Determination of Capsaicinoids in Foods Using Ultra ... - Springer Link

Food Sci. Biotechnol. 19(4): 1005-1009 (2010) DOI 10.1007/s10068-010-0141-8

RESEARCH ARTICLE

Determination of Capsaicinoids in Foods Using Ultra High Performance Liquid Chromatography Jaeho Ha, Hye-Young Seo, You-Shin Shim, Dong-Won Seo, Homoon Seog, Masahito Ito, and Hiroaki Nakagawa

Received: 4 March 2010 / Revised: 19 April 2010 / Accepted: 19 April 2010 / Published Online: 31 August 2010 © KoSFoST and Springer 2010

Abstract A sensitive, precise, and specific ultra high performance liquid chromatographic (u-HPLC) method was developed for the analysis of capsaicin in foods. The method validation parameters yielded good results, including linearity, precision, accuracy, and recovery. The u-HPLC separation was performed on a reversed column C18 (particle size 2 µm, i.d. 2 mm, length 50 mm, followed by fluorescence detection-excitation 280 nm, emission 325 nm). The recovery of capsaicin in gochujang was more than 91%, and the detection limit and lower determination limit of u-HPLC analysis were 0.054 and 0.163 µg/mL for capsaicin and 0.053 and 0.160 µg/mL for dihydrocapsaicin, respectively. The calibration graph for capsaicin and dihydrocapsaicin was linear from 0.2 to 10.0 µg/mL for uHPLC analysis. The inter-day and intra-day precisions (relative standard deviations) were 90%, C18H29NO3, Fw 307.42, CAS 19408-84-5; Sigma-Aldrich) and dissolve in 10 mL 95% ethanol in a volumetric flask to produce a solution of approximately 1 mg/mL. Ethanol (95%, ACS reagent grade), acetonitrile (chromatographic grade), and acetic acid (ACS reagent grade) were used. All solvents were of chromatographic or HPLC grade, and other reagents were ACS reagent grade. Analysis of capsaicinoids The concentration of capsaicinoids

J. Ha et al.

was determined using u-HPLC. The u-HPLC system (LaChromUltra L-2000 U Series; Hitachi-High Technologies Corp., Hitachinaka, Japan) was equipped with an eluant reservoir, u-HPLC pump (Model L-2200U), an autoinjection system of 10 µL injection at a fixed volume. LaChromUltra C18 (2 µm, 2 mm i.d.×50 mm L, Hitachi-High Technologies Corp.) was used as an analytical column. Mobile phase was acetonitrile: 1% acetic acid in water (6:4, v/v) and flow rate was 0.6 mL/min. Detector was L-2485U fluorescence detector (Hitachi-High Technologies Corp.), set at excitation wavelength 280 nm, emission wavelength 325 nm. Calibration graph Calibration graph for u-HPLC were based on peak area and were prepared by injecting 2 µL of the 0.2, 0.5, 1.0, 2.5, and 10.0 µg/mL solutions prepared by dilution of capsaicin and dihydrocapsaicin stock solutions with 95% ethanol.

Results and Discussion Effect of extraction time Extraction time plays an important role for efficient extraction depending on the concentration of capsaicinoids and sample matrix. It is necessary to optimize the time of extraction to achieve high extraction efficiency. In general, the extraction ratio of capsaicinoids in foods was increased in concomitant with increase of extraction time, the experiments were performed with gochujang (one of the traditional fermented foods) as a test sample by different extraction time (1, 3, 5, 6, and 7 hr) to evaluate the optimum extraction time. The results indicated that 95% extraction ratio of capsaicinoids could be achieved within 6 hr extraction as illustrated in Fig. 1 and 2. In the gochujang, the concentration of capsaicin is higher than that of dihydrocapsaicin, showing that extraction time of capsaicin required longer than that of dihydrocapsaicin. It seemed that the penetration of extraction solvent into gochujang was affected by the concentration of capsaicinoids due to its high amount of sugars (approximate 20%) and the viscous property of gochujang. In consequence, we purchased the 3 kinds of gochujang products (highcapsaicinoid gochujang, middle-capsaicinoid gochujang and low-capsaicinoid gochujang) and the effect of capsaicinoids concentration in gochujang on extraction ratio has been evaluated. In the case of the low concentration of capsaicinoids in gochujang (Low), shorter time required for extraction of capsaicinoids as 3 hr, however, in the case of the high concentration (High), the longer time required up to 6 hr or more, indicating that the higher concentration of capsaicinoids requires the longer extraction time as illustrated in Fig. 1 and 2.

1007

Determination of Capsaicinoids Using u-HPLC

Table 1. Calibration and sensitivity data of the u-HPLC method for the determination of capsaicinoids (µg/kg) Capsaicinoids

Linear range

R2

LOD

LOQ

Capsaicin Dihydrocapsaicin

0.20-10.0 0.20-10.0

0.9995 0.9999

0.054 0.053

0.163 0.160

Table 2. Intra-day repeatability data of the u-HPLC method for the determination of capsaicinnoids in gochujang (mg/kg) Number of sample

1)

Capsaicin2) Dihyderocapsicin3)

5.00881) 5.0030 5.0084 5.0019 5.0016 5.0037 5.0078 5.0046 5.0043 5.0831 5.0656 5.0379 5.0192 5.0325

28.1 27.1 27.8 30.4 28.4 28.6 29.2 28.3 28.5 30.8 29.5 31.8 31.1 29.8

14.2 13.7 14.3 15.5 14.6 14.6 15.2 14.5 14.6 16.1 15.4 16.7 16.3 15.3

Mean

5.0202

29.2

15.1

SD

0.0249

1.33

0.84

RSD, %

0.50

4.54

5.55

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Fig. 1. Extraction ratio of capsaicin as a function of time. Amount of capsaicin in the initial sample was estimated to be 15.6 (Low), 35.3 (Middle), and 98.6 (High) mg/kg.

Amount of sample (g)

1)

Values represent the mean of triplicate analyses; SD, standard deviation; RSD, relative standard deviation

Fig. 2. Extraction ratio of dihydrocapsaicin as a function of time. Amount of dihydrocapsaicin in the initial sample was estimated to be 14.8 (Low), 22.2 (Middle), and 44.1 (High) mg/kg.

Linearity To determine the linearity, 5 different concentrations of capsaicin standards were used in a working range from 0.2 to 10.0 µg/mL for the u-HPLC method. Each solution was injected 3 times and the average values of triplicate analysis were represented in Table 1. The calibration graph for capsaicin and dihydrocapsaicin was linear from 0.2 to 10.0 µg/mL for the uHPLC method. Regression analysis revealed a good relation (correlation coefficient r=0.9995 for capsaicin and 0.9999 for dihydrocapsaicin). The graph showed a negligible intercept, which was calculated by the leastsquare method’s regression equation. Limit of detection (LOD) and limit of quantitation (LOQ) The LOD and LOQ values were estimated at an

SD/b ratio of 3 and 10, where SD and b stand for the standard deviation of the intercept and slope of the regression line, respectively. LOD was 0.054 µg/mL for capsaicin and 0.053 µg/mL for dihydrocapsaicin. LOQ was 0.163 µg/mL for capsaicin and 0.160 µg/mL for dihydrocapsaicin. Repeatability Intra-day repeatability (n=14) test was performed on capsaicin and dihydrocapsaicin for the uHPLC methods. The relative standard deviations for intraday repeatability were revealed 4.54 for capsaicin and 5.55 for dihydrocapsaicin as shown in Table 2. Precision and accuracy Intra- and inter-day precision data of the u-HPLC method were given in Table 3, indicating that the relative standard deviation of intra- and inter-day are better than 5.21% for capsaicin and 9.79% for dihydrocapsaicin. In order to study the accuracy of the uHPLC methods, recovery experiments were carried out with the standard addition method. The recovery of the added standard to the assay samples was calculated from:

1008

J. Ha et al.

Table 3. Intra- and inter-day precision and accuracy data of the u-HPLC method for the determination of capsaicinoids in gochujang Spiked amount Intra-day Component (mg/kg) (%) Capsaicin 19.7 3.07 39.4 4.51 98.4 2.42 Dihydrocapsaicin 08.8 6.51 17.5 5.81 43.8 5.00 1)

Table 4. Applicability data of the u-HPLC method for the determination of capsaicinoids in various foods (mg/kg) Food

Inter-day Recovery (%) (%) 5.21 94.0 4.53 94.8 3.27 91.1 9.79 97.0 5.18 93.4 6.63 91.4

Gochujang 1 Gochujang 2 Gochujang 3 Gochujang 4 Chilli oil Kimchi Snack

Capsaicin 1)

Dihydrocapsaicin 1)

16.3±0.06 35.8±0.05 95.0±0.06 28.5±0.90 290.0±0.500 02.3±0.15 04.5±0.02

15.7±0.03 22.6±0.02 44.4±0.07 14.6±0.60 146.0±0.500 1.30±0.06 3.64±0.06

1)

1)

Sample weight approximately 5.0 g; Concentration of capsaicin and dihydrocapsaicin in the initial sample was 29.3 and 15.1 mg/kg, respectively.

Recovery, %=[(Ct−Cu)/Ca]×100 where Ct is the total concentration of the analyte found, Cu is the concentration of the present analyte in the original gochujang, and Ca is the concentration of the pure analyte added to the original gochujang. The results were given in Table 3. The average recoveries obtained were quantitative (91.1-94.8% for capsaicin, 91.4-97.0% for dihydrocapsaicin), indicating good accuracy of the u-HPLC methods. Applicability u-HPLC method has been known to be economical and environmentally friendly due to extremely rapid analysis. In concomitant with the fast analysis, the consumption of solvent for mobile phase can be reduced

Values represent the mean±SD (n=3).

up to 5 to 10 fold, comparing with the conventional HPLC method. To apply the u-HPLC method to various food products, 7 samples, containing a different amount of capsaicin, were analyzed by the u-HPLC methods. The results showed that the u-HPLC method can be applicable to the wide variety of foods containing chilli power, demonstrating the good separation without hindrance of interferences in foods as shown in Fig. 3. Acknowledgments We especially thank Hitachi-High Technologies Corporation for its financial support and also appreciate the technical advice received from Analytical Systems Design Department, Naka Division.

Fig. 3. Separation of capsaicinoids in various foods using u-HPLC-FLD.

1009

Determination of Capsaicinoids Using u-HPLC

References 1. Todd PH, Bensinger MG, Riftu T. Determination of pungency due to capsaicum by gas-liquid chromatography. J. Food Sci. 42: 660665 (1977) 2. Iwai K, Suzuki T, Fujiwake H. Simultaneous microdetermination of capsaicin and its four analogs by using high-performance liquid chromatography and gas chromatography-mass spectrometry. J. Chromatogr. 172: 303-311 (1979) 3. Reilly CA, Crouch DJ, Yost GS. Quantitative analysis of capsaicinoids in fresh peppers oleoresin capsicum and pepper spray products. J. Forensic Sci. 46: 502-509 (2001) 4. Chiang GH. HPLC analysis of capsaicins and simultaneous determination of capsaicins and pipeine by HPLCECD and UV. J. Food Sci. 51: 499-503 (1986) 5. Perucka I, Oleszek W. Extraction and determination of capsaicinoids in fruit of hot pepper Capsicum annuum L. by spectrophotometry and high-performance liquid chromatography. Food Chem. 71: 287291 (2000) 6. AOAC. Official Method of Analysis of AOAC Intl. 18 ed. Method 995.03. Association of Official Analytical Chemists, Gaithersburg, MD, USA (2005) 7. Kim KS, Park JB, Kim SA. Quality characterisitics of gochujang prepared with Korean single-harvested pepper (Capsicum annuum L.). J. Korean Soc. Food Sci. Nutr. 36: 759-765 (2007) 8. Chae IS, Kim HS, Ko YS, Kang MH, Hong SP, Shin D. Effect of citrus concentrate on the physicchemical properties of gochujang. Korean J. Food Sci. Technol. 40: 626-632 (2008) 9. Kwon DJ. Quality improvement of gochujang using Cordyceps sp. th

Korean J. Food Sci. Technol. 36: 81-85 (2004) 10. Oh HI, Park JM. Changes in quality characteristics of traditional gochujang prepared with a meju of different fermentation period during aging. Korean J. Food Sci. Technol. 29: 1166-1174 (1997) 11. Reilly CA, Crouch DJ, Yost GS. Quantitative anlaysis of capsaicinoids in fresh peppers, oleoresin capsicum, and pepper spray products. J. Forensic Sci. 46: 502-509 (2001) 12. Ha J, Han KJ, Kim KJ, Jeong SW. Gas chromatographic analysis capsaicin in gochujang. J. AOAC Int. 91: 387-391 (2008) 13. Hawer W, Ha J, Hwang J, Nam Y. Effective separation and quantitative analysis of major heat principles in red pepper by gas chromatography. Food Chem. 49: 99-103 (1994) 14. Estella-Hermoso MA, Campanero MA, Mollinedo F, Blanco-Prieto MJ. Comparative study of A HPLC-MS assay versus an UHPLCMS/MS for anti-tumoral alkyl lysophospholipid edelfosine determination in both biological samples and in lipid nanoparticulate systems J. Chromatogr. B. 877: 4035-4041 (2009) 15. Marin JM, Gracia-Lor E, Sancho JV, Lopez FJ, Hernandez, F. Application of ultra-high-pressure liquid chromatography-tandem mass spectrometry to the determination of multi-class pesticides in environmental and wastewater samples study of matrix effects. J. Chromatogr. A. 1216: 1410-1420 (2009) 16. Hoffman PG, Lego MC, Galetto WG. Separation and quantitation of red pepper major heat principles by reverse high performance liquid chromatography. J. Agr. Food Chem. 31: 1326-1330 (1983) 17. Saria A, Lembeck F, Skofitsch G. Determination of capsaicin analogues by high performance liquid chromatography. J. Chromatogr. 208: 41-46 (1981)