Capsaicinoids, Tocopherol, and Sterols Content in Chili (Capsicum sp.) by Gas Chromatographic-Mass Spectrometric Determination

Abstract

A gas chromatography-mass spectrometry method was developed for the quantitative determination of capsaicinoids, vitamin E, and phytosterols in chili peppers using a simple extraction technique for rapid screening. These components were extracted with acetonitrile and were injected into gas chromatography attached with mass spectrometry. The mean recovery values for triplicate analysis were between 90.6–99.7%. Besides major capsaicinoids (capsaicin, dihydrocapsaicin, nordihydrocapsaicin), four more minor capsaicinoids (nonivamide, nornordihydrocapsaicin, homodihydrocapsaicin I and II) were detected in chili samples. α-Tocopherol was also detected in the same run along with three phytosterols (campesterol, γ-sitosterol, and stigmasterol). Variations were observed across the chili sample in all of the constituents except a few minor capsaicinoids. The method was suitable for simultaneous estimation of capsaicinoids, vitamin E, and phytosterols in a single run from any types of pepper. Thus, the method is effective for rapid screening of peppers for its nutraceutical composition using single solvent extraction.

Keywords:

• Capsicum sp.
• Capsaicinoids
• Vitamin E
• Phytosterols
• GC-MS

INTRODUCTION

Hot peppers are intensively used as food additives for their pungency, aroma, and colour. The pungent principle of chili fruit is a group of compounds called capsaicinoids, in which the most important components are capsaicin, dihydrocapsaicin, and nordihydrocapsaicin (Fig. 1). Capsaicinoids are vanillylamides of branched chain fatty acids, with 9–11 carbons, of which capsaicin (vanillylamide of 8-methylnontrans-6-enoic acid) and dihydrocapsaicin (vanillylamide of 8 methylnonanoic acid) occur in quantities greater than 80% in chili. These two compounds are the most pungent capsaicinoids, with the equivalent value of 16.1 × 10⁶ Scoville heat unit (SHU).^[1] The remaining derivatives are found in very small amounts.^[1,2] Capsaicinoids are being produced in glands on the pepper’s placenta and the white ribs present along the sides of a pepper skin. Capsaicinoids are synthesized through the cinnamic acid pathway, and it is thought that their degradation is aided by the action of peroxidase.^[3]

FIGURE 1 Structures of capsaicinoids, α-tocopherol, and sterols estimated by the GC-MS method.

In addition to its therapeutic effects as a stimulant and counter irritant, it is being used as a neuropharmacological tool. Capsaicin is known to have great medical value and it has been reviewed to evaluate its effect in treatment of painful conditions, such as rheumatic diseases, cluster headache, painful diabetic neuropathy, postherpetic neuralgia, etc.^[4] In recent years, capsaicinoids have been studied and found to be an effective treatment for a number of sensory nerve fiber disorders, including arthritis, cystitis, human immunodeficiency virus, etc.^[5,6] They have also been reported as having an antioxidant and antibacterial effect on a certain group of bacteria.^[7]

Reverse phase liquid chromatographic method has been used for determination of capsaicin analogues.^[8–11] Gas chromatographic (GC) methods were also reported in the literature using packed^[12,13] or capillary columns.^[14,15] But these methods usually require extensive and long sample cleanup prior to analysis. Most of these methods require derivatization in order to increase the volatility of capsaicinoids, thus increasing the total time of estimation. A gas chromatography-mass spectrometry (GC-MS) method has been reported to analyse these compounds,^[16] which include an extra clean-up step for total analysis.

Phytosterols present in vegetables are known to be hypocholesterolemic. These phytochemicals and their derivatives are also potent antioxidants.^[17] Phytosterol content in vegetable extract is generally unnoticed. But due to emerging importance of phytosterols in human nutrition, it gained importance. α-Tocopherol content in chili is quite high and it is known to be a significant antioxidant. Thus, the estimation protocol for tocopherol and phytosterols holds a valid reason.^[18]

A simple and rapid method was developed including extraction of capsaicinoids as well as sterols and tocopherol with acetonitrile, followed by GC/MS analysis. This procedure allows separation of capsaicinoids, tocopherol, and sterols. Capsaicinoids included three major capsaicinoids: capsaicin, dihydrocapsaicin, and nonivamide, as well as identification of other minor capsaicinoids viz. nordihydrocapsaicin, nornorcapsaicin, homocapsaicin, and homodihydrocapsaicin, reported in the literature.^[19] The proposed method does not require derivatization for the estimation and is a compromise between short time analysis and better sensitivity due to inclusion of GC-MS. The present method was validated for determination of capsaicinoids, phytosterols, and tocopherols in chili.

MATERIALS AND METHODS

Reagents and Chemicals

Capsaicin and dihydrocapsaicin were purchased from Sigma Chemical Co. and were of the highest purity. Solvents for GC-MS were chromatographic grade and were obtained from Merck® (India). Water was purified to 18.2 MΩ-cm by means of a bench-top water purifier (Elix, Millipore).

Standards

To prepare stock solution of capsaicin and dihydrocapsaicin, weigh 50 mg of capsaicin, 25 mg dihydrocapsaicin, and put into a 50-mL volumetric flask. Dilute to volume with acetonitrile to give concentrations of 1000 and 500 μg mL⁻¹ capsaicin and dihydrocapsaicin, respectively. Concentrations of working solutions of capsaicin (50–250 ng μL⁻¹) and dihydrocapsaicin (25–250 ng μL⁻¹) were prepared for further analysis.

GC-MS Apparatus and Configuration

GC-MS analysis was carried out using a 7890A GC (Agilent Co., USA) equipped with a HP-5MS column (30 m × 0.25 mm; 0.25 μm; Agilent Co., USA), which was directly connected to a triple axis HED-EM 5975C mass spectrometer (Agilent Co., USA). The injection volume was 1 μl with flow mode in split control. The carrier gas flow was set at 1 ml min⁻¹ helium. Helium (high purity, New Delhi, India) was used as carrier gas at a head pressure of 10 psi. The oven temperature was initially held at 90°C for 1 min. Hereafter, the temperature was raised with a gradient of 5°C min⁻¹ until the temperature reached to 290°C. Other settings were as follows: 250°C interface temperature, 200°C ion source temperature, and electron impact ionization (EI) at 70 eV. Mass spectra were analyzed by both full scan mode and selected ion monitoring (SIM) mode. Raw MS data were processed using the program MSD productivity Chemstation to obtain a purified spectrum by removing residual background contaminants, partially eluting peaks, and column bleed from the spectrum. Structures were confirmed using the library of the instrument. The MS acquisition parameters were: interface temperature, 280°C; ion source temperature, 200°C; electron ionization, 70 eV; full scan mode, 50–550 mass units; solvent delay, 3 min; and E.M voltage, 889.

Sample Preparation

Chili samples were lyophilized before extraction. Samples not subjected to direct analysis were stored at 4°C. One dried chili pepper (1 g) was extracted with 10 mL acetonitrile using a probe ultrasonicator (Sonics®, Vibra cell, ultrasonic processor, 750 Watt, 20 kHz) for 10 min at medium amplitude. An ultrasonicator was run at 50% amplitude and the energy used was 7830 Joules. Repeat the step two more times to get total extraction of capsaicinoids and other phytochemicals. Combine the extracts and centrifuge at 10,000 rpm for 10 min. The extracts were then filtered through a 0.45 μ nylon syringe filter (Millex, India) before chromatographic analysis.

Recovery Study

Recovery study was done by fortifying the chili matrix, known for its content of capsaicin and dihydrocapsaicin with known amounts of capsaicin and dihydrocapsaicin. Fortified samples were extracted following a previous procedure after allowing for completion of capsaicinoid-matrix interaction. Extracted samples were injected in a GC-MS system for quantification. Levels of spiking and recovery values are given in Table 1. Results are expressed as percentage of recovery of amounts added for fortification.

TABLE 1 Analytical recoveries of capsaicinoids (mean) added to a chili matrix

Compound	Correlation coefficient (r)	Linear range (ng μl^−1)	LOD (ng)	LOQ (ng)	Recovery (%)
Capsaicin	0.923	25–500	0.25	0.80	92.5
Dihydrocapsaicin	0.987	25–500	0.30	0.90	99.7
Tocopherol	0.979	0.5–100	0.1	0.3	90.6

RESULTS AND DISCUSSION

Extraction

Acetonitrile was used as extraction solvent because it achieved complete extraction of capsaicinoids along with tocopherol and sterols without extracting much of the interfering materials. The solvent is preferable because the same solvent can be used after centrifugation/filtration for analysis. Another advantage of the extraction method was time saving and it results in extracts suitable for chromatography, especially when using MS detection as it produces clean total ion chromatogram (TIC). Boonkird et al.^[20] also used microwave-assisted extraction for analysis of capsaicinoids. Manirakiza et al.^[16] used the same solvent for the extraction purpose, whereas Pino et al.^[21] used acetonitrile for the same purpose. Kozukue et al.^[22] used methanol for extraction of capsaicinoids. Several solvents were evaluated to finalize the solvent for extraction of capsaicinoids.^[23] Enzymatic treatments were also used by other investigators to improve the extraction of capsaicinoids from chili fruits.^[24]

Many techniques for the extraction of capsaicinoids from peppers have been studied, such as maceration, magnetic stirring, Soxhlet, ultrasound-assisted extraction, extraction by means of supercritical fluids, extraction by pressurized liquids, enzymatic extraction, and microwave-assisted extraction.^[8] These techniques are employed in response to the increasing demand for methods that allow the extraction process to be automated, extraction times to be shortened, the consumption of solvents to be reduced, and the exposure of operative personnel to solvents to be minimised. Capsaicinoid and tocopherol was simultaneously estimated using supercritical CO₂ and sub-critical pentane extraction followed by HPLC analysis.^[25]

GC-MS Characterization

Capsaicinoids were easily separated with the HP-5 MS column, with normal order of elution for a nonpolar stationary phase. Figure 2 showed the representative total ion chromatogram of an extract from a chili sample. Mass spectra of the identified capsaicinoids are given in Fig. 3. No great difference in fragmentation patterns appeared between capsaicinoids except molecular ion peak. Although the most intense ion is m/z = 137, corresponding to the vanillyl moiety, the quantitation was performed using the molecular ions of each compound (m/z = 293, 293, 279, 305, 307, for nordihydrocapsaicin, nonivamide, nornordihydrocapsaicin, capsaicin, and dihydrocapsaicin, respectively).

FIGURE 2 TIC of chili sample estimated by the GC-MS method.

FIGURE 3 EI-mass spectra of major and minor capsaicinoids present in chili sample.

In a LC-MS study, 3 major and 12 minor capsaicinoids, including nornordihydrocapsaicinoids, were detected in chili pods.^[26] Capsaicinoids were identified using GC-MS earlier as such or by derivatization using a different extraction procedure.^[16] The present study identified eight capsaicinoids in pepper extracts using gas chromatography/mass selective detection (Table 2). GC-MS characterizations of capsaicinoids were reported earlier also.^[13,16]

Along with capsaicinoid, three sterols (stigmasterol, campesterol, and β-sitosterol) were also identified and relative content was estimated. Simultaneous gas chromatographic separation of cholestanol, desmosterol, lathosterol, campesterol, and β-sitosterol was achieved using a 60-m length capillary column.^[27–30] Sterols were eluted at a temperature above 270°C, which is consistent with the report by Dutta and Normen.^[27] Keller and Jahreis^[29] reported a GC-MS method for quantitative determination of cholesterol, coprostanol, coprostanone, cholestanol, and bile acids. Another GC-MS method was described by Chevy et al.^[30] for determination of cholesterol, lathosterol, 8-dehydrocholesterol, and 7-dehydrocholesterol from amniotic fluid. Tocopherol was also identified and simultaneously analysed in chili extracts by its fragmentation pattern without derivatization. Mass spectra of the identified phytosterols and tocopherols are given in Fig. 4. Tocopherol was estimated earlier after derivatization using the same column.^[31] Recently, both GC and HPLC method were used for the estimation of sterols and tocols in vegetable oil.^[32]

FIGURE 4 EI-mass spectra of α-tocopherol and sterols present in chili sample.

Recovery Study

Calibration graphs were constructed by plotting the ratio between peak area of analyte and analyte concentration. Equations of plotted curves and correlation coefficients for capsaicin and dihydrocapsaicin are as follows:

where y = area of compound and x = concentration (ng μL⁻¹). Recoveries of the extraction procedure after initial screening of different methods ranged from 90.6 to 99.7% (Table 1). The method was applied to chili peppers of India. Relative concentrations found are given in Table 3. Capsaiciniod content reported in the literature ranged on a broad scale (10–1400 μg g⁻¹), with capsaicin and dihydrocapsaicin accounting for more than 90% in almost all species.^[16,24,33] Because of the impossibility of having a capsaicinoids-free matrix, the recovery of the procedure was estimated by spiking capsicum samples with a known amount of capsaicin and dihydrocapsaicin following the method by Manirakiza et al.^[16] Recovery was estimated calculating initial and fortified amount into account.

TABLE 2 Retention time and mass fragmentation pattern of capsaicinoids, tocopherol, and phytosterols

Compound	Rt	Identification	m/z	SIM
NDHC	32.814	MS	293	55 + 94 + 137 + 253
CRC-I	32.935	MS	293	—
CRC-II	33.310	MS	299	—
NVM	33.437	MS	293	55 + 73 + 105 + 137 + 195 + 253
NNDHC	33.950	MS	279	57 + 113 + 137 + 149 + 207 + 239
C	34.193	MS, STD	305	55 + 94 + 137 + 195 + 262
DHC	34.550	MS,STD	307	55 + 94 + 137 + 195 + 264
DHC-i	35.042	MS	307	55 + 94 + 137 + 195
HDHC-I	35.994	MS	321	55 + 95 + 137 + 178 + 207 + 252
HDHC-II	36.114	MS	321	55 + 95 + 137 + 178 + 207 + 252
α-tocopherol	42.113	MS, STD	430	57 + 121 + 165 + 205 + 281 + 341
Campesterol	43.576	MS	400	105 + 145 + 207 + 255 + 289 + 327 + 367
Stigmasterol	44.125	MS	412	55 + 91 + 133 + 165 + 207 + 239 + 281 + 341 + 379
γ-Sitosterol	45.853	MS	414	57 + 105 + 145 + 178 + 213 + 255 + 329 + 381

Quantification

Capsaicinoids, tocopherols, and phytosterols in four chili samples were indicated by the relative content and by comparing the mass spectra between standards and samples. Four major capsaicinoids (capsaicin, dihydropcapsaicin, nonivamide, and nordihydrocapsaicin) were identified and quantified (Table 3). Usual variation was observed between chili samples. Two CRCs (capsaicinoid-related compounds) were detected in Chili-I and Chili-II samples. Nornordihydrocapsaicin was identified in Chili-I only. In the other sample, it was found undetectable. Interestingly, DHC-i (isomer of DHC) was also found only in one sample (Chili-II). In terms of α-tocopherol content, Chili-I was found to possess the maximum and the least was found in Chili-II. Campesterol and γ-sitosterol were also detected and variations were detected in chili samples. But, while stigmasterol content was maximum in Chili-I and in the other three samples, it was found not quantifiable (NQ). The present method described in this study was able to estimate major capsaicinoids, tocopherol, and phytosterols in a single run besides identification of minor capsaicinoids. Single solvent extraction, minimum clean up, and quick estimation of major phytochemicals known to be antioxidants made the present investigation novel. Furthermore, no derivatization was required for the estimation of the compounds. The method will be useful for quick screening of chili germplasm for their phytochemical content.

TABLE 3 Quantity and relative content* of capsaicinoids in fresh chili varieties analysed by GC-MS

	Rt (min)	m/z	Chili-I	Chili-II	Chili-III	Chili-IV
NDHC	32.814	293	nd	1.05	0.42	2.94
CRC-I	32.935	293	q	nd	nd	nd
CRC-II	33.310	299	q	q	nd	nd
NVM	33.437	293	0.32	1.51	nd	1.55
NNDHC	33.950	279	d	nd	nd	nd
C	34.193	305	1.29	0.55	0.14	0.65
DHC	34.550	307	0.89	0.33	0.23	0.18
DHC-i	35.042	307	nq	q	nq	nq
HDHC-I	35.994	321	2.18	4.38	nd	0.70
HDHC-II	36.114	321	nq	nq	nq	nq
Tocopherol	42.113	430	19.10	nd	8.93	4.39
Campesterol	43.576	400	2.54	0.57	nd	0.70
Stigmasterol	44.125	412	q	nq	nq	nq
γ-Sitosterol	45.853	414	nd	4.05	2.40	2.69

d: Detectable; q: quantifiable; nq: non quantifiable; nd: non detectable.

*Relative content = (area of sample-area of sample with least value)/area of sample with least value.

CONCLUSIONS

The method described in this study was simple, rapid, and allows detection of a range of compounds by using simple extraction followed by direct GC-MS analysis without derivatization. Thus, the simultaneous estimation method of capsaicinoid, tocopherol, and phytosterols made this investigation unique and ideal for quick screening of chili lines used for breeding programme or nutraceutical exploitation. The method ensured high reproducibility. The use of GC-MS for quantitation gives information about the possible interferences, minor constituents, and allows a more accurate quantitation of the capsaicinoid, tocopherol, and sterol content of the chili peppers. The method is suitable for capsaicinoids determination from any type of peppers and is effective for rapid screening of peppers for its nutraceutical composition using single solvent extraction.

ACKNOWLEDGMENTS

The authors are thankful to the Head, Division of Agricultural Chemicals for providing the necessary facilities.

FUNDING

The authors are grateful to the National Agricultural Innovation Project (I.C.A.R.), New Delhi, India for research funding.