Food Anal.
Methods
DOI 10.1007/s12161-014-9980-x
GC–MS Characterization of Volatile Compounds in Habanero
Pepper (Capsicum chinense Jacq.) by Optimization of Headspace
Solid-Phase Microextraction Conditions
Luis F. Cuevas-Glory & Odri Sosa-Moguel & Jorge Pino &
Enrique Sauri-Duch
Received: 29 May 2014 / Accepted: 21 August 2014
# Springer Science+Business Media New York 2014
Abstract The habanero pepper (Capsicum chinense Jacq.) is
very aromatic and is the hottest pepper in the world. In this
study, a headspace solid-phase microextraction/gas chroma-
tography–mass spectrometry (HS-SPME/GC–MS) method
was developed to evaluate the volatile compounds that con-
tribute to the aroma of Yucatan habanero pepper. The optimi-
zation of the extraction conditions by HS-SPME/GC–MS was
carried out by using a fractional factorial design (2k-2V) and
univariate analysis. Eight factors were evaluated: equilibrium
time, extraction temperature, extraction time, salt addition,
pH, fibre, vial size and agitation. Optimal results were as
follows: 14 min of equilibrium, 53 °C and 46 min of extrac-
tion, polydimethylsiloxane/divinylbenzene/carboxen
(PDMS/DVB/CAR) fibre and a 15-mL vial. Fifty-three com-
pounds were identified in habanero pepper employing the
optimized extraction conditions.
Keywords Capsicum chinense . Experimental design .
Headspace solid-phase microextraction . Optimization .
Volatile compounds
Abbreviations
HS-SPME Headspace solid-phase microextraction
PDMS Polydimethylsiloxane
PDMS/DVB/ Polydimethylsiloxane/divinylbenzene/
CAR carboxen
SDE Simultaneous steam-distillation-solvent
extraction
L. F. Cuevas-Glory (*) : O. Sosa-Moguel : J. Pino : E. Sauri-Duch
Instituto Tecnologico de Merida, Merida, Yucatán, Mexico
e-mail: lfcuevas@yahoo.com
Introduction
The Yucatan region is the largest producer of the pepper
Capsicum chinense Jacq. in Mexico. This fruit is known as
the Yucatan habanero pepper, due to its origin designation
acquired in 2010. This pepper is one of the hottest peppers in
the world, owing to its high content of capsaicin. However,
parameters of quality such as colour and flavour which have
drawn attention in the food, cosmetic and pharmaceutical
industries have not been widely investigated in habanero
peppers from the Yucatan. Depending on the species and the
level of ripeness, the aroma of pepper changes due to differ-
ences in volatile compound profiles (Mazida et al. 2005).
The typical aroma of C. chinense peppers is one of the most
attractive properties, representing a quality parameter for the
consumer (Teixeira et al. 2006). The characterization of aroma
compounds in foods and other natural products is still a
challenge despite sophisticated techniques now available to
researchers. Aroma compounds usually present in food matri-
ces at very low concentrations, a wide range of polarities,
differing solubility, volatility and stability in temperature and
pH. Therefore, there is no single method for the identification
of aroma compounds. Ideally, the isolation methods should
not discriminate between polar and nonpolar compounds and
cause thermal degradation, oxidation or reduction of the aro-
ma compounds, pH changes or loss of highly volatile com-
pounds (Da Costa and Eri 2005).
The analytical work involved in characterizing the volatile
fraction of peppers can be summarized as isolation by differ-
ent techniques, separation, identification and quantification by
gas chromatography–mass spectrometry (GC–MS) (Bogusz
et al. 2011). For the extraction of the aroma from Capsicum
spp., some methodologies, such as simultaneous steam-
distillation-solvent extraction (SDE) (Pino et al. 2006, 2007),
steam distillation (Gahungu et al. 2011) and headspace solid-
 Food Anal. Methods

phase microextraction (HS-SPME) (Mazida et al. 2005;
Teixeira et al. 2006; Rodriguez-Burruezo et al. 2010;
Bogusz et al. 2011; Eggink et al. 2012), have been used with
variable success. However, solid-phase microextraction
(SPME) has shown to be an excellent sampling method,
allowing simultaneous extraction and concentration of
analytes from headspace of complex matrices. This technique
utilizes a fused silica optical fibre, coated with a thin polymer
layer, to extract the analytes from a liquid or a gaseous phase.
When compared to traditional methods, this technique offers
advantages such as reduced time study, no organic solvent is
employed, low cost, low sample volumes and can be easily
automated (Arthur and Pawliszyn 1990).
The SPME technique is very sensitive to experimental
conditions. Any change in the experimental parameters would
affect the coefficient of distribution, the absorption rate, as
well as the amount of compounds absorbed by the fibre
and reproducibility of the method (Camara et al. 2006).
Despite the substantial growth in applications of SPME
technique to analyse aroma and considering that SPME
analysis is affected by several factors, very little effort
has been made to optimize the SPME method (Carasek
and Pawliszyn 2006). The procedure utilized for opti-
mizing the extraction conditions has been traditionally
carried out by following a univariate approach in which
each factor is studied separately. However, when more
response variables are of interest to the analyst, factorial
design results can be complicated to analyse. These
effects are more easily investigated by using a multivar-
iate experimental design, which allows for simultaneous
variation of all evaluated factors, thereby, making it
possible to distinguish interactions among them that
would not be detectable by classical experimental de-
sign (Teixeira et al. 2006; Penteado et al. 2006).
Optimization of HS-SPME technique for analysing vol-
atile compounds of Capsicum spp. has been a previous-
ly reported design (Teixeira et al. 2006; Bogusz et al.
2011). However, their effort focused only on the extrac-
tion time and temperature. The objective of this study
was to optimize the factors that influence the efficiency
of HS-SPME for detection and quantification of volatile
compounds from Yucatan’s habanero pepper by GC–
MS.
Materials and Methods
Samples
Fresh habanero pepper fruits were obtained from INIFAP
(Instituto Nacional de Investigaciones Forestales, Agrícolas
y Pecuarias) in the Yucatan, Mexico. The habanero pepper
fruits were blended (Waring 51BL31, USA) with distilled
water (1:1v/v) during 30 s to facilitate the release of volatile
compounds by solid diffusion.

Fig. 1 Pareto charts of main

F
effects and interactions on the G
fractional factorial design (2k-2V) C
for total peak area. A Equilibrium D
FG
time. B Extraction temperature. C GH
Extraction time. D Salt addition. B
CF
E pH. F Fibre type. G Vial size. H DF
Agitation A
BF
AF
FH
H
AB
BG
AH
CE
EH
AC
BH
EF
AG
AE
DH
BD
CH
DE
CG
BE
EG
DG
BC
AD
E
CD

0 2 4 6 8
Food Anal. Methods

Optimization Strategy response the total area of the GC–MS chromatogram

The HS-SPME multivariate optimization was accomplished
in two stages. The first step was to carry out a screening test to
establish the relative influence of the factors and their interac-
tions on the total sum of peak areas obtained in the gas
chromatography–flame ionization detector (GC–FID) analy-
sis. A 2-level fractional factorial design, 2k-2v (where k = 8
effect), of two level, high (+) and low (−), was carried out. The
chosen factors were as follows: equilibrium time (30 and
60 min), extraction temperature (30 and 60 °C), extraction
time (30 and 60 min), salt addition (0 and 2 g of NaCl), pH
(3.5 with HCl 0.1 N and 7.5 with NaOH 0.1 N), fibre (poly-
dimethylsiloxane (PDMS) 100 μm and polydimethylsiloxane/
divinylbenzene (PDMS/DVB) 50/30 μm [Supelco,
Bellefonte, PA, USA]), vial size (7- and 15-mL vial
with a PTFE-lined screw cap) and magnetic stirring (0
and 800 min−1). Sixty-four experiments were performed
in a random order. The second step consisted in
performing a univariate analysis with five levels of the
factors chosen in the screening test to find the optimal
conditions of the extraction process by using as the
peaks. These experiments were carried out in triplicate.
One-way analysis of variance and Duncan’s multiple
range tests were used. Statgraphics Plus 5.1 (Statpoint
Technologies, Warrenton, VA, USA) software was uti-
lized to perform the statistical analyses.
GC–FID
Since the identities of the compounds were not necessary for
optimizing the SPME method and due to stability and easy
operation of the flame ionization detector (FID), this
first step of the work was carried using a GC–FID
PerkinElmer AutoSystem XL (Shelton, CT, USA). GC
conditions were as follows: injector, 250 °C; injection
mode, split/splitless (1 min); detector, 250 °C; chro-
matographic column, AT-5 MS (5 % phenyl-95 %
dimethylpolysiloxane as stationary phase, 30 m ×
0.25 mm ID and 0.25 μm of thickness); carrier, nitro-
gen (1 mL/min); oven programme, 50 °C (2 min),
4 °C/min, 250 °C (8 min).

7 mL 7 mL
1.2E+07 15 mL 1.2E+07
A B 15 mL
b
1.0E+07 1.0E+07
b
8.0E+06 8.0E+06
ab
Total area

Total area

6.0E+06 a 6.0E+06
a a
4.0E+06
a
4.0E+06
a
2.0E+06 2.0E+06

0.0E+00 0.0E+00
PDMS PDMS/DVB/CAR 0 800
Fibre Agitation (min-1)

30 min 0 g NaCl
1.2E+07 1.2E+07 2 g NaCl
60 min D
C
1.0E+07 1.0E+07
b b
8.0E+06 8.0E+06
ab ab
Total area
Total area

6.0E+06 6.0E+06
a a
4.0E+06 a 4.0E+06 a

2.0E+06 2.0E+06

0.0E+00 0.0E+00
PDMS PDMS/DVB/CAR PDMS PDMS/DVB/CAR
Fibre Fibre
Fig. 2 Double interaction on the screening by HS-SPME. a Fibre and vial size. b Agitation and vial size. c Fibre and extraction time. d Fibre and salt
addition. Bars with different superscripts are significantly different (p≤0.05)

GC–MS
Identification of volatile compounds from peppers was per-
formed by using a GC–MS PerkinElmer Clarus 500 (Shelton,
CT, USA). A fused silica AT-5 MS (5 % phenyl-95 %
dimethylpolysiloxane as stationary phase, 30 m×0.25 mm
ID and 0.5 μm thickness) and helium (1 mL/min) was used.
The chromatographic conditions were as those described
above for the GC–FID system. GC–MS was run in electron
impact (EI) mode at 70 eV with a scan range of 35–400 u.
Volatile compounds were identified by comparison of their
obtained mass spectra with those reported at the databases
Wiley 7, Adams 2001 and NIST 05 and their corresponding
linear retention index (LRI) values.
Results and Discussion
Fractional Factorial Design 2k-2V
The results of the screening test allowed us to detect those
factors with the greatest influence on the experimental re-
sponses. An analysis of variance (ANOVA) was performed
to determine statistical significance of each effect and interac-
tions between the different factors. Figure 1 shows a standard-
ized Pareto chart for the total peak area. The bar length in the
graph provides information about the importance of the con-
tribution to the model of each experimental factor and inter-
action. The vertical line in the chart sets the limit for signifi-
cance at 95 % confidence level. Fibre type, vial size, extrac-
tion time, salt addition and extraction time were the most
significant parameters. It is known that the HS-SPME effi-
ciency is also affected by a double interrelation of factors,
Fig. 3 Effect of extraction
temperature on the efficiency
extraction by HS-SPME. Bars
with different superscripts are
significantly different (p≤0.05)
Total area
Food Anal. Methods
such as fibre and vial size, agitation and vial size, extraction
time and fibre and salt addition and fibre type (Fig. 2). The
type of fibre was the principal influential factor that affected
interrelated parameters. PDMS/DVB/CAR fibre was the most
effective SPME fibre for extraction of the volatile compounds
from habanero pepper. This is a remarkable fibre, highly
efficient in comparison to others utilized in aroma studies by
HS-SPME (Jelén et al. 2012). Its efficacy is probably due to a
higher affinity for compounds present in the sample (esters,
aldehydes and alcohols) and to its intrinsic polar nature. Fibre
selection is primarily based on the polarity and volatility of the
specific analyte. PDMS/DVB/CAR was a highly efficient
fibre when compared to other fibres for optimizing the extrac-
tion of volatile compounds from C. frutescens var. malagueta
(Bogusz et al. 2011). This fibre is considered a bipolar type
which contains meso-macroporous structures with a wide
range of pore sizes. In addition, a combination of DVB-
CAR coatings increases both the porosity distribution and
the polarity of the fibre, which in turn improves retention of
analytes into the fibre when compared to a PDMS fibre
(Carasek and Pawliszyn 2006). In a complex matrix, the
limiting step for this process is the diffusion of the analytes
through the system (Steffen and Pawliszyn 1996). On HS-
SPME non dynamic, the mass transfer between the sample
and the headspace is affected by the size of the contact surface
between the two phases. Agitation and vial size showed
optimal extraction of compounds using a higher vial size
without agitation. This effect can be attributed mainly to the
vial size, as observed in Fig. 2b; the effect of agitation in the
vial of the same size did not reveal a significant difference.
Stirring the diluted sample facilitates mass transport between
the bulk of the aqueous sample and the fibre (Pawliszyn
1999). However, when analysing highly volatile compounds
by HS-SPME, most of the analytes found in the headspace are
3.5E+08
b
b
3.0E+08
2.5E+08
a
2.0E+08 a
a
1.5E+08
1.0E+08
5.0E+07
0.0E+00
32 39 46 53 60
Extraction temperature (°C)
Food Anal. Methods

Fig. 4 Effect of extraction time 3.0E+08

on the efficiency extraction by c
HS-SPME. Bars with different
superscripts are significantly 2.5E+08
different (p≤0.05) b

2.0E+08
a

Total area
a a
1.5E+08

1.0E+08

5.0E+07

0.0E+00
32 39 46 53 60

Extraction time (min)

readily extracted, even when the solution is not stirred. The
interaction between fibre type and extraction time demon-
strates that a long extraction time of 60 min favours the
extraction of a higher number of volatile compounds when
PDMS/DVB/CAR fibre is used (Fig. 2c). This is probably due
to the polarity of volatile compounds in the headspace and a
longer time which favours adherence of analyte molecules to
additional sites in the fibre. Nevertheless, the interaction be-
tween the factors fibre type and salting out demonstrated a
significant increase in sensitivity when the PDMS/DVB/CAR
fibre, and without NaCl addition, was evaluated. The effect of
salting out improves extraction efficiency due to decreasing
the solubility of analytes in solution, thus, increasing the
amount of adsorbed analytes on the fibre (Wu et al. 2000).
However, the polar molecules may play a role in electrostatic
interactions with the salt ions in solution, thereby, reducing
their ability to move into the fibre coating (Camara et al.
2006). Since the fibre type and vial size are two qualitative
variables, they were discarded from double interrelation.
Extraction time, salt addition and extraction temperature were
the variables required to study HS-SPME optimization.
Optimization
Effect of Extraction Temperature
The substance distribution constant between the fibre and the
sample depends also on the temperature. Figure 3 shows the
efficiency of the extraction, which was determined by the total
volatile compounds of habanero pepper extracted at different

Fig. 5 Effect of salt addition on 4.5E+08

the efficiency extraction by HS- a
SPME. Bars with different 4.0E+08
superscripts are significantly a
different (p≤0.05) 3.5E+08

3.0E+08
Total area

b
2.5E+08
b b
2.0E+08

1.5E+08

1.0E+08

5.0E+07

0.0E+00
0.0 0.5 1.0 1.5 2.0

Salt (g)
 Food Anal. Methods

Table 1 Volatile compounds in habanero pepper cultivars at the ripening stages green and orange by HS-SPME and GC–MS

Compound LRIa Greenb Orangeb Significant differencec

Esters
Isobutyl isopentanoate 1004 0.1 t
Isopentyl 2-methylbutanoate 1094 0.1 t
Isopentyl isopentanoate 1103 0.8 0.6 *
2-Methylbutyl isopentanoate 1107 0.2 0.1 *
(Z)-3-Hexenyl isobutanoate 1145 tc nd
Pentyl isopentanoate 1148 0.5 0.5
Hexyl isobutanoate 1151 0.2 0.1
Isoprenyl pentanoate 1152 0.2 0.1
Methyl salicylate 1190 t t
Hexyl 2-methylbutanoate 1237 2.0 2.5
Hexyl isopentanoate 1240 14.8 33.1 *
Heptyl isobutanoate 1248 nd 0.1
(Z)-3-Hexenyl 2-methylbutanoate 1293 0.1 t
(Z)-3-Hexenyl isopentanoate 1295 14.8 0.7 *
Hexyl pentanoate 1298 20.3 17.7
(E)-2-Hexenyl pentanoate 1299 3.2 1.2 *
Heptyl butanoate 1300 nd t
Hexyl tiglate 1328 t t
Heptyl 2-methylbutanoate 1332 0.9 0.8
Heptyl isopentanoate 1338 6.3 8.6 *
Octyl isobutanoate 1348 0.1 0.1
(Z)-3-Hexenyl hexanoate 1375 nd 0.1
Hexyl hexanoate 1382 0.1 0.4 *
Benzyl pentanoate 1396 1.0 0.3 *
Octyl 2-methylbutanoate 1418 t 0.5
Octyl isopentanoate 1440 2.6 3.2 *
Heptyl hexanoate 1448 nd 0.1
Hexyl benzoate 1558 0.1 0.1
Citronellyl isopentanoate 1577 0.1 0.2
Terpenes
Limonene 1035 0.1 0.1
α-Copaene 1384 t 0.6
β-Cubebene 1392 1.5 0.6 *
(E)-α-Ionone 1430 t t
(E)-β-Farnesene 1458 0.2 t
β-Chamigrene 1475 0.5 0.3 *
Germacrene 1480 t nd
γ-Himachalene 1483 8.2 3.9 *
(E)-β-Ionone 1489 t t
α-Murolene 1490 0.3 0.1 *
γ-Cadinene 1515 0.1 0.1
Aldehydes
(E)-2-Hexanal 853 0.3 0.2
(E)-2-Nonenal 1164 t nd
Tetradecanal 1611 0.1 0.1
Pentadecanal 1707 0.2 0.3
Hexadecanal 1816 0.1 0.1
Alcohols
Food Anal. Methods
Table 1 (continued)
Compound LRIa
(E)-2-Hexenol 861
3,3-Dimethylcyclohexanol 1390
Alkanes
2-Methyl-1-tetradecene 1445
2-Methyl-tetradecane 1462
1-Hexadecane 1600
1-Pentacosane 2500
Others
2-Isobutyl-3-methoxypyrazine 1174
Oxacyclotridecan-2-one 1577
t represents the compound at trace level (<0.1)
nd not detected
a
LRI, retention indices obtained using a 5 % phenyl-95 % dimethylpolysiloxane capillary column
b
Percent relative area
c
Significant difference at p≤0.05 (n=3)
temperatures (32, 39, 46, 53 and 60 °C). Optimal results were
at 53 and 60 °C. Optimal temperature depends on matrix
composition, its components and the stationary phase used
(Wardencki et al. 2004). In that context, 64°C was the maxi-
mum extraction temperature for C. chinense pepper, since an
increase on the temperature can motivate the desorption of
analytes from the fibre due to a competing mechanism of
thermal desorption of the volatile compounds (Teixeira et al.
2006).
Effect of Extraction Time
The fibre time exposure is a parameter that strongly influences
quantitative adsorption on the stationary phase of the fibre.
Figure 4 shows the effect of extraction time (32–60 min) of the
extracted volatile compounds from habanero pepper; optimal
result was obtained at the longest extraction time. However, a
slight decrease in the response was observed at 53 °C.
Therefore, extraction time of 46 min was ultimately selected
for carrying out the analysis. In general, longer times favour
filling up of more sites on the fibre by the analyte molecules.
However, a more prolonged time, when all sites have been
occupied, occasionally causes desorption (Zhang and
Pawliszyn 1993). This effect was also observed in the effi-
ciency of the extraction of certain monoterpenes, such as
limonene and β-caryophyllene (Hamm et al. 2003). Almost
invariably, the extraction time profile depends on the individ-
ual analyte. In case of multicomponent systems, competition
occurs for the active sites on the coating of the SPME fibre
(Keszler and Heberger 1999). By increasing the extraction
time, higher-boiling compounds might possibly displace the
previously adsorbed lower-boiling compounds.
Greenb Orangeb Significant differencec
nd 0.1
16.7 21.7 *
1.5 t
1.4 0.7 *
0.3 0.2 *
nd 0.1
t nd
0.1 0.1
Effect of Salt Addition
The effect of salting out on the extraction of volatiles from
habanero pepper was studied at the range of 0 to 2 g of NaCl
(Fig. 5). The experiment response decreased when the quantity
of salt was increased. Generally, salt addition led to an increase
in extraction performance due to the salting out effect.
However, the polar molecules could participate in electrostatic
interactions with the salt ions in solution, thereby, reducing their
ability to move to the fibre coating (Camara et al. 2006).
Application of the HS-SPME–GC–MS Method
for the Analysis of Volatile Compounds from Mayapan
Habanero Pepper
By utilizing the optimized HS-SPME technique, it was possi-
ble to identify 53 volatile compounds from habanero peppers,
green and orange varieties (Table 1). The number of volatile
compounds here reported was higher than those previously
reported in yellow and purple C. chinense peppers from Brasil
(34 volatiles) design with PDMS 100 μm fibre (Teixeira et al.
2006), in P1152 (31 volatiles) and C. chinense Scotch bonnet
of Cuba (20 volatiles) with PDMS/DVB/CAR 50/30 μm fibre
(Moreno et al. 2012). However, 155 volatiles were identified
in farolillo pepper (C. chinense) from Ecuador with
PDMS/DVB/CAR 50/30 μm fibre (Rodriguez-Burruezo
et al. 2010), and 77 compounds were reported in murupi
pepper (C. chinense) from Brasil with PDMS/DVB/CAR
50/30 μm fibre (Bogusz et al. 2012). Moreover, HS-SPME
has been the technique utilized for some other varieties of
Capsicum such as C. annuum jalapeño (76 volatiles),
C. annuum serrano (66 volatiles), C. frutescens tabasco (124

volatiles), C. frutescens malagueta (83 volatiles), C. baccatum
var. pendulum (49 volatiles) and C. pubescens (63 volatiles)
(Rodriguez-Burruezo et al. 2010; Bogusz et al. 2011;
Kollmannsberger et al. 2011). The most abundant chemical
classes of the volatile compounds detected in habanero green
and orange peppers were as follows: esters, terpenes, alcohols,
aldehydes and alkanes. The abundance of aliphatic esters in
C. chinense has been studied by different isolation methods
(Buttery et al. 1969; Teixeira et al. 2006; Gahungu et al. 2011;
Rodriguez-Burruezo et al. 2010; Kollmannsberger et al.
2011). Volatiles such as hexyl isopentanoate, hexyl
pentanoate, (Z)-3-hexenyl isopentanoate and heptyl
isopentanoate were the most abundant esters found in both
varieties. Hexyl isopentanoate was previously described as an
analyte with a powerful fruity odour (Forero et al. 2009). A
total of 11 terpenoids, five aldehydes, four alcohols and four
alkanes were identified in two different stages of maturation of
habanero pepper: γ-himachalene, β-cubebene and 3,3-
dimethylcyclohexanol were the principal analytes. The con-
stituents of the volatile fraction in habanero pepper can differ
according to the maturation grade; the compounds can in-
crease, decrease or even disappear (Pino et al. 2006).
Compounds such as (Z)-3-hexenyl isopentanoate, (E)-2-
nonenal, 2-methoxy-3-isobutylpyrazine and
oxaciclotridecan-2-one were absent in green habanero pepper.
The levels of (E)-2-hexenal and (Z)-3-hexenol decreased dur-
ing maturation of habanero pepper, while levels of (E)-2-
hexenol and 4-methylhexanol increased.
Concluding Remarks
HS-SPME methodology is a simple, fast and solvent-free
method which, when used simultaneously with a multivariate
experimental design, makes possible the identification of the
most significant parameters and their interactions on the aro-
ma extraction process. Optimal extraction conditions for ha-
banero pepper by using HS-SPME were as follows: 15 mL of
vial size, extraction time of 46 min, extraction temperature of
53 °C and PDMS/DVB/CAR fibre. Fifty-three compounds
were identified in the volatile fraction in green and orange
habanero peppers. The most significant volatile compounds in
both habanero pepper varieties were hexyl isopentanoate, 3,3-
dimethylcyclohexanol, hexyl pentanoate and (Z)-3-hexenyl
isopentanoate.
Conflict of Interest Odri Sosa-Moguel declares that she has no conflict
of interest. Jorge Pino declares that he has no conflict of interest. Enrique
Sauri-Duch declares that he has no conflict of interest. Luis Cuevas-Glory
declares that he has no conflict of interest. This article does not contain
any studies with human or animal subjects.
Food Anal. Methods
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