl  benzoate  and 
coumarin. 

Eugenol  acetate  and  anethole  were  included  in  the  development  and  in-house  validation  of  the  HS-GC-MS 
method as markers for the presence of clove and pepper, respectively. In most of the cinnamon samples the 
content of anethole was below the LOQ and even LOD of the method. Eleven samples had levels of anethole 
that were outliers among the anethole results. For eight of the samples the anethole content (between 1 and 3 
times  the  LOQ  of  the  method)  could  be  due  to  cross  contamination.  For  the  remaining  three  samples,  the 
anethole content is so high (14 to 42 times the LOQ of the method), that it would be difficult to explain by cross 
contamination. Out of the three, the one with the highest anethole content was sold as stick, without information 
about botanical or geographical origin. One of the two ground cinnamon samples had also a eugenol acetate 
content that was an outlier among the eugenol acetate results (most of them below the LOQ of the method). 
Neither  pepper  nor  clove  could  be  detected  in  any  of  the  mentioned  samples,  but  Allium  cepa  (onion),  was 
detected by NGS and qPCR in the ground sample containing eugenol acetate, what could also explain the high 
content of anethole in that sample. No explanation could be found for the extremely high anethole content in 
the stick cinnamon sample.  

The maximum total ash content allowed by the ISO standards (ISO 6539:2014, and ISO 6538:1997), depends 
on the geographical origin of the product. Therefore, strictly speaking, only test materials with a geographical 
origin declaration could be evaluated for their compliance with the ISO standards. Eight samples, seven coming 
from  Sri  Lanka  and  one  from  Vietnam,  did  not  met  the  international  quality  standards  regarding  total  ash 
content. On top of those, one sample labelled as aromaticum, without geographical origin information, would 
not respect the ISO standard if it would come from China, but it would if it would come from Vietnam. The same 
applies to eight Ceylon cinnamon samples without geographical origin declaration, that would not respect the 
quality ISO standard if coming from any place where Ceylon cinnamon is produced, other than Madagascar. 

The  measurement  carried  out  by  Raman,  EDXRF,  HS-GC-MS  and  DNA-based  methods,  revealed  some 
irregularities next to the ones described above, described here after.  

Ten samples (2 Ceylon cinnamon, 2 cassia and 6 without botanical origin information) were not compliant with 
Commission Regulation (EU) 2023/915, that sets a maximum level of lead in bark species of 2.0 mg kg-1, what 
corresponds to 9.6 % of the samples analysed. 

Another safety concern is raised by the coumarin content in some samples of Cassia cinnamon. The compliance 
with legislation can only be tested in the final products containing cinnamon as ingredient; nevertheless, some 
discussion needs to be done keeping in mind the TDI of 0.1 mg/kg bodyweight set by EFSA (EFSA, 2008).  A 

9 

 
 
 
teaspoon of cinnamon weights approximately 5 g. The TDI would have been surpassed for children aged 6 to 
18 months (weight between 7 and 10 kg) when consuming in one day 0.5 g (one eighth of a teaspoon) of the 
Cassia sample with the highest coumarin concentration. The limit would be surpassed for the same group of 
population consuming 1 g of the thirteen Cassia cinnamon samples containing more than 838 mg kg-1 coumarin. 
Out of those 13 samples, five are not suspicious of adulteration and are compliant with existing legislation. 
Some studies propose the use of up to 6 g day-1, for up to 4 months to reduce fasting plasma glucose levels 
(Hlebowicz et al, 2007), and the consumption in recent years of smoothies and milk shakes with recipes that 
include one or more teaspoon cinnamon as flavouring agent has increased. The use of one teaspoon of the two 
Cassia cinnamons with the highest coumarin content in this study, would surpass the TDI value for adults with 
a  weight  of  up  to  80  kg  and  60  kg,  respectively.  Thirty-one  more  samples  (6  Cassia  cinnamons,  21  of  the 
samples without botanical information, and 1 of the samples labelled as Ceylon cinnamon that turn out to be 
totally or partially substituted by Cassia cinnamon), would have the same effect when the milk shake/smoothie 
would be consumed by children younger than 10 years old (estimated weight 20 kg or less). Therefore, 29.8 % 
of the total, 35 % of the Cassia cinnamon and 48.9 % of the samples without botanical origin information, 
could represent a safety problem for children younger than ten years. The validation of the HS-GC-MS method 
used to quantify coumarin did not include recovery studies, and therefore, the number could be higher because 
the amount detected could not correspond to the total content of coumarin. 

Low levels of peanut DNA were found in one Ceylon sample from Sri Lanka. This study did not include validation 
of  a  DNA-based  method  to  detect  peanuts  in  cinnamon  and  therefore,  this  finding  would  need  further 
experiments and confirmation by alternative techniques. In one sample, rice DNA was found in amounts high 
enough not to be explained by cross-contamination. Seven samples (three Ceylon cinnamons, two cassia and 
three without botanical origin information) were flagged as suspicious by qPCR due to the presence of a large 
number of extraneous species, among others onion, fenugreek, and rice. However, these findings would need 
further  investigation  with  methods  fully  validated  for  the  detection  of  the  mentioned  species,  before  been 
considered conclusive. 

Thirteen samples had a high sulphur content (between 1.4 and 4 times the median of the results for the sulphur 
concentrations covered by the 95 % confidence interval) and were outliers among the sulphur results. Those 
samples could be not compliant with Commission Regulation (EU) 1129/2011 on food additives that sets a 
maximum level of 150 mg SO2/kg in cinnamon. Nevertheless, in this study only total sulphur was measured, 
the samples should be analysed with a method allowing the specific determination of SO2. 

Six test materials (5 Ceylon cinnamons and one without information on geographical indication) had quite high 
levels of camphor (2-bornanone), (between 5 and 40 times the highest content covered by the 95 % confidence 
interval). In cinnamon, camphor is present in the roots of the plant and therefore, an explanation for the high 
camphor  concentration  could  be  that  bark  has  been  partially  substituted  by  the  root  of  the  plant.  Another 
explanation could be that Ceylon cinnamon has been substituted by Cinnamomum camphora, another cinnamon 
species of less quality. Unfortunately, it was not possible to find C. camphora on market to verify this suspicion, 
so this theory remains a hypothesis. Samples high in camphor were also high in one or several of the following 
elements  Al,  Si,  Ti,  Fe,  Cr,  and  Zr.  Those  elements  are  normally  present  in  inorganic  materials  at  higher 
concentrations than in organic matrices. The higher content in those elements could also explain why most of 
those  samples  have  a  high  total  ash  content.  One  of  the  Ceylon  cinnamon  samples  with  a  high  camphor 
concentration was produced in Madagascar, in the remaining samples from Madagascar the camphor content 
was low and covered by the 95 % confidence interval. For this reason, information on that sample is given in 
Annex  1.  Twelve  more  samples  contained  camphor  at  concentrations  not  covered  by  the  95  %  confidence 
interval, some of them also characterised by relatively high levels of Al, Si, Ti, Fe and/or Zr, but they were not 
taken into consideration because those levels could be due to cross-contamination. 

10 

 
 
 
 
 
  
 
Figure 3: Box and whisker plot for outlier and non-outlier cinnamon test materials included in the study based 
on their camphor content. 

Source: JRC 

Next to the test items mentioned in the paragraph above, some other samples with normal camphor levels, had 
contents of Al, Si, Ti, Cr, Fe and Zr higher than those covered by the 95 % confidence interval for one or several 
of the mentioned elements. The origin of the mentioned elements could also be the material used to process 
the cinnamon, for instance to mill it. All the test materials in which high levels of Al, Si, Ti, Cr, Fe, Zr and Pb were 
found,  were  commercialised  ground,  and  all  of  them  were  considered  anomalous  also  by  other  techniques 
different than EDXRF, such as HS-GC-MS and Raman. 

Methyl esters of some fatty acids such as myristic, palmitic, linoleic, oleic, and stearic acids were detected in 
eight samples. Methyl esters of these fatty acids could be formed by partial methylation of the free acids by 
the internal standard solution containing methanol during the extraction of volatiles that occurred at 150 °C for 
15 min. Seeds are rich in fatty acids (Ribeiro et al, 2019) and the presence of detectable amounts of some fatty 
acids  in  the  mentioned  samples  would  suggest  that  they  contain  not  only  cinnamon  bark  but  also  seeds. 
Quantification of those compounds could not be carried out due to the elevated price of those substances. The 
identification was done through their respective masses. Further research would be needed to reach a definitive 
conclusion. 

In one sample Mucor was detected by NGS, this fungus is a causal agent of mould in several fruits. That sample 
had a volatile profile different from the other test materials and was one of the samples non-compliant with 
legislation due to the high levels of lead. 

On  top  of  the  anomalies  related  to  the  contents  of  anethole  and  eugenol  acetate  described  earlier  in  this 
chapter, some samples were outliers for the composition of some of the volatile compounds quantified by HS-
GC-MS, what points at the substitution of the bark by other spices or even parts of the cinnamon plant. For 
instance, some samples are outliers with a too high content of eugenol, which is the most abundant volatile 
compounds in Ceylon cinnamon leaves, 70 to 95 % in comparison with the 5 to 10 % in bark (Rao et al, 2014), 
some samples are outliers with a high content of cinnamyl acetate, predominant compound in fruit, flowers, 
and fruit stalks of Ceylon cinnamon (Jayaprakasha et al, 2011), some others are outliers with a high content of 
caryophyllene  oxide  that  is  particularly  present  in  Ceylon  cinnamon  flowers  (Rao  et  al,  2014)  and  other  in 
caryophyllene present in the fruit. Some samples have a low hydroxicinnamaldehyde content with less than one 
third the median of all samples, while other three have 3 to 4 times more than the median. Those deviations 
from the normal composition in volatile compounds could represent the normal variations among cinnamon 
(for instance depending on the grade Alba, Continental, Mexican and Hamburg), cross-contamination issues that 
could  be  normal  or  due  to  poor  processing  practices,  or  in  extreme  cases  could  be  the  result  of  fraudulent 
substitution of the cinnamon bark. With the exception of four of the samples mentioned in this paragraph (3 
Ceylon cinnamons and 1 without information about botanical variety), several of the anomalies described earlier 

11 

 
 
 
 
 
had also been observed in the same test materials, and so those samples were declared suspicious for other 
reasons. The four exceptions were labelled as “Suspicious?” and would need further investigation. The same 
label was used with three Ceylon cinnamon samples with high levels of S as only outlying component.  

Table 1 and Figure 4 summarise the outcome of the study. 

Table 1: Summary of the findings obtained on commercially available cinnamon samples. 

Type of irregularity 

Ceylon 
cinnamon (%) 

Cassia (%) 

? (%) 

Total (%) 

Rate of samples legally non-compliant, lacking quality, suspicious of adulteration, outliers due to unusual chemical 
composition 

Legally non-compliant, Pb > 2 mg kg-1 (n= 10) 

Likely presence of peanut (n= 1) 

Potentially hazardous for children due to high levels 
of coumarin (n= 31) 

Substitution of Ceylon cinnamon by cassia (n= 4) 

Substitution of Ceylon cinnamon by cinnamon 
camphora or by cinnamon roots (n= 6) 

Total ash content not compliant with international 
standards, and/or cross-contamination with many 
different species (n= 10) 

Suspicious (strong outliers for several techniques 
based on composition information) (n= 24) 

Suspicious? (statistical outliers due to unusual 
composition, further investigation needed) (n=7) 

Non suspicious samples 

Samples not covered by any of the groups above and 
found non-suspicious (n= 35) 

Ceylon cinnamon from Madagascar not evaluated 
(n=5) 

Source: JRC 

4.5 

2.3 

- 

9.1 

11.4 

11.8 

14.0 

9.6 

1 

35 

48.9 

29.8 

- 

- 

- 

2.3 

3.8 

5.8 

13.6 

11.8 

4.7 

9.6 

31.8 

11.8 

18.6 

23.1 

13.6 

- 

2.3 

7.7 

4.8 

58.8 

58.1 

33.7 

11.4 

- 

- 

4.8 

12 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4: Summary of the findings obtained on commercially available cinnamon samples. 

Source: JRC 

13 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4  Conclusions 

The main outcome of this study is the high rate of commercially available cinnamon samples that were either 
legally non-compliant due to high levels of lead (close to 10 % of the total amount of samples analysed, and 
even higher, 14 %, for the samples with no information about botanical origin), and/or suspicious of poor quality 
and of different types of fraudulent practices, mostly substitutions by other plants or by parts of the cinnamon 
tree other than the bark. 

Only around 40 % of the samples analysed were free of any suspicion of fraud or lack of quality by the different 
techniques used. The lower rate of non-suspicious samples was found among the Ceylon cinnamon samples, at 
just 5 % (all sold in stick form). The higher rate of irregularities in Ceylon cinnamon samples is understandable, 
as this cinnamon is about twice as expensive as cassia, and therefore the economic profit for fraudsters is 
higher.  Nevertheless,  before  extracting  final  conclusions  about  adulteration  rates,  mainly  based  on  outlying 
composition of volatile compounds, Ceylon cinnamon material with full traceability records, including samples 
from different geographical origins and different grades, should be analysed and used as reference to detect 
adulteration. Otherwise, there is the risk of declaring exceptional quality samples as suspicious, simply because 
their composition is different from that of most samples available on the European market. 

The screening methods based on Raman and EDXRF were able to detect 100 % of the test materials labelled 
as Ceylon cinnamon that were totally or partially substituted by cassia, as confirmed by the volatile composition 
of the samples determined by HS-GC-MS. Given the high rate of suspicious Ceylon cinnamon samples, it was 
not possible to carry out a study to test if both screening methods were able to detect 5 % substitution. To 
carry out that study, pure Ceylon cinnamon test materials with full traceability records would be needed.  

The EDXRF screening method was able to flag as suspicious 87 % of all samples lacking quality, those suspicious 
of substitution by cassia, other plants and different parts of the cinnamon tree, as well as those with anomalous 
volatile composition. The method is the only one among the different analytical approaches used in this study, 
able to detect samples legally non-compliant due to a high content of lead, and to flag those with an outlying 
concentration  of  sulphur,  pointing  to  the  abusive  use  of  SO2.  Four  non-suspicious  samples  were  flagged  as 
suspicious by EDXRF, what corresponds to a false positive rate of 6.7 %. No clustering of samples depending 
on whether they were issue of the organic or conventional agriculture was observed in the PCA scores plots 
based on elemental analysis.  

The Raman-based screening method detected 53 % of irregular test materials; the method could not detect 
neither the samples lacking quality due to too high ash content, nor those with lead content higher than 2 mg 
kg-1 when that was the only irregularity found. Only one non-suspicious sample was flagged as suspicious by 
Raman, corresponding to a rate of false positives of only 1.7 %. 

A HS-GC-MS-based method was successfully validated in-house to confirm the results found by the screening 
methods. The method was able to detect 65 % of the test samples with irregularities. As in the case of the 
Raman  screening  method,  HS-GC-MS  failed  to  detect  some  samples  with  high  lead  content  and  total  ash 
content.  The  method  did  not  flagged  as  suspicious  any  of  the  samples  found  non-suspicious  by  the  other 
methods. Methods for the determination of the total content of coumarin in cinnamon should be standardised 
to help control laboratories to incorporate that type of analyses in their regular controls. 

Based on the results obtained by DNA-based methods, none of the samples included in the study contained 
neither clove nor pepper, however DNA from other plant(s) was found in some of the samples. In particular, a 
qPCR method should be developed and validated to detect and quantify the presence of peanuts in cinnamon, 
which could represent a high risk for people allergic to peanuts. Also, qPCR methods should be developed and 
validated not only to detect, but also to quantify rice, onion, coffee, fenugreek, and Bupleurum chinense, whose 
presence was detected in some of the cinnamon test materials included in this study (Annex 6). 

In summary, the types of fraudulent practises detected on samples commercially available in the European 
market are diverse. Adulteration cannot be identified exclusively with a single analytical technique and several 
approaches need to be followed to address the problem in a holistic way. The magnitude of the problem is such 
that it requires attention especially given the projected increase of the global cinnamon market. 

14 

 
 
5  Annexes 

5.1  Annex 1: Test Samples 

Table 2: Summary of information about the test samples analysed, the decisions based on the results of the 
different analytical techniques and the final decision. Empty cells in the decisions for the different techniques: 
not suspicious. 

Purchase 
form 

Origin 

Total ash  

TGA (%) 

ED-XRF 

HS-GC-
MS 

NGS 

qPCR 

Raman 

Final 
conclus
ion 

BIO 

Ground 

S 

S 

S 

ND 

BIO/ 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

BIO 

BIO 

BIO 

BIO 

Sample id 

Label 
variety 

Cin001 

Cin002 

Cin003 

Cin004 

Cin005 

Cin006 

Cin007 

Cin008 

Cin009 

Cin010 

? 

? 

? 

? 

? 

? 

? 

? 

? 

? 

Cin011 

Ceylon 

Cin012 

Ceylon 

Cin013 

? 

Cin014 

Ceylon 

Cin015 

Cin016 

Burma
nni 

Aroma
ticum 

Cin017 

? 

Cin018 

Ceylon 

Cin019 

Ceylon 

Cin020 

Ceylon 

Cin021 

Ceylon 

Cin022 

Ceylon 

Cin023 

Cassia 

Ground 

Ground 

Ground 

Ground 

Ground 

Ground 

Ground 

Ground 

Ground 

? 

? 

? 

? 

? 

? 

? 

? 

? 

? 

Ground 

Madagascar 

Ground 

Sri Lanka 

Ground 

NON EU 

Ground 

Madagascar 

BIO 

Ground 

BIO 

Ground 

? 

? 

NON 
BIO 

BIO 

BIO 

BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

Ground 

Ceylan and 
Indonesia 

Ground 

NON EU 

Ground 

NON EU 

Ground 

NON EU 

? 

? 

Ground 

Ground 

Ground 

S 

S 

S 

NS 

NS 

NS 

NS 

S 

NS 

NS 

NS 

NS 

S 

NE 

S? 

NS 

NE 

NS 

S* 

S 

S 

S 

S 

S 

S 

NS 

NS 

S 

S 

S 

S 

S 

S 

S 

S 

S  

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

Cin024 

Cassia 

BIO 

Ground 

Vietnam 

Cin025 

Ceylon 

NON 
BIO 

Ground 

? 

S 

S 

S 

15 

 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
 
 
  
  
  
  
  
  
  
  
  
  
  
  
 
 
 
 
 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
  
  
 
  
  
  
  
  
 
  
  
  
  
  
Purchase 
form 

Origin 

Total ash  

TGA (%) 

ED-XRF 

HS-GC-
MS 

NGS 

qPCR 

Raman 

Final 
conclus
ion 

Ground 

Vietnam 

S 

S 

BIO 

Ground 

Ground 

Ground 

Ground 

Ground 

Ground 

? 

? 

? 

? 

? 

? 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

Sample id 

Label 
variety 

Cin026 

? 

Cin027 

Cin028 

Burma
nni 

Burma
nni 

Cin029 

Ceylon 

Cin029 
batch 2 

Ceylon 

Cin030 

Cassia 

Cin030 
batch 2 

Cassia 

Cin031 

? 

Cin032 

Ceylon 

Cin033 

Ceylon 

Cin034 

Ceylon 

Cin035 

Ceylon 

Cin036 

Ceylon 

Cin037 

Ceylon 

Cin039 

Ceylon 

BIO/ 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

BIO 

BIO 

BIO 

NON 
BIO 

Ground 

Sri Lanka 

Stick 

Sri Lanka 

Stick 

Sri Lanka 

S 

Stick 

Sri Lanka 

Stick 

Stick 

Stick 

Sri Lanka 

Sri Lanka 

? 

Stick 

Sri Lanka 

S 

S 

S 

S 

S 

S 

Cin040 

Ceylon 

BIO 

Ground 

? 

Cin041 

Ceylon 

Cin042 

Ceylon 

NON 
BIO 

NON 
BIO 

Ground 

Sri Lanka 

Stick 

Sri Lanka 

Cin043 

Ceylon 

BIO 

Ground 

? 

Ground 

Madagascar 

Ground 

Sri Lanka 

Ground 

India 

Stick 

Stick 

? 

Sri Lanka 

Ground 

? 

Ground 

Sri Lanka 

Ground 

Sri Lanka 

Cin044 

Ceylon 

Cin045 

Ceylon 

Cin046 

Ceylon 

Cin047 

Ceylon 

Cin048 

Ceylon 

Cin049 

Ceylon 

Cin050 

Ceylon 

Cin051 

Ceylon 

Cin052 

Cassia 

Cin053 
batch1 

? 

NON 
BIO 

NON 
BIO 

BIO 

BIO 

BIO 

BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

NS 

NS 

S 

S 

NS 

S 

NS 

NS 

S* 

S? 

S* 

S* 

S* 

S 

S 

S 

S* 

S 

NE 

S 

S 

S 

NS 

S 

S 

S 

NS 

S? 

NA 

NA 

NA 

NA 

NA 

NA 

NA 

NA 

NA 

NA 

NA 

NA 

NA 

NA 

S 

S 

S 

NA 

NA 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

NA 

NA 

NA 

NA 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

S 

Stick 

Indonesia 

NA 

NA 

Ground 

Ceylan and 
Indonesia 

S 

S 

S 

16 

 
 
  
  
  
  
  
 
  
  
  
  
  
 
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
 
  
 
  
  
 
  
  
 
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
 
 
 
 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Purchase 
form 

Origin 

Total ash  

TGA (%) 

ED-XRF 

HS-GC-
MS 

NGS 

qPCR 

Raman 

Final 
conclus
ion 

Ground 

Sri Lanka 
(Ceylan) and 
Indonesia 

Stick 

Stick 

Stick 

? 

Ground 

S 

S 

? 

? 

? 

? 

S 

S 

S 

NA 

NA 

NA 

NA 

S? 

NA 

NA 

BIO/ 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

BIO 

BIO 

BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

Sample id 

Label 
variety 

Cin053 
batch2 

Cin054 
batch1 

Cin054 
batch2 

Cin054 
batch3 

Cin055 

Cin056 

? 

Ceylon 

Ceylon 

Ceylon 

? 

? 

Cin057 

Ceylon 

Cin058 

Ceylon 

Cin059 

Ceylon 

Cin060 

Cin061 

Cin062 

Cin063 

? 

? 

? 

? 

burma
nni/ 

Cin064 

cassia/ 

loureir
oi 

S 

S 

S 

S 

S 

Stick 

Indonesia 

Ground 

Sri Lanka 

Ground 

Sri Lanka 

Stick 

Sri Lanka 

Ground 

Stick 

Stick 

? 

? 

? 

Ground 

Tropics 

S 

Ground 

? 

S 

S 

S 

S? 

S 

S 

S 

S 

S 

S? 

Cin065 

Ceylon 

BIO 

Ground 

Sri Lanka 

Cin066 

Cin067 

Cin068 

? 

? 

? 

Cin069 

Ceylon 

Cin070 

Cassia 

Cin071 

Ceylon 

Cin072 

Cassia 

Cin073 

Ceylon 

Cin074 

Cassia 

Cin075 

Ceylon 

Cin076 

Ceylon 

Cin077 

Ceylon 

Cin078 

Cin079 

? 

? 

NON 
BIO 

NON 
BIO 

NON 
BIO 

BIO 

BIO 

BIO 

? 

? 

BIO 

BIO 

BIO 

BIO 

NON 
BIO 

NON 
BIO 

Stick 

Vietnam 

Ground 

Vietnam 

Ground 

Outside EU 

Stick 

Outside EU 

Ground 

Vietnam 

Ground 

Outside EU 

Ground 

Ground 

Ground 

? 

? 

India 

Ground 

Madagascar 

Ground 

Sri Lanka 

Stick 

Sri Lanka 

Stick 

Indonesia 

Stick 

? 

S 

S 

S 

S 

17 

S* 

S? 

S? 

S? 

S 

NS 

S 

S 

NS 

NS 

NS 

NS 

S 

NS 

S* 

S* 

S 

S 

S 

S 

NA 

NA 

S 

NA 

NA 

NA 

NA 

NA 

NA 

S 

S 

S 

S 

S 

S 

NA 

NA 

S 

S 

NA 

NA 

S? 

NS 

S 

S 

S 

S 

S 

S 

NS? 

NA 

NA 

NS? 

NA 

NA 

NA 

NA 

S 

S 

S 

S 

S 

NE 

S? 

NS 

NS 

NS 

S 

S 

S 

S 

S 

S 

S 

S 

 
 
  
  
 
  
  
 
  
  
  
  
  
 
  
  
  
 
  
  
  
  
  
  
  
  
 
  
  
  
 
  
  
  
  
  
 
  
  
  
 
  
  
 
  
  
  
  
 
  
  
  
  
  
  
 
  
  
  
 
  
  
  
 
 
  
  
  
  
 
  
  
  
  
  
  
  
  
  
 
 
 
 
 
  
  
  
  
  
 
  
  
 
  
  
  
 
  
Sample id 

Label 
variety 

Cin080 

Cin081 

Cin082 

? 

? 

? 

Cin083 

Cassia 

Cin084 

Cin085 

Cin086 

Cin093 

Cin094 

Cin095 

Cin096 

Cin097 

? 

? 

? 

? 

? 

? 

? 

? 

Cin098 

Cassia 

Cin099 

Cassia 

Cin100 

Ceylon 

Cin101 

Cin102 

Cin 103 

Cin 104 

Cin 105 

Cin 106 

? 

? 

? 

? 

? 

? 

Source: JRC 

BIO/ 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

NON 
BIO 

Purchase 
form 

Origin 

Total ash  

TGA (%) 

ED-XRF 

HS-GC-
MS 

NGS 

qPCR 

Raman 

Final 
conclus
ion 

NS 

S 

NS 

NS 

NS 

S 

S 

NS 

S 

NS 

NS 

NS 

S* 

NS 

S 

NS 

NS 

S 

S 

NE 

S 

Stick 

Vietnam 

Ground 

Vietnam 

S 

Ground 

Stick 

Ground 

Ground 

? 

? 

? 

? 

Ground 

Stick 

Ground 

Stick 

Ground 

? 

? 

? 

? 

? 

NS 

NA 

NA 

S 

S 

S 

S 

S 

S 

S 

NA 

NA 

S 

S 

S? 

NA 

NA 

S 

S 

S 

S? 

NA 

NA 

Stick 

China 

NA 

NA 

Ground 

Stick 

Stick 

Stick 

Ground 

? 

? 

? 

? 

? 

Stick 

Sri Lanka 

Stick 

Sri Lanka 

Stick 

Madagascar 

S 

S 

S 

S 

S? 

S 

S 

S 

S 

NA 

NA 

S? 

NA 

NA 

S? 

NA 

NA 

NA 

NA 

S 

S? 

NA 

NA 

S 

Ground 

Madagascar 

S 

S 

S 

18 

 
 
  
  
 
  
  
  
  
  
  
  
  
  
  
 
  
  
  
  
 
  
 
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
 
  
 
  
  
  
  
  
 
  
  
  
 
  
  
 
  
  
  
 
 
 
 
 
 
 
  
  
  
 
 
 
5.2  Annex 2: X-ray fluorescence based method 

The elemental composition (Na, Mg, Al, Si, P, Cl, S, K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, As, Br, Rb, Sr, Zr, Cd, Ba, Pb, V, 
Co, Se, Mo, Sn and Sb) of the cinnamon samples was determined by EDXRF, using an Epsilon 4 spectrometer 
(PANalytical, Almelo, The Netherlands). The instrument is equipped with a Silver (Ag) anode X-ray tube with a 
Beryllium (Be) side window (50 µm thickness) and a silicon drift detector SDD30. The maximum high tension, 
anode current and power are 50 kV, 3 mA and 15 W respectively. The system has six filters (Cu-500, Ti, Al-50, 
Al-200, Cu-300 and Ag, with thickness of 500, 7, 50, 200, 300 and 100 µm respectively) that generate the 
excitation X-rays. The selection of the appropriate filter depends on the element to be analysed and was done 
according  to  the  recommendations  of  the  instrument  manufacturer.  The  system  is  air-cooled  and  does  not 
require  the  use  of  liquid  nitrogen.  The  instrument  was  calibrated  every  month  with  the  CRM  INCT-OBTL-5, 
Oriental  Basma  Tobacco  Leaves  (Department  of  Analytical  Chemistry,  Institute  of  Nuclear  Chemistry  and 
Technology)  correcting  in  this  way  the  normal  drift  of  the  instrument.  The  performance  of  the  EDXRF 
spectrometer was checked monthly with the CRM INCT-PVTL-6, Polish Virginia Tobacco Leaves (Department of 
Analytical  Chemistry,  Institute  of  Nuclear  Chemistry  and  Technology).  The  ED-XRF  was  controlled  by  the 
PANalytical Epsilon 4 Software, Version 2.0N/ICSW2.11 

The EDXRF method was validated using an approach previously described (Fiamegos et al, 2018). Summarising, 
a single calibration curve covering different types of organic matrices was built for each element analysed using 
the following Certified Reference Materials (CRM) and Reference Materials (RM): BCR 191 (Brown bread), NIST 
3233 (Fortified breakfast cereal), DUWF-1 (Durum wheat flour), NIST 1567b (Wheat flour), BRAN-1 (Corn bran), 
IRMM 804 (Rice flour), IRMM PT-43 (Rice flour), NIST 1568b (Rice flour), NIST 3234 (Soy flour), BCR 679 (White 
cabbage), IAEA 359 (Cabbage), NIST 1570a (Spinach leaves), NIST 1573a (Tomato leaves), OBTL-5 (Tobacco), 
PVTL-6 (Tobacco), RT3 (Tobacco), RT5 (Tobacco ), AJJA-17 (Tobacco), NIST 3287 (Blueberry), ERBM-CD 281 (Rye 
grass), BCR 129 (Hay powder), BCR 402 (White clover), NIST 1575a (Pine needles), BCR 482 (Lichen), IAEA 336 
(Lichen), IMEP 119  (Vegetable feed), NIST 3290 (Dry cat food), IAEA 392 (Algae),  IAEA 413 (Algae), MIJ 7405-
a (Seaweed), BCR 414 (Plankton), DORM-5 (Fish protein), ERM-BB 422 (Fish muscle), NIST 1566b (Oyster tissue), 
ERM-BB 184 (Bovine muscle), ERM-BB 185 (Bovine liver), ERM-BB 186 (Pig kidney), NIST 1548a (Mixed diet), 
NIST  1549a  (Whole  milk  powder),  NIST  3252  (Protein  drink  mix).  Those  CRMs  and  RMs  for  which  the  ratio 
between the calculated and the certified or indicative mass fraction was outside the range 70-130 %, were left 
out of the calibration curve. The remaining mass fractions were used to set the working ranges for the different 
elements, Table 3.  

To evaluate the accuracy of the method, trueness, and precision, the following CRMs and RMs, not included in 
the calibration curves, were measured: SPIN-1 (Spinach), KINO-1 (Kinoa flour), REDS-1 (Hard red spring wheat 
flour),  BOVM-1  (Bovine  muscle),  DOLT-5  (Dogfish  liver),  TORT-3  (Lobster  hepatopancreas),  VITA-1 
(Multivitamin).  One pellet of each of the CRM/RM was measured in triplicate in three different measurement 
sessions  spread  over  three  days.  The  results  obtained  were  used  to  evaluate  the  within  and  between-day 
variation. The intermediate precision calculated with Analysis of Variance (ANOVA), confidence interval 95 %, 
using the software Statistica (TIBCO, version 13.5.0.17), was taken as expanded uncertainty (k=2).  

The fit-for-purpose of the trueness was evaluated with z-scores (ISO 13528: 2015), using the equation:  

Z=(x-X)/σ 

Where:   

x: mass fraction obtained with the method under validation  

X: certified or indicative mass fraction provided in the certificate of analyses by the CRM or RM producer  

σ: Standard deviation calculated with the Horwitz equation modified by Thompson (Thompson, 2000).  

The z-score interpretation was done as follows:  

|z| ≤ 2 satisfactory score  

2 < |z| < 3 questionable result  

|z| ≥ 3 unsatisfactory result  

19 

 
 
 
 
 
Table 3 summarises the results obtained in the trueness study. The numbers on a blue background are the 
certified  (black),  and  indicative  values  (white-italic)  provided  by  the  producer  of  the  CRM/RM.  Satisfactory, 
questionable  and  unsatisfactory  z-scores  are  given  in  cells  with  a  green,  orange  and  red  background, 
respectively.  

The  limits  of  quantification  for  the  different  elements,  LOQ,  were  set  to  the  lowest  mass  fraction  in  the 
calibration curve for which satisfactory results were obtained with an intermediate precision ≤ 25 %. Table 3 
includes also the thresholds used in this study to consider a cinnamon sample as outlier although they are only 
indicative and should be established in a collaborative trial. 

Table  3:  Accuracy expressed as z-score of the EDXRF method obtained using Certified Reference Materials 
(CRMs). All certified reference values, and indicative values are expressed in mg kg-1. 

Na 

375 

Mg 

313 

P 

671 

9.5 

10 

8 

375-
18210 

313-
36700 

671-
17210 

S 

30 

15 

30-
9600 

>2000 

Cl 

K 

301 

1282 

Ca 

118 

5 

4.5 

4 

Cr 

0.6 

12 

222-
16500 

1282-
47500 

118.4-
57100 

0.6-377 

Mn 

1.78 

12 

1.78-
828 

Fe 

9.4 

10 

9.4-
3560 

>800 

1313 

-1.06 

1000 

18500 

19000 

222 

12100 

28600 

10.2 

389 

3560 

0.62 

0.08 

-2.01 

<LOQ 

16.12 

0.81 

0.50 

-0.95 

-1.61 

2100 

960 

8360 

1900 

15200 

145 

0.37 

71.2 

0.09 

0.61 

2.99 

-1.40 

0.30 

1.52 

<LOQ 

0.23 

9900 

940 

11500 

14400 

550 

2.35 

8.91 

1070 

-1.35 

-0.82 

7.14 

-0.28 

-1.75 

2.78 

-3.09 

0.17 

Sample 
Identification 

LOQ (mg kg-1) 
Expanded 
Uncertainty 
(%)(k=2) 
Working range  
(mg kg-1) 
Threshold  used  for 
outliers (mg kg-1) 

SPIN-1 (Spinach) 

z-score 

VITA-1 
supplement) 

(Vitamin 

z-score  

BOVM-1 
muscle) 

z-score  

(Bovine 

DOLT-5  (Dogfish 
liver)  

z-score  

KINO-1 
Flour) 

z-score 

(Quinoa 

37 

1970 

<LOQ 

-0.66 

6010 

720 

-1.14 

0.40 

84.1 

0.71 

REDS-1 (Hard Red 
Wheat 
Spring 
Flour) 

7 

365 

1370 

1830 

500 

1150 

143 

0.026 

4.50 

31 

z-score  

<LOQ 

1.17 

0.51 

0.77 

1.15 

<LOQ 

-0.66 

<LOQ 

1.21 

-3.07 

TORT-3 
(Lobster 
hepatopancreas) 

z-score  

Sample 
Identification 

LOQ (mg kg-1) 
Expanded 
Uncertainty 
(%)(k=2) 
Working range  
(mg kg-1)  
Threshold used for 
outliers (mg kg-1) 

SPIN-1 (Spinach) 

z-score 

1.95 

15.6 

179 

1.89 

-0.86 

-1.12 

Ni 

Cu 

Zn 

As 

Se 

Rb 

Sr 

Mo 

Cd 

Hg 

Pb 

0.57 

2.84 

6.49 

0.18 

0.37 

2.76 

1.19 

0.38 

0.56 

0.32 

0.75 

11 

15 

3 

7 

13 

4 

4 

19 

9 

7 

11 

0.57-
113 

2.8-
598 

6.5-
4560 

0.18-
127 

0.37-
10.9 

2.76-
50 

1.19-
1470 

0.38-
36.2 

0.56-
204 

0.32-
53.2 

40.7 

-0.63 

0.70 

1.08 

0.062 

<LOQ 

140.8 

1.72 

0.265 

0.01 

<LOQ 

<LOQ 

20 

0.75-
242 

>2 

2.33 

0.62 

 
 
 
 
 
 
 
 
 
 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
 
 
 
 
 
 
 
 
 
 
  
  
  
  
  
  
  
  
Sample 
Identification 

LOQ (mg kg-1) 
Expanded 
Uncertainty 
(%)(k=2) 
Working range  
(mg kg-1)  
VITA-1  (Vitamin 
supplement) 

z-score  

BOVM-1  (Bovine 
muscle) 

z-score  

DOLT-5  (Dogfish 
liver)  

Ni 

Cu 

Zn 

As 

Se 

Rb 

Sr 

Mo 

Cd 

Hg 

Pb 

0.57 

2.84 

6.49 

0.18 

0.37 

2.76 

1.19 

0.38 

0.56 

0.32 

0.75 

11 

15 

3 

7 

13 

4 

4 

19 

9 

7 

11 

0.57-
113 

2.8-
598 

6.5-
4560 

0.18-
127 

0.37-
10.9 

2.76-
50 

1.19-
1470 

0.38-
36.2 

0.56-
204 

0.32-
53.2 

98 

2360 

0.046 

4.2 

9 

18.1 

0.02 

-1.73 

1.67 

<LOQ 

-0.53 

0.28 

0.17 

<LOQ 

2.84 

142 

0.009 

0.076 

28.7 

0.052 

0.08 

0.013 

2.01 

0.48 

<LOQ 

<LOQ 

-0.75 

<LOQ 

<LOQ 

<LOQ 

0.75-
242 

0.064 

<LOQ 

0.38 

<LOQ 

1.71 

35.0 

105.3 

34.6 

8.3 

3.73 

1.41 

14.5 

0.44 

0.162 

z-score  

0.01 

-0.28 

-0.17 

0.04 

-0.93 

1.21 

-0.43 

0.05 

-0.01 

<LOQ 

KINO-1  (Quinoa 
Flour) 

z-score 

REDS-1 
Red 
Wheat Flour) 

(Hard 
Spring 

30.6 

0.00100 

0.17 

-0.59 

<LOQ 

<LOQ 

2.49 

1.14 

0.063 

0.00097 

0.0028 

<LOQ 

<LOQ 

<LOQ 

0.2 

2.0 

10.6 

4 

0.55 

0.02 

z-score  

<LOQ 

<LOQ 

-0.93 

-6.68 

3.49 

<LOQ 

TORT-3  (Lobster 
hepatopancreas) 

5.3 

497 

136 

59.5 

10.9 

36.5 

3.44 

42.3 

0.292 

0.225 

z-score  

-0.46 

-1.71 

-0.76 

1.53 

-0.97 

0.70 

0.07 

0.54 

<LOQ 

-0.68 

Source: JRC 

No full validation could be conducted for the elements Al, Ba, Br, Si, Ti, and Zr because the few CRMs/RMs used 
in  the  study  with  certified/reference  mass  fractions  for  those  elements  had  to  be  used  to  construct  the 
calibration curves and none was left for the trueness study. The LOQ, intermediate precision and the working 
range  provided  in  Table  4  for  those  elements  are  merely  indicative  and  based  on  the  calibration  curves 
constructed with a limited number of CRMs/RMs.  

Table 4: Working range, LOQs and expanded uncertainties for the elements for which a full validation could 
not be carried out. All mass fractions expressed as mg kg-1. 

Element 

Threshold for 
suspicious  
(mg kg-1) 

Working range  

LOQ  

Expanded 
Uncertainty 
(%)(k=2) 

Al 

Ba 

Br 

Si 

Ti 

Zr 

>1050 

>1300 

>80 

>10 

310-3137 

6.4-117 

0.35-1300 

2370*-6720 

12.3-490.73 

10.24-24.2 

310 

6.4 

0.35 

2370 

12.3 

10.24* 

7 

6 

19 

5 

1 

7 

*Lowest mass fraction in the CRMs/RMs available in our laboratory. The LoQ could be lower than that. 

Source: JRC 

Only two or three CRMs/RMs had certified/reference mass fractions for V, Co, Sn, and Sb; for that reason, the 
LOQ,  intermediate  precision and  working  range  could  not  be  determined.  The  mass fractions  obtained  were 
used as semi-quantitative information for modelling purposes.  

21 

 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
 
  
 
 
Principal Component Analysis (PCA), a non-supervised multivariate analysis tool that reduces the amount of 
variables  (elemental  and  volatile  mass  fractions)  into  a  reduced  number  of  non-correlated  principal 
components, was used  to  visualise  clusters  in  function  of  their  botanical  species.  Also  PLS-DA a  supervised 
multivariate analysis tool that maximises the differences between categories was used for the same purpose. 
Concentrations were normalised by unit-variance scaling. No further pre-processing was applied. The amount 
of principal components was always set to three to avoid overfitting.  For modelling purposes, two different 
models were built-up for ground and stick samples, respectively, because it was observed that the milling of 
cinnamon had an impact on the elemental profile of cinnamon. As an example, the PCA score plot for ground 
cinnamon samples is shown in Figure 4. 

Figure 4: Score plot based on elemental profiles determined by PCA for ground cinnamon samples 

Source: JRC 

22 

 
 
 
 
 
 
 
 
 
5.3  Annex 3: Thermogravimetric analysis (TGA)  

The ash content of the cinnamon test materials was determined with a ThermoGravimetric Analyzer (Mettler 
Toledo). Test materials were measured in duplicate with test portion sizes ranging from 35 to 64 mg. The test 
portions were heated in an oxidative atmosphere to 550 °C and held at that temperature until the weight of 
the residue stayed constant. The weight % of the residue was reported as the ash content. 

23 

 
 
 
 
 
5.4  Annex 4: Vibrational spectroscopy based method 

Vibrational  spectroscopy  has  been  extensively  used  to  study  cinnamon  speciation  as  well  as  potential 
substitution  of  Ceylon  cinnamon  by  cassia.  In  particular,  Fourier  Transform  Infra-Red  (FT-IR)  spectroscopy 
combined with chemometrics has been previously applied on thin films coated with cinnamon essential oil (Li 
et al., 2013) and directly on cinnamon powder samples (Lixourgioti et al, 2022 & Lopes et al., 2022 & Yasmin 
et al., 2019) to study the differences among cassia and Ceylon species as well as the adulteration of the latter 
by  the  former.  In  addition,  FT-IR  was  also  used  for  the  first  time  to  determine  the  adulteration  of  Ceylon 
cinnamon by cinnamon spend (waste) by preparing several admixtures of the individual cinnamon species at 
varying ratios (Lixourgioti et al., 2022). Other studies reported the use of FT-NIR spectroscopy combined with 
chemometrics to detect adulteration of Ceylon cinnamon by cassia (Yasmin et al.,2019 & Shawky and Selim, 
2018 & Cantarelli et al., 2020) as well as by extraneous substances such as black pepper and/or clove (Castro 
et al., 2023). On the other hand, Nelson et al. applied surface enhanced Raman spectroscopy (SERS) on the 
essential oil extracted from Ceylon and cassia cinnamon samples in order to investigate the differences among 
the volatile compounds present in both species that could be subsequently used as markers to detect cinnamon 
adulteration. 

However, most of the aforementioned studies were largely conducted on a big number of cinnamon mixtures 
that were formed by a small number of real world cinnamon samples or on real world cinnamon samples that 
were  purchased  from  a  limited  number  of  places  in  the  market  and  they  are  mainly  based  on  infrared 
spectroscopy (Mid-IR and Near-IR). In this study, we report the development of a Raman screening method that 
uses  a  benchtop  Fourier  Transform  (FT)  instrument  for  the  detection  of  cinnamon  adulteration  on  a  large 
number of powder samples (>100) that were purchased from several European countries. To the best of our 
knowledge, this is the first time that Raman spectroscopy is applied to study cinnamon adulteration directly on 
powder samples that cover a substantial portion of the European market. In addition, a transferability study 
from the benchtop FT-Raman to a Raman handheld device is conducted for a selection of cinnamon samples 
to assess the feasibility of the latter device as a fast screening tool for the detection of cinnamon adulteration 
by control laboratories.  

Raman screening method using benchtop instrument  

Overview of Raman spectra on cinnamon species and reference compounds 

For all the Raman measurements, a benchtop Vertex 70 Fourier Transform (FT) spectrometer (Bruker, Belgium) 
pumped Nd:YAG laser 
equipped with a RAM II module was used in backscattering mode. An air
operating at 1064 nm was used for the excitation and a liquid N2 cooled ultrahigh sensitivity Ge detector for 
the detection of Raman light. The excitation laser power was kept at 150 mW for the measurements to avoid 
sample damaging, while the spectra were collected with a scanning range of 200–3200 cm-1, a resolution of 4 
cm-1 and 250 scans that were averaged for each spectrum. All samples were placed in glass vials and measured 
directly using the RAMII sample holder that was kept steady for all measurements. Figure 5a shows a glass vial 
containing cinnamon powder, while Fig. 5b illustrates the same glass vial placed on the RAMII sample holder 
for measurement.  

cooled diode

‐

‐

Figure 5. (a). Cinnamon powder in glass vial for Raman measurement. (b). Glass vial placed on the RAMII 
sample holder for spectrum acquisition. White arrow shows the laser light heading towards the sample and 
red arrow shows the collected backscattered light after interaction with the sample.

 Source: JRC 

24 

 
 
 
 
 
Prior  to  the  measurements  of  the  cinnamon  samples,  reference  spectra  of  the  most  abundant  chemical 
compounds present in the essential oil were recorded to assist the interpretation of the cinnamon spectra. Most 
of the reference compounds were purchased in liquid form, while a few of them were bought as solids. Figure 
6 shows the Raman spectra of these substances. 

Figure 6: Raman spectra of the most abundant chemical compounds present in cinnamon essential oil. Note 
that the assignment ‘’v’’ refers to the stretching vibration. 

Source: JRC 

The spectral profiles of these compounds exhibit peak overlapping across the fingerprint region (300 - 1800 
cm-1), albeit with significant relative intensity variations. Considering that the measurement settings for the 
Raman spectra of all substances were kept the same, it can be clearly shown that cinnamaldehyde is by far the 
strongest Raman scatterer among them. This can be explained due to the conjugated nature of cinnamaldehyde 
that contains several double bonds resulting in high Raman scattering cross section (Bock & Gierlinger, 2019). 
This  observation  in  combination  with  the  fact  that  it  is  the  most  abundant  volatile  found  in  cinnamon  bark 
indicates that cinnamaldehyde is expected to be the main Raman scatterer among all volatile compounds, even 
though its concentration in the powder sample would be less than 10%. 

Figure 7 shows Raman spectra of cassia and Ceylon labelled cinnamon samples together with the reference 
spectrum of pure cinnamaldehyde for comparison. The graph clearly demonstrates striking similarities between 
cassia cinnamon and cinnamaldehyde spectra, whereas such similarities are much less evident for Ceylon and 
cinnamaldehyde. It is interesting to note that the relative intensity ratio of the two sharp peaks located at 1597 
and 1625 cm-1 in both cassia and Ceylon cinnamon are somewhat different to the corresponding intensity ratio 
in pure cinnamaldehyde, due to the fact that for the cinnamon samples there is also additional contribution 
from other aldehydes (e.g. coniferyl aldehyde) present in the lignin, the latter being one of the most prominent 
compounds in cinnamon (Agarwal et al., 2011). However, upon comparison of the 1597/1625 cm-1 peak ratio 
values for cassia and Ceylon to the 1597/1625 cm-1 peak ratio for pure cinnamaldehyde, cassia shows much 
better correlation to cinnamaldehyde compared to Ceylon, which indicates that the presence of cinnamaldehyde 
is more prominent in cassia cinnamon species, in agreement with previous works (Pages-Rebull et al., 2024 & 
Wang et al., 2020). Additional Raman bands at 1002, 1125 and 1253 cm-1 characteristic for cinnamaldehyde 
are also evident in the spectrum of cassia cinnamon. Scattering from coumarin could also contribute at this 
frequency range (see Fig. 6), although only in cassia samples, where the latter compound is more prominent. 

Even though the Raman bands above 1500 cm-1 are mainly assigned to cinnamaldehyde, coniferyl aldehyde to 
a lesser extent and to a minor contribution from coumarin, the signal observed in the spectral range from 900 

25 

 
 
 
 
 
 
 
 
  
to 1500 cm-1 contains additional scattering from cellulose/hemicellulose structures that are also largely present 
in cinnamon bark (Agarwal et al., 2011). Hence, cinnamon spectra exhibit several overlapping features that are 
characteristic  of  lignin,  cellulose  and  cinnamaldehyde  and  coumarin.  Although  coumarin  concentration  is 
estimated not to exceed 1% in cassia cinnamon powder, the weak contribution to the Raman spectrum of cassia 
is possible, if taken into account that it is the second strongest scatterer among the volatile compounds after 
cinnamaldehyde (see Fig. 6).      

Figure 7. Raman spectra of cassia and Ceylon labelled cinnamon samples together with the reference spectrum 
of pure cinnamaldehyde for comparison. Spectra are normalized to the 1597 cm-1 peak for better visualization. 

Source: JRC 

Multivariate statistical analysis of Raman spectra 

Figure 8 shows an overview of the Raman spectra recorded for all the cinnamon powder samples that were 
investigated in this study. Every spectrum in the graph represents the average of 3 replicate measurements 
taken for every sample. Between every replicate measurement, the glass vial was shaken to achieve a different 
measurement point and maximum degree of homogeneity.  

Figure 8. Overview of all the Raman spectra taken on the cinnamon samples. 

Source: JRC 

26 

 
 
           
 
 
 
 
 
 
Principal component analysis (PCA) was applied after mean centering the Raman spectra in order to explore 
differences among samples, using the SIMCA 18 software (Umetrics, Sweden). Prior to this, the spectra were 
pre-processed using Standard Normal Variate (SNV). Figure 9 shows an overview of the PCA investigation for 
all  cinnamon  samples,  where  the  first  two  principal  components  explain  approximately  80%  of  the  total 
variation.  

Figure 9. PCA scores plot (p2 vs p1) of all cinnamon samples. Cassia cinnamon samples are depicted in green, 
while Ceylon labelled samples are shown in red. 

Source: JRC 

According to the PCA scores plot, the Ceylon cinnamon labelled samples (shown in red) appear highly scattered 
along the first principal component. The left part of the scores plot is mainly characterised by Ceylon samples 
that show very weak cinnamaldehyde Raman signal (shown in blue dashed circle), whereas the central part 
denotes higher cinnamaldehyde presence. On the other hand, cassia cinnamon samples are largely projected at 
the upper (central) right part of the scores plot where cinnamaldehyde is even more prominent (shown in black 
circle), while a minor number of cassia samples exhibiting predominant cinnamaldehyde Raman signal is found 
at the bottom right part of the score plot (shown in dotted purple circle). The PCA findings are corroborated by 
the loadings plots for the two principal components and illustrated in Figures 10a and 10b.  

Figure 10: (a). Loading plot for p1. (b). Loading plot for p2.  

The loadings plot for the first principal component reveals that the most prominent contribution originates from 
the  strong  doublet  found  at  1597  and  1625  cm-1,  where  the  major  part  of  the  intensity  in  these  bands  is 

Source: JRC 

27 

 
 
 
 
 
 
 
assigned  to  ring  and  C=C  stretching  vibrations  in  cinnamaldehyde,  with  additional  scattering  from  coniferyl 
aldehyde that is present in lignin (Bock & Gierlinger, 2019) and a minor contribution from coumarin, while the 
loadings plot for the second principal component shows that there is also significant enhancement from the 
1000 – 1500 cm-1 spectral range where scattering from cellulose is also observed (Agarwal et al., 2011).  

An interesting finding is that the weak presence of cinnamaldehyde depicted at the left cluster of the PCA scores 
plot  is  not  only  observed  for  Ceylon  labelled  samples,  but  also  for  a  small  number  of  cassia  cinnamon 
specimens, which is somehow counterintuitive according to literature. However, an increased amount of either 
camphor (by GC-MS analysis) and/or some mineral elements (by XRF analysis) was detected for some of these 
samples, which could indicate partial or total substitution by Cinnamomum camphora (rich in camphor and poor 
in cinnamaldehyde) and/or inorganic matter rich in these minerals, respectively. An alternative explanation for 
the presence of camphor could entail the partial or total substitution of the cinnamon bark by the root of the 
tree,  which  also  contains  camphor  in  substantial  amounts  (Rao  &  Gan,  2014).  The  substitution  of  bark  by 
another part of the plant can be also corroborated by the high ash content found in some of these samples. 
Both scenarios can explain the very low (or absent) Raman signal of cinnamaldehyde seen in these spectra, 
which provides clear evidence that these samples are suspicious.   

The remaining Ceylon labelled samples located in the center of the PCA plot are generally separated by the 
main cluster of cassia samples (in black circle) along both principal components. However, four Ceylon labelled 
samples (19, 40, 46, 50) appear within the cassia cluster indicating potential substitution of the former by the 
latter, which is in agreement with the GC-MS results. It is observed that most of the samples within the cassia 
cinnamon cluster appear shifted to the right compared to the majority of the Ceylon labelled samples located 
in the center in PC1 due to the higher cinnamaldehyde content present in the former, whereas an upshift across 
PC2  is  detected  as  a  result  of  relative  spectral  differences  originating  from  additional  Raman  scattering  of 
cellulose, lignin and coumarin. Further to the aforementioned observations, an extra spectral contribution could 
also arise from cinnamic acid which is one of the most abundant phenolic acids in cinnamon and it is found in 
cassia at higher concentrations than Ceylon (Klejdus & Kováčik, 2016), exhibiting some characteristic Raman 
peaks in the 1000 – 1700 cm-1 range (Kalinowska et., 2017), albeit with relative intensity differences compared 
to  cinnamaldehyde,  coumarin  and  lignin.  However,  if  this  phenol  acid  could  indeed  enhance  the  variations 
between cassia and Ceylon species along PC2, such an enhancement would be relatively weak, due to the low 
concentration of cinnamic acid in cinnamon powder.  

To further elucidate these findings, additional sample variation is assessed by investigating the third principal 
component,  as  shown  in  Figure  11.  Similar  to  the  PCA  scores  plot  in  Fig.  9,  it  is  observed  that  the  Ceylon 
cinnamon labelled samples (shown in red) are spread across the first principal component. The left part of the 
scores plot is mainly ascribed to Ceylon samples that exhibit very weak cinnamaldehyde Raman signal (shown 
in  blue  dashed  circle),  whereas  moving  towards  the  centre  the  presence  of  cinnamaldehyde  becomes  more 
prominent. On the other hand, cassia cinnamon samples are now projected at the centre and bottom right part 
of the scores plot, where cinnamaldehyde is systematically increasing (shown in black circle).  

In this PCA plot, cassia cinnamon samples form a well-defined cluster and the four Ceylon labelled samples 
(19, 40, 46 and 50) that are suspicious for partial or total substitution by cassia are more clearly observed now. 
The  Ceylon  and  cassia  samples  located  at  the  left  of  the  scores  plot  are  suspicious  for  substitution  by 
Cinnamomum camphora and/or cinnamon root, which is also demonstrated in the previous PCA plot. The rest 
of the Ceylon labelled samples are now located at the upper central part of the plot and they are separated 
from the main cassia cluster.    

28 

 
 
 
 
 
 
     
 
 
 
 
 
 
 
 
 
 
Figure 11: PCA scores plot (p3 vs p1) of all cinnamon samples. Cassia cinnamon samples are depicted in green, 
while Ceylon labelled samples are shown in red.  

Source: JRC 

To  provide  more  insight  to  the  qualitative assessment  of Ceylon  substitution  by  cassia,  a  semi-quantitative 
approach was applied using admixtures of Ceylon and cassia cinnamons at various relative concentrations. For 
the mixing, the Ceylon matrix is composed of a few Ceylon samples that were not flagged as suspicious from 
either Raman or other analytical techniques shown in this report. Hence, the PCA plot is constructed again with 
the inclusion of these admixtures, as seen in Figure 12.  

Cinnamon mixtures were prepared for Raman investigation by spiking Ceylon cinnamon with increasing amounts 
of cassia cinnamon, as shown in Table 5. In this investigation, sample M1 was produced by mixing three pure 
Ceylon samples together and M8 was made by mixing three pure cassia samples. The three samples used to 
prepare mixtures M1 and M8 were chosen based on their purity as seen by principal component analysis (PCA). 

Table 5. Cinnamon mixtures with nominal concentrations. 

Mixtures 

M1 

M2 

M3 

M4 

M5 

M6 

M7 

M8 

100 

95 

91 

80 

70 

50 

30 

0 

0 

5 

9 

20 

30 

50 

70 

100 

Ceylon 
(% w/w) 

Cassia 
(% w/w) 

Source: JRC 

From the PCA plot, it can be seen that the scores corresponding to the admixtures (shown in blue and expressed 
as % of cassia) are homogeneously spread mainly across the third principal component from the Ceylon region 
(center of the plot) towards the cassia region (right part of the plot). It is also observed that between 5 and 9% 
as well as 20 and 30% cassia concentrations, the relevant scores appear very close to each other due to minor 
spectral variations detected.  

It  is evident  from  the  graph that  the  5  and  9%  admixtures  fall within  the Ceylon  range,  indicating  that the 
detection of less than 10% cassia in Ceylon was not feasible. On the other hand, the 20 and 30% scores seem 
to separate somehow from the Ceylon range, although still remaining close to it and far away from the cassia 
cluster region, whereas the 50 and 70% scores are found clearly inside the cassia cluster. Taking into account 
this information, it is estimated that the level of detection (LOD) of cassia in Ceylon can be set to 20%.                   

29 

 
 
 
 
 
 
 
 
 
Figure 12: PCA scores plot (p3 vs p1) of all cinnamon samples and admixtures. Cassia cinnamon samples are 
depicted in green, Ceylon labelled samples are shown in red and admixtures are in blue. 

Source: JRC 

Overall, it was demonstrated that Raman spectroscopy combined with PCA can be a valuable screening method 
to detect the substitution of Ceylon by other species (e.g. Cinnamomum camphora), different part of the plant 
(e.g. root) or inorganic matter containing high concentrations of minerals (also in some cassia samples) through 
the absence (or nearly absence) of cinnamaldehyde as demonstrated in p1 as well as the substitution of Ceylon 
cinnamon by cassia as a result of the spectral differences determined in p1, p2 and p3 components. The method 
also provided a semi-quantitative approach to study the adulteration of Ceylon by cassia by using admixtures 
of the two cinnamon species at various relative concentrations, which showed that an LOD value of 20% can 
be used as a realistic threshold. This is the first study reporting on the development of an analytical method 
based  on  Raman  spectroscopy  combined  with  multivariate  statistics  for  screening  cinnamon  adulteration 
directly on powder samples. A general overview of the samples denoted as suspicious in the PCA plots shown 
in Figs. 9, 11, and 12 is provided in Table 2 together with the findings from other analytical techniques.   

Feasibility study using a handheld Raman device 

A Progeny (Rigaku Analytical Devices, France) equipped with a 1064 nm excitation laser and a TE-cooled InGaAs 
512 pixel detector was employed for the measurements on the cinnamon powder. The recorded spectral range 
was 200–2500 cm-1, with a spectral resolution of 8 cm-1, 150 mW power, 3000 ms acquisition time and 30 
averages. Before each measurement, a background was acquired and a baseline correction was automatically 
done.  A  daily  check  is  required  prior  to  its  use  and  is  achieved  by  acquiring  the  spectrum  of  a  benzonitrile 
reference standard.  

Similar  to  the  measurements  performed  with  the  benchtop  Raman  spectrometer,  the  samples  were  kept  in 
sealed glass vials and measured through the container, which means that no sample preparation was required. 
The glass vials were placed on the Rigaku sample holder and the measurements were carried out through the 
side of the vial, adjusting the focusing of the laser to maximize the signal. The sample holder was kept steady 
for all measurements and no interference from the glass vials was observed. 

As this is intended to be only a feasibility study, a selection of 48 (approximately 50% of the total number of 
samples)  samples  was  used  to  perform  Raman  measurements  with  the  handheld  device  for  a  preliminary 
assessment  on  cinnamon  speciation.    Figures  13a  and  13b  show  a  glass  vial  containing  cinnamon  powder 
mounted  on  the  Rigaku  handheld  device  for  Raman  measurements  and  an  overview  of  the  Raman  spectra 
recorded for the selected cinnamon powder samples, respectively.   

30 

 
 
 
 
 
 
 
 
 
Figure 13. (a). The Rigaku handheld device with a glass vial containing cinnamon powder mounted for Raman 
measurements (b). Overview of the Raman spectra taken on the selected cinnamon samples using the Rigaku 
handheld device. 

Source: JRC 

The Raman spectra presented above were pre-processed in the same manner as the one described for the FT-
Raman method and PCA was applied using the SIMCA 18 software. Figure 14 shows an overview of the PCA 
investigation for the selected cinnamon samples. 

According to the PCA scores plot, the Ceylon cinnamon labelled samples (shown in red) appear highly scattered 
along the first principal component. The left part of the scores plot is mainly characterised by Ceylon samples 
that show very weak cinnamaldehyde Raman signal, whereas cassia cinnamon samples are largely projected 
at the right part of the scores plot where cinnamaldehyde is more prominent (shown in green circle). It can be 
observed that although cassia specimens appear somehow projected together and away from the majority of 
Ceylon labelled samples, there are four Ceylon specimens projected with cassia, as shown from the black frame 
in Fig. 14. These are the 19, 40, 46 and 50 samples that were found as suspicious by FT-Raman for containing 
cassia either partially or totally. In addition, the Ceylon labelled samples that are located at the very left part 
of the PCA plot as a result of low cinnamaldehyde signal were also flagged as suspicious by the FT-Raman 
method due to their high content of either camphor or some other mineral, as demonstrated by XRF and GC-
MS analyses. However, there are still two cassia samples that appear separated from their main cluster and 
close to Ceylon species, which is somehow in contradiction to the FT-Raman findings.  

Overall, the Raman measurements taken with the Rigaku handheld device seem to be in general correlation to 
the FT-Raman results, even if not all the samples were investigated. It must be emphasized though that this is 
only a feasibility study and more experiments need to be carried out to validate these observations. In general, 

31 

 
 
 
 
 
 
 
 
the signal to noise ratio is a critical factor to ensure reliable spectroscopic measurements, which must be taken 
into consideration in order to further optimize the experimental conditions with the handheld device. 

Figure 14: PCA scores plot (p2 vs p1) of the selected cinnamon samples. Cassia cinnamon samples are depicted 
in  green  and  ceylon  labelled  samples  are  shown  in  red.  Black  circles  shows  a  cluster  for  cassia  samples 
containing some ceylon labelled samples. 

Source: JRC 

32 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5.5  Annex 5: Gas chromatography-based method 

A  method  based  on  the  use  of  Head  Space-Gas  Chromatography-Mass  Spectrometry  (HS-GC-MS)  was 
developed and in-house validated for the determination of 14 volatile compounds present in cinnamon. The 
selection of compounds to be included in the study was based on previous research on cinnamon adulteration 
carried out by unit F.4 Food Integrity, of the JRC (Ghidotti et al, 2023) and were pinene, limonene, benzyl alcohol, 
camphor,  hydrocinnamaldehyde,  anethole,  cinnamaldehyde,  eugenol,  caryophyllene,  coumarin,  cinnamyl 
acetate, eugenol acetate, caryophyllene oxide, benzyl benzoate. 

The following standards α-pinene (≥ 97%), D-limonene (≥ 95%), benzyl alcohol (≥ 99%), camphor (2-bornanone) 
(≥ 98%), hydrocinnamaldehyde (3-phenylpropanal) (≥ 95%), anethole (4-Allylanisole) (≥ 98%), cinnamaldehyde 
(≥ 99%), eugenol (≥ 99%), β-caryophyllene (purity not specified), coumarin (99+%), cinnamyl acetate (≥ 97%), 
eugenol acetate (≥ 98%), caryophyllene oxide (≥ 95%), benzyl benzoate (≥ 99%) and 2-methoxy-4-propylphenol 
(≥ 99%) were used for calibration. 

Stock standard solutions were prepared by dissolving weighted amounts of the standards in acetonitrile at 10, 
50, 100,  500 and  1000  mgL-1,  except  for  cinnamaldehyde,  for  which  the  concentrations  of  stock standard 
solutions were 100, 500, 1000, 10000, 50000 and 100000 mgL-1. 

The calibration samples were prepared by weighing aliquots of 10 µL of stock standard solutions and 10 µL of 
internal standard solutions into 20 mL screw top headspace vials. For cinnamaldehyde calibration, 20 µL of 
internal standard solutions were added instead. 

Cinnamon  sample  preparation  was  performed  by  weighing  50  mg  in 20  mL  screw top  headspace  vials  and 
spiking  the  sample  with  10  µL  internal  standard  solution  (2-methoxy-4-propylphenol).  The  analysis  of 
cinnamaldehyde required a dedicated method. Therefore, a sample replicate was prepared by weighing 50 mg 
cinnamon and spiking with 20 µL of internal standard solution. 

The samples were incubated at 150°C for 15 minutes in a ThermoPal autosampler (PAL COMBI-xt). An aliquot 
of 1mL headspace gas was injected into a Trace1310 GC by means of a gas syringe kept at 150°C. The injector 
was kept at 200°C and the injection was performed with a split ratio of 10 (Topaz Split liner 1x6.3x78 mm). 
The chromatographic separation was performed on a DB-5MS capillary column (30-m length, 0.25-mm inner 
diameter, 0.25-µm film thickness) with helium carrier gas at 1 ml min−1. The GC oven was kept at 50°C for 2 
minutes and then ramped at 5°C/min to 300°C. Mass spectra were acquired with a TSQ Quantum XLS Ultra 
mass spectrometer scanning the mass range 45-450 with scan time of 0.2 seconds.    

Cinnamaldehyde analysis was performed with a dedicated method. The vials were incubated at 150°C for 15 
minutes and 250 µL of headspace were injected into the GC, with a split ratio of 100. The oven temperature 
was kept at 50°C for 2 minutes and then ramped to 300°C at 30°C/min. Mass spectra were acquired at the 
same conditions as the other volatile compounds. 

A cinnamon mix containing 1 g of 52 cinnamon samples was prepared. A repeatability study was performed by 
analysing the cinnamon mix in three different days. In each one of the three days the analysis was performed 
in triplicate. The intermediate precision of the method calculated from the within- and between-pellet ANOVA, 
was taken as the expanded uncertainty (confidence interval 95 %, k=2) associated to the results. 

Limits of detection and quantification were estimated based on the analysis of ten blanks. For each compound, 
the average peak area of the ten blanks was compared to that of the lowest calibration point to calculate the 
LOD. LOQs valued were estimated as three times the LOD levels. 

Table 6 summarises the LOD, LOQ, the expanded uncertainty of the method covered by the calibration curves 
for the 14 compounds analysed. The thresholds used to declare a sample as outlier due to the content of a 
certain  compound  is  also  included.  Those  thresholds  are  only  indicative  and  should  be  established  in  a 
collaborative trial. 

33 

 
 
 
 
 
 
 
Table 6: LOD, LOQ, expanded uncertainty (95 % confidence interval, k=2) and thresholds used for detection of 
outliers for the 14 compounds included in the study. 

Camphor 

Hydrocinnamaldehyde 

Anethole 

Eugenol 

Pinene 

Limonene 

0.01 

0.03 

0.06 

0.18 

30.01 

27.75 

Benzyl 
alcohol 
0.41 

1.24 

19.59 

0.78 

2.34 

28.65 

0.25 

0.76 

16.00 

10.7 - 779 

12.5 - 953 

9.33 - 953 

11.8 - 969 

>150 

- 

>98 

>800 

>20 

1.45 

4.36 

26.93 

9.05 - 
943 

0.87 

2.60 

4.42 

9.20 - 915 

>5 for for 
susbstitution 
of Ceylon by 
cassia 
>100 for 
substitution 
of bark with 
leaves 

Caryophyllen
e 
0.17 

0.52 

26.02 

Coumarin 

0.50 

1.50 

26.15 

20.2 - 867 

9.81 - 883 

Cinnamyl 
acetate 
0.50 

Eugenol 
acetate 
0.17 

Caryophyllene 
oxide 
0.17 

Benzyl 
benzoate 
0.14 

1.49 

10.24 

10.1 - 
1090 

0.52 

51.44 

11.1 - 
1107 

0.51 

8.20 

9.96 - 960 

0.42 

10.96 

10.1 - 
1091 

Cinnamaldehyde 

0.52 

1.56 

21.66 

106 - 48619 

>2000 

>20 for 
substitution 
of Ceylon 
by cassia 
<20 for 
substitution 
of cassia 
bark by 
other plant 

>300 

>0.52 

>40 

>30 for 
substitution 
of Ceylon 
by cassia 

>7000 
<18000 

5.87 -
810 

>200 

LOD (mg L-1) 

LOQ (mg L-1) 
Uncertainty 
(k=2) 
Working 
range 
(mg kg-1) 

Threshold 
used for 
outliers  
(mg kg-1) 

LOD (mg L-1) 

LOQ (mg L-1) 
Uncertainty 
(k=2) 
Working 
range 
(mg kg-1) 

Threshold 
used for 
outliers  
(mg kg-1) 

Source: JRC 

Figure 15 shows the PCA score plots based on the volatile compounds included in the validation of the HS-GC-
MS method, obtained for Ceylon cinnamon, cassia and samples without indication of botanical variety (?). 

Figure 15: PCA score plot for Ceylon cinnamon, cassia and samples without botanical variety indication (?), 
based on the volatile profile of the samples. 

PCA score plot. Ground samples

5

4

3

2

1

0
-1

-2

-3

-4

-5

]
2
[
t

-10

-5

0

5

10

15

t[1]

?

Cassia

Ceylon

PCA score plot. Stick samples

8

6

4

2

0

-2

-4

-6

]
2
[
t

-6

-4

-2

0

t[1]

2

4

6

?

Cassia

Ceylon

Source: JRC 

34 

 
 
 
 
 
  
5.6  Annex 6: Feasibility study for the identification of adulterants by 

DNA-based methods 

Quantification of pepper and clove in admixed samples 

Two quantitative real-time PCR (qPCR) methods were in-house developed and validated to quantify clove and 
black pepper as known adulterants of cinnamon (Castro et al., 2023; Gopu et al., 2008). In-house validation for 
cinnamon admixed with pepper or clove is presented in this annex. Briefly, the methods are able to detect 1.1% 
m/m of clove and 0.8% m/m of pepper in cinnamon. ISO 6539-2014 (Ceylon cinnamon) and ISO 6538-1997 
(Cassia cinnamon) both tolerate up to 1% m/m of extraneous matter (leaves, stems, chaff and other vegetable 
matter  together  with  sand,  earth  and  dust)  in  whole  cinnamon.  Intermediate  precision,  LOQ,  repeatability, 
trueness and expanded measurement uncertainty set the dynamic range from 3.8% m/m to 100% m/m (clove) 
and from 2.5% m/m to 100% m/m (pepper) in a cinnamon matrix. The trueness for both methods is presented 
in Table 7. 

Table 7: Measurements and bias associated with trueness in cinnamon samples admixed with five 
concentrations of clove or pepper 

Syzygium aromaticum (clove)  

Piper nigrum (black pepper)  

adulterant 
(m/m)  

2%  

5%  

10%  

50%  

100%  

Source: JRC 

measured (% m/m)  

bias  

measured (% m/m)  

bias  

2.09  

4.96  

10.42  

38.20  

123.46  

4.7%  

-0.8%  

4.2%  

-
23.6%  

23.5%  

1.89  

4.98  

11.01  

51.85  

93.48  

-5.6%  

-0.3%  

10.1%  

3.7%  

-6.5%  

Syzygium  aromaticum  and  Piper  nigrum  were  detected  at  traces  level  in  one  commercial  sample  each. 
Additional analytical methods would be needed to confirm this observation. 

Extension to other adulterants in commercial cinnamon samples 

In addition to the detection of clove and pepper, we performed an untargeted NGS (Next Generation Sequencing) 
analysis to identify species unrelated to cinnamon in the same commercial samples, obtained by convenient 
sampling. Given the low likelihood of substitution in products sold as bark, this analysis was limited to powder 
products.  Four  molecular  markers  (rbcL,  trnL,  psbA,  ITS2),  recommended  either  by  the  Consortium  for  the 
Barcode of Life (CBOL) Plant Working Group or scientific literature (CBOL Plant Working Group et al., 2009; Zhou 
et al., 2023), were amplified by PCR and sequenced. The sequences (DNA reads) were analysed against two 
different reference databases: one with a high sensitivity but low specificity (NCBI nucleotide collection) and 
one  with  a  high  specificity  but  low  sensitivity  (BOLDv4).  The  ITS2  marker  was  found  to  be  unsuitable  for 
cinnamon samples, consistent with observations in the Lauraceae family (Liu et al., 2012). qPCR was used to 
confirm the presence of adulterants and contaminants found by NGS.  

A total of 61 commercial samples from nine different Member States were analysed. The majority of these 
samples (36/61, 59%) did not contain any suspicious species that could indicate potential adulteration. Several 
known herbs and spices adulterants were detected and confirmed by qPCR. However, these positive detections 
cannot be accurately quantified without validated methods, which are out of the scope of this agreement. 

The suspected species are grouped into three categories:  

35 

 
 
 
  
 
 
Known cinnamon adulterant, such as Coffea spp.  
Species commonly used as adulterant/filler for herbs and spices.  
Species that could potentially be used as adulterant due to their physical or chemical similarities to 
cinnamon as well as their availability in the market. 

The resulting list was compiled based on their likelihood of being an adulterant in cinnamon (Table 8). 

Table 8: Potential adulterants in commercial samples by NGS analysis 

Species 

Detection NGS 

qPCR confirmation  

(>5% reads, NCBI) 

(Cq <35) 

Category 

Coffea spp. 

Yes (trnL) 

Allium spp. 

Yes (psbA, trnL) 

Brassica spp. 

Yes (psbA, rbcLa) 

Capsicum spp. 

Yes (trnL) 

Oryza sativa 

Yes (trnL, rbcLa) 

Sinapis alba 

Yes (psbA) 

Arachis hypogaea 

No 

Bupleurum chinense 

Yes (rbcLa) 

Melilotus spp. 

Yes (psbA, trnL, 
rbcLa) 

Trigonella spp. 

Yes (psbA, trnL) 

* no primers available 

Source: JRC 

No 

Yes 

Yes 

No 

Yes 

Yes 

Yes 

No* 

No 

No 

known cinnamon adulterant 

known herbs and spices adulterant 

known herbs and spices adulterant 

known herbs and spices adulterant 

known herbs and spices adulterant 

known herbs and spices adulterant 

suspicious contaminant 

suspicious contaminant 

suspicious contaminant 

suspicious contaminant 

Litsea spp. and Ocotea spp. were identified in over 40 samples through DNA read assignment. However, due to 
their  phylogenetic  proximity  to  Cinnamomum  spp.  within  the  Laurales  order,  it  is  likely  that  their  barcode 
sequences  exhibit  a  high  degree  of  similarity,  leading  to  potential  misidentification.  As  a  result,  these 
assignments were not taken as conclusive evidence of the presence of these species.  

Known cinnamon adulterants 

Coffee  husk  (Coffea  arabica)  is  a  known  adulterant  in  cinnamon,  already  detected  in  commercial  samples 
(Coqueiro et al., 2024; Pages-Rebull et al., 2024). Low signal was detected in our NGS analysis for Coffea spp 
but none were confirmed by qPCR (Ct >35). The study by Coqueiro et al. (2024) claims that 60% of commercial 
cinnamon samples in Brazil were adulterated with coffee husk, with contamination levels ranging from 8% to 
46%. Although not confirmed in this small sample set, it is plausible that coffee husk may also be used as 
cinnamon adulterant in cinnamon products available on the European market. 

Known herbs and spices adulterants 

One commercial sample shows DNA reads associated to Oryza sativa (rice), and the qPCR analysis confirms its 
presence,  suggesting  an  adulteration  rather  than  a  contamination.  Rice  has  been  identified  as  a  potential 
adulterant in spices, including turmeric and black pepper, where it is used as a filler due to its low cost and 

36 

 
 
 
 
similar appearance to ground spices (Maquet et al., 2021; Zhou et al., 2023). Rice husk or rice grain could be 
used as a filler in cinnamon. Furthermore, other starch-based products, such as corn, have been found to be 
used as substitutes for cinnamon (Coqueiro et al., 2024). NGS/qPCR analysis revealed the presence of Allium 
cepa (onion), detected at a level (Cq<30), suggesting adulteration. Onion has been previously detected in red 
pepper / paprika commercial samples (Kang, 2018; Maquet et al., 2021).  

Other  known  herbs  and  spices  adulterants  were  detected  at  levels  suggesting  contamination  during  the 
processing or packaging stages rather than adulteration. These species include chilli/paprika (Capsicum spp.), 
Allium sativum (garlic), Brassica species and Sinapis alba (white mustard). No prior reports of these species in 
cinnamon exist, requiring further investigation.  

Suspicious contaminants 

Several species are considered as suspicious contaminants due to their physical or chemical similarities with 
cinnamon, as they were detected in traces amounts. Among these, the root of Bupleurum chinense is used in 
traditional Chinese medicine. Peanut (Arachis hypogaea) is a food allergen, as such it may trigger mild to serious 
reactions in sensitive people. Fenugreek (Trigonella foenum-graecum) is commercially available on the European 
market as a food supplement, sometimes combined with cinnamon. Common sweet clovers (Melilotus spp.) are 
adulterant of vanilla pods (Negi et al., 2021) and contain significant amount of coumarin (European Food Safety 
Authority, 2012). The coumarin content differs between and within Melilotus species and genotypes (Nair et al., 
2010). For instance, M. indicus and M. officinalis may contain up to 0.98% and 0.61% (dry weight) of coumarin, 
respectively (Nair et al., 2010), which is comparable to the coumarin content found in non-Ceylon cinnamon 
species, such as Indonesian cinnamon (Cinnamomum burmannii) and Vietnamese cinnamon (C. loureiroi) (Wang 
et al., 2013). The tolerable daily intake for coumarin is 0.1 mg / kg body weight (European Food Safety Authority, 
2008), equivalent to 1.5 g of a plant containing 0.5 % coumarin for a 75 kg-body. RASFF notification 2022.3913 
from  Estonia  official  control  on  the  market  flagged  a  coumarin  content  of  23,6  ±  4,7  mg/kg  in  a  batch  of 
cinnamon sticks, higher than the maximal limit of 15 mg/kg. 

Standard operating procedures 

Material & Methods for DNA-based analysis 

DNA extraction and QC 

DNA  extraction  protocol,  optimised  for  cinnamon  bark  (adapted  from  Swetha  et  al.,  2014),  was  performed 
manually using a CTAB extraction buffer and included cleaning steps with chloroform/isoamyl alcohol (24/1 v/v) 
combined  with  3M  sodium  acetate  (pH  5.2).  DNA  was  precipitated  overnight  at  -20°C  with  isopropanol  and 
washed thrice with ethanol 70% v/v before being resuspended in elution buffer. 

The DNA extract was quantified using a Qubit 4 fluorometer (Invitrogen) with Broad Range chemistry. Purity 
was assessed with a NanoDrop Eight spectrophotometer (ThermoFisher Scientific). 

Untargeted DNA analysis by Next-Generation Sequencing 

Four molecular markers (rbcL, trnL, psbA, and ITS2; Table 9) were selected based on recommendations from 
the Consortium for the Barcode of Life Plant Working Group or their discriminative power (Zhou et al., 2023; 
CBOL  Plant  Working  Group  et  al.,  2009).  Each  marker  was  amplified  by  PCR  using  the  Invitrogen  Platinum 
SuperFi II PCR Master Mix, following the manufacturer's instructions. PCR products were purified using the Mag-
Bind  TotalPure  NGS  magnetic  beads  (Omega  BIO-TEK)  and  quantified  by  fluorometry  using  a  Qubit  4 
fluorometer,  according  to  manufacturer’s  instructions.  Library  preparation  was  performed  according  to 
manufacturer’s instructions, either using the Ion Chef System for trnL, psbA and ITS2, or the Native Barcoding 
Kit 96 V14 for rbcLa.  

37 

 
 
 
 
 
 
 
Table 9: Primers of the markers used in this report. Sequences are indicated 5’ 3’. 

Marker  

Forward Primer 

Reverse Primer 

rbcLa 

ATGTCACCACAAACAGAGACTAAAGC  GTAAAATCAAGTCCACCRCG 

trnL 

psbA 

ITS2 

CGAAATCGGTAGACGCTACG 

GGGGATAGAGGGACTTGAAC 

GTTATGCATGAACGTAATGCTC 

CGCGCATGGTGGATTCACAATCC 

ATGCGATACTTGGTGTGAAT 

GACGCTTCTCCAGACTACAAT 

Source: JRC 

The sequencing of the molecular markers was performed using two different platforms. Specifically, the trnL, 
psbA, and ITS2 markers were sequenced on the Ion GeneStudio S5 System (ThermoFisher) utilizing an Ion Chip 
520 with the EXT chemistry, while the rbcL marker was sequenced on the MinION platform (Oxford Nanopore 
Technologies) with a R10.4.1 flowcell. This sequencing effort generated a total of 18 million reads, distributed 
as follows: 8 million reads for the rbcL marker, 4 million reads for the trnL marker, 3.7 million reads for the 
ITS2 marker, and 1.75 million reads for the psbA marker. Although ITS2 marker is reported in the scientific 
literature  as  a  discriminative  marker  for  culinary  herbs,  our  results  showed  that  it  was  unsuitable  for 
distinguishing cinnamon samples. This finding aligns with a previous study on the Lauraceae family, highlighting 
ITS2 limitations (Liu et al., 2012).  

Following  sequencing,  the  generated  reads  underwent  quality  checking  to  ensure  their  suitability  for 
downstream  analysis.  High-quality  reads  were  analysed  using  custom  Snakemake  pipelines,  which  enabled 
species  identification  through  two  complementary  approaches.  Firstly,  reads  were  assigned  to  the  NCBI  nt 
reference database using Kraken2 v2.1.3, a taxonomic classification tool (Wood et al, 2019). Secondly, reads 
were mapped to the BOLD (Barcode of Life Data) reference database for species identification based on their 
DNA barcodes (Ratnasingham et al., 2024).  

Quantitative real-time PCR 

Quantitative real-time PCR (qPCR) was used i) to confirm the presence of adulterants and contaminants 
found by NGS, ii) for the in-house validation of methods quantifying Piper nigrum (black pepper) and Syzygium 
aromaticum (clove) in a cinnamon matrix, and iii) to screen the presence of these two species in the commercial 
samples  purchased  in  powder.  qPCR  amplification  was  carried  out  in  a  20  µL  reaction  volume  using  10  µL 
PowerUp™ SYBR™ Green Master Mix, 400 nM of each forward and reverse primer and 25 ng of extracted DNA 
(whenever possible) as template. For each primer pair, a non-template control consisting in 25 ng of pure Ceylon 
cinnamon DNA was included. The amplifications were performed with a QuantStudio 7 qPCR system (Applied 
Biosystems) in 96-well plates. Primer pairs were tested for cross-reactivity with other species by qPCR using 
SYBR™ Green chemistry. The following targets were investigated (Table 10).  

38 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 10: qPCR primers used in this report. 

Species  

Allium cepa  

Target  

Maturase K  

Allium sativum  

Alliinase  

Arachis hoehnei / hypogaea 

Arah3 C  

Primers (fw / rv), 5’3’  

CACGAATACCATAATTGGAATAATCTTTA  

AGCACTAATAAAATTTCGCACTCAAA 

GCCTCATTACAGCCCAATCA 
CATCCTTTATCAACGCCCAC 

TGTTTGTCCCTCACTACAACA 
GAAGCTCCTCGTCGTACA 

Brassica spp. 

Cruciferin 

CAGCTCAACAGTTTCCAAACGA 

CGACCAGCCTCAGCCTTAAG 

Capsicum spp.  

Pun1 (acyltransferase)  

ACCTCGTCTACCACGAAATGCC  

Coffea spp.  

ATP-dependent Clp 
protease  

TGCCACCTCAACTTCCTTCCT 

TTCCGAAGTCCTGGAGAG  

CGGAGGATATCTCAATCG 

Melilotus spp.  

GA3ox1  

GTGCCGACGATACAAATGAC  

ACCCTTTCATTCTTCCAAGG 

Oryza sativa 

Phospholipase D 

TGGTGAGCGTTTTGCAGTCT 

CTGATCCACTAGCAGGAGGTCC 

Piper nigrum 

Hydroxycinnamoyl 
transferase 

GCCGCAGATTCTCAAGGA 

CGAAGTCGCCGAAGTCAT 

Sinapis alba  

MADS D  

TGAAAACTCTCTTCCCCTCTTAGG 
ACAAATGCACACAAGACAGAGATATAGA 

Syzygium aromaticum 

Elongation factor 1a 

TAGGGAGCATGCTTTGCTTG 

Trigonella foenum-graecum  

Centromere CenH3  

CCAGATACGACACTGACACGTA 

GCCAGGAATTTACCTTGTTGC 

CAAACCTATGTCGGTGTCTGA 

Reference gene  

GCTCAGTTGGTTAGAGCGTTG 

(tRNA-Ile-TAT) 

GCGACCTTCGGCTCATAA 

Source: JRC 

39 

 
 
 
 
 
6  References  

ANSES,  Opinion  of  the  French  Agency  for  Food,  Environmental  and  Occupational  Health  &  Safety  on  the 
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42 

 
 
 
 
List of abbreviations and definitions  

ATR 

CRM 

Attenuated total reflectance 

Certified Reference Materials 

EDXRF 

Energy Dispersive x-Ray Fluorescence 

EU-CCP 

EU-Coordinated Control Plan  

FDA 

FT-IR 

Food and Drug Administration 

Fourier Transform Infra-Red 

HS-GC-MS  Head Space-Gas Chromatography-Mass Spectrometry 

LOD 

LOQ 

NA 

NE 

NS 

NGS 

NIR 

PCA 

Limit of Detection 

Limit of Quantification 

Not analysed 

Not evaluated 

Not suspicious 

New Generation Sequencing 

Near Infra Red 

Principal Component Analyses  

PLS-DA 

Partial Least Square-Discriminant Analyses 

qPCR 

Quantitative real-time Polymerase Chain Reaction 

S 

SNV 

TGA 

Suspicious 

Standard Normal Variate 

Thermogravimetric analyses 

43 

 
 
 
 
 
 
List of figures  

Figure 1: Summary of information about the cinnamon test materials included in this study. 

Figure 2: Box and whisker plot for eugenol, benzyl benzoate and coumarin in Ceylon cinnamon and cassia test 
materials according to their label. 

Figure 3: Box and whisker plot for outlier and non-outlier cinnamon test materials included in the study based 
on their camphor content. 

Figure 4: Score plot based on elemental profiles determined by PCA for ground cinnamon samples. 

Figure 5. a. Cinnamon powder in glass vial for Raman measurement. b. Glass vial placed on the RAMII sample 
holder for spectrum acquisition. White arrow shows the laser light heading towards the sample and red arrow 
shows the collected backscattered light after interaction with the sample. 

Figure 6: Raman spectra of the most abundant chemical compounds present in cinnamon essential oil. 

Figure 7: Raman spectra of cassia and ceylon labelled cinnamon samples together with the reference spectrum 
of pure cinnamaldehyde for comparison. Spectra are normalized to the 1597 cm-1 peak for better visualization. 

Figure 8. Overview of all the Raman spectra taken on the cinnamon samples. 

Figure 9: PCA scores plot (t2 vs t1) of all cinnamon samples. Cassia cinnamon samples are depicted in green, 
while ceylon labelled samples are shown in red. 

Figure 10: (a). Loading plot for PC1. (b). Loading plot for PC2.  

Figure 11: PCA scores plot (t3 vs t1) of all cinnamon samples. Cassia cinnamon samples are depicted in green, 
while ceylon labelled samples are shown in red.  

Figure 12: PCA scores plot (t3 vs t1) of all cinnamon samples and admixtures. Cassia cinnamon samples are 
depicted in green, ceylon labelled samples are shown in red and admixtures are in blue. 

Figure 13. (a). The Rigaku handheld device with a glass vial containing cinnamon powder mounted for Raman 
measurements (b). Overview of the Raman spectra taken on the selected cinnamon samples using the Rigaku 
handheld device. 

Figure 14: PCA scores plot (t2 vs t1) of the selected cinnamon samples. Cassia cinnamon samples are depicted 
in green and ceylon labelled samples are shown in red. 

Figure 15: PCA score plot for Ceylon cinnamon, cassia and samples without botanical variety indication (?), 
based on the volatile profile of the samples. 

44 

 
 
 
 
List of tables  

Table 1: Summary of the findings obtained on commercially available cinnamon samples. 

Table 2: Summary of information about the test samples analysed, the decisions based on the results of the 
different analytical techniques and the final decision. Empty cells in the decisions for the different techniques: 
not suspicious. 

Table  3:  Accuracy expressed as z-score of the EDXRF method obtained using Certified Reference Materials 
(CRMs). All certified reference values, and indicative values are expressed in mg kg-1. 

Table 4: Working range, LOQs and expanded uncertainties for the elements for which a full validation could 
not be carried out. All mass fractions expressed as mg kg-1 

Table 5. Cinnamon mixtures with nominal concentrations. 

Table 6: LOD, LOQ, expanded uncertainty (95 % confidence interval, k=2) and thresholds used for detection of 
outliers or the 14 compounds included in the study. 

Table  7:  Measurements  and  bias  associated  with  trueness  in  cinnamon  samples  admixed  with  five 
concentrations of clove or pepper 

Table 8: Potential adulterant in commercial samples by NGS analysis 

Table 9: Primers of the markers used in this report. Sequences are indicated 5’ 3’ 

Table 10: qPCR primers used in this report. 

45 

 
 
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