Environ Monit Assess (2021) 193: 539

https://doi.org/10.1007/s10661-021-09302-9

GC–MS investigations of VOCs in South Indian honey

samples as environmental biomarkers
Anbalagan Srinivasan · Thiyagarajan Maruthavanan ·
Rajagopal Mayildurai · Alagunambi Ramasubbu

Received: 13 January 2021 / Accepted: 12 July 2021 / Published online: 30 July 2021
© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2021

Abstract Natural honey is a viscous liquid composed The results show the presence of nearly 540 VOCs and
of a supersaturated solution of glucose and fructose. semi-VOCs comprising alcohols, carboxylic acids, halo-
Honeybees collect nectar and convert them into honey genated hydrocarbons, furan and pyran derivatives, and
through biochemical reactions. These small creatures pyridine and pyrazine derivatives. Malonic acid (0.01–
are the major contributors to pollination and food pro- 0.18%), n-hexa decanoic acid (0.02–8.69%), 9-octa
duction for humans. At the same time, they are the worst decanoic acid (0.03–4.01%), propanoic acid (1.01%),
victims of urbanization and irrational use of pesticides, oleic acid (6.15%), and benzoic acid (1.48%) were found
insecticides, and other hazardous materials. Any distur- to be present in some of the samples. This investigation
bance to the existence of honeybees is a serious threat to would pave the way to identifying the geographical loca-
the biodiversity. The quality of a honey sample is largely tion of floral honey based on the specific VOCs present
affected by the contamination of volatile organic com- in the samples.
pounds (VOCs) due to environmental pollution. The
present study analyzes systemically 25 samples of honey Keywords Honey · Volatile organic compounds ·
harvested from the southern part of the Western Ghats Liquid–liquid extraction · Carboxylic acids · Aroma
for the probable existence of traces of toxic substances. profile
The samples were subjected to a liquid–liquid extrac-
tion process, followed by gas chromatography-mass
spectrometry (GC–MS) to identify and characterize Introduction
the hazardous substances/volatile organic compounds.
Honey, a natural carbohydrate-enriched sweetening
agent, is collected from the nectars of flowers by
A. Srinivasan · R. Mayildurai · A. Ramasubbu (*)
honeybees and is converted and stored in combs for
Post Graduate and Research Department of Chemistry,
Government Arts College (Autonomous), ripening (Buba et al., 2013). Apart from sugars and
Coimbatore 641 018, India water as the major chemical constituents (> 95%),
e-mail: [email protected] honey possesses free amino acids, vitamins, pig-
ments, proteins, and numerous other volatile
T. Maruthavanan
Department of Chemistry, SONASTARCH, Sona College organic compounds (VOCs) (Cordella et al., 2003).
of Technology (Autonomous), Salem 636 005, India Owing to the presence of such nutritional and
medicinal ingredients, honey has been employed
R. Mayildurai
as a medicinal supplement for the treatment of
Department of Chemistry, Kumaraguru College
of Technology (Autonomous), Coimbatore 641 049, India asthma, obesity, diabetes, phlegm, eye infections,

Vol.:(0123456789)
13
539 Page 2 of 11 Environ Monit Assess (2021) 193: 539

cough, thirst, hiccups, haematemesis, leprosy, worm However, to the best of our knowledge, there are
infestation, vomiting, diarrhoea, etc. Furthermore, no reports on the systematic investigation of pesti-
honey, known as madhu, in Ayurveda, is used as an cides, fungicides, and insecticides in Indian honey
external application to treat certain ailments and as samples. With regard to this fact, in the present inves-
a natural preservative and sweetener. The fatty acids tigation, 25 honey samples from the Western Ghats,
and dicarboxylic acids present in honey (royal jelly) South India, one of the top 10 world hotspot biodi-
can prevent the expansion of transplantable AKR versity zones, were collected and investigated for the
leukaemia (Townsend et al., 1961). The strong anti- existence of various VOCs by GC–MS analysis. The
microbial activity of honey is due to the presence sites are only about 20–40 km from cities with intense
of methylglyoxal, hydrogen peroxide, polyphenols, industrial activities, and hence, they were found suit-
ascorbic acid, complex carbohydrates, and proteins able to study the impact of pollutants on the quality
such as defensin-1 (Alvarez-Suarez et al., 2014). of honey sourced from these areas. The annual pre-
However, due to the modernization of agriculture, cipitation of the Western Ghats region ranges from
the excessive usage of herbicides, pesticides, fungi- 3000 to 4000 mm. The sample site is a home for Apis
cides, etc. has caused numerous problems while har- dorsata, Apis cerana, Apis florea, Apis species, and
vesting honey from flowering nectar plants. In par- Trigona iridipennis. It is hypothesized that the honey
ticular, the nectars of flowers secreted by plants are samples collected from the sampling sites contain
inadvertently contaminated with these toxic pollut- VOCs and the type and quantity of VOCs can be used
ants, and hence, the honey harvested from honeybees as a marker to identify the geographical location of
contains traces of these pollutants (Campbell et al., the floral honey and the level of pollution of the col-
2016). For instance, honeybees come into contact lection site.
with chemicals used to control the fungal growth in
plants rich in nectar (Legard et al., 2001; Mullin et al.,
2010; Pettis et al., 2012). This contamination leads to Materials and methods
a drop in honey production and affects honey quality
(DeGrandi-Hoffman et al., 2015; Simon-Delso et al., All the reagents employed in this investigation were
2014; Zhu et al., 2014). Hence, the determination of of Analar grade. The solvents (dichloromethane
such pollutants present in honey is very important. and 2-pentanol) were used after proper distillation.
Radovic et al. reported approximately 110 VOCs Honey samples were collected from 25 sites, viz.,
from 43 honey samples using dynamic headspace GC Pollachi, Marudhamalai, Thekkampatti, Karama-
MS analysis (Radovic et al., 2001). Recently, a study dai, Thirumalayampalayam, Anaimalai, Thalavadi,
found that exposure to fungicides, viz., 3-pyridinecar- Hasanoor, Dhimbam, Puliampatti, Kollathur, Kol-
boxamide derivatives and pyraclostrobin, by foraging limalai, Chakkarapatti, Coonoor, Dharapuram, Udu-
bees inhibited the electron transport chain (Campbell malaipettai, Chamraj Nagar, Gajanur, Kollegal, Bal-
et al., 2016). Approximately 99 common pesticides ligave, Nanjangud, Walayar, Ottapalam, Lakkidi, and
have been reported in honeybee wax, beebread, and Meenakshipuram (Table 1). Selected samples were
honey samples, which are threatened by honeybee analyzed and labelled. The listed honey samples were
colony disorders, bee losses, etc. (Simon-Delso et al., collected with the help of local beekeepers and were
2014). Sudden decline in honeybees due to the exces- subjected to analyses.
sive usage of pesticides, especially neonicotinoids, has
been reported (Connolly, 2017; Mitchell et al., 2017). Sample preparation: liquid–liquid extraction
The profile of VOCs in honey has been also pro-
posed as a way to detect honey adulteration (Arroyo- Fifty grammes of each of 25 different virgin honey
Manzanares et al., 2019). samples was carefully admixed with 100 mL dichlo-
The volatile characteristics of honey have been romethane ­(CH2Cl2) and shaken vigorously at
found to be dependent on the various fuels used dur- 1000 rpm for 3 h at room temperature. Fifty micro-
ing smoking of the honeycomb. Honey can be con- ­ −1) was also added as an
litres of 2-pentanol (1.0 g L
taminated by the smoke used as it can absorb vapours internal standard, and the entire matrix was kept aside
from the air (Tananaki et al. 2009). for 2 h in a separating funnel. The clear supernatant

13
Environ Monit Assess (2021) 193: 539 Page 3 of 11 539

Table 1  Honey sample S. no. Sample Location Sample zone

code and its origin of code
collection in the Western 1 HS 1 Pollachi, Coimbatore, Tamil Nadu
Ghats 2 HS 2 Marudhamalai, Coimbatore, Tamil Nadu
3 HS 3 Thekkamatti, Coimbatore, Tamil Nadu
4 HS 4 Karamadai, Coimbatore, Tamil Nadu
5 HS 5 Thirumalayampalayam, Coimbatore, Tamil Nadu
6 HS 6 Anaimalai, Coimbatore, Tamil Nadu
7 HS 7 Thalavadi, Erode, Tamil Nadu
8 HS 8 Hasanoor, Erode, Tamil Nadu
9 HS 9 Dhimbam, Erode, Tamil Nadu
10 HS 10 Puliampatti, Erode, Tamil Nadu
11 HS 11 Kollathur, Salem, Tamil Nadu
12 HS 12 Kollimalai, Salem, Tamil Nadu
13 HS 13 Chakkarapatti, Dharmapuri, Tamil Nadu
14 HS 14 Coonoor, The Nilgiris, Tamil Nadu
15 HS 15 Dharapuram, Tirupur, Tamil Nadu
16 HS 16 Udumalaipettai, Tirupur, Tamil Nadu
17 HS 17 Chamraj Nagar, Karnataka
18 HS 18 Gajanur, Shimoga, Karnataka
19 HS 19 Kollegal, Chamraj Nagar, Karnataka
20 HS 20 Balligave, Shimoga, Karnataka
21 HS 21 Nanjangud, Mysore, Karnataka
22 HS 22 Walayar, Palakkad, Kerala
23 HS 23 Ottapalam, Palakkad, Kerala
24 HS 24 Lakkidi, Palakkad, Kerala
25 HS 25 Meenakshipuram, Palakkad, Kerala

organic layer was collected and subjected to GC–MS peaks. However, as per the USEPA, WHO, and CPCB
analysis (Castro-Vázquez et al., 2003). standards, the compounds classified as VOCs were
A Shimadzu QP 2010 GC MS instrument equipped singled out selectively and were grouped into various
with an Rtix 5 MS column has been used to analyze categories according to their functional groups. The
the honey samples. The samples were introduced to compounds were categorized as carboxylic acids (12
the column using a split/splitless injector. The carrier different acids), furans and pyrans (7 compounds),
gas at a linear velocity of 36.8 cm/s using the line- alcohols and phenols (13 compounds), halogen-
of-sight interface was kept at 260 °C, whereas the ated hydrocarbons (12 compounds), and pyridine
ion-source temperature was set to 250 °C. The liquid and pyrazine derivatives (3 compounds). The aroma
extraction of honey was manually injected and main- description of honey samples based on the obtained
tained at 1.0 mL/min. The flowrate was maintained VOCs is shown in Table 7. All the mass spectra of
in splitless mode, the column oven temperature was identified or characterized VOCs were compared with
maintained at 80 °C, and the injection temperature Wiley MS (McLafferty & Stauffer, 1989) and the
was maintained at 250 °C. The mass spectral data of NIST library.
VOCs were acquired in the range 50–800 m/z with an
electron impact source of 70 eV. Aroma profile

Determination of VOC by GC–MS The other main characteristic features of honey are
aroma and flavour, caused by the presence of VOCs at
The honey samples were subjected to GC–MS analy- low concentrations. In general, a light-coloured honey
ses for the detection and determination of various is mild in flavour, and a darker honey has a more pro-
VOCs present in them, and the results are given in nounced flavour (Castro-Vázquez et al., 2006). Since
Tables 2, 3, 4, 5, and 6. The liquid–liquid extraction the aroma and flavour are largely dependent upon the
followed by GC MS analysis indicated that there VOCs (Baroni et al., 2006), they were predicted by
were 540 compounds with an overall retention of 883 comparing with standards reported in the literature.

13
539 Page 4 of 11 Environ Monit Assess (2021) 193: 539

Table 2  Distribution of carboxylic acids in honey samples (in peak area percentage)
S. no VOCs HS 1 HS 2 HS 3 HS 4 HS 5 HS 6 HS 7 HS 8 HS 9 HS 10 HS 11 HS 12 HS 13

1 Acetic acid - - 0.89 - 2.15 - - - 0.95 0.58 - - -

2 Propanoic acid 1.01 - - - - - - - - - - - -
3 Butanoic acid 6.05 0.02 - - - - 0.03 0.13 - - 0.02 2.2 1.7
4 Oxalic acid - 0.02 - - 1.21 0.54 0.04 - - - 0.04 0.1 -
5 Malonic acid - - - - - - 0.18 - - - - 0.03 0.01
6 Ascorbic acid - - - - - 1.35 - - - -
7 Benzoic acid 1.48 - - - - - - - - - - - -
8 Phthalic acid 0.4 - - 2.6 - - - - - - - - -
9 Tetradecanoic acid - - - - - - - - - - - 0.07 -
10 Hexadecanoic acid - 0.86 - - - - 1.82 0.02 - - - 8.69 -
11 Octadecanoic acid - 3.2 - - - 0.25 4.01 1 - 1.07 - 1.71 -
12 Oleic acid - - - - - - - - - - - 6.15 -
S. no VOCs HS 14 HS 15 HS 16 HS 17 HS 18 HS 19 HS 20 HS 21 HS 22 HS 23 HS 24 HS 25

1 Acetic acid - - - - - - - 0.33 0.77 0.24 1.23 -

2 Propanoic acid - - - - - - - - - - - -
3 Butanoic acid 0.3 2 - 1.3 - - 0.89 - - - - -
4 Oxalic acid - - - - - - - - - - -
5 Malonic acid - - - - - - - - - - - -
6 Ascorbic acid - - - - - - - - - - -
7 Benzoic acid - - - - - - - - - - - -
8 Phthalic acid 0.07 - - - - - 1.03 - - - - -
9 Tetradecanoic acid - - - - - - - - - - - -
10 Hexadecanoic acid - 0.2 - 2.19 0.56 - - - - - 1.68
11 Octadecanoic acid 0.38 0.03 0.97 0.52 0.18 - - 0.65 0.88 0.59 - -
12 Oleic acid - - - - - - - - - - - -

Results and discussion (0.01–0.18%), n-hexadecanoic acid (0.02–8.69%),

9-octa decanoic acid (0.03–4.01%), propanoic acid
Carboxylic acids have different aromas ranging from (1.01%), oleic acid (6.15%), and benzoic acid (1.48%)
spicy to rancid depending on the length of the chain. were identified, which have not been reported earlier.
Honey contains a range of carboxylic acids, and Most of the animal and vegetable fats, especially
the type of acids is responsible for the characteris- butterfat, coconut, palm, and nutmeg oils, contain
tic aroma (Shimoda et al., 1996). Butanoic acid was tetradecanoic acid, which is a saturated 14-carbon
found in all the samples, and the percentage (relative fatty acid. As lower homologues are destroyed in
to the total VOCs) ranged between 0.02 and 6.05%. It the due course of storage, a higher fatty acid such as
can result in pungent smells in honey samples. Apart hexadecanoic acid might be produced by the oxida-
from the butanoic acid and derived compounds, tion of hexadecanal (Moreira et al., 2010). Oxalic
other acids, such as acetic and 2-ethyl-hexanoic acids acid is naturally present in many naturally occurring
were also identified in all the samples. The aliphatic organic acids and was found in HS 2, HS 5, HS 6, HS
acids such as ascorbic acid and acetic acid present 7, HS 11, and HS 12 at low levels. Phthalic acid was
in honey samples may be due to bee metabolism. observed in honey samples HS 1, HS 4, HS 14, and
These acids have been identified as VOCs (De Maria HS 20. However, it should be noted that phthalic acid
& Moreira, 2003; DelaFuente et al., 2007). Interest- is not a natural component. Therefore, it was prob-
ingly, in our study, organic acids such as malonic acid ably a contaminant (Kaskoniene et al., 2010). The

13
 Table 3  Distribution of furan and pyran derivatives in honey samples (in peak area percentage)
S. no VOCs HS 1 HS 2 HS 3 HS 4 HS 5 HS 6 HS 7 HS 8 HS 9 HS 10 HS 11 HS 12 HS 13
Environ Monit Assess (2021) 193: 539

1 2-Furan-carboxaldehyde 8.39 1.21 3.21 - 1.54 - - - - - 44.86 - 0.02

2 2-Furan methanol 3.15 - - - - - 0.71 - - - - -
3 1,3-Diphenyl-isobenzofuran 1.83 - - - 0.56 - - - - - - -
4 2,5-Furan-dicarboxaldehyde - - - - - - - - - 1.82 - - -
5 2-N-Butyltetrahydrofuran - - - - - - - - - - - - -
6 Pyrano(3,2-B)-pyran-2(3H)-one-3D 25.65 - 0.53 1.58 - 0.48 - -
7 2-Hydrogen-pyran-2-methanol - - 0.21 - - - - -
S. no VOCs HS 14 HS 15 HS 16 HS 17 HS 18 HS 19 HS 20 HS 21 HS 22 HS 23 HS 24 HS 25

1 2-Furan-carboxaldehyde - 39.84 - - - 0.54 0.75 - - - 2.3 1.36

2 2-Furan methanol - - - - 0.41 - 2.89 - - - - -
3 1,3-Diphenyl-isobenzofuran - - - - - - - - - - - -
4 2,5-Furan-dicarboxaldehyde - 1.23 - - - - - - - - - -
5 2-N-Butyltetrahydrofuran - - - 0.3 - - - - - - - -
6 Pyrano(3,2-B)-pyran-2(3H)-one-3D - - - 9.21 - - - - - - - -

7 2-Hydrogen-pyran-2-methanol - - - 5.01 - - - - - - - -
Page 5 of 11

13
539
539 Page 6 of 11 Environ Monit Assess (2021) 193: 539

Table 4  Distribution of alcohols in honey samples (in peak area percentage)

S.no VOCs HS 1 HS 2 HS 3 HS 4 HS 5 HS 6 HS 7 HS 8 HS 9 HS 10 HS 11 HS 12 HS 13

1 Phenol 13.92 0.69 0.98 2.22 - - 0.38 0.15 - - 0.71 0.03 4.1
2 1-Pentacosanol - 0.46 - 0.56 - - 0.96 - - - 0.21 0.95 -
3 1-Eicosanol - - - - 1.52 - - - - - 0.05 0.42 -
4 o–o-Biphenol - - - - - - - - - - 0.01 - 0.27
5 N-Pentadecanol - 0.13 - - - - - - - - - - -
6 1-Hexadecanol - - - - - - - - 0.27 - - - -
7 1-Heptacosanol - - - - - - - - - - - 1.81 -
8 1-Tetracosanol - - - - - - - 1.67 - - - 2.81 -
9 1-Pentacosanol - - - - - - - 2.65 - - - - -
10 Ethanol - - - - - - - - - 0.66 0.64 - -
11 2-Butanol - - - - - - - - - - - - 0.31
12 3-Pentanol 11.2 - - - - - - - - - - - -
13 3-Tetradeccanol 1.48 - - - - - - - - - - - -
S.no VOCs HS 14 HS 15 HS 16 HS 17 HS 18 HS 19 HS 20 HS 21 HS 22 HS 23 HS 24 HS 25

1 Phenol 0.12 1.21 0.68 2.44 - - 0.84 - - - - 1.02

2 1-Pentacosanol - - - 0.66 - - - - - - - -
3 1-Eicosanol 0.57 0.17 0.38 0.09 - - - - - - 0.53 -
4 o–o-Biphenol - - - 0.54 - - - - 0.41 - - -
5 N-Pentadecanol - 0.06 - - 0.52 - - 0.11 - - - 1.58
6 1-Hexadecanol - - - - - - - - - - - -
7 1-Heptacosanol - - 0.13 0.3 - - - - - - - -
8 1-Tetracosanol - - - - - 1.23 - - - 0.91 - -
9 1-Pentacosanol 0.15 - 0.46 - - - - - - 0.02 - -
10 Ethanol - 0.15 - - - - - - - - - 0.95
11 2-Butanol - - - - - - - - - - - -
12 3-Pentanol - - - - - - - - - - - -
13 3-Tetradeccanol - - - - - 1.23 - - - - - -

aromatic acids of honey contribute flavours to honey. Ando, 1979). 2-Furan-carboxaldehyde (0.02–44.86%);
The presence of free and bound aromatic acids was 2-furan methanol (0.71–3.15%); 1,3-diphenyl-isobenzo
reported in monofloral (from a single type of flower) furan (0.56–1.83%); and 2-n-butyl tetrahydrofuran
varieties of honey. Their presence is used to describe (0.3%) are furan-derived compounds that were identi-
the sources of honey. fied in the honey samples.
Furan and pyran analogues may be produced due to The existence of furan derivatives indicates a pos-
firing. However, the various investigated honey sam- sible loss in freshness with a long period of storage at
ples did not contain any amino acid residues; hence, the high temperature (Sancho et al., 1992). Previous stud-
existence of furan/pyran derivatives might be high due ies (Barra et al., 2010; Castro-Vázquez et al., 2007)
to the thermal decomposition of fructose or any other also support the above observation.
sugars (Cuevas-Glory et al., 2007; Seok et al., 2015). The pyran derivative, pyrano(3,2-B)-pyran-
1,3-Diphenyl-isobenzofuran was found in samples 2(3H)-one-3D, was present in the range from 0.48
HS1 and HS5. Although the inference of VOCs was to 25.65% in samples except for HS3, HS5, HS6,
aimed at pollutants, this shed light on the existence of and HS10. 2-H-pyran-2-Methanol was found in HS7
various vital antioxidant compounds, e.g. 1,3-diphenyl- (5.01%,). Apart from this, 2–3-dihydro-3,5-dihydroxy-
isobenzofuran, as free radical scavengers (Kobayashi & 6-methyl-4H-pyran-4-one was also identified. The

13
 Table 5  Distribution of halogenated hydrocarbons in honey samples (in peak area percentage)
S. no VOCs HS 1 HS 2 HS 3 HS 4 HS 5 HS 6 HS 7 HS 8 HS 9 HS 10 HS 11 HS 12 HS 13

1 Acetic acid, dichloro- - - - - - - - - 12.79 14.94 - - -

2 1-Chloroheptacosane - - - - - - - - - - - - 1.2
3 2-Bromotetradecane - - - - - - - - - - - - 0.09
4 22,23-Dibromostigmasterol acetate - - - - - - - - - - - - 0.23

5 1-Bromodocosane - - - - - - - - - - - - 0.01
6 Triacontane, 1-bromo- - - - - - - - - - - - - 0.01
Environ Monit Assess (2021) 193: 539

7 Nonane, 2-bromo-5-ethyl- 0.07 - - - - - - - - - - -

8 Docosane, 1,22-dibromo- 0.06 - - - - - - - - - - - -

9 Octadecane, 1-iodo- 0.14 - - - - - - - - - - - -

10 Nonadecyltrifluoroacetate - - - - - - - - - - - 2.25 -

11 Heptacosylheptafluorobutyrate - - - - - - - - - - - 3.28 -

12 Hexatriacontylpentafluoropropionate - - - - - - - 0.05 - - - -

S. no VOCs HS 14 HS 15 HS 16 HS 17 HS 18 HS 19 HS 20 HS 21 HS 22 HS 23 HS 24 HS 25

1 Acetic acid, dichloro- - - - - - - - 16.01 9.88 18.61 13.62 -

2 1-Chloroheptacosane - - - - - - - - - - - -
3 2-Bromotetradecane - - - - - - - - - - - -
4 22,23-Dibromostigmasterol acetate - - - - - - - - - - - -

5 1-Bromodocosane - - - - - - - - - - - -
6 Triacontane, 1-bromo- - - - - - - - - - - - -
7 Nonane, 2-bromo-5-ethyl- - - - - - - - - - - - -

8 Docosane, 1,22-dibromo- - - - - - - - - - - - -

9 Octadecane, 1-iodo- - - - - - - - - - - - -
10 Nonadecyltrifluoroacetate - - - - - - - - - 2.2 1.3 -
Page 7 of 11

13
539

11 Heptacosylheptafluorobutyrate - - 1.45 1.52 - - - - - - - -

 Table 5  (continued)
S. no VOCs HS 14 HS 15 HS 16 HS 17 HS 18 HS 19 HS 20 HS 21 HS 22 HS 23 HS 24 HS 25
12 Hexatriacontylpentafluoropropionate - - - 2.3 - - - - - - - -

13
539 Page 8 of 11

Table 6  Distribution of pyridine and pyrazine derivatives in honey samples (in peak area percentage)
S. no VOCs HS 1 HS 2 HS 3 HS 4 HS 5 HS 6 HS 7 HS 8 HS 9 HS 10 HS 11 HS 12 HS 13

1 3-Pyridinecarboxamide, n-methyl - - 8.34 - 0.03 - - - - - 0.02 - 1.40

2 2-Acetoxy-5-methylpyrazine - - - - - - - - - - - 5.19

3 Pyrazine,2,5-dimethyl- - - - - - - - - - - - - -
S. no VOCs HS 14 HS 15 HS 16 HS 17 HS 18 HS 19 HS 20 HS 21 HS 22 HS 23 HS 24 HS 25

1 3-Pyridinecarboxamide, n-methyl - 0.6 - - - - - - - - - 0.96

2 2-Acetoxy-5-methylpyrazine - - - - - - - - - - - -

3 Pyrazine,2,5-dimethyl- - 0.1 - - - - - - - - - -
Environ Monit Assess (2021) 193: 539
Environ Monit Assess (2021) 193: 539 Page 9 of 11 539

Table 7  Aroma description S. no VOCs identified Aroma descriptions Samples

of honey samples based on
obtained VOCs 1 Carboxylic acid Spicy All the samples
Spicy to rancid (HS1 to HS25)
2 Alcohols and aldehydes Sweet, almond, marzipan
(Cosanal, Eicosanol, etc.) Aldehyde, citrus, fatty floral green,
spiny green, sweet, oily
3 Hydrocarbons Sweet, mild orange
4 Furfural Sweet, fruit, cherry, soft almond HS 1, 2, 3, 5, 8, 10,
11, 13, 15, 17,
24, 20

production of pyran-derived compounds from the The GC MS analyses revealed that a few honey
Maillard reaction indicates the alteration of taste and samples were contaminated with traces of halogenated
aroma of the honey samples (Castro-Vázquez et al., hydrocarbons, which might be attributed to the introduc-
2006; Clarke & Ndip, 2011). Furan and pyran com- tion of pesticidal/insecticidal pollutants in the environ-
pounds lead to the formation of dioxane. ment. 1-Chloroheptacosane (HS13), 1-bromodocosane,
Phenols and alcohols are important compounds in were present in the honey samples. 2-Bromotetradecane;
honey. Phenol ranging from 0.15 to 5.06% was identified 22,23-dibromostigmasterol acetate; and heptafluorobu-
in all the samples. Apart from phenol, other alcohols, tyrate were detected in the samples HS13 and HS17
such as 1-penta1-hexacosanol, butanol, ethanol, 1-eicos- (Chowdhury et al., 2013).
anol, and X-decanol (X = N-penta, 1-hepta, 1-penta, Table 7 presents the correlation between the VOC
1-hexa, and 1-un), were observed at reasonable percent- and aroma of honey. Carboxylic acids were observed
ages by GC–MS. Alcohols are associated with the fresh (both long- and short-chain acids) in all the honey
flavour of honey (Castro-Vázquez et al., 2007). Reports samples irrespective of the geography of the site.
on volatile alcohols, namely, phenyl–ethyl alcohol and This implies that the honey samples had a spicy fla-
propanol, are found in the literature. However, no earlier vour and/or spicy to rancid flavour. Furfural was also
reports on the existence of such alcohols under investiga- observed in most of the honey samples, which leads
tion have been reported. Researchers have suggested that to the sweet, fruity, cherry, and soft almond flavour
the presence of alcoholic compounds in honey depends of honey.
on the extraction method (Alissandrakis et al., 2005; De As beeswax contains mostly higher homologous
Maria & Moreira, 2003; Serra Bonvehí & Ventura Coll, hydrocarbons and their respective fatty acid esters, it was
2003). The extraction methods such as hydro distilla- expected that honey samples would be no exception and
tion (HD), liquid–liquid extraction (LLE), simultaneous would be enriched with fatty acids and fatty acid esters.
steam distillation extraction (SDE), or Likens-Nicker- Earlier reports have suggested that honey has excellent
son simultaneous distillation extraction (LNSDE) and antimicrobial, antifungal, and anticancer activities. These
micro-simultaneous steam distillation-solvent extrac- biological activities are attributed to the existence of
tion (MSDE) can produce furan and pyran derivatives various fatty acids and their esters. However, under the
(Cuevas-Glory et al., 2007). Furthermore, essential com- pretext of modern agriculture and the green revolution,
pounds that contribute to the aroma may be decomposed agricultural industries are completely hampered by the
on heating (Alissandrakis et al., 2005). excessive usage and exploitation of fertilizers, pesticides,
Volatile organic compounds like hexane, decane, fungicides, and herbicides, etc. The apiculture industries
benzene, and benzaldehyde identified in several and honeybee rearing are completely affected. Owing to
honey samples (Serra Bonvehí & Ventura Coll, 2003; these anti-natural cultures by most of the farming indus-
Shimoda et al., 1996) are considered the major con- tries and farmers, it is feared that the honey samples har-
tributors to the characteristic aroma of honey sam- vested from the southwestern zone of the Western Ghats
ples. To the best of our knowledge, this is the first (honey samples under investigation) might lose their
attempt in South India to profile locally produced purity and be contaminated with these dangerous VOCs
honey based on its VOC content. and other chemicals.

13
539 Page 10 of 11 Environ Monit Assess (2021) 193: 539

Conclusion microextraction−gas chromatography−mass spectrom-

etry coupled to chemometrics. Journal of Agricultural
and Food Chemistry, 54(19), 7235–7241. https://​doi.​
Gas chromatography-mass spectral studies revealed org/​10.​1021/​jf061​080e
the presence of more than 540 volatile and semi- Barra, M. P. G., Ponce-Díaz, M. C., & Venegas-Gallegos, C.
volatile organic compounds in the honey samples (2010). Volatile compounds in honey produced in the
collected from the Western Ghats of South India central valley of Ñuble Province, Chile. Chilean Journal
of Agricultural Research, 70, 75–84.
through a liquid–liquid solvent extraction procedure. Buba, F., Gidado, A., & Shugaba, A. (2013). Physicochemi-
The VOCs present in the samples were of five cat- cal and microbiological properties of honey from North
egories, namely, alcohols, carboxylic acids, halogen- East Nigeria. Biochemistry and Analytical Biochemistry,
ated hydrocarbons, furan and pyran derivatives, and 2(142), 61–67.
Campbell, J. B., Nath, R., Gadau, J., Fox, T., DeGrandi-Hoffman,
pyridine and pyrazine derivatives. The GC–MS data G., & Harrison, J. F. (2016). The fungicide Pristine® inhib-
were correlated with the Wiley MS and NIST librar- its mitochondrial function in vitro but not flight metabolic
ies. Additionally, the study established the presence rates in honeybees. Journal of Insect Physiology, 86, 11–16.
of malonic acid, n-hexadecanoic acid, 9-octa deca- https://​doi.​org/​10.​1016/j.​jinsp​hys.​2015.​12.​003
Castro-Vázquez, L., Díaz-Maroto, M. C., & Pérez-Coello, M.
noic acid, propanoic acid, oleic acid, and benzoic acid S. (2006). Volatile composition and contribution to the
hitherto unreported in honey samples. This prelimi- aroma of Spanish honeydew honeys. Identification of a
nary study, on further extension, would help identify new chemical marker. Journal of Agricultural and Food
the botanical and geographical origin of a honey sam- Chemistry, 54(13), 4809–4813. https://​doi.​org/​10.​1021/​
jf060​4384
ple if a database is created based on large number of Castro-Vázquez, L., Díaz-Maroto, M. C., & Pérez-Coello, M.
samples. Thus, the type and amount of VOCs could S. (2007). Aroma composition and new chemical markers
act as an environmental biomarker. of Spanish citrus honeys. Food Chemistry, 103(2), 601–
606. https://​doi.​org/​10.​1016/j.​foodc​hem.​2006.​08.​031
Acknowledgements Dr. A.R. gratefully acknowledges UGC, New Castro-Vázquez, L., Pérez-Coello, M. S., & Cabezudo, M.
Delhi, India, for financial assistance in the form of the Major Research D. (2003). Analysis of volatile compounds of rosemary
Project (UGC MRP: 39-723/2010(SR) dated 11/01/2011). Mr. R.M. honey. Comparison of Different Extraction Techniques.
expresses his heartfelt gratitude to the Management of Kumaraguru Chromatographia, 57(3–4), 227–233. https://​doi.​org/​10.​
College of Technology (Autonomous), Coimbatore, 641049, India, 1007/​BF024​91721
for constant encouragement. Chowdhury, M., Razzaque, M., Sultana, N., Mustafiz, S.,
Akter, S., Akter, A., et al. (2013). Chlorinated pesticide
residue status in some winter vegetables. Intl. J. Agric.
Data availability The datasets generated and analyzed dur- Crop Science, 6(11), 667–675.
ing the current study are available from the corresponding Clarke, A. M., & Ndip, N. (2011). Identification of volatile
author on reasonable request. compounds in solvent extracts of honeys produced in
South Africa. African Journal of Agricultural Research,
6(18), 4327–4334.
Connolly, C. N. (2017). Nerve agents in honey. Science,
References 358(6359), 38–39. https://​doi.​org/​10.​1126/​scien​ce.​aao60​00
Cordella, C. B. Y., Militão, J. S. L. T., Clément, M. C., &
Alissandrakis, E., Tarantilis, P. A., Harizanis, P. C., & Polissiou, Cabrol-Bass, D. (2003). Honey characterization and
M. (2005). Evaluation of four isolation techniques for honey adulteration detection by pattern recognition applied on
aroma compounds. Journal of the Science of Food and Agri- HPAEC-PAD profiles. 1. Honey floral species charac-
culture, 85(1), 91–97. https://​doi.​org/​10.​1002/​jsfa.​1934 terization. Journal of Agricultural and Food Chemistry,
Alvarez-Suarez, J. M., Gasparrini, M., Forbes-Hernández, T. 51(11), 3234–3242. https://​doi.​org/​10.​1021/​jf021​100m
Y., Mazzoni, L., & Giampieri, F. (2014). The composition Cuevas-Glory, L. F., Pino, J. A., Santiago, L. S., & Sauri-Duch, E.
and biological activity of honey: A focus on manuka honey. (2007). A review of volatile analytical methods for determin-
Foods, 3(3), 420–432. https://​doi.​org/​10.​3390/​foods​30304​20 ing the botanical origin of honey. Food Chemistry, 103(3),
Arroyo-Manzanares, N., García-Nicolás, M., Castell, A., Campillo, 1032–1043. https://​doi.​org/​10.​1016/j.​foodc​hem.​2006.​07.​068
N., Viñas, P., López-García, I., & Hernández-Córdoba, M. Delafuente, E., Sanz, M., Martinezcastro, I., Sanz, J., &
(2019). Untargeted headspace gas chromatography—Ion Ruizmatute, A. (2007). Volatile and carbohydrate
mobility spectrometry analysis for detection of adulterated composition of rare unifloral honeys from Spain. Food
honey. Talanta, 205, 120123. https://​doi.​org/​10.​1016/j.​talan​ta.​ Chemistry, 105(1), 84–93. https://​doi.​org/​10.​1016/j.​
2019.​120123 foodc​hem.​2007.​03.​039
Baroni, M. V., Nores, M. L., Díaz, M. P., Chiabrando, G. A., De Maria, C. A. B., & Moreira, R. F. A. (2003). Compostos
Fassano, J. P., Costa, C., & Wunderlin, D. A. (2006). voláteis em méis florais. Química Nova, 26(1), 90–96.
Determination of volatile organic compound patterns https://​doi.​org/​10.​1590/​S0100-​40422​00300​01000​16
characteristic of five unifloral honey by solid-phase

13
Environ Monit Assess (2021) 193: 539 Page 11 of 11 539

DeGrandi-Hoffman, G., Chen, Y., Watkins Dejong, E., Chambers, GC–MS analysis of aroma compounds to authenticity test-
M. L., & Hidalgo, G. (2015). Effects of oral exposure to fun- ing of honey. Food Chemistry, 72(4), 511–520. https://​doi.​
gicides on honey bee nutrition and virus levels. Journal of org/​10.​1016/​S0308-​8146(00)​00263-6
Economic Entomology, 108(6), 2518–2528. https://​doi.​org/​ Sancho, M. T., Muniategui, S., Huidobro, J. F., & Simal
10.​1093/​jee/​tov251 Lozano, J. (1992). Aging of honey. Journal of Agricul-
Kaškonienė, V., Venskutonis, P. R., & Čeksterytė, V. (2010). tural and Food Chemistry, 40(1), 134–138. https://​doi.​org/​
Carbohydrate composition and electrical conductivity of 10.​1021/​jf000​13a026
different origin honeys from Lithuania. LWT – Food Sci- Seok, Y. J., Her, J. Y., Kim, Y. G., Kim, M. Y., Jeong, S. Y.,
ence and Technology, 43(5), 801–807. https://​doi.​org/​10.​ Kim, M. K., & Lee, K. G. (2015). Furan in thermally pro-
1016/j.​lwt.​2010.​01.​007 cessed foods—A review. Toxicological Research, 31(3),
Kobayashi, S., & Ando, W. (1979). Co-oxidations of 241–253. https://​doi.​org/​10.​5487/​TR.​2015.​31.3.​241
1,3-diphenylisobenzofuran by the Haber-Weiss reaction. Serra Bonvehí, J., & Ventura Coll, F. (2003). Flavour index
Is singlet oxygen concerned in this oxidation? Biochemi- and aroma profiles of fresh and processed honeys. Journal
cal and Biophysical Research Communications, 88(2), of the Science of Food and Agriculture, 83(4), 275–282.
676–681. https://​doi.​org/​10.​1016/​0006-​291X(79)​92101-6 https://​doi.​org/​10.​1002/​jsfa.​1308
Legard, D. E., Xiao, C. L., Mertely, J. C., & Chandler, C. K. Shimoda, M., Wu, Y., & Osajima, Y. (1996). Aroma com-
(2001). Management of botrytis fruit rot in annual winter pounds from aqueous solution of haze (Rhus succedanea)
strawberry using captan, thiram, and iprodione. Plant Dis- honey determined by adsorptive column chromatogra-
ease, 85(1), 31–39. https://​doi.​org/​10.​1094/​PDIS.​2001.​ phy. Journal of Agricultural and Food Chemistry, 44(12),
85.1.​31 3913–3918. https://​doi.​org/​10.​1021/​jf960​1168
McLafferty, F. W., & Stauffer, D. B. (1989). The Wiley/NBS Simon-Delso, N. San Martin, G. Bruneau, E., Minsart, L. A.,
registry of mass spectral data, 1. Wiley. Mouret, C., & Hautier, L. (2014). Honeybee colony disor-
Mitchell, E. A. D., Mulhauser, B., Mulot, M., Mutabazi, A., der in crop areas: the role of pesticides and viruses. PloS
Glauser, G., & Aebi, A. (2017). A worldwide survey of one, 9(7), e103073.
neonicotinoids in honey. Science, 358(6359), 109–111. Tananaki, C., Gounari, S., & Thrasyvoulou, A. (2009). The
https://​doi.​org/​10.​1126/​scien​ce.​aan36​84 effect of smoke on the volatile characteristics of honey.
Moreira, J. D., Knorr, L., Ganzella, M., Thomazi, A. P., de Journal of Apicultural Research, 48(2), 142–144. https://​
Souza, C. G., de Souza, D. G., Souza, D. O. (2010). doi.​org/​10.​3896/​IBRA.1.​48.2.​10
Omega-3 fatty acids deprivation affects ontogeny of gluta- Townsend, G. F., Brown, W. H., Felauer, E. E., & Hazlett, B.
matergic synapses in rats: Relevance for behavior altera- (1961). Studies on the in vitro antitumor activity of fatty
tions, Neurochemistry International, 56(6–7) (pp. 753– acids: iv. The esters of acids closely related to 10-hydroxy-
759), ISSN 0197–0186. https://​doi.​org/​10.​1016/j.​neuint. 2-decenoic acid from royal jelly against transplantable mouse
Mullin, C. A., Frazier, M., Frazier, J. L., Ashcraft, S., Simonds, leukemia. Canadian Journal of Biochemistry and Physiology,
R., VanEngelsdorp, D., & Pettis, J. S. (2010). High levels 39(11), 1765–1770. https://​doi.​org/​10.​1139/​o61-​195
of miticides and agrochemicals in North American apiar- Zhu, W., Schmehl, D. R., Mullin, C. A., & Frazier, J. L. (2014).
ies: Implications for honey bee health. PLoS One, 5(3), Four common pesticides, their mixtures and a formulation
e9754. https://​doi.​org/​10.​1371/​journ​al.​pone.​00097​54 solvent in the hive environment have high oral toxicity to
Pettis, J. S., Johnson, J., & Dively, G. Pesticide exposure in honeybee larvae. PLoS One, 9(1), e77547. https://​doi.​org/​
honey bees results in increased levels of the gut pathogen 10.​1371/​journ​al.​pone.​00775​47
Nosema. Naturwissenschaften, 99(2), 153–158. https://​
doi.​org/​10.​1007/​s00114-​011-​0881-1 Publisher’s Note Springer Nature remains neutral with regard
Radovic, B. S., Careri, M., Mangia, A., Musci, M., Gerboles, M., to jurisdictional claims in published maps and institutional
& Anklam, E. (2001). Contribution of dynamic headspace affiliations.

13