Journal of Chromatographic Science 2013;51:412– 418
doi:10.1093/chromsci/bms156 Advance Access publication October 18, 2012
Article
Simultaneous Quantification of Capsaicinoids and Ascorbic Acid from Pungent Peppers
Haejin Bae1, Guddadarangavvanahally K. Jayaprakasha1, Kevin Crosby1, John L Jifon1,2* and Bhimanagouda S. Patil1*
1
Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, College Station, TX 77845,
and 2Texas A&M AgriLife Research, Weslaco, TX 78596
*Authors to whom correspondence should be addressed. Email: [email protected]; [email protected]
Received 24 May 2012; revised 20 August 2012
The development of simultaneous extraction and determination
of bioactive molecules from natural products is becoming more
popular. The present study reports the development of a method for
the simultaneous extraction and determination of both capsaicinoids and ascorbic acid in peppers. Capsaicin (341.61 mg/g), dihydrocapsaicin (119.91 mg/g) and ascorbic acid (2,109.60 mg/g)
were extracted with 3% metaphosphoric acid–ethanol (2:8) as a
solvent. The efficient extraction of capsaicinoids (412.61 mg/g) and
ascorbic acid (2,785.93 mg/g) was achieved at a sample-to-solvent
ratio of 1:8 after 30 minutes of sonication. Simultaneous separation
of capsaicinoids and ascorbic acid was achieved using a Gemini
C18 column with a gradient elution of 0.03M phosphoric acid and
methanol. Capsaicinoids and ascorbic acid were simultaneously
detected at 282 and 254 nm, respectively. The recovery of capsaicinoids ranged from 96.21 to 108.71%, and the recovery of ascorbic
acid ranged from 97.01 to 98.83%. The limits of detection for
capsaicin, dihydrocapsaicin and ascorbic acid were 0.24, 0.21 and
0.26 mg, respectively. Relative standard deviation for the intra-day
and inter-day variability in the results was less than 3%, indicating
that the method produced highly reproducible results. Therefore,
this method enables the reproducible, simultaneous separation and
quantification of capsaicinoids and ascorbic acid from peppers.
Introduction
Peppers are a common part of the daily diet in many countries,
serving both as vegetables and as spices. Peppers also have
many health-promoting bioactive compounds such as capsaicinoids, ascorbic acid, carotenoids and flavonoids (1). In
pungent, or hot peppers, capsaicinoids and ascorbic acid
(Figure 1) are the most abundant components, and several
studies have shown that capsaicinoids and ascorbic acid have
anti-cancer and anti-proliferative effects (2 –4). Among pepper
capsaicinoid compounds, capsaicin and dihydrocapsaicin are
the most pungent capsaicinoids, and other capsaicinoids such
as nonivamide, nordihydrocapsaicin, homocapsaicin and homodihydrocapsaicin are relatively less pungent (5). As with many
bioactive compounds, the capsaicinoid and ascorbic acid contents of peppers vary substantially in different cultivars (6, 7)
and under different growth conditions; examination of this
variation is a key for the development of improved pepper varieties with higher levels of these health-promoting compounds.
For this, improved, rapid and reliable methods are essential to
analyze capsaicinoids and ascorbic acid. For example, current
methods use separate extractions and assays; therefore,
improved sample preparation methods will facilitate the
high-throughput analysis of capsaicinoids and ascorbic acid
simultaneously.
As a continuation of research on methods for the quantification of bioactive compounds from fruits and vegetables, the
present study aimed to simultaneously extract and quantify capsaicinoids and ascorbic acid from peppers. Different extractionassisted methods, such as ultrasound, supercritical fluid and
microwave techniques, have been tested for their effectiveness
in the isolation of capsaicinoids and ascorbic acid. The
ultrasound-assisted method has been used for the extraction of
capsaicinoids using methanol or ethanol, with extraction times
from one minute to one hour (8, 9). The supercritical fluid
method has been successfully used to extract capsaicinoids
(10), but this method is expensive, limiting its use in routine
analysis. Although the microwave-assisted method has increased
the extraction rate using energy (11), aqueous solvents are
required to obtain efficient extraction (12). Unlike capsaicinoids, extraction-assisted methods for ascorbic acid have not
been commonly applied (13) because ascorbic acid is not
stable during the extraction process. To avoid degradation or
oxidation of ascorbic acid, dilute metaphosphoric acid is commonly used for extraction (14, 15). In a previous study, Topuz
and Ozdemir (16) reported the quantification of carotenoids,
capsaicinoids and ascorbic acid in pepper cultivars, but the
samples were separately extracted and quantified using
different methods for each compound.
Capsaicinoids and ascorbic acid have also been analyzed using
colorimetry (17, 18), capillary electrophoresis (19, 20) and liquid
chromatography–mass spectrometry (LC–MS) techniques (21,
22). Colorimetric methods are cost-effective for the quantification of capsaicinoids and ascorbic acid, but separation and quantification of individual capsaicinoids and ascorbic acid is not
possible. Capillary electrophoresis requires the least amount of
organic solvent and needs only a small amount of sample for
analysis, but it shows poor sensitivity (23). Therefore, reversedphase high-performance liquid chromatography (HPLC)
methods have been developed for independent separation and
quantification of capsaicinoids and ascorbic acid. Few attempts
have been made to simultaneously extract and separate capsaicinoids and ascorbic acid to save time and to improve the efficiency of the analysis. In a previous study, Randall et al. (24)
successfully extracted ascorbic acid using 5% metaphosphoric
acid and ethanol. In the current study, the authors simultaneously
extracted capsaicinoids and ascorbic acid from peppers with 3%
metaphosphoric acid and ethanol. Various conditions for
sample preparation were optimized for the quantification,
including extraction solvents, solvent ratios, extraction times
and HPLC methods for the simultaneous separation and
# The Author [2012]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. Structures of capsaicin, dihydrocapsaicin and ascorbic acid quantified in the present study.
detection of capsaicinoids and ascorbic acid. To the best of the
authors’ knowledge, this is the first report of simultaneous
extraction and separation of capsaicinoids and ascorbic acid
from pungent peppers.
Experimental
Chemicals and sample material
Capsaicin and dihydrocapsaicin were purchased from SigmaAldrich (St. Louis, MO, USA). L-Ascorbic acid was purchased
from Mallinckrodt (Paris, KY, USA). HPLC-grade methanol was
purchased from Fisher Scientific (Fair Lawn, NJ, USA).
Mature serrano peppers (Capsicum annuum L. cv. ‘Tuxtlas’)
were harvested at a greenhouse at Texas A&M University
(College Station, TX, USA). Whole peppers, excluding the
stalks, were chopped and ground for the analysis.
Instrumentation and conditions
A Perkin Elmer (Shelton, CT, USA) HPLC system consisting of a
LC-250 B pump, a Nelson 900 autosampler and a 235C diode
array detector was used. The analysis was performed on a
C18 Gemini column (250 4.6 mm i.d., 5 mm particle size;
Phenomenex, Torrance, CA, USA) with gradient mobile phase of
solvent A (0.03M of phosphoric acid in water) and solvent B
[methanol (MeOH)] at a flow rate of 1 mL/min. The gradient
program was used for the separation of compounds as follows:
0% B (0 –5 min), 0 –100% B (5 –12 min), 100% B (12– 15 min)
and 100 –0% B (15 –20 min). Capsaicinoids and ascorbic acid
were simultaneously separated and detected at 282 and 254 nm,
respectively. The data were processed using TotalChrome
Navigator software (version 6.2.1) from Perkin Elmer.
Extraction solvents and ratio of sample to solvent
The pepper sample (5 g) was homogenized with 40 mL of
solvent for 30 s using a Polytron homogenizer (Brinkmann
Instruments, Westbury, NY, USA). Five different solvents, 3%
metaphosphoric acid (3% MPA), ethanol (EtOH), 3% MPA –EtOH
(8:2, v/v), 3% MPA –EtOH (1:1, v/v) and 3% MPA –EtOH (2:8,
v/v), were used to extract capsaicinoids and ascorbic acid. The
homogenates were sonicated (Cole-Parmer 8893; Cole-Parmer
Instrument Company, Vernon Hills, IL, USA) for 30 min and centrifuged (Marathon 16KM, Fisher Scientific, Fair Lawn, NJ, USA)
at 7,500 rpm for 10 min. The supernatants were filtered through
a 0.45 mm membrane filter and injected into the HPLC. To optimize the extraction efficiency of capsaicinoids and ascorbic
acid, different ratios of samples to solvents (1:3, 1:4, 1:6 and 1:8)
were compared. To optimize the solvent to sample ratio, pepper
samples (5 g) were homogenized with 15, 20, 30 and 40 mL of
3% MPA –EtOH (2:8, v/v). The extracts were sonicated for
30 min, centrifuged and filtered for HPLC analysis. Values shown
are mean + standard deviation (SD) from three independent
experiments.
Sonication and extraction time
To optimize the extraction time, pepper samples (5 g) were
homogenized with 40 mL of 3% MPA–EtOH (2:8, v/v). The
homogenates were extracted for different times (15, 30, 45 and
60 min) using a sonicator (Cole-Parmer 8893, Cole-Parmer
Instrument Company, Vernon Hills, IL, USA) without setting the
water temperature. Samples were further extracted for various
times (30 min, 3, 6, 12 and 24 h) in a shaker. Finally, the extracts
were centrifuged and filtered before HPLC analysis.
Recovery study
The recovery study was evaluated by spiking known concentrations of standard capsaicinoids and ascorbic acid into the
pepper samples. Three different concentrations of capsaicin
(4.17, 14.30 and 23.80 mg/mL), dihydrocapsaicin (8.03, 10.70
and 13.37 mg/mL) and ascorbic acid (12.60, 25.20 and
50.40 mg/mL) were added to the mixture of pepper sample
(5 g) and 40 mL of 3% MPA –EtOH (2:8, v/v) for the spiking
study. The mixture was sonicated, centrifuged and filtrated for
HPLC analysis. The recovery rate was calculated using expected
values obtained by adding known amounts of standards, and
actual values were obtained by HPLC analysis.
Calibration curve, limit of detection, and precision
The calibration curves for capsaicinoids and ascorbic acid were
constructed by serial dilution of a known standard to different
concentrations, followed by HPLC and measurement of the peak
areas. Standards for capsaicin (7.8, 15.6, 31.2, 62.5, 125 and
250 mg/mL), dihydrocapsaicin (3.4, 6.8, 13.7, 27.5, 55 and
110 mg/mL), and ascorbic acid (15.6, 31.2, 62.5, 125, 250 and
500 mg/mL) were prepared. The regression equation was
Simultaneous Quantification of Capsaicinoids and Ascorbic Acid from Pungent Peppers 413
calculated in the form of y ¼ ax + b, where x was concentration and y was the peak area of compounds. Linearity was established by the coefficient of determination (R 2). Limits of
detection (LOD) were determined by injecting serially diluted
standard solutions and obtaining the signal-to-noise ratio (3:1)
for LOD. The precision of the HPLC conditions was evaluated by
the intra-day (seven injections per day) and inter-day (five
consecutive days) analysis of standards. The precision was
expressed as relative standard deviation (RSD, %) of retention
time.
Statistical analysis
All experiments were performed using the SAS statistical
system 9.2 (SAS Institute, Cary, NC, USA) for data analysis. The
comparison of means was analyzed by Tukey’s test. Data are
presented as average and SD. Significant differences were determined at the P 0.05 level.
Results and Discussion
Selection of extraction solvents
Five solvents, 3% metaphosphoric acid (MPA), ethanol (EtOH),
3% MPA –EtOH (8:2), 3% MPA– EtOH (1:1) and 3% MPA –EtOH
(2:8), were tested for their efficiency of extraction of capsaicinoids and ascorbic acid (Figure 2A). The capsaicin contents
were not significantly different in extracts made with EtOH, 3%
MPA –EtOH (2:8) and 3% MPA: EtOH (1:1). The highest level of
dihydrocapsaicin was extracted in EtOH, followed by 3% MPA –
EtOH (2:8). For ascorbic acid, the extraction solvents also
showed different efficiencies, in order from most to least
efficient: 3% MPA (2,130.33 mg/g), 3% MPA –EtOH (8:2)
(2,119.26 mg/g), 3% MPA –EtOH (1:1) (2,113.00 mg/g) and 3%
MPA –EtOH (2:8) (2,109.60 mg/g). Although water-based solvents were not commonly used for capsaicinoid analysis, the
extraction efficiency of target compounds was increased by
using small amounts of water (25). Based on these results, 3%
MPA –EtOH (2:8) was a better combination of solvents for
Figure 2. Extraction efficiency of capsaicinoids (capsaicin, dihydrocapsaicin) and ascorbic acid. (A) Different extraction solvents: 3% MPA = 3% metaphosphoric acid; 8P:2E =
3% metaphosphoric acid:EtOH (8:2); 1P:1E = 3% metaphosphoric acid:EtOH (1:1); 2P:8E = 3% methaphosphoric acid:EtOH (2:8); and EtOH = ethanol. (B) Ratio of sample to
solvent using 3% metaphosphoric acid:EtOH (2:8). Different letters denote significant differences (P 0.05) within each group of capsaicinoids and ascorbic acid.
414 Bae et al.
efficient, simultaneous extraction of capsaicinoids and ascorbic
acid.
Selection of ratio of sample to solvent
Different ratios (1:3, 1:4, 1:6 and 1:8, w/v) of sample to solvent
were compared to determine the optimum extraction efficiency. The maximum amounts of capsaicinoids (412.61 mg/g)
and ascorbic acid (2,785.93 mg/g) were extracted from the
pepper samples with a sample-to-solvent ratio of 1:8 and the
lowest amounts were extracted with a ratio of 1:3 (Figure 2B).
Topuz and Ozdemir (16) used 1:6 and 1:4 ratios of sample to
solvent for the extraction of capsaicinoids and ascorbic acid,
respectively, but extraction and analysis were performed independently. The present study clearly demonstrated that the
quantity of extraction solvent is critical. The higher ratio of
sample to solvent is better for the maximum extraction of capsaicinoids and ascorbic acid, compared to the previously
reported method (16).
Comparison of sonication and extraction time
Sonication is one of the key methods for better extraction of
capsaicinoids and ascorbic acid. Pepper samples were sonicated for 15, 30, 45 and 60 min to optimize the extraction
time. The levels of capsaicinoids and ascorbic acid varied at different sonication times (Figure 3A). The content of capsaicinoids was the highest (391.38 mg/g) at 30 min of sonication
time. Low levels of capsaicinoids and ascorbic acid were
observed when the sonication time was shorter or longer than
30 min. Ascorbic acid was significantly higher at 30 min
(2,719 mg/g), followed by 45 and 60 min. This result clearly
demonstrates that the full contents of ascorbic acid were not
fully extracted within 15 min of sonication.
To find the degradation rate of capsaicinoids and ascorbic
acid, different extraction times were compared (Figure 3B).
Because 30 min was the best time for extraction (within
60 min, based on previous data) a much wider range of times
(30 min, 3, 6, 12 and 24 h) was compared in this experiment.
Concentrations of capsaicinoids and ascorbic acid were
Figure 3. Levels of capsaicinoids and ascorbic acid using 3% MPA –EtOH (2:8) at (A) sonication time and (B) extraction time. Different letters denote significant differences
(P 0.05) within each group of capsaicinoids and ascorbic acid.
Simultaneous Quantification of Capsaicinoids and Ascorbic Acid from Pungent Peppers 415
Figure 4. Simultaneous HPLC separation and quantification of standards and pepper sample. Capsainoids and ascorbic acid were monitored at 282 and 254 nm, respectively.
significantly higher at 30 min, but the levels decreased during
the 3 –6 h extraction, by up to 10–13%, and further decreased,
up to 19%, for the 12 –24 h extraction. The extraction efficiency of ascorbic acid was diminished in this order: 3 h
(18%) . 6 h (22%) . 12 h (24%) . 24 h (26%). The degradation rate of ascorbic acid was faster than that of capsaicinoids.
A previous study reported maximum capsaicinoids at 60 min of
extraction using acetonitrile with a dry sample-to-solvent ratio
of 1:33 (26). In another study, ascorbic acid was extracted from
dry pepper powder by shaking for 15 min with 3% metaphosphoric acid at a 1:100 ratio of sample to solvent (27). In the
present study, the maximum amount of capsaicinoids and
ascorbic acid was simultaneously extracted in 30 min.
Development of HPLC method
The maximum amounts of capsaicin, dihydrocapsaicin and ascorbic acid were simultaneously extracted with 3% MPA–EtOH
(2:8), as determined by HPLC (Figure 4). This method was
applied to separate and quantify capsaicinoids and ascorbic acid
within 20 min using the mobile phase of phosphoric acid
(0.03M) and methanol. The HPLC-separated capsaicinoids and ascorbic acid were detected at 282 and 254 nm, respectively.
Previous studies reported the quantification of capsaicinoids and
ascorbic acid independently (28, 29), but in the present study,
the simultaneous separation and detection of capsaicinoids and
416 Bae et al.
Table I
Recovery of Capsaicin, Dihydrocapsaicin and Ascorbic Acid from Peppers
Compounds
Capsaicin
Dihydrocapsaicin
Ascorbic acid
Standard added
(mg)
Recovery (mg)
Expected
Actual*
4.17
14.30
23.80
8.03
10.70
13.37
12.60
25.20
50.40
101.82
111.95
121.45
51.34
54.01
56.68
507.99
520.59
545.79
99.28 + 0.12
121.24 + 0.14
126.78 + 0.21
51.64 + 0.58
53.16 + 0.44
62.00 + 1.34
492.79 + 8.10
513.08 + 3.24
539.38 + 1.53
Recovery
(%)
RSD
(%)
96.21
104.97
102.38
100.59
98.44
108.71
97.01
98.56
98.83
0.38
0.26
0.49
1.13
0.83
2.16
1.64
0.63
0.28
*Values are means + SD of triplicate samples.
ascorbic acid was accomplished using an optimized combination
of solvents. The developed method provided good separation and
quantification of capsaicinoids and ascorbic acid from pepper
varieties.
Recovery study
The recovery study was conducted by adding three different
concentrations of each standard to samples and then measuring
how much was recovered after extraction. The recovery ranged
from 96.21 to 104.97% for capsaicin, 98.44 to 108.71% for
Table II
Regression Equation, Linear Range, LODs and Precision Data of Extracted Capsaicin, Dihydrocapsaicin and Ascorbic Acid
Compounds
Regression equation
(y ¼ ax + b)
R2
Linear
(mg/mL)
LOD
(mg/mL)
Intra-day
RSD (%)
Inter-day
RSD (%)
Capsaicin
Dihydrocapsaicin
Ascorbic acid
y ¼ (430.32)x þ 8.8939
y ¼ (427.78)x þ 6.70181
y ¼ (1869.7)x þ 14.023
0.9998
0.9993
0.9981
1.95– 62.50
1.72– 55.00
3.91– 62.50
0.24
0.21
0.26
1.16
1.16
0.86
1.34
1.30
2.93
dihydrocapsaicin and 97.01 to 98.83% for ascorbic acid
(Table I). It is clear from this experiment that recovery of capsaicinoids was very high, and in some cases, recovery was
greater than 100%. The recovery data support the reliability
and accuracy of this newly developed analytical method.
Calibration curve, LOD and precision
A method for linear regression equations were determined by
plotting the concentration against peak area for capsaicin, dihydrocapsaicin and ascorbic acid. Good linearity and correlation
coefficients (R 2 . 0.99) were obtained for each analyte
(Table II). LODs for capsaicin, dihydrocapsaicin and ascorbic
acid were 0.24, 0.21 and 0.26 mg, respectively. The precision of
the developed HPLC system was evaluated by inter-day and
intra-day injection of capsaicin, dihydrocapsaicin and ascorbic
acid (Table II). Intra-day variability was found to be 1.16% for
capsaicinoids and 0.86% for ascorbic acid. For the reproducibility, inter-day (five days) variability ranged from 1.30 to 1.34%
for capsaicinoids, and was 2.93% for ascorbic acid. These low
RSD values (,3%) indicate that this method is precise for the
simultaneous extraction and analysis of capsaicinoids and ascorbic acid in peppers.
Conclusions
A method for simultaneous extraction and quantification of two
different categories of bioactive components in peppers was
developed. The efficient extraction conditions were achieved by
optimization of extraction solvents, sample-to-solvent ratios and
extraction time. This, one-step extraction and HPLC analysis
proved to be convenient, simple, efficient, sensitive, accurate
and reliable. The validated HPLC method demonstrated good linearity and precision. The developed method can be successfully
be used for the determination of capsaicinoids and ascorbic acid
in large numbers of pepper samples.
Acknowledgments
The present research report is based upon work supported by
Designing Foods for Health through the Vegetable & Fruit
Improvement Center, USDA-NIFA No. 2010-34402-20875.
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