Introduction

Capsaicinoids are a group of pungent compounds found mostly

in capsicum fruits, the structures of which are acid amides of
vanillylamine and C9 C11 branched-chain fatty acids. There
are five naturally occurring capsaicinoids which have been
reported, namely, capsaicin, nordihydrocapsaicin,
dihydrocapsaicin, homocapsaicin and homodihydrocapsaicin.
1,2
Of these, capsaicin and dihydrocapsaicin are the major
components of most capsicum species. Capsaicinoid
compounds have been considered as a major indicator of the
chili product qualities. Besides their pungent properties, the
capsaicinoid compounds have also been studied and used for
medical and military purposes, such as analgesic creams and
defensive spray.
3,4
The first reliable reported measurement of
chili pungency was the Scoville Organoleptic Test.
5
An
accurate determination of the levels of various capsaicinoids has
become important because of the increasing demand by
comsumers for foods, and the increasing use in
pharmaceuticals.
6,7
Similarly, scientists in the area of genetics,
biogenesis, food chemistry and physiology, also need reliable,
safe, and reproducible standard analytical procedures and the
rapid methods for the separation and quantitation of these
capsaicinoid compounds that are useful for comparing
pungency levels among different samples. Therefore, the
Scoville Organoleptic Test has since been replaced by
instrumental methods. The analysis of capsaicinoids has been
conducted by using spectrophotometric,
811
gas
chromatographic,
1216
micellar electrokinetic capillary
chromatographic,
17
high-performance liquid chromatographic
procedures,
1825
and liquid chromatographic-mass spectrometric
procedures.
26
Techniques using high-performance liquid
chromatography provide accurate and efficient analysis of
content and type of capsaicinoids present in a chili sample.
However, it is still necessary to optimize the method for each
chromatographic system in order to identify each of the
remaining closely related capsaicinoids in the extract. The
literature regarding the optimization of this techniques for the
determination of capsaicinoid compounds of Thai capsicum
fruits is still inadequate. Therefore, this study was conducted to
optimize the sample preparation, separation, detection and
identification for Thai capsicum fruits and to achieve a
convenient, rapid and efficient analysis of capsaicinoid
compounds.
Experimental
Plant material
The matured chili pods with stems removed were dried in a
hot-air oven under 55C for 24 30 h, ground with seeds to pass
through a 60 mesh sieve and stored in sealed plastic bags at 5C
until examined. Some of the chili pods used for this study had
been grown under farming practices at Lampang Agricultural
Research and Training Center (LARTC) and some were
purchased from local markets.
Reagents
Acetonitrile (HPLC-grade, J. T. Baker, USA), methanol
(HPLC-grade, Carlo Erba, Italy) and acetone (ACS-grade,
Fluka, Switzerland) were used. Deionized, double distilled
water was used throughout. All solvents for the
chromatographic system were filtered and degassed using a 0.45
m pore size polyamide (nylon) filter.
Capsaicinoid standards
Standards of capsaicin (CAPS, 8-methyl-N-vanillyl-6-
nonenamide), and dihydrocapsaicin (DHC, 8-methyl-N-vanillyl-
661 ANALYTICAL SCIENCES JUNE 2002, VOL. 18
2002 The Japan Society for Analytical Chemistry
Optimization of High-Performance Liquid Chromatographic
Parameters for the Determination of Capsaicinoid
Compounds Using the Simplex Method
Rachaneewan KARNKA,*
,
** Mongkon RAYANAKORN,*

Surasak WATANESK,*
and Yuthsak VANEESORN*
*Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand
**Lampang Agricultural Research and Training Center, Lampang 52000, Thailand
A high-performance liquid chromatographic method was developed for the analysis of capsaicinoid compounds, the
pungent principles of capsicum fruits. A sequential simplex method was applied to optimize the chromatographic
response function used to assess the quality of separation by varying the chromatographic parameters. The separation
was achieved in 11 min using a C-8 column of 15-cm length and 4.6 mm diameter using a UV detector. A flow rate of
1.15 ml min
1
at a column temperature of 43.5C using 63.7% methanol in water gave the most efficient separation. The
method was found to be suitable for the determination of the major capsaicinoid compounds in the capsicum samples.
(Received November 21, 2001; Accepted March 22, 2002)

To whom correspondence should be addressed.

E-mail: mongkon@chiangmai.ac.th
nonanamide) were purchased from Sigma (Sigma-Aldrich, St.
Louis, MO, USA). The solutions of all standards were prepared
in acetonitrile. Appropriate dilutions of the initial solution were
prepared in order to obtain a calibration curve.
Apparatus
The HPLC system consisted of an HPLC Shimadzu pump
10AD, an SPD-M10AV variable wavelength UV detector and
CLASS-LC10 Software for data processing.
An Inertsil RP-8 column was used with a controlled-
temperature oven.
Octadecyl (C18) 40 m Prep LC packing for solid phase
extraction (SPE) was obtained from J. T. Baker (Phillipsburg,
USA).
Optimization of capsaicinoids extraction by ultrasonication
The extraction efficiencies for the capsaicinoids from chili
peppers in various organic solvents (acetone, acetonitrile and
methanol) were compared, and it was found that acetonitrile
gave the highest extraction rate with the fewest impurities. The
optimum volume of the solvent and the time of sonication were
then determined using only acetonitrile. A 0.3 g sample of chili
was sonicated for 60 min with 10, 15, 20, 30 and 40 ml of
acetonitrile and another 0.3 g of chili was sonicated for 10, 30,
45, 60, 75 and 105 min with 10 ml acetonitrile, respectively.
The amounts of the individual extracted capsaicinoids were
determined and the peak areas were calculated.
Clean-up
A C18 Sep-pak (200 mg) was washed with 0.5 ml of double-
distilled water and 0.5 ml of methanol, and then conditioned
with about 0.5 ml of acetonitrile. A 0.5 ml portion of extract
was injected into the conditioned Sep-pak. After the
capsaicinoids were eluted with 0.5 ml of acetonitrile, the
cartridge was washed three times with 0.5 ml of acetonitrile and
all washed solutions were collected in the same tube. The
solution was filtered through a 0.45 m filter membrane with a
syringe filter into a small glass vial. The obtained filtrate was
later used for an HPLC analysis with each injection volume of 5
l.
Chromatographic optimization
An Inertsil RP-8 column was used with a controlled-
temperature oven. The overall temperature control was
maintained within 0.5C with a variation of from 26.0C to
50.0C. The flow rate used varied from 0.7 to 1.2 ml min
1
.
The mobile phase was a mixture of methanolwater with
varying percentages of methanol from 55% to 70% for the RP-8
column. Percentages below 55% were not used because of the
excessively high column pressure obtained with a flow rate of
1.2 ml min
1
. The detector wavelength was set at 280 nm.
Chromatographic response function
The separation quality of capsaicinoid compounds for
achieving the maximum resolution with the minimum assay
time was assessed at the end of the chromatogram by
calculating the value of a chromatographic response function
(CRF). The CRF is a flexible function that allows desirable
time and resolution criteria to be specified. The corresponding
terms in the chromatogram are then compared to these criteria
and the function is maximized by changing the experimental
variables. It is represented by the following equation:
27,28
CRF = Ri + L
a
b|TM TL| c(T0 T1) (1)
where Ri is the resolution between adjacent pairs of peaks. In
practice it is limited to a maximum value of 2.00 so that all pairs
of well-resolved peaks make no further contribution to the CRF.
L is the total number of peaks detected, TM is an acceptable
analysis time, TL is the retention time of the last eluted peak, T1
is the elution time of the first peak, T0 is a specified minimum
retention time, and a, b, c are the arbitrary weighting factors (a
value of 1 was used in the this work).
Results and Discussion
Ultrasonic extraction
In this work ultrasonic solvent extraction was used as a simple
and inexpensive method applicable to capsicum samples. The
goal of the optimization procedure was to improve the
extraction efficiency with minimum solvent consumption and
minimum time needed for the extraction procedure. The
efficiency of the extraction procedure was verified by the peak
area of the same samples. From the results of the extraction
efficiencies of various organic solvents (acetone, acetonitrile
and methanol), acetonitrile was used, because it gave a
reasonably high extraction rate and fewer impurities for the
capsicum studied.
The best extract of capsaicinoids from capsicum samples was
obtained with 10 ml of acetonitrile in one extraction step for 60
min with a controlled column temperature of 43.0 0.5C. The
results are shown in Figs. 1 and 2. The reproducibility of the
ultrasonic extraction of 0.3 g-capsicum samples with 10 ml of
acetonitrile for 60 min sonication was evaluated using 10
consecutive analyses. The reproducibility of the peak areas of
capsaicin and dihydrocapsaicin was found to be satisfactory
662 ANALYTICAL SCIENCES JUNE 2002, VOL. 18
Fig. 1 Peak areas of capsaicinoids as a function of the volume of
acetonitrile.
Fig. 2 Peak areas of capsaicinoids as a function of the time of
sonication.
with 1.05% RSD (n = 10) and 1.08% RSD (n = 10),
respectively.
The recovery of clean-up column
There was almost a complete elimination of interferents when
SPE-C18 was used with acetonitrile as the eluent. The worst
interferents, especially pigments, were reduced. The percent
recovery for capsaicin was found to be 98.91 and for
dihydrocapsaicin it was 99.23 with standard deviations of 0.09
and 0.19, respectively.
Chromatographic optimization
The chromatographic parameters were optimized using a
chemometric approach based on the use of the simplex
method.
27,28
When using a simplex, each vertex corresponds to
a set of experimental conditions. In this work, the factors which
were varied to improve the separation of capsaicinoid
compounds included the mobile-phase composition, the flow
rate and the column temperature. From the resulting
chromatogram under each set of conditions the chromatographic
response function (CRF) was calculated and the relative
responses were ranked. The advantage of the CRF for the
simplex method is that it allows a weighting of important
chromatographic features (Ri and TM) for simplex movements
that result in a higher CRF value with increased resolution,
reasonably short analysis time (TM TL) and good retention (T1
> T0). The modified simplex was started after introducing upper
and lower boundary conditions for the above three variables. In
this process, the CRF value is calculated for m sets of starting
conditions, where m is the number of factors to be optimized
plus 1. In this case m is 4. The corresponding initial
experimental conditions and CRF values are given in Table 1.
The point corresponding to the lowest value of CRF was then
reflected about the surface (hyperface) defined by the remaining
three points to give a fifth set of conditions to evaluate and rank.
Expansion and contraction for the simplex was allowed, based
on the usual rules.
2729
To select whether expansion, contraction
or keeping the reflection steps, by using the differences in the
responses at the vertices to estimate the vertex with a better
response when the vertex violated a boundary condition on one
of the experimental factors.
30
The next vertex and process were
repeated sequentially until an apparent optimum had been
obtained.
The results of the sequential simplex progress are given in
Table 2. The simplex was halted after 30 experiments, since
there was no further significant improvement towards the
maximization of the CRF value after vertex 21. Figure 3 shows
the variation in CRF with the experiment number; it can be seen
that an optimum response was achieved rapidly. Although
experiment number 7 had the highest CRF value, because nearly
the same set of conditions reappeared near vertices 22 30, the
early high CRF value was considered to be fortuitous. This
return to the optimum value increases ones confidence that the
method is rugged and efficient. Chromatograms obtained under
optimum conditions selected as vertex number 7 are presented
in Fig. 4, showing excellent resolution among the three expected
peaks. The data indicate that the variation of the column
temperature has a more significant effect on the CRF value than
do the other two parameters.
663 ANALYTICAL SCIENCES JUNE 2002, VOL. 18
55.0 70.0 70.0 60.0 60.0 55.0
0.70 1.20 1.00 1.00 0.70 1.20
26.0 50.0 31.0 26.0 43.0 50.0
Table 1 Boundary conditions of the experimental parameters
and the initial conditions used for simplex optimization with the
C8 column
Boundary condition Minimum Maximum
Experiment No.
1 2 3 4
Mobile phase
composition/%
methanol in water
Flow rate/ml min
1
Temperature/C
1 B 70.0 1.00 31.0 3.981
2 W 60.0 1.00 26.0 3.709
3 N 60.0 0.70 43.0 0.702
4 N 55.0 1.20 50.0 0.971
5 CR 62.5 0.95 48.9 4.518
6 CW 60.9 1.10 33.7 0.937
7 CR 63.7 1.15 43.5 5.970
8 CW 61.3 0.88 43.1 2.239
9 CW 60.2 1.12 45.5 3.922
10 CW 66.1 1.03 38.5 4.170
11 R 68.0 0.98 41.8 3.845
12 CW 62.2 1.07 44.6 4.762
13 CR 61.2 1.07 49.3 4.562
14 CW 64.5 1.05 42.1 4.780
15 CR 64.0 1.20 40.7 5.527
16 CW 63.0 1.00 46.2 5.168
17 CR 65.0 1.19 40.9 5.777
18 CW 63.1 1.11 43.4 5.887
19 CW 64.0 1.11 42.3 5.821
20 R 63.3 1.08 45.4 5.487
21 CW 63.8 1.17 41.9 5.716
22 CR 63.3 1.18 43.2 5.906
23 CW 63.8 1.13 42.6 5.805
24 R 63.0 1.16 44.8 5.859
25 CR 63.2 1.16 44.1 5.895
26 CW 63.3 1.15 43.5 5.894
27 R 63.7 1.20 42.7 5.965
28 CW 63.4 1.17 43.3 5.907
29 R 63.9 1.20 43.2 5.934
30 CW 63.6 1.18 43.2 5.960
Table 2 Relationship between chromatographic response
function and vertex number during simplex optimization with
the C8 column
Vertex
No.
a
% MeOH
Flow rate/
ml min
1
Column temperature/
C
CRF
a. B, best; N, next to the worst; W, worst; R, reflection; CR, contraction
on the R side; CW, contraction on the W side.
Fig. 3 Relationship between the chromatographic response
function (CRF) and the experiment number during simplex
optimization with the C8 column.
Calibration
The determination of the two capsaicinoids in the fruit
extracts was performed using the external standard method.
The calibration graphs were expressed as chromatographic peak
areas of standard capsaicinoids versus corresponding
concentrations of the standards in the concentration range of 1
100 mg l
1
. The definition of the limit of detection
31,32
used here
is the concentration corresponding to a signal of the blank
(calculated from the extrapolation of the regression line of the
data rather than from separate measurements) plus three
standard deviations of the noise, assuming a normally
distributed variation around the regression line derived from the
actual data over the concentration range studied. Although the
signals for 1 mg l
1
samples were measured in this work, the
limits of detection for capsaicin and dihydrocapsaicin were
found to be 1.65 and 1.87 mg l
1
, respectively, using the above
definition. The regression lines, correlation coefficients, limits
of detection and limits of quantitation are summarized in Table
3.
Reproducibility
The reproducibility was evaluated by 8 consecutive analyses
with both capsaicin and dihydrocapsaicin in a standard solution.
The relative standard deviations of the retention time for
capsaicin and dihydrocapsaicin were excellent with a mean
retention time of 7.76 min, 1.26% RSD for capsaicin and 1.32%
RSD with a mean retention time of 10.46 min for
dihydrocapsaicin. The instrument repeatability data for the
corrected area calculation for a standard solution of a
concentration of 50 mg l
1
for capsaicin and 50 mg l
1
for
dihydrocapsaicin were satisfactory with the mean of the
calculated concentration being 48.79 mg l
1
with 1.58% RSD for
capsaicin and 3.49% RSD with a mean calculated concentration
of 51.60 mg l
1
for dihydrocapsaicin.
Determination of capsaicinoids in capsicum extractions
The optimal conditions were determined for the standard
solution. A peak just before capsaicin and after
dihydrocapsaicin appeared to be impurities contained in
capsaicin and dihydrocapsaicin, and probably represented minor
capsaicinoids, such as nordihydrocapsaicin, homocapsaicin and
homodihydrocapsaicin. The peak just before capsaicin is likely
to be nordihydrocapsaicin, as reported in another study.
33
Using the optimum condition, the results were obtained for
individual capsaicinoid peaks of capsicum species tested,
including capsicum samples collected from farming practices at
Lampang Agricultural Research and Training Centre (LARTC)
and some from local markets, as shown in Table 4. As for the
recovery rate of the column clean-up, it was found to be 93.3%
0.6 (n = 3) and 89.6% 0.2 (n = 3) for capsaicin and
dihydrocapsaicin, respectively.
Conclusion
The determination of the optimum conditions for sample
preparation and capsaicinoid extraction was relatively
straightforward. Optimization of the column temperature, the
flow rate and the mobile phase composition to achieve good
analytical separation was achieved rapidly using the simplex
method. In this study, it was found that the effect of changing
the column temperature was more important than changes in the
mobile phase or flow rate. The separation of compounds with a
similar structure appears to be particularly sensitive to
temperature changes. In summary, the optimum
chromatographic separation of capsaicinoid compounds with
good resolution in a short time was accomplished using the
simplex method. It has proved to be a useful tool for
developing the analysis method.
664 ANALYTICAL SCIENCES JUNE 2002, VOL. 18
Fig. 4 Chromatogram of capsaicinoids obtained with the C8
column using the optimum conditions (1, nordihydrocapsaicin; 2,
capsaicin; 3, dihydrocapsaicin).
Table 3 Calibration range, regression lines, correlation
coefficients, limits of detection and limits of quantitation for
capsaicinoids studied
Parameter Capsaicin Dihydrocapsaicin
1 100 1 100
Y = 2374.6X + 953.82 Y = 1969.3X + 1066.9
0.9998 0.9998
1.65 1.87
5.49 6.23
Calibration
range/mg l
1
Regression line
Correlation
coefficient
Limit of
detection/mg l
1a
Limit of
quantitation/mg
1b
a. yL = yB + 3sB.
31,32
b. yL = yB + 10sB.
31,32
Table 4 Various capsicum samples and their contents of
capsaicin and dihydrocapsaicin
Capsicum
sample
a
Capsaicin content Dihydrocapsaicin content
%g g
1
SD
b
%g g
1
SD
b
C1 0.0209 0.0002 0.0170 0.0010
C2 0.0362 0.0010 0.0437 0.0003
C3 0.0146 0.0001 0.0125 0.0008
C4 0.0515 0.0012 0.0510 0.0012
C5 0.0214 0.0004 0.0228 0.0007
C6 0.0244 0.0005 0.0285 0.0007
C7 0.3289 0.0082 0.2439 0.0057
C8 0.5038 0.0020 0.4641 0.0008
C9 0.6985 0.0156 0.4075 0.0072
C10 0.5398 0.0007 0.3251 0.0013
a. C1 C5, from LARTC farming practices; C6 C10, from local
markets.
b. SD: standard deviation, n = 3.
Acknowledgements
The authors gratefully acknowledge Professors S. N. Deming
and R. L. Deming for their initial suggestion concerning an
approach to the simplex method of optimization and the
Postgraduate Education and Research Program in Chemistry
(PERCH) of Thailand and the Graduate School of Chiang Mai
University for their partial support.
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