Article pubs.acs.org/JAFC
Fractionation Analysis of Manganese in Turkish Hazelnuts (Corylus avellana L.) by Inductively Coupled Plasma−Mass Spectrometry Umran Seven Erdemir* and Seref Gucer Department of Chemistry, Faculty of Arts and Sciences, University of Uludag, Gorukle Campus, 16059 Nilufer-Bursa, Turkey ABSTRACT: In this study, an analytical fractionation scheme based on water, diethyl ether, n-hexane, and methanol extractions has been developed to identify manganese-bound fractions. Additionally, in vitro simulated gastric and intestinal digestion, noctanol extraction, and activated carbon adsorption were used to interpret the manganese-bound structures in hazelnuts in terms of bioaccessibility. The total content of manganese in the samples was determined by inductively coupled plasma−mass spectrometry after microwave-assisted digestion, and additional validation was performed using atomic absorption spectroscopy. Water fractions were further evaluated by high-performance liquid chromatography hyphenated to inductively coupled plasma− mass spectrometry for the identification of water-soluble manganese fractions in hazelnut samples. The limits of detection and quantification were 3.6 and 12.0 μg L−1, respectively, based on peak height. KEYWORDS: hazelnut (Corylus avellana L.), manganese, fractionation, bioaccessibility, ICP-MS
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INTRODUCTION Hazelnut (Corylus avellana L.), a nut species widely consumed worldwide, is mainly distributed along the coasts of the Black Sea region of Turkey1 and has great economic importance.2 Turkey is the major global hazelnut producer, supplying 65% of the world’s total production,3 followed by Italy, the United States, Azerbaijan, and Spain.4 Hazelnuts can be consumed as a natural (raw) or roasted fruit5 and are an important additive in the food industry for a variety of products.4,6 Hazelnuts are a good source of major and trace elements that are vital to human health, and its chemical composition has been widely described in the literature.3,4,7 Hazelnut plays a major role in human nutrition and health because of its special nutritional value.7,8 It is well-known that environmental factors, such as climate, the presence of fertilizers, soil conditions, harvest year, and cultivation type, may affect the mineral content of hazelnuts. In addition, mineral composition has been shown to be related to the hazelnut variety.2,9 The hazelnut is an excellent source of manganese, with a high elemental content.2,7,10 Manganese activates many enzymes as a cofactor and is required for protein and fat metabolism and for a healthy nervous system.11 Manganese deficiency affects the nervous system, which can result in epilepsy. High concentrations or overdoses of manganese can cause a neurological disorder called manganism, the symptoms of which are similar to those of Parkinson’s disease.12−14 Additionally, manganese deficiency inhibits carbohydrate and lipid metabolism and also affects glucose tolerance and serum high-density lipoprotein cholesterol degradation.15 In this regard, manganese is an essential microelement for organisms. The recommended daily intake of manganese ranges from 2.2 to 4.6 mg day−1.16 Consumption of the recommended daily amount of 42.5 g of hazelnut from different varieties provides at least 40% of the recommended manganese intake for adults.10 The bioaccessibility of trace metals depends on many factors, including food composition and gastrointestinal conditions. © XXXX American Chemical Society
Additionally, fractionation studies can address the mobility and bioaccessibility of metals. Manganese can exist as the free metal ion in plants or as a complex with organic acids, proteins, polyphenols, and polysaccharides.17 These free levels or complex structures may influence the bioaccessibility or biotoxicity of manganese. The amounts of manganese in food samples are not sufficient to assess the daily intake amounts in terms of human health, nutrition, and disease.17 However, manganese bioaccessibility in food samples is not well characterized. In recent literature, fractionation studies with HCl and NaOH or sodium phosphate buffer solutions have been evaluated for use with protein structures from various nut samples (Brazil nut, pine nut, etc.).11,18 Elements that could be related to these structures, such as Mn, Zn, Ni, and Cu, were analyzed.11 Additionally, Goncalves et al. applied sequential extractions to evaluate lipids, proteins, and low molecular weight fractions of Brazil nuts for barium fractionation analysis.19 We could not find a detailed examination related to the fractionation and bioaccessibility of manganese in hazelnut samples. Consequently, in vitro digestion methods, together with fractionation studies, will provide valuable insights into manganese bioaccessibility. The aim of the study was to propose an analytical method for the bioaccessibility assessment of manganese in hazelnut samples via fractionation and in vitro digestion methods. Different solvent extractions were applied to characterize the manganese-bound structures in the matrix, and simulated digestion methods were evaluated in terms of sufficient intake interactions by inductively coupled plasma−mass spectrometry (ICP-MS). Additionally, a hyphenated technique based on reversed-phase liquid chromatography separation with inductively coupled plasma detection was applied for the rapid Received: July 2, 2014 Revised: October 10, 2014 Accepted: October 13, 2014
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dx.doi.org/10.1021/jf503145t | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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spray chamber, and a standard glass torch. The sampler and skimmer cones were made of nickel. The operating conditions of the ICP-MS were as follows: RF power, 1000 W; plasma argon flow rate, 17.0 L min−1; nebulizer flow rate, 0.85 L min−1; sample uptake rate, 1.5 mL min−1. The other plasma conditions and acquisition parameters were the same as those reported in our earlier studies.12 Additionally, total manganese analyses were performed by a Varian (Australia) AA240FS fast sequential atomic absorption spectrometer (AAS) for validation purposes. The optimum parameters for manganese determination were as follows: wavelength, 279.5 nm; HCL current, 10 mA; acetylene flow rate, 2.0 L min−1; slit width, 0.2 mm; and flame height, 7 mm. The fatty acid profiles in the hazelnut samples were identified by gas chromatography with flame ionization detection (GC-FID, Shimadzu, Kyoto, Japan) consisting of a DB-WAX analytical column (30 m × 0.25 mm × 0.25 μm). The injector and detector temperatures were 250 °C. The GC oven was maintained at 210 °C (isothermal). The injection volume was 1 μL, and the injection mode was split (1:20 ratio). Helium was used as the carrier gas with a flow rate of 1 mL min−1. HPLC was coupled online with ICP-MS through a Rheodyne switching valve apparatus (PerkinElmer SCIEX), which accomplished sample introduction from the outlet of the column to the nebulizer of the ICP-MS instrument via high-purity fluoropolymer tubing (PFA, Poly Fluorinated Alkoxy, 0.019 in. i.d., 1/16 in. o.d., and 580 mm length, PerkinElmer SCIEX). The software Chromera version 1.2.254.0 (PerkinElmer SCIEX) was used to assess the online HPLC-ICP-MS chromatograms and data. The 55Mn isotope was measured on the basis of peak height using three replicates. Sample Preparation. Three packages (90−250 g) of unshelled and processed (peeled, roasted, and ready-to-eat) Giresun-quality hazelnut samples obtained from the same manufacturer were mixed, and a portion of the homogenized sample was ground to a fine powder in a porcelain mortar and then transferred to polyethylene bottles. This preparation procedure described in detail above was separately applied to three different samples of the same quality purchased at different times and markets. Then, the obtained hazelnut samples were coded as samples 1, 2, and 3. The following sample preparation procedures were applied to preweighed samples. Major Constituents Analysis. The moisture contents were determined according to ISO 771:1997.20 The oil contents of the samples were determined according to TS 765:196921 after diethyl ether extraction. Additionally, chloroform/methanol (2:1 v/v) extraction22 was applied to the samples to compare their lipid contents. The dietary fiber, crude fiber, crude ash, and sugar contents were determined according to AOAC 985.29 1986, ISO 5498:1981, and ISO 5984:2002 standard methods and the Luff Scroll method,23−26 respectively. Methyl esters of the fatty acids were prepared and analyzed according to methods TS 4504 EN ISO 550927 and TS 4664 EN ISO 5508,28 respectively, with some modifications. Briefly, transesterification was performed with 0.1 g of oil residue remaining from the samples after the application of method TS 765:1969.21 Isooctane (4 mL) and methanolic KOH (0.8 mL) solutions were used for the transesterification. After vortexing for 30 s at room temperature, the upper isooctane layers of the samples were injected into the GC-FID column. Total Manganese Validation Analysis. To minimize contamination, all of the glassware, polyethylene bottles, polypropylene centrifuge tubes, and pipet tips were soaked in 10% (v/v) nitric acid for at least 48 h and then rinsed several times with ultrapure water before drying in an oven at 40 °C. The hazelnut samples were digested prior to AAS and ICP-MS analyses. The detailed sample preparation procedure based on the DS/EN 14084 method,29 together with the microwave digestion operating conditions, was reported in our earlier studies.12 Briefly, 0.5 g of homogenized samples was digested with 6 mL of HNO3 and 1 mL of H2O2. The microwave digestion operating conditions were as follows: 250 W (2 min), 0 W (2 min), 250 W (6 min), 400 W (5 min), and finally 600 W (5 min). An open-wet digestion method with the same proportion of nitric acid and hydrogen peroxide used in the microwave digestion was also applied. After cooling to room temperature, the samples were transferred to
screening of free and/or bound water-soluble manganese fractions from samples with minimal sample preparation.
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MATERIALS AND METHODS
Reagents and Samples. A manganese(II) standard solution at a concentration of 1000 μg mL−1 was purchased from Inorganic Ventures (Lakewood, NJ, USA). Suprapure HNO3 (65%, v/v), HCl (30%, v/v), and all other reagents and chemicals used in the fractionation studies were of liquid chromatography (LiChrosolv) or analytical reagent grade and were obtained from Merck (Darmstadt, Germany). The digestive enzymes, pepsin (P7000, from porcine gastric mucosa), pancreatin (P1750, from porcine pancreas), and bile extract (B8631), were obtained from the local suppliers of SigmaAldrich (St. Louis, MO, USA) and used for the simulation of in vitro gastric and intestinal digestions. Sodium hydrogen carbonate was a Carlo Erba RPE reagent (Rodano, Milan, Italy) and was used for the preparation of the digestion solutions. Activated carbon (AOX Batch, PPTRE0049, Euroglas, Okehampton, UK) in powder form was used to evaluate the adsorption capabilities of manganese. All of the standards and samples were filtered through Double Rings 103 (12.5 cm filter paper) blue ribbon filter paper, followed by filtration through 0.45 μm hydrophilic polyvinylidene fluoride (PVDF) syringe filters (Millex-HV, Millipore) prior to analysis. Certified reference materials, NIST 1567a wheat flour (Gaithersburg, MD, USA) and Conostan (S21-10 μg g−1) oil-based multielement calibration standard (LGC Standards, London, UK), were used for validation purposes. A FAME (fatty acid methyl ester) Mix C4−C24 (Supelco 18919-1AMP, SigmaAldrich) standard mixture was used for fatty acid analysis. The water used was of ultrapure grade (18.3 MΩ·cm, Zeener Power I, Human Corp., Seoul, Korea). Highly pure (99.999%) argon and nitrogen gases were purchased from Orsez (Bursa, Turkey). Helium gas (99.999%) was supplied by Linde Gas A.Ş. (Bursa, Turkey). Among the many varieties of hazelnuts that are cultivated in Turkey, Tombul hazelnuts are known as Giresun or premium quality and are mainly grown in Giresun, Turkey.9 Thus, Fiskobirlik Efit A.Ş. (hazelnut processing company in Giresun) brand or Giresun quality unshelled and processed (Tombul) hazelnut samples were purchased from markets in Giresun and Bursa. Apparatus and Instrumentation. Total manganese (Mn) analyses were performed after sample digestion using a microwave digestion system (Microwave Labstation MLS 1200 mega, Milestone, Italy). An Elma LC-30H model ultrasonic bath (Singen, Germany) operated at an ultrasonic frequency of 35 kHz and a power of 240 W was used for the sample preparation. A pH meter (Minilab IQ125 Isfet pH tester, IQ Scientific Instruments Inc., Carlsbad, CA, USA) was obtained from PerkinElmer (PerkinElmer SCIEX, Shelton, CT, USA). An unstirred thermostatic water bath (Clifton NE1-22, Nickel Electro Ltd., UK) and an MSE Mistral 2000 centrifuge (MSE Scientific Instruments, UK) were used for the bioaccessibility studies. A PerkinElmer 200 series high-performance liquid chromatography (HPLC) system consisting of a series 200 quaternary pump, a series 225 autosampler, a series 200 column oven, and a vacuum degasser was used for the chromatographic analyses. The separation and identification of manganese fractions was achieved using a Brownlee DB Aq. C-18 (150 mm × 4.6 mm i.d. and 5 μm film thickness) analytical column (PerkinElmer SCIEX) equipped with a guard column (40 mm) with the same stationary phase material. The column was maintained at 26 °C, and the elution was performed with filtered and degassed ultrapure water. A methanol/water mixture as a gradient mobile phase was also used to evaluate the separation. The gradient program began with 100:0 (A%/B%, v/v) for 0−1 min, then the following steps were applied: 95:5 (A%/B%, v/v) for 1 min, 97:3 (A %/B%, v/v) for 2 min, and 98:3 (A%/B%, v/v) for 2 min. The elution was performed using a 1.5 mL min−1 flow rate, and the injection volume was 30 μL. The determination of the total manganese in the samples/fractions and the monitoring of manganese fractions in the extracts were performed by an Elan 9000 ICP-MS (PerkinElmer SCIEX) composed of a PerkinElmer Ryton cross-flow nebulizer, a Scott-type double-pass B
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polypropylene centrifuge tubes and diluted to 25 mL. Three independent replicates were prepared for each of the samples, spiked samples, and reagent blanks. Working solutions of manganese were prepared on a daily basis by appropriate dilution of a single stock solution and used for external calibration. The calibration curve was constructed from eight data points for standard solution concentrations ranging from 0.5 to 80 μg L−1 for ICP-MS analysis and from six data points ranging from 0.1 to 2.0 mg L−1 for AAS analysis. Because no hazelnut type certified reference material was available, each of the three hazelnut samples was spiked with aqueous manganese standard solutions at one level (0.1 mg L−1) prior to microwave digestion for recovery studies. The calibration methods using aqueous standards and standard addition were compared on the basis of their slopes. Additionally, wheat flour certified reference material (for the defatted hazelnut samples) and the Conostan S-21 oil analysis standard (for the oil fraction of the hazelnut samples) were used to determine the accuracy of the manganese determinations by ICP-MS after the microwave and/or open-wet digestion methods. All microwave-digested samples were analyzed by AAS, followed by ICPMS. The open-wet digested samples were analyzed only by ICP-MS. Fractionation Studies. Manganese was extracted from 0.1 g of hazelnut sample using 15 mL of various solvents, including water, diethyl ether, n-hexane, and methanol. The extractions were performed in closed clear glass vials for 1.5 h at room temperature in an ultrasonic bath. Reagent blanks were also prepared in the same manner. The organic solvents in each fraction were filtered through blue ribbon filter paper and evaporated to dryness under nitrogen gas, and the residue was analyzed by ICP-MS after dissolving in 10 mL of 1% (v/v) HNO3. All of the fractionation studies were performed in triplicate. The final extracts were then analyzed for manganese content by ICPMS. The fractionation scheme is summarized in Figure 1. Additionally,
per 100 g was determined on the basis of mass loss. The fractionation approach based on activated carbon adsorption was performed using the method introduced by Kowalewska et al. with some modifications.30 Briefly, a mixture of 30 mL of ultrapure water, 2.5 mL of methyl isobutyl ketone, and 0.03 g of activated carbon was added to each 0.1 g of sample. After extraction in an ultrasonic bath for 1 h at room temperature, the samples were filtered through blue ribbon filter paper and dried. The manganese contents in the samples were leached with 10 mL of 10% (v/v) nitric acid and determined by ICP-MS after appropriate dilution. HPLC-ICP-MS Analysis. The water-soluble fractions were subjected to online analysis by HPLC-ICP-MS. After fractionation with water, all of the solutions were filtered through 0.45 μm PVDF filters and stored in clear polypropylene centrifuge tubes at 4 °C until analysis. The calibration standards ranged in concentration from 136 to 595 μg L−1. The retention times of the standards and the samples, together with the peak heights, were compared to screen the manganese levels and the fractions. Bioaccessibility Assessments. The bioaccessible manganese levels were determined after an in vitro enzymatic digestion procedure with appropriate solutions of pepsin and pancreatin. The simulated gastric digestion was initiated by adding 2 mL of freshly prepared pepsin−HCl solution (0.2 g of pepsin in 5 mL of 0.1 M HCl) to 0.03 g of sample. A 15 mL volume of ultrapure water was then added to the samples. The pH values of the solutions were adjusted to 1.9 with 0.1 M HCl. The solutions were incubated in a water bath at 37 °C for 1 h. The enzyme residues were removed by centrifugation at 2135g for 5 min, and the manganese contents of the samples determined by ICPMS were designated the pepsin-digestible amounts. For the simulated intestinal digestion, 2.5 mL of freshly prepared enzyme solution (pancreatin/bile: 0.45 g of bile extract and 0.075 g of pancreatin in 37.5 mL of 0.1 M NaHCO3) was added to 15 mL of the solutions containing 0.03 g of the samples. The pH values of the solutions were adjusted to 6.9 with 0.1 M NaHCO3 or 0.1 M HCl. The solutions were incubated in a water bath at 37 °C for 2 h, and the manganese contents of the samples were determined by ICP-MS after the removal of the enzyme residues. The manganese levels in these solutions were designated the pancreatin-digestible amounts. The simulated gastrointestinal digestion was designated as the pepsin+pancreatin-digestible amount and was prepared by adding 4.5 mL of water to 3 mL of gastric extract. After pH adjustment, 3 mL of the pancreatin/bile solution was added to the samples. The pH values of the solutions were adjusted to 6.9, and the solutions were incubated in a water bath at 37 °C for 2 h.12,17,31 The gastrointestinal digests were centrifuged at 2135g for 5 min. The supernatants were filtered immediately to remove any enzyme residue through PVDF syringe filters prior to being stored at 4 °C and analyzed by ICP-MS. The contents of the digests were analyzed to ensure the inactivity of any remaining enzyme residue. The manganese contents of the enzymatic digestions, together with the total element digestions, were used to calculate the percent bioaccessibility according to the equation
Figure 1. Fractionation scheme of manganese in hazelnut samples.
bioaccessibility (%) = ([Mn 2 +]2 /[Mn 2 +]1 ) × 100
the lipids were extracted using a chloroform/methanol (2:1 v/v) extraction. Two grams of the samples was subjected to extraction in an ultrasonic bath for 30 min using 15 mL of the solvent mixture. The samples were filtered through blue ribbon filter paper, and 15 mL of fresh solvent mixture was added to the filtered sample. The entire procedure was applied in triplicate for one sample (hazelnut 1), and the number of independent applications for this sample was five. After this sequential extraction, the samples were dried and the fat content
where [Mn2+]2 and [Mn2+]1 are the total manganese contents determined by ICP-MS after in vitro digestion and microwave-assisted acid digestion, respectively.17 Additionally, 0.1 g samples were extracted with 15 mL of n-octanol to compare with the results obtained from intestinal pancreatic digestion. The extraction was performed in an ultrasonic bath for 1.5 h. Subsequently, a liquid− liquid extraction was performed using 5 mL of 1% (v/v) HNO3 (in
Table 1. Major Matrix Components of the Three Different Hazelnut Samplesa
a
sample
moisture (%)
dietary fiber (%)
crude fiber (%)
crude protein (%)
oil (%)
sugar (%)
crude ash (%)
1 2 3
2.03 ± 0.01 1.65 ± 0.02 1.89 ± 0.03
11.90 ± 0.41 10.48 ± 0.45 10.29 ± 1.05
5.45 ± 0.24 5.97 ± 0.42 5.95 ± 0.40
18.2 ± 0.8 18.3 ± 0.2 17.0 ± 0.2
64.6 ± 0.7 61.2 ± 0.7 64.7 ± 0.6
2.22 ± 0.24
2.20 ± 0.18 2.13 ± 0.17 2.33 ± 0.07
Results represent the mean ± standard deviation (N = 3). C
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triplicate), and the nitric acid fractions were analyzed by ICP-MS. To evaluate its compatibility, n-octanol extraction was applied to the spiked samples (at two levels) for recovery studies.17
Table 3. Fatty Acids in the Three Hazelnut Samples Based on the Methyl Ester Compositionsa
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RESULTS AND DISCUSSION Fractionation Studies. Manganese fractionation studies are important for establishing the relationship between metal mobility in organisms and nutrition. Additionally, understanding the role of major components of hazelnut is important for predicting manganese-binding food structures, which will provide valuable insights for further bioaccessibility studies. The major food components of hazelnut samples are outlined in Table 1. According to this table, most of the manganese could be present in oil-, protein-, or fiber-bound structures, with the hazelnuts consisting primarily of oil (60%) and protein (18%). The organic solvents selected for fractionation studies should represent these components, and these fractions are important for bioaccessibility assessments. The oil contents were 64.6, 61.2, and 64.7% for samples 1, 2, and 3, respectively. We focused only on the oily matrix for fractionation purposes; the protein contents were discussed within an online method by HPLC-ICP-MS. For fractionation purposes, diethyl ether, nhexane, and methanol extractions were applied to remove the neutral, free, and bound polar lipids,17,22 respectively. The manganese contents in these fractions ranged from 0.67 to 0.79 mg kg−1 in the diethyl ether fractions, from 0.36 to 0.67 mg kg−1 in the n-hexane fractions, and from 0.15 to 0.42 mg kg−1 in the methanol fractions (Table 2). As discussed in the literature,
water
diethyl ether
n-hexane
methanol
1 2 3
10.89 ± 2.62 13.38 ± 0.02 7.33 ± 0.32
0.78 ± 0.15 0.79 ± 0.24 0.67 ± 0.05
0.36 ± 0.03 0.67 ± 0.02 0.40 ± 0.02
0.15 ± 0.05 0.23 ± 0.01 0.42 ± 0.04
a
sample 1 (%)
sample 2 (%)
sample 3 (%)
myristic (C14:0) palmitic (C16:0) palmitoleic (C16:1) stearic (C18:0) oleic (C18:1) linoleic (C18:2) linolenic (C18:3) arachidic (C20) eicosenoic (C20:1) behenic (C22:0) erucic (C22:1)
