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High-performance liquid chromatography method to measure α- and γ-tocopherol in leaves, flowers and fresh beans from Moringa oleifera
Journal of Chromatography A, 1105 (2006) 111–114
High-performance liquid chromatography method to measure ␣- and ␥-tocopherol in leaves, flowers and fresh beans from Moringa oleifera D.I. S´anchez-Machado, J. L´opez-Cervantes ∗ , N.J. R´ıos V´azquez Departamento de Biotecnolog´ıa y Ciencias Alimentarias, Instituto Tecnol´ogico de Sonora, P.O. Box 541, Antonio Caso s/n, Col. Villa ITSON, 85139 Cd. Obreg´on, Sonora, Mexico Available online 10 August 2005
Abstract A high-performance liquid chromatography method for the microscale determination of ␣- and ␥-tocopherol in leaves, flowers and fresh beans from Moringa oleifera is reported. The method includes microscale saponification and extraction with n-hexane. Optimized conditions for reversed-phase HPLC with UV detection were as follows: column, 25 cm × 0.46 cm, Exil ODS 5-m; column temperature, 25 ◦ C; mobile phase, a 20:80 (v/v) mixture of methanol:acetonitrile; flow rate, 1.0 ml/min. With these conditions, method precision (relative standard deviation) was 5.6% for ␣-tocopherol and 4.9% for ␥-tocopherol. We used this method to measure ␣- and ␥-tocopherol in samples from M. oleifera as part of nutritional studies in edible plants cultivated in the Northwest M´exico. © 2005 Elsevier B.V. All rights reserved. Keywords: Food analysis; ␣-Tocopherol; ␥-Tocopherol; HPLC; Moringa oleifera
1. Introduction Ten to 12 species of the Moringaceae family have been reported to belong to one genus called Moringae. Almost all Moringa species are native to India, from where they have been introduced in many warm countries [1,2]. The tree ranges in height from 5 to 10 m and sometime even 15 m. This tree grows rapidly even in poor soil and is little affected by drought. The flowers and the fruits appear twice a year, and seeds or cuttings can propagate the tree; the latter is more preferred. The leaves, flowers, fruits and roots of the tree are used as vegetables and the trunk is used in the paper industry [3,4]. The fresh beans after roasting also make a palatable dish. Seed are also consumed after frying and reported to taste like peanuts. A number of medicinal and therapeutic properties have been ascribed to various parts of this multipurpose tree, which included the treatment of ascites rheumatism, and use as cardiac and circulatory stimulants [5–7]. In India and Philippine, village people use the fresh leaves to prepare fat foods, and it has been found that there is a significant increase in the shelf life of this foods due that moringa leaves can be a ∗
Corresponding author. Tel.: +52 644 4109000; fax: +52 644 4109001. E-mail address: [email protected] (J. L´opez-Cervantes).
0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.07.048
good source of natural antioxidants [8]. Accordingly, there is a strong need for effective antioxidants from natural source because they may be safer for humans. In recent years, the physiological functionality of foods has received much attention due to the increasing interest in human health and has been studied in vitro and in vivo by many researchers. The antioxidative action, one of the important physiological functions of foods, is supposed to protect living organisms from oxidative damages, resulting in the prevention of various diseases such as cancer, cardiovascular diseases, and diabetes [9,10]. The tocopherol homologues (␣-, -, ␥-, and ␦-tocopherol) are natural antioxidants [11]. ␣-Tocopherol is the primary vitamer with biological activity; however, the other vitamers have been shown to have decreased activity. ␥-Tocopherol has 10% of the activity of ␣-tocopherol, and ␦-tocopherol has 1% of the activity of ␣tocopherol [12]. It has been reported that the retention of natural Vitamin E is at least double that of the synthetic form. Method to extract vitamins in foods is time consuming and tedious [14]. Traditionally, fast-soluble vitamin analysis is performed by alkaline saponification of the entire sample matrix, or of an isolated lipid fraction, followed by liquid extraction with organic solvents like diethyl ether or n-hexane. A few methods analyse these vitamins directly
112
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without saponification in various matrices [15,16]. HPLC methods for the determination of ␣- and ␥-tocopherol have largely replaced direct spectrophotometric and fluorometric procedure. HPLC had been used with fluorescence detection, with UV detection, and most recently with detection by evaporative light scattering. Method precision is in all cases high [17]. The species Moringa oleifera has a considerable amount of quantitative nutritional information [18]. Additionally, a full characterization of the oil produced from their seeds has been reported [4]. However, the available information on the ␣- and ␥-tocopherol content in samples of this edible plant is very limited [19]. Therefore, the aim of the present work was the optimization of the extraction and chromatographic conditions for measuring tocopherols from M. oleifera, the validation of the method, and the measurement of representative sample obtaining reliable date.
plants 2, 4, 6, and 24 months old. Soon after collection, the samples were dried at 60 ◦ C for 8 h and stored in darkness at room temperature until the analysis. 2.4. Standards and quantification Stock standard solutions of ␣-tocopherol (0.4 mg/ml) and ␥-tocopherol (0.2 mg/ml) were prepared daily by dissolution in 100% methanol and stored at −10 ◦ C in the dark. Working solutions were prepared from these solutions and diluted with methanol prior analysis. For determination of ␣- and ␥-tocopherol in leaves, flowers and fresh beans, the stock solution was in all cases analyzed together with the samples, and analyte concentrations in samples were estimated on the basis of peak areas. All samples were analyzed in duplicate. ␣- and ␥-Tocopherol contents are cited as mean ± standard deviation. 2.5. Sample preparation
2. Experimental 2.1. Chemicals and reagents HPLC-grade methanol, hexane and acetonitrile (EMD Chemicals, Darmstadt, Germany). Analytical-grade pyrocatechol (Aldrich, St. Louis, USA), potassium hydroxide (J.T. Baker, Xalostoc, M´exico) and (±)-␣- and ␥-tocopherol (Fluka, Steinheim, Switzerland). Ultrapure water was prepared using a NANOpure Diamond UV system (Barnstead International, Buduque, Iowa, USA). KOH solution was prepared in methanol (0.5 M). The pyrocatechol solution (1 g in 5 ml of methanol) was prepared fresh daily, and stored at about 4 ◦ C in the dark. 2.2. Equipment The HPLC–UV system (GBC, Dandenong, Australia) was equipped with an auto injector LC 1650, an on line solvent degasser LC1460, a system controller WinChrom, a pump LC1150, a column oven LC1150, a 20 l injection loop (Rheodyne, Cotati, CA, USA), and a photodiode array detector LC5100. Chromatographic analysis was performed using an analytical scale (25 cm × 0.4 cm I.D.) SS Exil ODS column with a particle size 5 m (SGE, Dandenong, Australia). HPLC conditions were as follow: mobile phase 20:80 (v/v) methanol:acetonitrile; a flow rate of 1.0 ml/min and column temperature 25 ◦ C. The UV detection wavelength was set at 208 nm. The total time between injections was 18 min. Identification of tocopherols was based on retention time, co-injection with standards, and UV spectra. 2.3. Samples M. oleifera leaves, flowers and fresh beans were collected in Cajeme, Sonora, M´exico. The leaves were harvested of
Determination of water content. Water content of leaves, flowers and fresh beans was determined by weighing before and after drying to constant weight in a vacuum oven at 40 ◦ C. Saponification and extraction. ␣- and ␥-Tocopherol were extracted from the leaves, flowers and fresh beans by the method of S´anchez-Machado et al. [17] with minor modifications. A subsample of 0.40 g (±0.001 g) was weighed out in a screw-top assay tube. Two hundred microlitres of pyrocatechol solution and 5 ml of KOH solution (0.5 M in methanol) were added, and immediately vortexed for 20 s. The tubes were placed in a water bath at 80 ◦ C for 15 min, removing them every 5 min and vortexing again for 15 s. After cooling in iced water, 1 ml of distilled water and 5 ml of hexane was added, and the mixture was rapidly vortexed for 1 min, then centrifuged for 2 min at 425 × g. Three ml of the upper phase was transferred to another test tube and dried under nitrogen. The residue was redissolved in 3 ml of the HPLC mobile phase (20:80, v/v, methanol:acetonitrile), then membranefiltered (pore size 0.50 m; Whatman, Clifton, New Jersey, USA). Finally, an aliquot of 20 l was injected into the HPLC column. Before injection, the extracts were maintained at −10 ◦ C in the dark.
3. Results and discussion 3.1. Sample preparation There have been many studies of optimum conditions for the extraction of ␣- and ␥-tocopherol, from foods of different types with saponification, or of an lipid fraction [16,20], but there are small-scale applications concerning simultaneous extraction of this analytes from different products [13,21]. The large-scale extraction methods are expensive in terms of apparatuses required, cost of solvents, and time. We therefore decided to develop a micro-scale method of the type described
D.I. S´anchez-Machado et al. / J. Chromatogr. A 1105 (2006) 111–114
113
Table 1 Linearity for ␣- and ␥-tocopherol analyzed Compound
Range (g/ml)
Equation
r
␣-Tocopherol ␥-Tocopherol
7–140 0.4–8
y = 68686x − 68871 y = 84903x − 11980
0.9999 0.9998
x, amount (g/ml); y, peak area; r, correlation coefficient.
Fig. 1. Typical chromatogram of a standard mixture. Peak identification: (1) ␥-tocopherol and (2) ␣-tocopherol.
by Botsoglou et al. [22] and S´anchez-Machado et al. [17]. To determine optimal sample amounts, preliminary assays were performed with 0.1, 0.2, 0.3, 0.4 and 0.6 g of sample and with different amount of 0.5 M KOH in methanol (3, 5, 7 and 9 ml); the results indicated that good results are achievable with 0.40 g of sample and 5 ml 0.5 M KOH in methanol. 3.2. Tocopherols identification ␣- and ␥-Tocopherol in the leaves, flowers and fresh beans were identified by comparison of retention times and UV absorption spectra with those obtained for corresponding standards. For determination of retention times, the reference standards were injected both individually and as a mixture. Typical chromatogram of a standard mixture is shown in Fig. 1. Peaks were observed at 9.17 ± 0.035 min for ␥tocopherol, and 10.52 ± 0.050 min for ␣-tocopherol. Typical chromatogram of flowers from M. oleifera is shown in Fig. 2. 3.3. Method validation The linearity of standard curves (Table 1) was expressed in terms of the correlation coefficient (r) from plots of the integrated peak area versus concentration of the standard
(g/ml). These equations were obtained over a wide concentration range, in concordance with the level of ␣- and ␥-tocopherol found in leaves, flowers and fresh beans. All curves are based on analyses of at least four dilutions of the corresponding standard. In all cases the relationships between concentration and peak area were linear, with coefficients of determination greater than 0.999. Method precision was evaluated on the basis of the relative standard deviation (RSD) of ␣- and ␥-tocopherol determinations in eight samples of leaves from M. oleifera, with very good results for both tocopherols; both values are low by comparison with those obtained in related previous studies [23]. The Table 2 shows precision results. These results indicate that the present method can be used for routine analyses of ␣- and ␥-tocopherol in quality control laboratories. Determination of detection limits on the basis of signal-to-noise ratio (3:1) as per American Chemical Society guidelines [24] were 140 ng/ml for ␥-tocopherol and 160 ng/ml for ␣tocopherol, which are smaller than those presented in other works [14,25,26]. Accuracy was estimated by means of recovery assays. The recovery of the method was evaluated by analysis of six samples of leaves, 6 old months, from M. oleifera spiked before saponification with ␣-tocopherol (628 g/g of sample), and ␥-tocopherol (20 g/g of sample), and estimated as 90.3% and 89.1%, respectively. Table 2 shows the recovery percentages obtained. 3.4. α- and γ-Tocopherol in samples from Moringa oleifera The analytical method was applied to the determination of ␣- and ␥-tocopherol content in leaves, flowers and fresh beans of the tree, that are eaten as vegetables. Table 3 shows ␣- and ␥-tocopherol content of the samples considered in the present study. ␥-Tocopherol contents ranged from 5.7 (leaves of adult plants) to 27.8 (leaves of 6-month-old plants) g/g of dry mass. Important variations can be observed for the different samples with values for ␣-tocopherol ranging from 95.9 (fresh beans) to 744.5 (leaves from adult plants) g/g of dry Table 2 Precision and recovery of the HPLC proposed method Precision (n = 8)
␣-Tocopherola Fig. 2. Typical chromatogram of flowers from Moringa oleifera. Peak identification: (1) ␥-tocopherol and (2) ␣-tocopherol.
␥-Tocopherola a
Recovery (n = 6)
Mean ± SD
RSD (%)
Percent
RSD (%)
243.4 ± 13.7 6.8 ± 0.3
5.6 4.9
90.3 89.1
4.1 5.4
Results expressed as g/g dry mass.
D.I. S´anchez-Machado et al. / J. Chromatogr. A 1105 (2006) 111–114
114
References
Table 3 ␣- and ␥-tocopherol content in Moringa oleifera Compound
␣-Tocopherola (mean ± SD)
Leaves 2 months 4 months 6 months Adults
731.8 399.3 530.7 744.5
Flowers Fresh beans
305.7 ± 23.9 95.9 ± 7.3
± ± ± ±
75.6 43.6 44.7 95.4
␥-Tocopherola (mean ± SD) 9.5 8.4 27.8 5.7
± ± ± ±
0.9 0.5 3.7 0.5
9.1 ± 0.9 N.D.
N.D.: not detected. a Results expressed as g/g dry mass.
mass. The difference could be explained by variation between the old of the plants as well as by variation between the different parts of plant. The highest measures were obtained for leaves and the lowest for flowers and fresh beans. It is interesting to observe the high ␣-tocopherol content of plants with proved antioxidant activity, indicating the possible relationship between tocopherol content and functional activity [21]. In general terms, these results are in agreement with previous works in moringa leaves [19].
4. Conclusions The method described allows the rapid isolation and quantitative determination of ␣- and ␥-tocopherol in samples of the different parts from tree M. oleifera. Due to the ease of sample preparation, the method may be used in routine analysis of vegetable foods. Our results suggest that moringa, particularly their leaves, might be useful nutritional source of tocopherols.
Acknowledgements This work was financed under project no. ITSON-EXB35 from Program of Profesor’s Improvement from the Public Education Department (PROMEP).
[1] S. Lalas, J. Tsaknis, J. Food Comp. Anal. 15 (2002) 65. [2] G. Vlahov, P.K. Chepkwony, P.K. Ndalut, J. Agric. Food Chem. 50 (2002) 970. [3] J. Tsaknis, S. Lalas, V. Gergis, V. Dourtoglou, V. Spiliotis, J. Agric. Food Chem. 47 (1999) 4495. [4] J. Tsaknis, S. Lalas, J. Food Comp. Anal. 15 (2002) 79. [5] N. Sreeramulu, G.D. Ndossi, K. Mtotomwema, Food Chem. 10 (1983) 205. [6] A. Caceres, A. Saravia, S. Rizzo, L. Zabala, E. De Leon, F. Nave, J. Ethnopharmacol. 36 (1992) 233. [7] F. Anwar, M.I. Bhanger, J. Agric. Food Chem. 51 (2003) 6558. [8] P. Siddhuraju, K. Becker, J. Agric. Food Chem. 51 (2003) 2144. [9] K. Azuma, M. Nakayama, M. Koshioka, K. Ippoushi, Y. Yamaguchi, K. Kohata, Y. Yamauchi, H. Ito, H. Higashio, J. Agric. Food Chem. 47 (1999) 3993. [10] R. Aquino, A. C´aceres, S. Morelli, L. Rastrelli, J. Nat. Orod. 65 (2002) 1773. [11] C.M. Seppanen, M. Rahmani, S. Csallany, J. Food Sci. 68 (2003) 1644. [12] C.J. Wyatt, S. P´erez Carballido, R.O. M´endez, J. Agric. Food Chem. 46 (1998) 4657. [13] D.J.M. G´omez-Coronado, C. Barbas, J. Agric. Food Chem. 51 (2003) 5196. [14] G.W. Chase Jr., A.R. Long, J. AOAC Int. 81 (1998) 582. [15] R. Bruni, A. Medici, A. Guerrini, S. Scalia, F. Poli, C. Romagnoli, M. Muzzoli, G. Sacchetti, Food Chem. 77 (2002) 337. [16] B. Rodas Mendoza, S.M. Pons, A.I. Castellote Bargall´o, M.C. L´opezSabater, J. Chromatogr. A 1018 (2003) 197. [17] D.I. S´anchez-Machado, J. L´opez-Hern´andez, P. Paseiro-Losada, J. Chromatogr. A 976 (2002) 277. [18] R.P.H. McBurney, C. Griffin, A.A. Paul, D.C. Greenberg, J. Food Comp. Anal. 17 (2004) 277. [19] L.S. Ching, S. Mohamed, J. Agric. Food Chem. 49 (2001) 3101. [20] M. Ryyn¨anen, A.-M. Lampi, P. Salo-V¨aa¨ n¨anen, V. Ollilainene, V. Piironene, J. Food Comp. Anal. 17 (2004) 749. [21] D.J.M. G´omez-Coronado, E. Ib´an˜ ez, F.J. Rup´erez, C. Barbas, J. Chromatogr. A 1054 (2004) 227. [22] N. Botsoglou, D. Fletouris, I. Psomas, A. Mantis, J. AOAC Int. 81 (1998) 1177. [23] P. Salo-V¨aa¨ n¨anen, V. Ollilainen, P. Mattila, K. Lehikoiner, E. Salmela-M¨ols¨a, V. Piironen, Food Chem. 71 (2000) 535. [24] American Chemical Society (ACS), Subcommittee of Environmental Analytical Chemistry, Anal. Chem. 52 (1980) 2242. [25] A. Rodr´ıguez-Bernaldo de Quir´os, J. L´opez-Hernandez, J. SimalLozano, Eur. Food Res. Technol. 212 (2001) 687. [26] M.G. Paulo, H.M. Cabral, J.A.G. Morais, A.J. Almeida, J. Pharm. Biomed. Anal. 21 (1999) 399.
High-performance liquid chromatography method to measure ␣- and ␥-tocopherol in leaves, flowers and fresh beans from Moringa oleifera D.I. S´anchez-Machado, J. L´opez-Cervantes ∗ , N.J. R´ıos V´azquez Departamento de Biotecnolog´ıa y Ciencias Alimentarias, Instituto Tecnol´ogico de Sonora, P.O. Box 541, Antonio Caso s/n, Col. Villa ITSON, 85139 Cd. Obreg´on, Sonora, Mexico Available online 10 August 2005
Abstract A high-performance liquid chromatography method for the microscale determination of ␣- and ␥-tocopherol in leaves, flowers and fresh beans from Moringa oleifera is reported. The method includes microscale saponification and extraction with n-hexane. Optimized conditions for reversed-phase HPLC with UV detection were as follows: column, 25 cm × 0.46 cm, Exil ODS 5-m; column temperature, 25 ◦ C; mobile phase, a 20:80 (v/v) mixture of methanol:acetonitrile; flow rate, 1.0 ml/min. With these conditions, method precision (relative standard deviation) was 5.6% for ␣-tocopherol and 4.9% for ␥-tocopherol. We used this method to measure ␣- and ␥-tocopherol in samples from M. oleifera as part of nutritional studies in edible plants cultivated in the Northwest M´exico. © 2005 Elsevier B.V. All rights reserved. Keywords: Food analysis; ␣-Tocopherol; ␥-Tocopherol; HPLC; Moringa oleifera
1. Introduction Ten to 12 species of the Moringaceae family have been reported to belong to one genus called Moringae. Almost all Moringa species are native to India, from where they have been introduced in many warm countries [1,2]. The tree ranges in height from 5 to 10 m and sometime even 15 m. This tree grows rapidly even in poor soil and is little affected by drought. The flowers and the fruits appear twice a year, and seeds or cuttings can propagate the tree; the latter is more preferred. The leaves, flowers, fruits and roots of the tree are used as vegetables and the trunk is used in the paper industry [3,4]. The fresh beans after roasting also make a palatable dish. Seed are also consumed after frying and reported to taste like peanuts. A number of medicinal and therapeutic properties have been ascribed to various parts of this multipurpose tree, which included the treatment of ascites rheumatism, and use as cardiac and circulatory stimulants [5–7]. In India and Philippine, village people use the fresh leaves to prepare fat foods, and it has been found that there is a significant increase in the shelf life of this foods due that moringa leaves can be a ∗
Corresponding author. Tel.: +52 644 4109000; fax: +52 644 4109001. E-mail address: [email protected] (J. L´opez-Cervantes).
0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.07.048
good source of natural antioxidants [8]. Accordingly, there is a strong need for effective antioxidants from natural source because they may be safer for humans. In recent years, the physiological functionality of foods has received much attention due to the increasing interest in human health and has been studied in vitro and in vivo by many researchers. The antioxidative action, one of the important physiological functions of foods, is supposed to protect living organisms from oxidative damages, resulting in the prevention of various diseases such as cancer, cardiovascular diseases, and diabetes [9,10]. The tocopherol homologues (␣-, -, ␥-, and ␦-tocopherol) are natural antioxidants [11]. ␣-Tocopherol is the primary vitamer with biological activity; however, the other vitamers have been shown to have decreased activity. ␥-Tocopherol has 10% of the activity of ␣-tocopherol, and ␦-tocopherol has 1% of the activity of ␣tocopherol [12]. It has been reported that the retention of natural Vitamin E is at least double that of the synthetic form. Method to extract vitamins in foods is time consuming and tedious [14]. Traditionally, fast-soluble vitamin analysis is performed by alkaline saponification of the entire sample matrix, or of an isolated lipid fraction, followed by liquid extraction with organic solvents like diethyl ether or n-hexane. A few methods analyse these vitamins directly
112
D.I. S´anchez-Machado et al. / J. Chromatogr. A 1105 (2006) 111–114
without saponification in various matrices [15,16]. HPLC methods for the determination of ␣- and ␥-tocopherol have largely replaced direct spectrophotometric and fluorometric procedure. HPLC had been used with fluorescence detection, with UV detection, and most recently with detection by evaporative light scattering. Method precision is in all cases high [17]. The species Moringa oleifera has a considerable amount of quantitative nutritional information [18]. Additionally, a full characterization of the oil produced from their seeds has been reported [4]. However, the available information on the ␣- and ␥-tocopherol content in samples of this edible plant is very limited [19]. Therefore, the aim of the present work was the optimization of the extraction and chromatographic conditions for measuring tocopherols from M. oleifera, the validation of the method, and the measurement of representative sample obtaining reliable date.
plants 2, 4, 6, and 24 months old. Soon after collection, the samples were dried at 60 ◦ C for 8 h and stored in darkness at room temperature until the analysis. 2.4. Standards and quantification Stock standard solutions of ␣-tocopherol (0.4 mg/ml) and ␥-tocopherol (0.2 mg/ml) were prepared daily by dissolution in 100% methanol and stored at −10 ◦ C in the dark. Working solutions were prepared from these solutions and diluted with methanol prior analysis. For determination of ␣- and ␥-tocopherol in leaves, flowers and fresh beans, the stock solution was in all cases analyzed together with the samples, and analyte concentrations in samples were estimated on the basis of peak areas. All samples were analyzed in duplicate. ␣- and ␥-Tocopherol contents are cited as mean ± standard deviation. 2.5. Sample preparation
2. Experimental 2.1. Chemicals and reagents HPLC-grade methanol, hexane and acetonitrile (EMD Chemicals, Darmstadt, Germany). Analytical-grade pyrocatechol (Aldrich, St. Louis, USA), potassium hydroxide (J.T. Baker, Xalostoc, M´exico) and (±)-␣- and ␥-tocopherol (Fluka, Steinheim, Switzerland). Ultrapure water was prepared using a NANOpure Diamond UV system (Barnstead International, Buduque, Iowa, USA). KOH solution was prepared in methanol (0.5 M). The pyrocatechol solution (1 g in 5 ml of methanol) was prepared fresh daily, and stored at about 4 ◦ C in the dark. 2.2. Equipment The HPLC–UV system (GBC, Dandenong, Australia) was equipped with an auto injector LC 1650, an on line solvent degasser LC1460, a system controller WinChrom, a pump LC1150, a column oven LC1150, a 20 l injection loop (Rheodyne, Cotati, CA, USA), and a photodiode array detector LC5100. Chromatographic analysis was performed using an analytical scale (25 cm × 0.4 cm I.D.) SS Exil ODS column with a particle size 5 m (SGE, Dandenong, Australia). HPLC conditions were as follow: mobile phase 20:80 (v/v) methanol:acetonitrile; a flow rate of 1.0 ml/min and column temperature 25 ◦ C. The UV detection wavelength was set at 208 nm. The total time between injections was 18 min. Identification of tocopherols was based on retention time, co-injection with standards, and UV spectra. 2.3. Samples M. oleifera leaves, flowers and fresh beans were collected in Cajeme, Sonora, M´exico. The leaves were harvested of
Determination of water content. Water content of leaves, flowers and fresh beans was determined by weighing before and after drying to constant weight in a vacuum oven at 40 ◦ C. Saponification and extraction. ␣- and ␥-Tocopherol were extracted from the leaves, flowers and fresh beans by the method of S´anchez-Machado et al. [17] with minor modifications. A subsample of 0.40 g (±0.001 g) was weighed out in a screw-top assay tube. Two hundred microlitres of pyrocatechol solution and 5 ml of KOH solution (0.5 M in methanol) were added, and immediately vortexed for 20 s. The tubes were placed in a water bath at 80 ◦ C for 15 min, removing them every 5 min and vortexing again for 15 s. After cooling in iced water, 1 ml of distilled water and 5 ml of hexane was added, and the mixture was rapidly vortexed for 1 min, then centrifuged for 2 min at 425 × g. Three ml of the upper phase was transferred to another test tube and dried under nitrogen. The residue was redissolved in 3 ml of the HPLC mobile phase (20:80, v/v, methanol:acetonitrile), then membranefiltered (pore size 0.50 m; Whatman, Clifton, New Jersey, USA). Finally, an aliquot of 20 l was injected into the HPLC column. Before injection, the extracts were maintained at −10 ◦ C in the dark.
3. Results and discussion 3.1. Sample preparation There have been many studies of optimum conditions for the extraction of ␣- and ␥-tocopherol, from foods of different types with saponification, or of an lipid fraction [16,20], but there are small-scale applications concerning simultaneous extraction of this analytes from different products [13,21]. The large-scale extraction methods are expensive in terms of apparatuses required, cost of solvents, and time. We therefore decided to develop a micro-scale method of the type described
D.I. S´anchez-Machado et al. / J. Chromatogr. A 1105 (2006) 111–114
113
Table 1 Linearity for ␣- and ␥-tocopherol analyzed Compound
Range (g/ml)
Equation
r
␣-Tocopherol ␥-Tocopherol
7–140 0.4–8
y = 68686x − 68871 y = 84903x − 11980
0.9999 0.9998
x, amount (g/ml); y, peak area; r, correlation coefficient.
Fig. 1. Typical chromatogram of a standard mixture. Peak identification: (1) ␥-tocopherol and (2) ␣-tocopherol.
by Botsoglou et al. [22] and S´anchez-Machado et al. [17]. To determine optimal sample amounts, preliminary assays were performed with 0.1, 0.2, 0.3, 0.4 and 0.6 g of sample and with different amount of 0.5 M KOH in methanol (3, 5, 7 and 9 ml); the results indicated that good results are achievable with 0.40 g of sample and 5 ml 0.5 M KOH in methanol. 3.2. Tocopherols identification ␣- and ␥-Tocopherol in the leaves, flowers and fresh beans were identified by comparison of retention times and UV absorption spectra with those obtained for corresponding standards. For determination of retention times, the reference standards were injected both individually and as a mixture. Typical chromatogram of a standard mixture is shown in Fig. 1. Peaks were observed at 9.17 ± 0.035 min for ␥tocopherol, and 10.52 ± 0.050 min for ␣-tocopherol. Typical chromatogram of flowers from M. oleifera is shown in Fig. 2. 3.3. Method validation The linearity of standard curves (Table 1) was expressed in terms of the correlation coefficient (r) from plots of the integrated peak area versus concentration of the standard
(g/ml). These equations were obtained over a wide concentration range, in concordance with the level of ␣- and ␥-tocopherol found in leaves, flowers and fresh beans. All curves are based on analyses of at least four dilutions of the corresponding standard. In all cases the relationships between concentration and peak area were linear, with coefficients of determination greater than 0.999. Method precision was evaluated on the basis of the relative standard deviation (RSD) of ␣- and ␥-tocopherol determinations in eight samples of leaves from M. oleifera, with very good results for both tocopherols; both values are low by comparison with those obtained in related previous studies [23]. The Table 2 shows precision results. These results indicate that the present method can be used for routine analyses of ␣- and ␥-tocopherol in quality control laboratories. Determination of detection limits on the basis of signal-to-noise ratio (3:1) as per American Chemical Society guidelines [24] were 140 ng/ml for ␥-tocopherol and 160 ng/ml for ␣tocopherol, which are smaller than those presented in other works [14,25,26]. Accuracy was estimated by means of recovery assays. The recovery of the method was evaluated by analysis of six samples of leaves, 6 old months, from M. oleifera spiked before saponification with ␣-tocopherol (628 g/g of sample), and ␥-tocopherol (20 g/g of sample), and estimated as 90.3% and 89.1%, respectively. Table 2 shows the recovery percentages obtained. 3.4. α- and γ-Tocopherol in samples from Moringa oleifera The analytical method was applied to the determination of ␣- and ␥-tocopherol content in leaves, flowers and fresh beans of the tree, that are eaten as vegetables. Table 3 shows ␣- and ␥-tocopherol content of the samples considered in the present study. ␥-Tocopherol contents ranged from 5.7 (leaves of adult plants) to 27.8 (leaves of 6-month-old plants) g/g of dry mass. Important variations can be observed for the different samples with values for ␣-tocopherol ranging from 95.9 (fresh beans) to 744.5 (leaves from adult plants) g/g of dry Table 2 Precision and recovery of the HPLC proposed method Precision (n = 8)
␣-Tocopherola Fig. 2. Typical chromatogram of flowers from Moringa oleifera. Peak identification: (1) ␥-tocopherol and (2) ␣-tocopherol.
␥-Tocopherola a
Recovery (n = 6)
Mean ± SD
RSD (%)
Percent
RSD (%)
243.4 ± 13.7 6.8 ± 0.3
5.6 4.9
90.3 89.1
4.1 5.4
Results expressed as g/g dry mass.
D.I. S´anchez-Machado et al. / J. Chromatogr. A 1105 (2006) 111–114
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References
Table 3 ␣- and ␥-tocopherol content in Moringa oleifera Compound
␣-Tocopherola (mean ± SD)
Leaves 2 months 4 months 6 months Adults
731.8 399.3 530.7 744.5
Flowers Fresh beans
305.7 ± 23.9 95.9 ± 7.3
± ± ± ±
75.6 43.6 44.7 95.4
␥-Tocopherola (mean ± SD) 9.5 8.4 27.8 5.7
± ± ± ±
0.9 0.5 3.7 0.5
9.1 ± 0.9 N.D.
N.D.: not detected. a Results expressed as g/g dry mass.
mass. The difference could be explained by variation between the old of the plants as well as by variation between the different parts of plant. The highest measures were obtained for leaves and the lowest for flowers and fresh beans. It is interesting to observe the high ␣-tocopherol content of plants with proved antioxidant activity, indicating the possible relationship between tocopherol content and functional activity [21]. In general terms, these results are in agreement with previous works in moringa leaves [19].
4. Conclusions The method described allows the rapid isolation and quantitative determination of ␣- and ␥-tocopherol in samples of the different parts from tree M. oleifera. Due to the ease of sample preparation, the method may be used in routine analysis of vegetable foods. Our results suggest that moringa, particularly their leaves, might be useful nutritional source of tocopherols.
Acknowledgements This work was financed under project no. ITSON-EXB35 from Program of Profesor’s Improvement from the Public Education Department (PROMEP).
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