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A small-scale sample preparation method with HPLC analysis for determination of tocopherols and tocotrienols in cereals
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JOURNAL OF FOOD COMPOSITION AND ANALYSIS
Journal of Food Composition and Analysis 17 (2004) 749–765 www.elsevier.com/locate/jfca
Original Article
A small-scale sample preparation method with HPLC analysis for determination of tocopherols and tocotrienols in cereals M. Ryyn.anen, A.-M. Lampi*, P. Salo-V.aa. n.anen, V. Ollilainen, V. Piironen Department of Applied Chemistry and Microbiology, Food Chemistry, Latokartanonkaari 11, P. O. Box 27, University of Helsinki, FIN-00014 Helsinki, Finland Received 21 February 2003; received in revised form 8 August 2003; accepted 22 September 2003
Abstract A small-scale sample preparation method was developed for reliable and economic analysis of tocopherols and tocotrienols in rye and other cereals. The saponification parameters were optimized using two experimental design protocols with statistical analyses. Three critical factors were optimized for hot saponification: time, temperature and amount of potassium hydroxide (KOH). The amount of KOH had the greatest effect. Two direct solvent extraction methods without saponification were tested and found to yield in comparable values than with saponification. Finally, three solvent mixtures for extracting tocopherols and tocotrienols after the optimized saponification step were studied in order to examine the effect of solvent polarity on their recovery. Adding a polar modifier to the solvent, tocopherol and tocotrienol values of rye were significantly increased. The optimized sample preparation method included saponification with 0.5 mL KOH at 100 C for 25 min followed by extraction with n-hexane:ethyl acetate (8:2). The repeatability of the recommended analytical procedure was good and the recoveries of added tocopherols from rye flour samples ranged from 90.3% to 94.3%. The procedure developed was applied to examine the amounts and distribution of tocopherols and tocotrienols in ten rye varieties. The average total tocopherol and tocotrienol content of rye grains was 48.8 mg/g with a 9.3% standard deviation between varieties. r 2003 Elsevier Inc. All rights reserved. Keywords: Tocopherols; Tocotrienols; HPLC; Rye; Cereals
*Corresponding author. Tel.: +358-919158412; fax: +358-919158475. E-mail address: anna-maija.lampi@helsinki.fi (A.-M. Lampi). 0889-1575/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2003.09.014
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1. Introduction Tocopherols and tocotrienols are a group of dietary plant constituents considered to have beneficial health effects. There are four tocopherol and four tocotrienol vitamers. All of them consist of a chromanol ring and a hydrophobic side chain, which is a phytyl in tocopherols and an isoprenyl with three double bonds in tocotrienols. The vitamers can be distinguished by the number and location of methyl groups on their chromanol ring. Generally, when the vitamin E activity of a food item is evaluated, a sum of the different vitamers is calculated taking into account their relative activities. For healthy people it is relatively easy to obtain sufficient tocopherols and tocotrienols from the diet to prevent well-defined vitamin E deficiency symptoms. However, higher intakes of E vitamers may also decrease the risk of several chronic diseases related to oxidative damage, e.g., coronary heart diseases and cancer. Most of the effects in the human body are due to their action as lipid-soluble antioxidants (e.g., Stampfer and Rimm, 1995; Stone and Papas, 1997; Theriault et al., 1999; Schwenke, 2002). Despite the fact that the latest North American guidelines consider a-tocopherol to be the only biologically active form of vitamin E and discount other vitamers (Anon, 2000), the biological importance of other E vitamers has gained attention. Recent studies have shown that tocotrienols have a variety of novel beneficial functions. For example, they may have a protective effect by lowering LDL cholesterol by inhibiting cholesterol biosynthesis (Qureshi et al., 1995; Hood, 1998; Theriault et al., 1999). There is growing evidence that the greatest advantage of a tocopherol- and tocotrienol-rich diet is achieved when various E vitamers are administered concurrently. It has been shown that a-tocopherol supplementation alone has little effect on mammary tumors, and evidently, other forms of vitamin E-like substances, such as a-, g- and d-tocotrienols, reduce the risk of breast cancer (Schwenke, 2002). Thus, it has been stated that the ratios of the individual tocopherols and tocotrienols play an important role in determining the hypocholesterolemic, antioxidant and antitumor properties of palm oil and rice bran (Qureshi et al., 2000, 2002). For example, a-tocopherol and a-tocotrienol had opposite effects on the cholesterol metabolism of chicks, a higher ratio of tocotrienols to tocopherols being optimal (Qureshi et al., 1989). Recently, the vitamers have also been shown to have non-antioxidant roles (Azzi and Stocker, 2000). The main sources of vitamin E-active compounds in the human diet are vegetable fats and oils and products derived from them, but cereals also contribute significantly to the dietary intake in the United States (14.6%, Murphy et al., 1990) and in Finland (18%, Heinonen and Piironen, 1991). In terms of amounts of tocopherols and tocotrienols, cereals are more important than in terms of vitamin E activity, since they are rich sources of the less vitamin E-active tocotrienols. For example, barley contains all four tocopherols and four tocotrienols. Other cereals such as wheat, oats and rye also contain more tocotrienols than tocopherols (Piironen et al., 1986; Balz et al., 1992; Peterson and Qureshi, 1993) and thus have a potentially beneficial distribution of the vitamers. Since cereals contain a number of E vitamers and each of them has a diverse biological and chemical character, they are a challenge for a food analyst, because it is essential to be able to analyse each vitamer separately. It is possible to accurately analyse all forms of tocopherols and tocotrienols with modern chromatographic methods (Eitenmiller and Landen, 1999; Abidi, 2000; Piironen, 2000; Rupe! rez
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et al., 2001; Lampi et al., 2002). There are three major sample preparation approaches for food samples: simple dilution of oil samples in organic solvent, direct extraction of the vitamers with organic solvents, and extraction of the vitamers after saponification. In cereals, tocopherols and tocotrienols are extracted by either a direct solvent method or by saponification followed by extraction. Extraction with organic solvents disrupts the structures to which the vitamers are attached and solubilizes them. n-Hexane is the commonly used solvent, but more polar solvent mixtures are also used, e.g., ethyl acetate in n-hexane or acetone (Ueda and Igarashi, 1987a, 1990; Eitenmiller and Landen, 1999; Rupe! rez et al., 2001). Saponification further assists in releasing the vitamers by degrading the food matrix, and removes the bulk of fat, which improves their chromatographic separation (Piironen, 2000; Rupe! rez et al., 2001). Tocopherols and tocotrienols are relatively unstable in alkaline conditions, and care must be taken to avoid their destruction. They are protected by using antioxidants, flushing the saponification vessel with nitrogen and working under subdued light (Eitenmiller and Landen, 1999). E vitamer analysis is most commonly conducted with normal-phase high-performance liquid chromatography with fluorescence detection (NP-HPLC-FLD). A fluorescence detector is preferred over an ultraviolet detector in analysing tocopherols and tocotrienols in complex food matrices because of its specificity and sensitivity (Piironen et al., 1986; Eitenmiller and Landen, 1999; Abidi, 2000). Other means of separation include reversed-phase HPLC, gas chromatography and, most recently, capillary electrochromatography (Abidi et al., 2002). Electrochemical detection is also applied as a sensitive detector for E vitamers after HPLC separation (Ueda and Igarashi, 1987b; Yamauchi et al., 2002), while mass spectrometry (MS) in . combination with HPLC assists in identification of compounds (Strohschein et al., 1999; Stoggl et al., 2001). As the sensitivity of the HPLC analysis has improved, it has become possible to scale down the food sample size, as has been done, e.g., for animal products (Salo-V.aa. n.anen et al., 2000). At the same time, a need for shorter sample preparation times and decreased amounts of chemicals has arisen. Since there are multiple factors to be optimized in the sample preparation method for E vitamer analysis, we chose an experimental design approach to develop a fast and reliable saponification method for cereal samples. Earlier, Lee et al. (2000) used response surface methodology to optimize an extraction procedure of tocopherols from tomato and broccoli. They used 70 C as the saponification temperature and varied the amounts of alkali and ethanol in the saponification mixture and the saponification time. In our study, a wider range of analytical conditions during saponification and extraction of E vitamers was investigated. Our hypothesis was that saponification is an efficient means to liberate tocopherols and tocotrienols from cereal matrix prior to lipid extraction, but the saponification conditions should be carefully examined and controlled to avoid decomposition of the analytes. In order to reliably and economically investigate the distributions of tocopherols and tocotrienols in rye and other cereals, a small-scale and rapid sample preparation method was developed. The saponification conditions were optimized using experimental design and solvent mixtures for extraction of tocopherols and tocotrienols were evaluated. Applicability of the recommended sample preparation method was tested by examining the amounts and distribution of tocopherols and tocotrienols in ten rye varieties grown in Finland. This was also performed in order to expose the most promising varieties with regard to the optimal ratio of tocopherols and tocotrienols.
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2. Materials and methods 2.1. Sample materials, standards, and reagents The material used for method development was rye flour (Myllyn paras; Helsingin mylly, Hyvink.aa. , Finland). Rapeseed oil was used as an in-house reference material for HPLC analysis (Kultasula; Raisio Group, Raisio, Finland). Samples of ten rye varieties were cultivated in the Agricultural Research Centre, Jokioinen, Finland, in 1999 (Boreal Plant Breeding Ltd, Jokioinen, Finland). Sample grains were milled with a hammer mill at the Department of Food Technology, University of Helsinki. All sample materials were stored in small portions in the dark at 20 C until analysed. HPLC-grade n-hexane, ethyl acetate and 1,4-dioxane were purchased from Rathburn (Walkerburn, UK). Milli Q water was of HPLC-grade. Ethanol was of spectrophotometric grade (AAS, 99.5%, Primalco, Finland). Ascorbic acid, p.a., was purchased from Merck (Darmstadt, Germany) and potassium hydroxide (KOH) pellets from EKA Nobel (Bohus, Sweden). Saponification solution was prepared by dissolving 50 g of KOH pellets in 100 mL MilliQ water. Sample extracts were filtered (GHP Acrodisc, 0.45 mm, Pall Corp., Anna Arbor, MI) before injecting to HPLC. a-, b-, g- and d-tocopherols (fur . biochemische Zwecke, Art no. 15496) were purchased from Merck. The standard stock solutions of the tocopherols were prepared to a concentration of approximately 500 mg/mL (in ethanol, AAS). The stock solutions were stored at 20 C. The concentrations were confirmed spectrophotometrically using the known absorption coefficients of each vitamer in ethanol Eð1% 1 cm Þ: 75.8 for a-tocopherol, 89.4 for b-tocopherol, 91.4 for g-tocopherol and 87.3 for d-tocopherol at 292, 296, 298 and 298 nm, respectively (Podda et al., 1996). The combined working solution was prepared by pooling suitable amounts of each tocopherol and diluting with n-hexane to obtain concentrations ranging from 1–100 ng/injection. Each tocotrienol vitamer was quantified with the respective tocopherol (e.g., AOCS, 1990; Kramer et al., 1997). Tokovid-palmoil extract (Hovid Sdn. Bhd., Malaysia) was used as a qualitative standard for tocotrienols. 2.2. Optimization of the sample preparation method The sample preparation method was based on the room temperature saponification method with n-hexane as an extraction solvent (Piironen et al., 1986). In this study a small scale and rapid procedure using hot saponification was developed for determining tocopherols and tocotrienols in cereals, using rye flour. To avoid destruction of labile vitamers, all work was carried out under subdued light, ascorbic acid was added as an antioxidant and reaction vessels were flushed with nitrogen before adding KOH. At first, sample amounts and solvent volumes were reduced from a previous method (Piironen et al., 1986) and hot saponification was conducted. Total tocopherol and tocotrienol values from rye flour obtained with the room temperature saponification and the hot saponification methods were comparable (24.571.0 mg/g and 23.872.2 mg/g) as were the recoveries of added tocopherols (range 58–129%). Thereafter we proceeded to optimize the hot saponification conditions. Three critical experimental factors of the hot saponification method were evaluated: saponification time,
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temperature and amount of KOH. The saponification method was developed and optimized using two experimental design protocols (Haaland, 1989). The first experiments included three critical factors with a wide range of analytical conditions, i.e., levels of factors, and the second included only two factors with a narrow range (Tables 1 and 2). All other sample preparation parameters remained constant. The results were analysed by using multiple linear regression analysis and visualized by response surface plots (Statgraphics Plus, 1997). The output factor for the trials was the total tocopherol and tocotrienol value and the target was to obtain as high a value as possible. Each trial was performed in duplicate and the mean value was used as the output factor. On the basis of the results of the experiments described above, it was further investigated whether saponification could be omitted in sample preparation. Thus two direct solvent extraction methods without saponification were tested. Solvent extraction method 1 included hot extraction with 2-propanol and n-hexane as extraction solvents (Koivu et al., 1997) and method 2 was the optimized hot saponification method excluding the addition of KOH. Optimization of the sample preparation method was finally fulfilled by investigating the effect of solvent polarity on extracting tocopherols and tocotrienols from the saponified mixture. Three extraction solvents were compared: n-hexane, n-hexane:ethyl acetate (9:1) and n-hexane:ethyl acetate (8:2). In order to evaluate the steps other than saponification in the sample preparation method as described above, all analyses were performed in triplicate at minimum. Comparisons of means or pairs were conducted with t-tests, and of three groups with one-way analysis of variances (ANOVA) followed by multiple range tests. All analyses were carried out using the same software as for multiple linear regression analyses (Statgraphics Plus, 1997).
Table 1 Analytical conditions as the input factors and total values of tocopherols and tocotrienols (mg/g) as the output factor in a Box-Behnken experiment to study the effects of saponification time (min), temperature ( C) and amount of KOH (mL) on the output factor (Haaland, 1989) (mean values from duplicate analyses) Trial
Temperature
Time
KOH
Total tocopherols and tocotrienols
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
60 60 100 100 60 60 100 100 80 80 80 80 80 80 80 80
20 60 20 60 40 40 40 40 20 20 60 60 40 40 40 40
2 2 2 2 1 3 1 3 1 3 1 3 2 2 2 2
21.7 18.5 21.5 18.6 21.2 14.9 23.6 16.5 22.6 17.1 20.1 18.1 22.1 18.6 19.0 21.4
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Table 2 Analytical conditions as input factors and total values of tocopherols and tocotrienols (mg/g) as the output factor in a Central Composite experiment to study the effects of saponification time (min) and the amount of KOH (mL) on the output factor (Haaland, 1989) (mean values from duplicate analyses) Trial
Time
KOH
Total tocopherols and tocotrienols
1 2 3 4 5 6 7 8 9 10 11
5 5 20 20 1.9 23.1 12.5 12.5 12.5 12.5 12.5
0.25 1 0.25 1 0.625 0.625 0.096 1.154 0.625 0.625 0.625
22.5 24.6 27.6 24.4 23.6 26.9 25.2 24.7 26.0 24.6 24.5
2.3. Recommended analytical procedure After method optimization, a recommended analytical procedure was established. The main steps are illustrated in Fig. 1. 2.3.1. Saponification Rye flour (0.5 g) was accurately weighed into a 30 mL Pyrex glass tube with a Teflon screw cap. Ascorbic acid (0.1 g), ethanol (5 mL) and water (2 mL) were added. After mixing the tube with a Vortex-mixer, the tube was flushed with nitrogen and KOH (0.5 mL) was added. The tube was capped and transferred to a boiling water bath for 25 min. The tube was mixed with the Vortexmixer after 10 min of boiling. The tubes were cooled in an ice-water bath. 2.3.2. Extraction Water (2.5 mL) and ethanol (2.5 mL) were added to the cooled tubes. Unsaponified lipids were extracted by using three portions (each 10 mL) of n-hexane:ethyl acetate (8:2). Tubes were shaken for 10 min with 500 strokes/min, and after separation of the phases, the organic layers were collected in a separation funnel. Organic extracts were washed three times with water and with addition of NaCl to avoid emulsion formation. The washed extract was transferred to a roundbottomed flask, and the organic phase was evaporated. Ethanol (2 mL) and n-hexane (2 mL) were added and evaporated to dryness. The residue was dissolved in n-hexane and transferred quantitatively to a 5 mL volumetric flask. Sample extracts were stored in Kimax test tubes in the dark at 70 C. Prior to HPLC analysis, extracts were filtered through a 0.45 mm filter. 2.3.3. Analytical HPLC Normal phase HPLC with fluorescence detection (excitation 292 nm, emission 325 nm) was used to analyse tocopherols and tocotrienols (Kamal-Eldin et al., 2000). The HPLC system consisted of a pump (Waters 510; Waters Corp., Milford, MA), an autosampler with a cooling
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Preparing the sample for saponification: 0.5 g sample 0.1 g ascorbic acid, 5 mL ethanol, 2 mL water flushing with nitrogen
Saponification: 0.5 mL KOH solution 100˚C, 25 min
Extraction and purification of non-saponifiable lipids: 2.5 mL ethanol, 2.5 mL water 3 x 10 mL n-hexane:ethyl acetate (8:2) washing the extract 3 x with water and evaporating to dryness
Preparing the sample for HPLC analysis: 2 mL ethanol and 2 mL n-hexane evaporating to dryness dissolving the residue in 5 mL n-hexane
NP-HPLC analysis with fluorescence detector
Fig. 1. Recommended analytical procedure.
module (Waters 712), a scanning fluorescence detector (Waters 474), and an Inertsil silica column (5 mm, 250 mm4.6 mm; Varian Chromapack, Middelburg, Netherlands) with a silica guard column (Guard-Pak Silica, Waters). The temperature of the column oven was 30 C. Separation of the vitamers was based on isocratic elution. The mobile phase contained 3% 1,4-dioxane and 97% n-hexane. The flow rate of the mobile phase was 2 mL/min. Tocopherols and tocotrienols were quantified with an external standard method in which quantification was based on peak areas. Calibration curves with six points were obtained daily. The method was validated by determining the following parameters: detection and determination limits, range of linearity and repeatability. Detection limits were defined as a signal three times the height of the noise. Determination limits were defined as three times the detection limit. The dayto-day repeatability of the HPLC method was also confirmed with in-house reference material (rapeseed oil) which was analysed daily during the 2-month period. 2.4. Evaluation and application of the recommended analytical procedure Verification of the vitamer identification was based on the HPLC-FLD data and HPLC-MS data. Full scan mass spectra and selected ion monitoring tracings derived from an atmospheric
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pressure chemical ionization (APCI) in positive mode were recorded using a quadrupole ion trap mass spectrometry (Esquire LC-MS, Bremen, Germany). The liquid chromatographic method used was the same as in analytical HPLC except that the eluent flow (2 mL/min) was split (1:10) prior ionization interface with a splitter (Acurate Post-Column Splitter, LC Packings, USA). The temperature of APCI interface and drying gas (nitrogen) was set at 300 C, and the potential of discharge corona needle was 4.5 kV. Mass spectra were recorded at the scan range of 100–700 m/z and the protonated molecular ions ([M+H]+) were measured. The reliability of the recommended analytical procedure was verified by recovery and repeatability tests with rye flour. Accuracy was verified by recovery tests, in which samples were spiked with tocopherols (10 mg/g of rye flour) before saponification. Spiked samples were prepared in duplicate on 3 days. Repeatability of the developed method was verified by analysing the tocopherol and tocotrienol contents of rye flour in duplicate over 3 days. The applicability of the recommended procedure to, e.g., whole grain samples was examined by analysing the tocopherol and tocotrienol contents of ten rye varieties, and by finding any variation in E vitamer composition. The varieties studied represent winter classes of rye cultivars grown in Finland and are thus a relevant set of samples to be analysed by the current method.
3. Results and discussion 3.1. Optimization of saponification conditions Three critical experimental factors were optimized in the small-scale hot saponification method for analysis of tocopherols and tocotrienols in cereals: saponification time, temperature and amount of KOH. Using the Box-Behnken design the total tocopherol and tocotrienol value of rye flour measured as the output factor ranged from 14.9 to 23.6 mg/g (Table 1). Two factors were shown to have a significant impact on the output factor, namely the saponification time (P ¼ 0:0877) and the amount of KOH (P ¼ 0:0002). Saponification temperature, however, was not an important factor (P ¼ 0:3634) and was excluded from the linear regression model. The influences of the two important factors are illustrated in Fig. 2. The coefficient of determination indicating the model’s ability to explain the relationships between the factors was 64.7%. The figure shows that the smaller the amount of KOH solution, the greater was the total tocopherol 26.8363 - 2.615*KOH - 0.047312*Time
T + T3, ug/g
24 22 20 18 16
1
1.4
1.8
2.2
KOH, mL
2.6
3
20
30
40
50
60
Time, min
Fig. 2. Response surface plot of effects of saponification time and amount of KOH on total value of tocopherols and tocotrienols.
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and tocotrienol value. The effect of saponification time on the output factor was less than that of the amount of KOH. For example, when the time increased from 20 to 60 min, the total tocopherol and tocotrienol value increased by 1.9 mg/g, but when the amount of KOH decreased from 3 to 1 mL, the output factor increased by 5.2 mg/g. Since the saponification temperature had an insignificant effect on tocopherol and tocotrienol values, the highest setting of 100 C was chosen for further analyses, e.g., to enhance decomposing of cereal matrix. In order to examine the effect of even lower levels of KOH and shorter saponification times, another experiment using the Central Composite design was carried out at a temperature stabilized to 100 C (Table 2). The total tocopherol and tocotrienol values were higher and the range smaller than in the first experiment, ranging from 22.5 to 26.9 mg/g. Here both the amount of KOH and the time had a significant effect on the output factor with P ¼ 0:0038 and P ¼ 0:0002; respectively. Moreover, there was an evident interaction between these two factors (P ¼ 0:0014) in the linear regression model (Fig. 3). The coefficient of determination of this model was even higher, 87.1%, than that of the first model. Again, the smaller the amount of KOH, the greater was the total tocopherol and tocotrienol value. However, at low levels of KOH, o1 mL, a longer saponification time was needed than at high KOH levels. The interaction between these two factors was so strong that at 0.096 mL of KOH, the total tocopherol and tocotrienol value was 19.8 mg/g higher at the longest saponification time (23.1 min) than at the shortest time (1.9 min) whereas at 0.625 mL of KOH, the difference was only 3.4 mg/g. When more than 1 mL of KOH was used, changes of saponification time within the range of the experiment had only minor effects on the output factor. Thus under these hot saponification conditions a compromise can be made between the amount of KOH and saponification time. Our finding that a small amount of KOH produces higher total tocopherol and tocotrienol values is supported by the results of Piironen et al. (1984), who investigated the effects of different amounts of KOH on E vitamer contents in a diet sample. They showed that treating samples with higher amounts of alkaline than used in this study partly destroyed tocopherols. In addition, high levels of soap might decrease the extractability of tocopherols and tocotrienols, because the soap produced during saponification might solubilize them into the alkaline media (Ueda and Igarashi, 1987b). However, saponification degrades the food matrix and purifies the lipid extract, which improves the efficacy of the sample preparation method. We also found that emulsions were formed less frequently when at least 0.25 mL of KOH was used than with less alkaline. With low levels of KOH, saponification times should be long enough as indicated in Fig. 3. Thus we
T + T3, ug/g
19.7418 + 5.19666*KOH + 0.444822*Time - 0.460444*KOH*Time
31 29 27 25 23 21 19
0
0.2
0.4
0.6
0.8
1
1.2
1620 8 12 0 4
24
Time, min
KOH, mL
Fig. 3. Response surface plot of effects of time and potassium hydroxide on the total amount of tocopherols and tocotrienols.
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concluded that the recommended saponification method should consist of a reasonable amount of KOH, 0.5 mL, with a saponification time of 25 min at 100 C. 3.2. Comparisons of direct solvent extraction and saponification The results of the previous experiments indicated that the total tocopherol and tocotrienol value from rye flour was greatest with the smallest amount of KOH. Thus it was further investigated whether saponification could be omitted and, instead, direct solvent extraction would be used in sample preparation where decomposition of matrix would be less efficient, but no losses due to hot alkaline would occur. The total value of tocopherols and tocotrienols determined by two direct extraction methods, i.e., hot extraction with 2-propanol and n-hexane, and optimized saponification of the method excluding addition of KOH, and by the saponification method gave statistically similar results (ANOVA, P > 0:05; n ¼ 3–5). The value was 24.270.8 mg/g for method 1, 23.970.9 mg/g for method 2, and 24.970.5 mg/g for the saponification method. Recoveries of added tocopherols from rye flour were 73.7–90.5% and 86.1–90.4% for the saponification and the direct extraction method 1, respectively. With saponification, recoveries of more polar tocopherols were lower than that of a-tocopherol, decreasing in the order of their polarity, while with solvent extraction with 2-propanol and n-hexane, no difference between the vitamers was observed. The different recoveries of added tocopherols in the saponification method are caused either by differences in their stabilities during saponification or extractabilities from the saponification mixture, and not from liberation from the matrix, because tocopherols were added as ethanol solutions. These findings illustrate one more important aspect of the sample preparation procedure, i.e., the polarity of the extraction solvent after saponification. Similarly, Ueda and Igarashi (1987b) found that soap formed from fat decreased the extractability of other tocopherols more than that of a-tocopherol when an apolar n-hexane was used as the extraction solvent. Hence, modification of extraction solvent following saponification was investigated to improve the recoveries of more polar tocopherols. Since the optimized saponification method was shown not to yield lower tocopherol and tocotrienol values in rye flour than direct extraction methods, it was concluded that saponification is beneficial for the efficient liberation of tocopherols and tocotrienols from cereals. Furthermore, resolution of the vitamers analysed by HPLC was better with the saponification method. Previously, comparisons of sample preparation methods consisting of saponification or direct extraction have yielded inconsistent results. Piironen et al. (1984) preferred saponification due to its higher total yield of tocopherols and tocotrienols, but Bonvehi et al. (2000) found ca 20% higher tocopherol values of oils and biscuits when samples were prepared without saponification than with saponification, and they therefore omitted saponification. One potential reason for the different results may be that precautions during sample preparation, including addition of antioxidants, working under subdued light and nitrogen atmosphere, are more important when the sample is subjected to saponification, because the vitamers are easily oxidized in the presence of alkali and the procedure must be carefully controlled. 3.3. Effect of solvent polarity on extraction of tocopherols and tocotrienols from saponified mixtures Development of the saponification step was conducted using n-hexane as the extraction solvent. A possible need for a more polar extraction solvent mixture to improve the extractability of
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tocopherols and tocotrienols was observed. The use of polarity modifying agents in extraction solvents has also been suggested earlier (Ueda and Igarashi, 1987a; Eitenmiller and Landen, 1999; Salo-V.aa. n.anen et al., 2000). Thus, after the saponification parameters had been obtained, three different extraction solvents were compared: n-hexane, n-hexane:ethyl acetate (9:1) and nhexane:ethyl acetate (8:2). The total tocopherol and tocotrienol values obtained from rye flour using these solvent mixtures were significantly different (ANOVA, Po0:001) giving values of 24.970.5, 28.770.6 and 29.570.3 mg/g (n ¼ 3–5), respectively. The multiple range tests showed that the mean with n-hexane as solvent was lower than with others (Po0:05). Thus polar modification of extraction solvent improved the extractability of tocopherols and tocotrienols present in the hot saponified rye flour mixture. At the same time, minor peaks eluting at the retention times of g-tocopherol, g-tocotrienol and d-tocotrienol also became detectable. When Ueda and Igarashi (1987a) investigated the effect of ethyl acetate concentration in n-hexane as an extraction solvent, they concluded that the addition of 10% ethyl acetate in n-hexane was optimal, because as the proportion of ethyl acetate increased above this level, some of it partitioned in the aqueous phase and decreased the volume of the organic layer. In our method, increasing the concentration of ethyl acetate did not have adverse effects, because the extraction was repeated three times and the organic layers were collected quantitatively to ensure the recovery of all vitamers. Thus 20% ethyl acetate in n-hexane was preferred instead of 10%. 3.4. Repeatability and accuracy of the recommended analytical procedure Finally, the recommended analytical procedure method was evaluated by repeating the analysis of rye flour and by spiking rye flour samples with tocopherols (Table 3). All analyses were performed in duplicate over 3 days. The total tocopherol and tocotrienol value was 28.770.1 mg/ g. b-tocotrienol was the major vitamer, followed by a-tocopherol, a-tocotrienol and b-tocopherol. The repeatability of the method was excellent, since the coefficients of variation (%CV) of these vitamers were 1.5–3.6%. Even the minor vitamers (g-tocopherol, g- and d-tocotrienols), that had contents near to the determination limit, had a low %CV of o19%. It should be remembered that this high repeatability was obtained with a highly homogenous 0.5 g sample of rye flour, and that greater variations may occur with more uneven materials. The average recovery of the added tocopherols at the 10 mg/g level was good. The highest recovery was obtained for a- and b-tocopherols, 94.3% and 93.6%, and the lowest for gtocopherol, 90.3% (Table 3). Thus recoveries were all >90% and the variation between vitamers
Table 3 Tocopherol (T) and tocotrienol (T3) contents in mg/g expressed as mean7SD (n ¼ 6) of (1) rye flour and (2) rye flour with added tocopherols. (3) Recoveries of added tocopherols determined by the recommended analytical procedurea
(1) (2) (3) a
a-T
a-T3
b-T
g-T
b-T3
g-T3
d-T
d-T3
Total
8.270.1 17.570.7 94.3
8.170.3 8.070.4
2.970.1 13.070.5 93.6
0.270.02 9.970.4 90.3
9.370.3 9.270.4
0.170.01 0.170.02
0.070.0 10.470.4 91.0
0.170.01 0.170.02
28.770.1 68.170.4
Identification of T and T3 was confirmed by high performance liquid chromatographic mass spectrometric data except for d-T3.
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was clearly lower than when n-hexane was used as the only solvent. This means that polar modification improves the extractability of the more polar tocopherols. In the study of Piironen et al. (1986), recoveries of a-, b-, g- and d-tocopherols were 94.3%, 95.2%, 96.5% and 95.7%, respectively, with saponification at room temperature. Despite optimization of the saponification method and careful carrying out of the procedure, hot saponification might destroy tocopherols and tocotrienols, and recoveries as high as those obtained with room temperature saponification might not be achieved. 3.5. Evaluation of the analytical HPLC method Identification of tocopherols and tocotrienols was confirmed on HPLC-MS data (Fig. 4). The m/z values of protonated molecular ions for a-, b- and g-tocopherols were 431.5, 417.4 and 417.4, and for a-, b-, g- tocotrienols were 425.4, 411.4 and 411.4, respectively. Verification of dtocotrienol was performed with HPLC-FLD derived from the Tokovid-palmoil extract, because the signal of protonated molecular ion of d-tocotrienol was too low for adequate identification using LC-MS. This is apparently due to the low ionization efficiency of d-tocotrienol in APCI process used in this study. However, the use of the mixture of n-hexane and 1,4-dioxane as a mobile phase enables the more uniform ionization efficiency for individual vitamers compared to . the results of Stoggl et al. (2001) when the eluent consisted of isooctane and diisopropyl ether. The quantitative HPLC method for analysis of tocopherols and tocotrienols was validated by several detector parameters. Its repeatability was confirmed with an in-house reference material and its stability by comparing the chromatograms during the 2-month period. Detection limits varied between 0.10 and 0.18 ng/injection. Determination limits of the vitamers ranged between 0.30 and 0.54 ng/injection. The detector response was linear in the tested ranges, 2–80 ng/injection (R2 ¼ 0:9998). The variation of the standard curve slopes varied from 0.2% to 0.3% (n ¼ 22), which showed that the stability of the detector response was excellent. The average tocopherol contents (n ¼ 13) of the in-house reference (rapeseed oil) were: a-tocopherol: α -T α -T3 30
25 β -T
β -T3
FLD
20
15
10 γ -T
5
0
2.5
5
7.5
δ -T3
γ -T3
10
12.5
15
17.5
20
Time [min]
Fig. 4. HPLC-chromatogram of rye flour (for details see Section 2).
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264716 mg/g, g-tocopherol 590728 mg/g and d-tocopherol 1977 mg/g, and the %CVs were 6.0%, 4.8% and 36.4%, respectively. These figures show that the HPLC method had good repeatability. The high variation of d-tocopherol was due to its low concentration, which was near to the determination limit. The silica column used proved to be very stable, because the %CVs of the retention times of tocopherols and tocotrienols over a two-month period were only approximately 0.5% (n ¼ 10). The retention time of void volume (to ) was 1.96 min (measured with dodecane) and all tocopherols and tocotrienols were well separated between retention times of 5.5 and 14.2 min. Resolution of the column was good, being 1.98 (n ¼ 10) for b- and g-tocopherols which are usually difficult to separate from each other completely. 3.6. Tocopherol and tocotrienol contents of rye The total combined content of tocopherols and tocotrienols of the rye flour used in the method development was 28.7 mg/g, of which the amounts of a- and b-tocopherols and -tocotrienols were 8.2, 8.1, 2.9 and 9.3 mg/g, respectively. In an earlier study, the corresponding amounts for whole meal rye flour were 10.2, 14.4, 3.1 and 11.3 mg/g, and for rye flour 5.5, 4.3, 2.6 and 6.4 mg/g, respectively (Piironen et al., 1986). Thus the values indicate that some parts of the kernel have been removed during the milling process of the rye flour used in this study. The average tocopherol and tocotrienol contents of the ten rye varieties commonly cultivated in Finland were 48.874.5 mg/g as analysed by the recommended analytical method (Table 4). Total E vitamer contents were higher than in the rye flour used, because whole grains were milled for samples without removing outer layers of the kernel. The best source of tocopherols and tocotrienols was the variety Akusti, 54.3 mg/g, and the poorest was Picasso, 39.9 mg/g. The overall variation between the varieties was 9.3%, which is slightly more than the variation in folate and plant sterol contents that were earlier shown to be 8% and 6%, respectively (Kariluoto et al., 2001; Piironen et al., 2002). There was an apparent although not significant (P ¼ 0:051) positive Table 4 Tocopherol (T) and tocotrienol (T3) contents of 10 rye varieties in mg/g. Mean of duplicate determinationsa
BOR9214 Akusti Riihi Picasso BOR9414 Esprit BOR7068 Elvi Anna Voima Average CV% a
a-T
a-T3
b-T
g-T
b-T3
g-T3
d-T3
Total
T
T3
T3/T-ratio
13.9 14.3 16.1 10.0 14.8 12.6 16.0 14.5 15.8 16.3 14.4 13.5
16.2 19.0 16.7 14.7 17.5 15.3 17.3 17.8 16.5 17.6 16.9 7.5
3.4 3.2 3.9 2.3 4.1 2.9 4.0 3.6 4.2 3.8 3.5 17.3
0.5 0.6 0.4 0.4 0.6 0.5 0.6 0.6 0.6 0.6 0.5 16.3
11.7 16.8 10.2 12.2 13.2 11.9 15.3 14.2 11.8 12.2 13.0 15.1
0.2 0.3 0.2 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.3 27.3
0.2 0.2 0.2 0.1 0.2 0.2 0.3 0.2 0.2 0.2 0.2 21.2
46.1 54.3 47.7 39.9 50.8 43.6 53.7 51.3 49.4 50.9 48.8 9.3
17.8 18.0 20.5 12.7 19.6 15.9 20.6 18.8 20.6 20.6 18.5 13.9
28.3 36.3 27.2 27.2 31.2 27.7 33.1 32.6 28.8 30.3 30.3 10.0
1.6 2.0 1.3 2.1 1.6 1.7 1.6 1.7 1.4 1.5 1.7 15.5
Identification of T and T3 was confirmed by high-performance liquid chromatographic, mass spectrometric data except for d-T3.
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correlation between E vitamer and plant sterol contents. As in the case of rye flour, a- and btocopherols and -tocotrienols were the major vitamers. Our values are in line with earlier studies, in which total a- and b-tocopherol and tocotrienol contents ranged between 23 and 49 mg/g (Morrison, 1978), between 32.4 and 54.7 mg/g (Barnes, 1982) and around 32.4 mg/g (Balz et al., 1992). In Poland, a lower level of 27.7 mg/g of tocopherols and tocotrienols in rye was found (Zielinski et al., 2001). The difference could be explained by variation between varieties as well as by variation between the methods of analysis. The authors extracted tocopherols and tocotrienols directly from rye with 80% methanol before NP-HPLC analysis. In the ten rye variety samples, tocopherol and tocotrienol contents varied between 17.8 and 20.6 mg/g and between 27.2 and 36.3 mg/g, respectively, and the tocotrienols to tocopherols ratio between 1.3 and 2.1 (Table 4). In earlier studies, the ratio was slightly lower at 1.6 (Balz et al., 1992) and 1.1 (Zielinski et al., 2001). The tocotrienols to tocopherols ratio did not correlate with the total E vitamer contents. For example, the highest ratio was measured for Picasso, which, on the other hand, had the lowest vitamer level. The best source of tocotrienols was Akusti with a content of 36.3 mg/g. Thus total vitamer contents and tocotrienols to tocopherol ratios could be separately considered when new varieties and/or cultivation techniques are developed and tested. Since other biological activities of tocopherols and tocotrienols have become evident in addition to vitamin E activity, and the role of tocotrienols as bioactive compounds has emerged, the tocotrienols to tocopherol ratio has become an important criterion for nutritional quality (Qureshi et al., 1989; Hood, 1998). This study illustrated that rye grains have a beneficial ratio of tocotrienols to tocopherols as well as a high level of tocopherols and tocotrienols, with evident variation between varieties.
4. Conclusions The optimized small-scale sample preparation method including hot saponification in combination with a NP-HPLC-FLD procedure was shown to be reliable for the determination of tocopherols and tocotrienols from cereals. Saponification under carefully controlled conditions to avoid degradation of the vitamers was shown to be an effective and sufficiently sensitive method for tocopherol and tocotrienol assay from cereals. Using hot saponification of 25 min, the time needed for sample preparation could significantly be reduced, and by scaling down the sample size to 0.5 g, the amounts of solvents needed were also reduced. Polar modification of nhexane with 20% ethyl acetate improved the extraction efficacy of the vitamers from the saponification mixture. With the optimized method, total tocopherol and tocotrienol content of rye flour was 27.870.1 mg/g, while those with cold saponification and direct extraction with hot 2propanol and hexane were lower being 24.571.0 and 24.17 0.8 mg/g, respectively. Thus with an optimized hot saponification method, higher amounts of tocopherols and tocotrienols were obtained than with the other methods studied. The NP-HPLC-FLD method was verified to be sensitive and reliable for the analysis of eight vitamers of tocopherols and tocotrienols. Finally, the repeatability and accuracy of the optimized procedure was confirmed by analysing rye flour and its applicability by analysing ten rye varieties for tocopherols and tocotrienols. This study showed that rye grains possess a beneficial ratio of tocotrienols to tocopherols as well as high amounts of tocopherols and tocotrienols, although with evident variation between varieties.
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Acknowledgements This study was financially supported by the Ministry of Agriculture and Forestry of Finland, the National Technology Agency of Finland and some Finnish food companies. It was part of the project ‘‘Bioactive compounds of rye’’ coordinated by Dr. Kirsi-Helena Liukkonen, VTT Biotechnology. The authors thank Seppo Hovinen from Boreal Plant Breeding Ltd. for the rye variety samples, Hanna Salminen for technical assistance as well as Dr. Afaf Kamal-Eldin (SLU, Uppsala, Sweden) for personal consultation.
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JOURNAL OF FOOD COMPOSITION AND ANALYSIS
Journal of Food Composition and Analysis 17 (2004) 749–765 www.elsevier.com/locate/jfca
Original Article
A small-scale sample preparation method with HPLC analysis for determination of tocopherols and tocotrienols in cereals M. Ryyn.anen, A.-M. Lampi*, P. Salo-V.aa. n.anen, V. Ollilainen, V. Piironen Department of Applied Chemistry and Microbiology, Food Chemistry, Latokartanonkaari 11, P. O. Box 27, University of Helsinki, FIN-00014 Helsinki, Finland Received 21 February 2003; received in revised form 8 August 2003; accepted 22 September 2003
Abstract A small-scale sample preparation method was developed for reliable and economic analysis of tocopherols and tocotrienols in rye and other cereals. The saponification parameters were optimized using two experimental design protocols with statistical analyses. Three critical factors were optimized for hot saponification: time, temperature and amount of potassium hydroxide (KOH). The amount of KOH had the greatest effect. Two direct solvent extraction methods without saponification were tested and found to yield in comparable values than with saponification. Finally, three solvent mixtures for extracting tocopherols and tocotrienols after the optimized saponification step were studied in order to examine the effect of solvent polarity on their recovery. Adding a polar modifier to the solvent, tocopherol and tocotrienol values of rye were significantly increased. The optimized sample preparation method included saponification with 0.5 mL KOH at 100 C for 25 min followed by extraction with n-hexane:ethyl acetate (8:2). The repeatability of the recommended analytical procedure was good and the recoveries of added tocopherols from rye flour samples ranged from 90.3% to 94.3%. The procedure developed was applied to examine the amounts and distribution of tocopherols and tocotrienols in ten rye varieties. The average total tocopherol and tocotrienol content of rye grains was 48.8 mg/g with a 9.3% standard deviation between varieties. r 2003 Elsevier Inc. All rights reserved. Keywords: Tocopherols; Tocotrienols; HPLC; Rye; Cereals
*Corresponding author. Tel.: +358-919158412; fax: +358-919158475. E-mail address: anna-maija.lampi@helsinki.fi (A.-M. Lampi). 0889-1575/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2003.09.014
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1. Introduction Tocopherols and tocotrienols are a group of dietary plant constituents considered to have beneficial health effects. There are four tocopherol and four tocotrienol vitamers. All of them consist of a chromanol ring and a hydrophobic side chain, which is a phytyl in tocopherols and an isoprenyl with three double bonds in tocotrienols. The vitamers can be distinguished by the number and location of methyl groups on their chromanol ring. Generally, when the vitamin E activity of a food item is evaluated, a sum of the different vitamers is calculated taking into account their relative activities. For healthy people it is relatively easy to obtain sufficient tocopherols and tocotrienols from the diet to prevent well-defined vitamin E deficiency symptoms. However, higher intakes of E vitamers may also decrease the risk of several chronic diseases related to oxidative damage, e.g., coronary heart diseases and cancer. Most of the effects in the human body are due to their action as lipid-soluble antioxidants (e.g., Stampfer and Rimm, 1995; Stone and Papas, 1997; Theriault et al., 1999; Schwenke, 2002). Despite the fact that the latest North American guidelines consider a-tocopherol to be the only biologically active form of vitamin E and discount other vitamers (Anon, 2000), the biological importance of other E vitamers has gained attention. Recent studies have shown that tocotrienols have a variety of novel beneficial functions. For example, they may have a protective effect by lowering LDL cholesterol by inhibiting cholesterol biosynthesis (Qureshi et al., 1995; Hood, 1998; Theriault et al., 1999). There is growing evidence that the greatest advantage of a tocopherol- and tocotrienol-rich diet is achieved when various E vitamers are administered concurrently. It has been shown that a-tocopherol supplementation alone has little effect on mammary tumors, and evidently, other forms of vitamin E-like substances, such as a-, g- and d-tocotrienols, reduce the risk of breast cancer (Schwenke, 2002). Thus, it has been stated that the ratios of the individual tocopherols and tocotrienols play an important role in determining the hypocholesterolemic, antioxidant and antitumor properties of palm oil and rice bran (Qureshi et al., 2000, 2002). For example, a-tocopherol and a-tocotrienol had opposite effects on the cholesterol metabolism of chicks, a higher ratio of tocotrienols to tocopherols being optimal (Qureshi et al., 1989). Recently, the vitamers have also been shown to have non-antioxidant roles (Azzi and Stocker, 2000). The main sources of vitamin E-active compounds in the human diet are vegetable fats and oils and products derived from them, but cereals also contribute significantly to the dietary intake in the United States (14.6%, Murphy et al., 1990) and in Finland (18%, Heinonen and Piironen, 1991). In terms of amounts of tocopherols and tocotrienols, cereals are more important than in terms of vitamin E activity, since they are rich sources of the less vitamin E-active tocotrienols. For example, barley contains all four tocopherols and four tocotrienols. Other cereals such as wheat, oats and rye also contain more tocotrienols than tocopherols (Piironen et al., 1986; Balz et al., 1992; Peterson and Qureshi, 1993) and thus have a potentially beneficial distribution of the vitamers. Since cereals contain a number of E vitamers and each of them has a diverse biological and chemical character, they are a challenge for a food analyst, because it is essential to be able to analyse each vitamer separately. It is possible to accurately analyse all forms of tocopherols and tocotrienols with modern chromatographic methods (Eitenmiller and Landen, 1999; Abidi, 2000; Piironen, 2000; Rupe! rez
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et al., 2001; Lampi et al., 2002). There are three major sample preparation approaches for food samples: simple dilution of oil samples in organic solvent, direct extraction of the vitamers with organic solvents, and extraction of the vitamers after saponification. In cereals, tocopherols and tocotrienols are extracted by either a direct solvent method or by saponification followed by extraction. Extraction with organic solvents disrupts the structures to which the vitamers are attached and solubilizes them. n-Hexane is the commonly used solvent, but more polar solvent mixtures are also used, e.g., ethyl acetate in n-hexane or acetone (Ueda and Igarashi, 1987a, 1990; Eitenmiller and Landen, 1999; Rupe! rez et al., 2001). Saponification further assists in releasing the vitamers by degrading the food matrix, and removes the bulk of fat, which improves their chromatographic separation (Piironen, 2000; Rupe! rez et al., 2001). Tocopherols and tocotrienols are relatively unstable in alkaline conditions, and care must be taken to avoid their destruction. They are protected by using antioxidants, flushing the saponification vessel with nitrogen and working under subdued light (Eitenmiller and Landen, 1999). E vitamer analysis is most commonly conducted with normal-phase high-performance liquid chromatography with fluorescence detection (NP-HPLC-FLD). A fluorescence detector is preferred over an ultraviolet detector in analysing tocopherols and tocotrienols in complex food matrices because of its specificity and sensitivity (Piironen et al., 1986; Eitenmiller and Landen, 1999; Abidi, 2000). Other means of separation include reversed-phase HPLC, gas chromatography and, most recently, capillary electrochromatography (Abidi et al., 2002). Electrochemical detection is also applied as a sensitive detector for E vitamers after HPLC separation (Ueda and Igarashi, 1987b; Yamauchi et al., 2002), while mass spectrometry (MS) in . combination with HPLC assists in identification of compounds (Strohschein et al., 1999; Stoggl et al., 2001). As the sensitivity of the HPLC analysis has improved, it has become possible to scale down the food sample size, as has been done, e.g., for animal products (Salo-V.aa. n.anen et al., 2000). At the same time, a need for shorter sample preparation times and decreased amounts of chemicals has arisen. Since there are multiple factors to be optimized in the sample preparation method for E vitamer analysis, we chose an experimental design approach to develop a fast and reliable saponification method for cereal samples. Earlier, Lee et al. (2000) used response surface methodology to optimize an extraction procedure of tocopherols from tomato and broccoli. They used 70 C as the saponification temperature and varied the amounts of alkali and ethanol in the saponification mixture and the saponification time. In our study, a wider range of analytical conditions during saponification and extraction of E vitamers was investigated. Our hypothesis was that saponification is an efficient means to liberate tocopherols and tocotrienols from cereal matrix prior to lipid extraction, but the saponification conditions should be carefully examined and controlled to avoid decomposition of the analytes. In order to reliably and economically investigate the distributions of tocopherols and tocotrienols in rye and other cereals, a small-scale and rapid sample preparation method was developed. The saponification conditions were optimized using experimental design and solvent mixtures for extraction of tocopherols and tocotrienols were evaluated. Applicability of the recommended sample preparation method was tested by examining the amounts and distribution of tocopherols and tocotrienols in ten rye varieties grown in Finland. This was also performed in order to expose the most promising varieties with regard to the optimal ratio of tocopherols and tocotrienols.
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2. Materials and methods 2.1. Sample materials, standards, and reagents The material used for method development was rye flour (Myllyn paras; Helsingin mylly, Hyvink.aa. , Finland). Rapeseed oil was used as an in-house reference material for HPLC analysis (Kultasula; Raisio Group, Raisio, Finland). Samples of ten rye varieties were cultivated in the Agricultural Research Centre, Jokioinen, Finland, in 1999 (Boreal Plant Breeding Ltd, Jokioinen, Finland). Sample grains were milled with a hammer mill at the Department of Food Technology, University of Helsinki. All sample materials were stored in small portions in the dark at 20 C until analysed. HPLC-grade n-hexane, ethyl acetate and 1,4-dioxane were purchased from Rathburn (Walkerburn, UK). Milli Q water was of HPLC-grade. Ethanol was of spectrophotometric grade (AAS, 99.5%, Primalco, Finland). Ascorbic acid, p.a., was purchased from Merck (Darmstadt, Germany) and potassium hydroxide (KOH) pellets from EKA Nobel (Bohus, Sweden). Saponification solution was prepared by dissolving 50 g of KOH pellets in 100 mL MilliQ water. Sample extracts were filtered (GHP Acrodisc, 0.45 mm, Pall Corp., Anna Arbor, MI) before injecting to HPLC. a-, b-, g- and d-tocopherols (fur . biochemische Zwecke, Art no. 15496) were purchased from Merck. The standard stock solutions of the tocopherols were prepared to a concentration of approximately 500 mg/mL (in ethanol, AAS). The stock solutions were stored at 20 C. The concentrations were confirmed spectrophotometrically using the known absorption coefficients of each vitamer in ethanol Eð1% 1 cm Þ: 75.8 for a-tocopherol, 89.4 for b-tocopherol, 91.4 for g-tocopherol and 87.3 for d-tocopherol at 292, 296, 298 and 298 nm, respectively (Podda et al., 1996). The combined working solution was prepared by pooling suitable amounts of each tocopherol and diluting with n-hexane to obtain concentrations ranging from 1–100 ng/injection. Each tocotrienol vitamer was quantified with the respective tocopherol (e.g., AOCS, 1990; Kramer et al., 1997). Tokovid-palmoil extract (Hovid Sdn. Bhd., Malaysia) was used as a qualitative standard for tocotrienols. 2.2. Optimization of the sample preparation method The sample preparation method was based on the room temperature saponification method with n-hexane as an extraction solvent (Piironen et al., 1986). In this study a small scale and rapid procedure using hot saponification was developed for determining tocopherols and tocotrienols in cereals, using rye flour. To avoid destruction of labile vitamers, all work was carried out under subdued light, ascorbic acid was added as an antioxidant and reaction vessels were flushed with nitrogen before adding KOH. At first, sample amounts and solvent volumes were reduced from a previous method (Piironen et al., 1986) and hot saponification was conducted. Total tocopherol and tocotrienol values from rye flour obtained with the room temperature saponification and the hot saponification methods were comparable (24.571.0 mg/g and 23.872.2 mg/g) as were the recoveries of added tocopherols (range 58–129%). Thereafter we proceeded to optimize the hot saponification conditions. Three critical experimental factors of the hot saponification method were evaluated: saponification time,
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temperature and amount of KOH. The saponification method was developed and optimized using two experimental design protocols (Haaland, 1989). The first experiments included three critical factors with a wide range of analytical conditions, i.e., levels of factors, and the second included only two factors with a narrow range (Tables 1 and 2). All other sample preparation parameters remained constant. The results were analysed by using multiple linear regression analysis and visualized by response surface plots (Statgraphics Plus, 1997). The output factor for the trials was the total tocopherol and tocotrienol value and the target was to obtain as high a value as possible. Each trial was performed in duplicate and the mean value was used as the output factor. On the basis of the results of the experiments described above, it was further investigated whether saponification could be omitted in sample preparation. Thus two direct solvent extraction methods without saponification were tested. Solvent extraction method 1 included hot extraction with 2-propanol and n-hexane as extraction solvents (Koivu et al., 1997) and method 2 was the optimized hot saponification method excluding the addition of KOH. Optimization of the sample preparation method was finally fulfilled by investigating the effect of solvent polarity on extracting tocopherols and tocotrienols from the saponified mixture. Three extraction solvents were compared: n-hexane, n-hexane:ethyl acetate (9:1) and n-hexane:ethyl acetate (8:2). In order to evaluate the steps other than saponification in the sample preparation method as described above, all analyses were performed in triplicate at minimum. Comparisons of means or pairs were conducted with t-tests, and of three groups with one-way analysis of variances (ANOVA) followed by multiple range tests. All analyses were carried out using the same software as for multiple linear regression analyses (Statgraphics Plus, 1997).
Table 1 Analytical conditions as the input factors and total values of tocopherols and tocotrienols (mg/g) as the output factor in a Box-Behnken experiment to study the effects of saponification time (min), temperature ( C) and amount of KOH (mL) on the output factor (Haaland, 1989) (mean values from duplicate analyses) Trial
Temperature
Time
KOH
Total tocopherols and tocotrienols
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
60 60 100 100 60 60 100 100 80 80 80 80 80 80 80 80
20 60 20 60 40 40 40 40 20 20 60 60 40 40 40 40
2 2 2 2 1 3 1 3 1 3 1 3 2 2 2 2
21.7 18.5 21.5 18.6 21.2 14.9 23.6 16.5 22.6 17.1 20.1 18.1 22.1 18.6 19.0 21.4
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Table 2 Analytical conditions as input factors and total values of tocopherols and tocotrienols (mg/g) as the output factor in a Central Composite experiment to study the effects of saponification time (min) and the amount of KOH (mL) on the output factor (Haaland, 1989) (mean values from duplicate analyses) Trial
Time
KOH
Total tocopherols and tocotrienols
1 2 3 4 5 6 7 8 9 10 11
5 5 20 20 1.9 23.1 12.5 12.5 12.5 12.5 12.5
0.25 1 0.25 1 0.625 0.625 0.096 1.154 0.625 0.625 0.625
22.5 24.6 27.6 24.4 23.6 26.9 25.2 24.7 26.0 24.6 24.5
2.3. Recommended analytical procedure After method optimization, a recommended analytical procedure was established. The main steps are illustrated in Fig. 1. 2.3.1. Saponification Rye flour (0.5 g) was accurately weighed into a 30 mL Pyrex glass tube with a Teflon screw cap. Ascorbic acid (0.1 g), ethanol (5 mL) and water (2 mL) were added. After mixing the tube with a Vortex-mixer, the tube was flushed with nitrogen and KOH (0.5 mL) was added. The tube was capped and transferred to a boiling water bath for 25 min. The tube was mixed with the Vortexmixer after 10 min of boiling. The tubes were cooled in an ice-water bath. 2.3.2. Extraction Water (2.5 mL) and ethanol (2.5 mL) were added to the cooled tubes. Unsaponified lipids were extracted by using three portions (each 10 mL) of n-hexane:ethyl acetate (8:2). Tubes were shaken for 10 min with 500 strokes/min, and after separation of the phases, the organic layers were collected in a separation funnel. Organic extracts were washed three times with water and with addition of NaCl to avoid emulsion formation. The washed extract was transferred to a roundbottomed flask, and the organic phase was evaporated. Ethanol (2 mL) and n-hexane (2 mL) were added and evaporated to dryness. The residue was dissolved in n-hexane and transferred quantitatively to a 5 mL volumetric flask. Sample extracts were stored in Kimax test tubes in the dark at 70 C. Prior to HPLC analysis, extracts were filtered through a 0.45 mm filter. 2.3.3. Analytical HPLC Normal phase HPLC with fluorescence detection (excitation 292 nm, emission 325 nm) was used to analyse tocopherols and tocotrienols (Kamal-Eldin et al., 2000). The HPLC system consisted of a pump (Waters 510; Waters Corp., Milford, MA), an autosampler with a cooling
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Preparing the sample for saponification: 0.5 g sample 0.1 g ascorbic acid, 5 mL ethanol, 2 mL water flushing with nitrogen
Saponification: 0.5 mL KOH solution 100˚C, 25 min
Extraction and purification of non-saponifiable lipids: 2.5 mL ethanol, 2.5 mL water 3 x 10 mL n-hexane:ethyl acetate (8:2) washing the extract 3 x with water and evaporating to dryness
Preparing the sample for HPLC analysis: 2 mL ethanol and 2 mL n-hexane evaporating to dryness dissolving the residue in 5 mL n-hexane
NP-HPLC analysis with fluorescence detector
Fig. 1. Recommended analytical procedure.
module (Waters 712), a scanning fluorescence detector (Waters 474), and an Inertsil silica column (5 mm, 250 mm4.6 mm; Varian Chromapack, Middelburg, Netherlands) with a silica guard column (Guard-Pak Silica, Waters). The temperature of the column oven was 30 C. Separation of the vitamers was based on isocratic elution. The mobile phase contained 3% 1,4-dioxane and 97% n-hexane. The flow rate of the mobile phase was 2 mL/min. Tocopherols and tocotrienols were quantified with an external standard method in which quantification was based on peak areas. Calibration curves with six points were obtained daily. The method was validated by determining the following parameters: detection and determination limits, range of linearity and repeatability. Detection limits were defined as a signal three times the height of the noise. Determination limits were defined as three times the detection limit. The dayto-day repeatability of the HPLC method was also confirmed with in-house reference material (rapeseed oil) which was analysed daily during the 2-month period. 2.4. Evaluation and application of the recommended analytical procedure Verification of the vitamer identification was based on the HPLC-FLD data and HPLC-MS data. Full scan mass spectra and selected ion monitoring tracings derived from an atmospheric
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pressure chemical ionization (APCI) in positive mode were recorded using a quadrupole ion trap mass spectrometry (Esquire LC-MS, Bremen, Germany). The liquid chromatographic method used was the same as in analytical HPLC except that the eluent flow (2 mL/min) was split (1:10) prior ionization interface with a splitter (Acurate Post-Column Splitter, LC Packings, USA). The temperature of APCI interface and drying gas (nitrogen) was set at 300 C, and the potential of discharge corona needle was 4.5 kV. Mass spectra were recorded at the scan range of 100–700 m/z and the protonated molecular ions ([M+H]+) were measured. The reliability of the recommended analytical procedure was verified by recovery and repeatability tests with rye flour. Accuracy was verified by recovery tests, in which samples were spiked with tocopherols (10 mg/g of rye flour) before saponification. Spiked samples were prepared in duplicate on 3 days. Repeatability of the developed method was verified by analysing the tocopherol and tocotrienol contents of rye flour in duplicate over 3 days. The applicability of the recommended procedure to, e.g., whole grain samples was examined by analysing the tocopherol and tocotrienol contents of ten rye varieties, and by finding any variation in E vitamer composition. The varieties studied represent winter classes of rye cultivars grown in Finland and are thus a relevant set of samples to be analysed by the current method.
3. Results and discussion 3.1. Optimization of saponification conditions Three critical experimental factors were optimized in the small-scale hot saponification method for analysis of tocopherols and tocotrienols in cereals: saponification time, temperature and amount of KOH. Using the Box-Behnken design the total tocopherol and tocotrienol value of rye flour measured as the output factor ranged from 14.9 to 23.6 mg/g (Table 1). Two factors were shown to have a significant impact on the output factor, namely the saponification time (P ¼ 0:0877) and the amount of KOH (P ¼ 0:0002). Saponification temperature, however, was not an important factor (P ¼ 0:3634) and was excluded from the linear regression model. The influences of the two important factors are illustrated in Fig. 2. The coefficient of determination indicating the model’s ability to explain the relationships between the factors was 64.7%. The figure shows that the smaller the amount of KOH solution, the greater was the total tocopherol 26.8363 - 2.615*KOH - 0.047312*Time
T + T3, ug/g
24 22 20 18 16
1
1.4
1.8
2.2
KOH, mL
2.6
3
20
30
40
50
60
Time, min
Fig. 2. Response surface plot of effects of saponification time and amount of KOH on total value of tocopherols and tocotrienols.
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and tocotrienol value. The effect of saponification time on the output factor was less than that of the amount of KOH. For example, when the time increased from 20 to 60 min, the total tocopherol and tocotrienol value increased by 1.9 mg/g, but when the amount of KOH decreased from 3 to 1 mL, the output factor increased by 5.2 mg/g. Since the saponification temperature had an insignificant effect on tocopherol and tocotrienol values, the highest setting of 100 C was chosen for further analyses, e.g., to enhance decomposing of cereal matrix. In order to examine the effect of even lower levels of KOH and shorter saponification times, another experiment using the Central Composite design was carried out at a temperature stabilized to 100 C (Table 2). The total tocopherol and tocotrienol values were higher and the range smaller than in the first experiment, ranging from 22.5 to 26.9 mg/g. Here both the amount of KOH and the time had a significant effect on the output factor with P ¼ 0:0038 and P ¼ 0:0002; respectively. Moreover, there was an evident interaction between these two factors (P ¼ 0:0014) in the linear regression model (Fig. 3). The coefficient of determination of this model was even higher, 87.1%, than that of the first model. Again, the smaller the amount of KOH, the greater was the total tocopherol and tocotrienol value. However, at low levels of KOH, o1 mL, a longer saponification time was needed than at high KOH levels. The interaction between these two factors was so strong that at 0.096 mL of KOH, the total tocopherol and tocotrienol value was 19.8 mg/g higher at the longest saponification time (23.1 min) than at the shortest time (1.9 min) whereas at 0.625 mL of KOH, the difference was only 3.4 mg/g. When more than 1 mL of KOH was used, changes of saponification time within the range of the experiment had only minor effects on the output factor. Thus under these hot saponification conditions a compromise can be made between the amount of KOH and saponification time. Our finding that a small amount of KOH produces higher total tocopherol and tocotrienol values is supported by the results of Piironen et al. (1984), who investigated the effects of different amounts of KOH on E vitamer contents in a diet sample. They showed that treating samples with higher amounts of alkaline than used in this study partly destroyed tocopherols. In addition, high levels of soap might decrease the extractability of tocopherols and tocotrienols, because the soap produced during saponification might solubilize them into the alkaline media (Ueda and Igarashi, 1987b). However, saponification degrades the food matrix and purifies the lipid extract, which improves the efficacy of the sample preparation method. We also found that emulsions were formed less frequently when at least 0.25 mL of KOH was used than with less alkaline. With low levels of KOH, saponification times should be long enough as indicated in Fig. 3. Thus we
T + T3, ug/g
19.7418 + 5.19666*KOH + 0.444822*Time - 0.460444*KOH*Time
31 29 27 25 23 21 19
0
0.2
0.4
0.6
0.8
1
1.2
1620 8 12 0 4
24
Time, min
KOH, mL
Fig. 3. Response surface plot of effects of time and potassium hydroxide on the total amount of tocopherols and tocotrienols.
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concluded that the recommended saponification method should consist of a reasonable amount of KOH, 0.5 mL, with a saponification time of 25 min at 100 C. 3.2. Comparisons of direct solvent extraction and saponification The results of the previous experiments indicated that the total tocopherol and tocotrienol value from rye flour was greatest with the smallest amount of KOH. Thus it was further investigated whether saponification could be omitted and, instead, direct solvent extraction would be used in sample preparation where decomposition of matrix would be less efficient, but no losses due to hot alkaline would occur. The total value of tocopherols and tocotrienols determined by two direct extraction methods, i.e., hot extraction with 2-propanol and n-hexane, and optimized saponification of the method excluding addition of KOH, and by the saponification method gave statistically similar results (ANOVA, P > 0:05; n ¼ 3–5). The value was 24.270.8 mg/g for method 1, 23.970.9 mg/g for method 2, and 24.970.5 mg/g for the saponification method. Recoveries of added tocopherols from rye flour were 73.7–90.5% and 86.1–90.4% for the saponification and the direct extraction method 1, respectively. With saponification, recoveries of more polar tocopherols were lower than that of a-tocopherol, decreasing in the order of their polarity, while with solvent extraction with 2-propanol and n-hexane, no difference between the vitamers was observed. The different recoveries of added tocopherols in the saponification method are caused either by differences in their stabilities during saponification or extractabilities from the saponification mixture, and not from liberation from the matrix, because tocopherols were added as ethanol solutions. These findings illustrate one more important aspect of the sample preparation procedure, i.e., the polarity of the extraction solvent after saponification. Similarly, Ueda and Igarashi (1987b) found that soap formed from fat decreased the extractability of other tocopherols more than that of a-tocopherol when an apolar n-hexane was used as the extraction solvent. Hence, modification of extraction solvent following saponification was investigated to improve the recoveries of more polar tocopherols. Since the optimized saponification method was shown not to yield lower tocopherol and tocotrienol values in rye flour than direct extraction methods, it was concluded that saponification is beneficial for the efficient liberation of tocopherols and tocotrienols from cereals. Furthermore, resolution of the vitamers analysed by HPLC was better with the saponification method. Previously, comparisons of sample preparation methods consisting of saponification or direct extraction have yielded inconsistent results. Piironen et al. (1984) preferred saponification due to its higher total yield of tocopherols and tocotrienols, but Bonvehi et al. (2000) found ca 20% higher tocopherol values of oils and biscuits when samples were prepared without saponification than with saponification, and they therefore omitted saponification. One potential reason for the different results may be that precautions during sample preparation, including addition of antioxidants, working under subdued light and nitrogen atmosphere, are more important when the sample is subjected to saponification, because the vitamers are easily oxidized in the presence of alkali and the procedure must be carefully controlled. 3.3. Effect of solvent polarity on extraction of tocopherols and tocotrienols from saponified mixtures Development of the saponification step was conducted using n-hexane as the extraction solvent. A possible need for a more polar extraction solvent mixture to improve the extractability of
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tocopherols and tocotrienols was observed. The use of polarity modifying agents in extraction solvents has also been suggested earlier (Ueda and Igarashi, 1987a; Eitenmiller and Landen, 1999; Salo-V.aa. n.anen et al., 2000). Thus, after the saponification parameters had been obtained, three different extraction solvents were compared: n-hexane, n-hexane:ethyl acetate (9:1) and nhexane:ethyl acetate (8:2). The total tocopherol and tocotrienol values obtained from rye flour using these solvent mixtures were significantly different (ANOVA, Po0:001) giving values of 24.970.5, 28.770.6 and 29.570.3 mg/g (n ¼ 3–5), respectively. The multiple range tests showed that the mean with n-hexane as solvent was lower than with others (Po0:05). Thus polar modification of extraction solvent improved the extractability of tocopherols and tocotrienols present in the hot saponified rye flour mixture. At the same time, minor peaks eluting at the retention times of g-tocopherol, g-tocotrienol and d-tocotrienol also became detectable. When Ueda and Igarashi (1987a) investigated the effect of ethyl acetate concentration in n-hexane as an extraction solvent, they concluded that the addition of 10% ethyl acetate in n-hexane was optimal, because as the proportion of ethyl acetate increased above this level, some of it partitioned in the aqueous phase and decreased the volume of the organic layer. In our method, increasing the concentration of ethyl acetate did not have adverse effects, because the extraction was repeated three times and the organic layers were collected quantitatively to ensure the recovery of all vitamers. Thus 20% ethyl acetate in n-hexane was preferred instead of 10%. 3.4. Repeatability and accuracy of the recommended analytical procedure Finally, the recommended analytical procedure method was evaluated by repeating the analysis of rye flour and by spiking rye flour samples with tocopherols (Table 3). All analyses were performed in duplicate over 3 days. The total tocopherol and tocotrienol value was 28.770.1 mg/ g. b-tocotrienol was the major vitamer, followed by a-tocopherol, a-tocotrienol and b-tocopherol. The repeatability of the method was excellent, since the coefficients of variation (%CV) of these vitamers were 1.5–3.6%. Even the minor vitamers (g-tocopherol, g- and d-tocotrienols), that had contents near to the determination limit, had a low %CV of o19%. It should be remembered that this high repeatability was obtained with a highly homogenous 0.5 g sample of rye flour, and that greater variations may occur with more uneven materials. The average recovery of the added tocopherols at the 10 mg/g level was good. The highest recovery was obtained for a- and b-tocopherols, 94.3% and 93.6%, and the lowest for gtocopherol, 90.3% (Table 3). Thus recoveries were all >90% and the variation between vitamers
Table 3 Tocopherol (T) and tocotrienol (T3) contents in mg/g expressed as mean7SD (n ¼ 6) of (1) rye flour and (2) rye flour with added tocopherols. (3) Recoveries of added tocopherols determined by the recommended analytical procedurea
(1) (2) (3) a
a-T
a-T3
b-T
g-T
b-T3
g-T3
d-T
d-T3
Total
8.270.1 17.570.7 94.3
8.170.3 8.070.4
2.970.1 13.070.5 93.6
0.270.02 9.970.4 90.3
9.370.3 9.270.4
0.170.01 0.170.02
0.070.0 10.470.4 91.0
0.170.01 0.170.02
28.770.1 68.170.4
Identification of T and T3 was confirmed by high performance liquid chromatographic mass spectrometric data except for d-T3.
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was clearly lower than when n-hexane was used as the only solvent. This means that polar modification improves the extractability of the more polar tocopherols. In the study of Piironen et al. (1986), recoveries of a-, b-, g- and d-tocopherols were 94.3%, 95.2%, 96.5% and 95.7%, respectively, with saponification at room temperature. Despite optimization of the saponification method and careful carrying out of the procedure, hot saponification might destroy tocopherols and tocotrienols, and recoveries as high as those obtained with room temperature saponification might not be achieved. 3.5. Evaluation of the analytical HPLC method Identification of tocopherols and tocotrienols was confirmed on HPLC-MS data (Fig. 4). The m/z values of protonated molecular ions for a-, b- and g-tocopherols were 431.5, 417.4 and 417.4, and for a-, b-, g- tocotrienols were 425.4, 411.4 and 411.4, respectively. Verification of dtocotrienol was performed with HPLC-FLD derived from the Tokovid-palmoil extract, because the signal of protonated molecular ion of d-tocotrienol was too low for adequate identification using LC-MS. This is apparently due to the low ionization efficiency of d-tocotrienol in APCI process used in this study. However, the use of the mixture of n-hexane and 1,4-dioxane as a mobile phase enables the more uniform ionization efficiency for individual vitamers compared to . the results of Stoggl et al. (2001) when the eluent consisted of isooctane and diisopropyl ether. The quantitative HPLC method for analysis of tocopherols and tocotrienols was validated by several detector parameters. Its repeatability was confirmed with an in-house reference material and its stability by comparing the chromatograms during the 2-month period. Detection limits varied between 0.10 and 0.18 ng/injection. Determination limits of the vitamers ranged between 0.30 and 0.54 ng/injection. The detector response was linear in the tested ranges, 2–80 ng/injection (R2 ¼ 0:9998). The variation of the standard curve slopes varied from 0.2% to 0.3% (n ¼ 22), which showed that the stability of the detector response was excellent. The average tocopherol contents (n ¼ 13) of the in-house reference (rapeseed oil) were: a-tocopherol: α -T α -T3 30
25 β -T
β -T3
FLD
20
15
10 γ -T
5
0
2.5
5
7.5
δ -T3
γ -T3
10
12.5
15
17.5
20
Time [min]
Fig. 4. HPLC-chromatogram of rye flour (for details see Section 2).
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264716 mg/g, g-tocopherol 590728 mg/g and d-tocopherol 1977 mg/g, and the %CVs were 6.0%, 4.8% and 36.4%, respectively. These figures show that the HPLC method had good repeatability. The high variation of d-tocopherol was due to its low concentration, which was near to the determination limit. The silica column used proved to be very stable, because the %CVs of the retention times of tocopherols and tocotrienols over a two-month period were only approximately 0.5% (n ¼ 10). The retention time of void volume (to ) was 1.96 min (measured with dodecane) and all tocopherols and tocotrienols were well separated between retention times of 5.5 and 14.2 min. Resolution of the column was good, being 1.98 (n ¼ 10) for b- and g-tocopherols which are usually difficult to separate from each other completely. 3.6. Tocopherol and tocotrienol contents of rye The total combined content of tocopherols and tocotrienols of the rye flour used in the method development was 28.7 mg/g, of which the amounts of a- and b-tocopherols and -tocotrienols were 8.2, 8.1, 2.9 and 9.3 mg/g, respectively. In an earlier study, the corresponding amounts for whole meal rye flour were 10.2, 14.4, 3.1 and 11.3 mg/g, and for rye flour 5.5, 4.3, 2.6 and 6.4 mg/g, respectively (Piironen et al., 1986). Thus the values indicate that some parts of the kernel have been removed during the milling process of the rye flour used in this study. The average tocopherol and tocotrienol contents of the ten rye varieties commonly cultivated in Finland were 48.874.5 mg/g as analysed by the recommended analytical method (Table 4). Total E vitamer contents were higher than in the rye flour used, because whole grains were milled for samples without removing outer layers of the kernel. The best source of tocopherols and tocotrienols was the variety Akusti, 54.3 mg/g, and the poorest was Picasso, 39.9 mg/g. The overall variation between the varieties was 9.3%, which is slightly more than the variation in folate and plant sterol contents that were earlier shown to be 8% and 6%, respectively (Kariluoto et al., 2001; Piironen et al., 2002). There was an apparent although not significant (P ¼ 0:051) positive Table 4 Tocopherol (T) and tocotrienol (T3) contents of 10 rye varieties in mg/g. Mean of duplicate determinationsa
BOR9214 Akusti Riihi Picasso BOR9414 Esprit BOR7068 Elvi Anna Voima Average CV% a
a-T
a-T3
b-T
g-T
b-T3
g-T3
d-T3
Total
T
T3
T3/T-ratio
13.9 14.3 16.1 10.0 14.8 12.6 16.0 14.5 15.8 16.3 14.4 13.5
16.2 19.0 16.7 14.7 17.5 15.3 17.3 17.8 16.5 17.6 16.9 7.5
3.4 3.2 3.9 2.3 4.1 2.9 4.0 3.6 4.2 3.8 3.5 17.3
0.5 0.6 0.4 0.4 0.6 0.5 0.6 0.6 0.6 0.6 0.5 16.3
11.7 16.8 10.2 12.2 13.2 11.9 15.3 14.2 11.8 12.2 13.0 15.1
0.2 0.3 0.2 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.3 27.3
0.2 0.2 0.2 0.1 0.2 0.2 0.3 0.2 0.2 0.2 0.2 21.2
46.1 54.3 47.7 39.9 50.8 43.6 53.7 51.3 49.4 50.9 48.8 9.3
17.8 18.0 20.5 12.7 19.6 15.9 20.6 18.8 20.6 20.6 18.5 13.9
28.3 36.3 27.2 27.2 31.2 27.7 33.1 32.6 28.8 30.3 30.3 10.0
1.6 2.0 1.3 2.1 1.6 1.7 1.6 1.7 1.4 1.5 1.7 15.5
Identification of T and T3 was confirmed by high-performance liquid chromatographic, mass spectrometric data except for d-T3.
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correlation between E vitamer and plant sterol contents. As in the case of rye flour, a- and btocopherols and -tocotrienols were the major vitamers. Our values are in line with earlier studies, in which total a- and b-tocopherol and tocotrienol contents ranged between 23 and 49 mg/g (Morrison, 1978), between 32.4 and 54.7 mg/g (Barnes, 1982) and around 32.4 mg/g (Balz et al., 1992). In Poland, a lower level of 27.7 mg/g of tocopherols and tocotrienols in rye was found (Zielinski et al., 2001). The difference could be explained by variation between varieties as well as by variation between the methods of analysis. The authors extracted tocopherols and tocotrienols directly from rye with 80% methanol before NP-HPLC analysis. In the ten rye variety samples, tocopherol and tocotrienol contents varied between 17.8 and 20.6 mg/g and between 27.2 and 36.3 mg/g, respectively, and the tocotrienols to tocopherols ratio between 1.3 and 2.1 (Table 4). In earlier studies, the ratio was slightly lower at 1.6 (Balz et al., 1992) and 1.1 (Zielinski et al., 2001). The tocotrienols to tocopherols ratio did not correlate with the total E vitamer contents. For example, the highest ratio was measured for Picasso, which, on the other hand, had the lowest vitamer level. The best source of tocotrienols was Akusti with a content of 36.3 mg/g. Thus total vitamer contents and tocotrienols to tocopherol ratios could be separately considered when new varieties and/or cultivation techniques are developed and tested. Since other biological activities of tocopherols and tocotrienols have become evident in addition to vitamin E activity, and the role of tocotrienols as bioactive compounds has emerged, the tocotrienols to tocopherol ratio has become an important criterion for nutritional quality (Qureshi et al., 1989; Hood, 1998). This study illustrated that rye grains have a beneficial ratio of tocotrienols to tocopherols as well as a high level of tocopherols and tocotrienols, with evident variation between varieties.
4. Conclusions The optimized small-scale sample preparation method including hot saponification in combination with a NP-HPLC-FLD procedure was shown to be reliable for the determination of tocopherols and tocotrienols from cereals. Saponification under carefully controlled conditions to avoid degradation of the vitamers was shown to be an effective and sufficiently sensitive method for tocopherol and tocotrienol assay from cereals. Using hot saponification of 25 min, the time needed for sample preparation could significantly be reduced, and by scaling down the sample size to 0.5 g, the amounts of solvents needed were also reduced. Polar modification of nhexane with 20% ethyl acetate improved the extraction efficacy of the vitamers from the saponification mixture. With the optimized method, total tocopherol and tocotrienol content of rye flour was 27.870.1 mg/g, while those with cold saponification and direct extraction with hot 2propanol and hexane were lower being 24.571.0 and 24.17 0.8 mg/g, respectively. Thus with an optimized hot saponification method, higher amounts of tocopherols and tocotrienols were obtained than with the other methods studied. The NP-HPLC-FLD method was verified to be sensitive and reliable for the analysis of eight vitamers of tocopherols and tocotrienols. Finally, the repeatability and accuracy of the optimized procedure was confirmed by analysing rye flour and its applicability by analysing ten rye varieties for tocopherols and tocotrienols. This study showed that rye grains possess a beneficial ratio of tocotrienols to tocopherols as well as high amounts of tocopherols and tocotrienols, although with evident variation between varieties.
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Acknowledgements This study was financially supported by the Ministry of Agriculture and Forestry of Finland, the National Technology Agency of Finland and some Finnish food companies. It was part of the project ‘‘Bioactive compounds of rye’’ coordinated by Dr. Kirsi-Helena Liukkonen, VTT Biotechnology. The authors thank Seppo Hovinen from Boreal Plant Breeding Ltd. for the rye variety samples, Hanna Salminen for technical assistance as well as Dr. Afaf Kamal-Eldin (SLU, Uppsala, Sweden) for personal consultation.
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