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Determination of essential elements (Cu, Fe and Zn) in juices of commercially available in Pakistan
Food and Chemical Toxicology 48 (2010) 2737–2740
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Determination of essential elements (Cu, Fe and Zn) in juices of commercially available in Pakistan Nusrat Jalbani a,*, Farooq Ahmed a,1, Tasneem Gul kazi b, Uzma Rashid a,1, Alia Bano Munshi a,1, Aftab Kandhro a,1 a b
Pakistan Council Scientific and Industrial Research Laboratories Complex, Karachi, Pakistan National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro, Pakistan
a r t i c l e
i n f o
Article history: Received 3 April 2010 Accepted 30 June 2010
Keywords: Juices Copper Iron Zinc FAAS Daily intake
a b s t r a c t In present work, the concentration of essential elements, copper (Cu), iron (Fe) and zinc (Zn) were measured in ten different juices, to evaluate whether the intakes of under study nutrients accomplished within recommended permissible levels. Flame atomic absorption spectrometry (FAAS) was used to estimate and evaluate the levels of essential elements such as Cu, Fe and Zn in ten juice samples. The elements were analyzed prior to microwave assisted acid digestion (MAD). For comparison purpose, wet acid digestion (CAD) method was used for determination of essential elements. The accuracy of proposed method was evaluated by performing addition-recovery experiments to prove the reliability of the method. All obtained analytical results by MAD and CAD showed a good agreement at a 95% confidence level. The detection limits of Cu, Fe and Zn were 22.7 lg/L, 3.85 lg/L and 3.05 lg/L. The dietary intakes of all three studied elements have also been estimated. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Juices are widely consumed by all age group due to flavor and nutritive value. The concentration of Cu and Zn in juice can vary widely depending on source, manufacturing process, and raw material (Licata et al., 2004). Copper and zinc are known to be essential nutrients for humans, stimulating the fundamental metabolic protein synthesis, but both can be toxic depending on the concentration (Underwood, 1977; Onianwa et al., 1999). Zinc constitutes about 33 lg/g of an adult body mass and it is essential as a constituent of many enzymes involved in several physiological functions, such as protein synthesis and energy metabolism (Onianwa et al., 2001). An adult human body contains about 1.5–2.0 lg/g of Cu (Nescentes et al., 2004), which is essential as a constituent of some metalloenzymes and is required in hemoglobin synthesis and in the catalysis of metabolic oxidation (Underwood, 1977). In this sense, considerable interest has been focused on Cu and Zn determination in foods and beverages (Onianwa et al., 2001). The availability of iron in foods (the proportion of the total iron content of a food which the gastro-intestinal tract and the organism are able to utilize) has added a most important point of view
* Corresponding author. Tel.: +92 3323387487; fax: +92 0213 4650785. E-mail addresses: [email protected] (N. Jalbani), farooqahmed@ yahoo.com (F. Ahmed), [email protected] (T.G. kazi), [email protected] (U. Rashid), [email protected] (A.B. Munshi), chemist_afi@yahoo.com (A. Kandhro). 1 Tel.: +92 3342231929; fax: +92 0213 4650785. 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.06.048
in the study of iron metabolism (Goyer, 1997; Sherman et al., 1934). The total iron content in the diet is of less importance than the amount of iron actually available for the use of the body in the foods employed (Olalla et al., 2000). The determination of elements in food samples traditionally been performed by digestion with acid (or acid mixtures), which is time consuming and involves some potential drawbacks such as analyte losses and sample contamined due to contaminated environment. The microwave-assisted digestion is also an adequate determinative technique, but it requires intensive sample handling, a long time to cool the reactor before opening and has a high operational cost (Soylak et al., 2005). The main advantage of microwave assisted samples pretreatment is its requirement of a small amount of mineral acids and a reduction in the production of nitrous vapors (Mesko et al., 2006; Jalbani et al., 2007). Flame atomic absorption spectrometry (FAAS) is the most extensively used for elemental determination (Ghaedi et al., 2005; Demirel et al., 2008), owing to its simple set up, low running costs, robustness, and good selectivity. For some applications FAAS is not suitable due to its relatively low sensitivity. This is mainly caused by the low efficiency of conventional pneumatic nebulizer and the short residence time of the sample in the flame (Afridi et al., 2006). The sensitivity of FAAS can be improved by increasing the efficiency of the aerosol generation/transport and by increasing the residence time of free analytic atoms in the absorption volume (Ghaedi et al., 2008). The determination of metals in foods and beverages is an important due to their essential or toxic action.
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N. Jalbani et al. / Food and Chemical Toxicology 48 (2010) 2737–2740
The main purpose of this work was to improve a simple and accurate method for evaluation of concentration of essential nutrients in juice. The analytical results were compared with those obtained by conventional wet acid digestion method on real samples by performing of spiking method. The accuracy of methodology was performed by standard addition method; because of the reference materials are commercially not available for juices. The duplicate samples of juices of different flavors was spiked with known amount of elements and subjected to both methods to perform a recovery test. The daily intake of the elements by people and children through different flavors of juice marketed in different cities of Pakistan.
2.3. Sampling Ten samples of juices (mango, pine apple, apple, tropical and orange) commercially available from the market of Karachi, Pakistan were collected randomly in the year of 2009. The ten collected samples were packed on different dates to observe the variation in the elemental contamination levels of the products. The results obtained for ten commercial samples of different juices are given in Table 1. According to a t-test, all determined and spiking values are in good agreement at a 95% confidence level; however the reference materials for juices are not commercially available.
2.4. Sample preparation 2. Material and methods 2.1. Reagents and glass wares All the reagents and chemicals Merck, (Darmstadt, Germany) were used of analytical grade. De-ionized water was used through out the work. Concentrated Nitric acid (65%), were spectroscopic grades Merck, (Darmstadt, Germany). Stock standard solutions (1000 lg/mL) of Cu, Fe and Zn were obtained from Fluka Kamica (Buchs, Switzerland) of corresponding metal ions. The laboratory glass wares were kept overnight in 10% (v/v HNO3) and washed with water and rinse with distilled water before use. After washing and rinsing, the glassware’s were dried in an oven at 80 °C for 1 h.
2.4.1. Microwave assisted-acid digestion method A microwave assisted-acid digestion procedure was carried out, in order to achieve a shorter digestion time and using minimum amount of acid. Duplicate of 2.0 mL of each juice samples were taken into microwave vessels but well shaken, opened and sampled by pouring directly from the original container into the microwave vessel. Added the samples to each vessels and 10 mL of a concentrated HNO3– H2O2 (2:1, v/v) and kept samples for 10 min at room temperature till the samples were homogenized, and then placed the vessels in covered PTFE container. This was then heated following a one-stage digestion programmed at 80% of total power (900 W), for 3–5 min. After cooling, the resulting solutions were evaporated to semidried mass to remove an excess acid, and then diluted upto 50.0 mL in volumetric flasks and kept as a stock sample solution.
2.2. Instrumentation A Hitachi Model 5000 Z flame atomic absorption spectrophotometer was used for determination of Cu, Fe and Zn in juice samples. The calibration curves (0– 1.2 lg/mL) for all three studied elements were established with solutions prepared from a 1000 lg/mL stock solution. The energy sources, Hollow cathode lamps (Perkin-Elmer) operating at recommended current. A milestone microwave oven (Osaka, Japan), programmable for time and microwave power from 100 to 900 W, was used for acid digestion of juice samples.
Table 1 Determination of Cu, Fe and Zn juice samples (lg/mL). S. no:
Name of Sample
Copper
Iron
Zinc
1 2 3 4 5 6 7 8 9 10
Mango Apple Pine apple Tropical Orange Frooto Classic Maza Shezan Poly
0.181 ± 0.011 0.174 ± 0.007 0.201 ± 0.011 0.222 ± 0.013 0.262 ± 0.011 0.184 ± 0.012 0.176 ± 0.009 0.203 ± 0.013 0.226 ± 0.015 0.264 ± 0.012
0.908 ± 0.051 0.413 ± 0.018 1.55 ± 0.087 1.03 ± 0.061 0.595 ± 0.031 0.912 ± 0.055 0.415 ± 0.022 1.62 ± 0.091 1.05 ± 0.064 0.598 ± 0.032
0.296 ± 0.015 0.187 ± 0.011 0.781 ± 0.049 0.407 ± 0.021 0.315 ± 0.013 0.298 ± 0.016 0.192 ± 0.011 0.784 ± 0.051 0.411 ± 0.022 0.318 ± 0.015
2.4.2. Conventional wet acid digestion method (CDM) Duplicate of 50 mL of sub samples of juices were placed into 250 mL Pyrex beaker. Added 10 mL volume of a concentrated HNO3 (65%) and 5 mL of H2O2 to each flask and solutions were heated on electric hot plate at 80 °C, for 2–3 h, till the clear transparent solutions were obtained. Finally the solutions were made up to 50 mL of volumetric flask. After making volume the prepared samples were subjected to FAAS for estimation of Cu, Fe and Zn. Blank digestions (without analyte) were also carried out. The contents of the sample solutions were evaporated, and the semidried mass and than filtered through what man No. 42 filter paper, and made up the final volume into 50 mL volumetric flasks with ultra pure water. The concentrations were obtained directly from calibration graphs after correction of the absorbance for the signal from an appropriate reagent blank. All experiments were conducted at room temperature (30 °C) following the well-established laboratory protocols. To establish the validity of the microwave assisted-acid digestion method was checked by performing standard addition method. In standard addition method, the known amount of standards (0.2, 0.4, 0.8 mg/L) were spiked with juice using replicate six samples of each, and applied both digestion methods.
2.5. Statistical analysis Statistical calculations were performed using computer program Minitab 13.2 (Minitab Inc., State College, PA) and Excel.
Table 2 The results of standard addition/recovery for Cu, Fe and Zn determination in juice (Apple) (n = 6).
a b
Added lg/mL
pffiffiffi ts= na MAD x
pffiffiffi ts= na CAD x
% Recovery
Paired t-testb Texperimental
Copper (Cu) 0.0 0.2 0.4 0.8
0.174 ± 0.007 0.368 ± 0.021 0.565 ± 0.034 0.963 ± 0.073
0.176 ± 0.009 0.372 ± 0.023 0.570 ± 0.032 0.973 ± 0.056
98.8 98.9 99.1 99.0
2.01 1.30 2.10 2.26
Iron (Fe) 0.0 0.2 0.4 0.8
0.413 ± 0.018 0.612 ± 0.037 0.812 ± 0.067 1.21 ± 0.077
0.415 ± 0.022 0.614 ± 0.035 0.813 ± 0.061 1.22 ± 0.078
99.5 99.7 99.9 99.8
2.08 2.34 1.07 2.04
Zinc (Zn) 0.0 0.4 0.6 0.8
0.187 ± 0.011 0.383 ± 0.022 0.584 ± 0.038 0.987 ± 0.082
0.190 ± 0.011 0.386 ± 0.021 0.590 ± 0.032 0.992 ± 0.078
98.4 99.2 99.0 99.5
1.65 2.08 1.87 2.03
Key = Average value ± confidence interval (P = 0.05). Paired t-test between MAD vs CAD, degree of freedom (n 1) = 5tCritical at 95% confidence limit = 2.571.
2.571
N. Jalbani et al. / Food and Chemical Toxicology 48 (2010) 2737–2740 Table 3 Daily intake of Cu, Fe and Zn by consumption of juices (mg of Cu, Fe and Zn based on 250 mL juices/person/day). Sample name
Copper
Iron
Zinc
Mango Apple Pine apple Tropical Orange Frooto Classic Maza Shezan Poly
0.045 0.044 0.050 0.056 0.066 0.046 0.044 0.051 0.057 0.066
0.227 0.103 0.388 0.259 0.149 0.228 0.104 0.405 0.264 0.150
0.074 0.047 0.195 0.102 0.079 0.075 0.048 0.196 0.103 0.080
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The spiking was performed on duplicate samples at three concentration levels 0.2, 0.4, 0.8 lg/mL for Cu, Fe and Zn, respectively. Average recovery of Cu, Fe and Zn spikes from juices matrixes as indicated in Table 2. The t-test was performed on spiked juice (Apple) samples as given in Table 2. According to t-test all the obtained values were statistically significant at 95% Confidence level. For the commercially available juice samples the concentration of studied elements were ranged as 0.181–0.262 lg/mL for Cu, 0.595–0.908 lg/mL for Fe, 0.296–0.315 lg/mL for Zn, respectively. In the Table 2 it is clearly seen that the concentration of Copper in poly was found higher, while in case of Fe and Zn the maximum concentration was observed in maza. The lower concentration was found for Fe and Zn in similar juice samples (Apple) as shown in Table 1.
2.6. Calibration and sensitivity Calibration and standard addition graphs were obtained for Cu, Fe and Zn. However, as the acid composition of both acid mixture and different matrixes of samples, the standard addition technique was used throughout the development of method.The detection and quantification limits, given by
LOD ¼ 3
s s and LOQ ¼ 10 m m
Respectively, where s is the standard deviation of ten measurements of reagent blank and m is the slope of the calibration or standard addition graph was obtained for Cu, Fe and Zn. Limit of detection (LOD) of studied three elements, Cu 22.7, Fe 3.85 and Zn 3.05 lg/L being reached, and LOQ for Cu 76.4, Fe 12.8 and for Zn 10.2 lg/L were calculated. Reproducibility of the method was assessed by using three replicates of juices (Apple) and taken from the same packages. The most reliable approach evaluating the accuracy of an analytical method requires analysis of certified reference materials with chemical composition and analytic concentration, which match those of the samples under investigation. The certified reference materials are not available, Hence the standard addition method was performed to obtain the recovery test prior to the microwave assisted-acid and conventional wet acid digestion methods. The spiking of juice was performed in duplicate at three concentration levels 0.2, 0.4, 0.8 lg/mL, respectively and in this experiment we make the volume up to 50.0 mL in volumetric flasks and were analyzed by FAAS. Average recovery of three studied essential elements spikes from juices matrixes. For recovery test the apple, was spiked with the known amount of standards of Cu, Fe, and Zn as presented in Table 2. The results of proposed method MAD showed the% recovery for Cu varied from 98.8% to 99.2%, Fe from 99.5% to 99.9% and for Zn the concentration is ranged as 98.4–99.5%. The significance of the effects was evaluated with the t-test (t of Student). A t Experimental values was calculated at degree of freedom n1 = 5, the experimental values are found lower than the critical (2.57), at a 95% of confidence level, indicate non significant difference were observed in obtained values of non essential elements by CAD and MAD method (Table 2).
3. Results The present study was carried out to determine the essential elements in ten juice samples. The concentrations of elements were determined in duplicate samples by using of FAAS after digestion by microwave closed vessels and conventional wet acid digestion method. The analyzed samples were categorized on the basis of flavors and type of fruits. The average concentration of minerals Cu 0.262 lg/mL was found higher in orange as compared to others juices (apple, pineapple, tropical and mango) as described in Tables 2. The mean concentration of Fe 1.55 lg/mL is observed maximum in pineapple as compared to other obtained juice samples. The % recoveries of understudied three elements were observed different by adding the three replicates of juice (Apple). The most reliable approach evaluating the accuracy of an analytical method requires analysis of certified reference materials with chemical composition and analytic concentration, which contest those of the samples under investigation. To the best of our knowledge, there are no any commercially available juice reference materials for understudy elements. Therefore the Standard addition method was performed by spiking of juice samples to obtain the recovery test prior to microwave assisted acid digestion method and conventional wet acid digestion method.
4. Discussion The packed fruit juices are usually consumed due to flavor and nutritive value. Fresh fruit juices are largely consumed in all age groups because of it is major source of nutrient for adult as well as older age group. Copper deficiency causes anemia, loss of weight, bone and cartilage with irregular physiological development. Abnormal ingestion causes neurological anomalies, hepatic and renal disturbances (Underwood, 1977). The iron intake of the Aberdeen poor (Franke et al., 2006; Davidson et al., 1933). Although some of the women and children were anemic, there was no correlation between the incidence of anemia and the iron content of the diet including diets affording only 6 mg. of iron daily over long periods of time. Shah et al., 2003, stated that dietary survey of various sections of the United States revealed a variation of daily iron intake from 7 mg. for the Alabama Negro to 31 mg. in the Northern lumber camps. In fruit juices, the concentration of Cu and Zn can vary from lg/ kg to mg/kg range (Fernandez et al., 2002). This aspect makes difficult the employment of conventional FAAS for direct determination of these elements. Moreover, direct aspiration of the fruit juices and milk can cause flame fluctuations and accumulation of solid deposits on the burner head (Bellido-Milla et al., 2000). The WHO (World Health Organization, WHO., 1973; World Health Organization, 1996; WHO, 2001) estimated that the daily average adult intake of copper (3.0 mg/person/day) may come from various types of foods. In case of iron, the values of daily intake is calculated as copper i.e. the major source of understudied elements are food including wheat, fruit and vegetable. The daily dietary intake of fruit is approximately (i.e. 0.334 mg/person/ day). From the study it was clearly shown that above determined elements were found under legal recommended values. The intake of above studied elements varies place to place. The variation of minerals in food is different due to the different food consumption habits and different environmental factors. The food consumption habit is depend on several socio-economic, physiological, environmental contamination where the population is living near to contaminated area whether living in non contaminated surrounding. The present study shows that the intake of Cu, Fe and Zn is different in different type of juice products in Pakistan and provides specific information on the average dietary intake of elements as shown in (Table 3). Furthermore, the present findings allowed the identification of the specific items most responsible for major and minor contribution to the intake of this nutritional essential element. The average consumption of these juices was estimated by the survey on the population of different areas of Karachi city, Pakistan. The daily intake of understudied element was calculated on the consumption of a minimum of 250 mL/person/day of the juices and compared with tolerance limits of recommended by
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WHO/FDA. Our results are consistent with reported values (Mesallam, 1987).
5. Conclusion The microwave-assisted digestion method is an inexpensive, rapid lesser amount of acid and shorter time is required for digestion of samples as compared to conventional wet acid digestion for the assessment of Cu, Fe and Zn. The juices with fruit ensure an important source of contributing the mineral with a light percentage of the DRIs of the human being. However this type of beverage is consumed by all age groups but mostly consumed by children. The Beneficial fruit juices include apple, carrots and sugar cane, grapes, cherries, grapefruit, blueberries, and lemons. Juices are not only supplies the massive amounts of nutrition to the body, but it also flushes and wash out the all organs of human body, aids in effective enzyme activity and maintain the metabolism. From the results it was clearly seen that there is no significant difference among the different type of juices. A juice does not pose a concern with regard to the prospect of iron-deficiency anemia, which remains a significant health problem in Karachi, Pakistan.
6. Conflict of interest The all authors assert that there are no conflicts of interest. References Afridi, H.I., Kazi, T.G., Jamali, M.K., Kazi, G.H., Arain, M.B., Jalbani, N., Shar, G.Q., 2006. Analysis of heavy metals in scalp hair samples of hypertensive patients by conventional and microwave digestion methods. Spectrosc. Lett. 39, 203–214. Bellido-Milla, D., Moreno-Perez, J.M., Hernandez-Artiga, M.P., 2000. Differentiation and classification of beers with flame atomic spectrometry and molecular absorption spectrometry and sample preparation assisted by microwaves. Spectrochim. Acta B 55 (7), 853–862. Davidson, L.S.P. et al., 1933. Observations on nutrition in relation to anemia. Brit. Med. J 1, 685. Demirel, S., Tuzen, M., Saracoglu, S., Soylak, M., 2008. Evaluation of various digestion procedures for trace element contents of some food materials. J. Hazard. Mater. 152, 1020–1026. Fernandez, P.L., Pablos, F., Martin, M.J., Gonzalez, A.G., 2002. Multi-element analysis of tea beverages by inductively coupled plasma atomic emission spectrometry. Food Chem. 76, 483–489.
Franke, S.I.R., Pra, D., Giulian, R., Dias, J.F., Yoneama, M.L., da Silva, J., Erdtmann, B., Henriques, J.A.P., 2006. Influence of orange juice in the levels and in the genotoxicity of iron and copper. Food Chem. Toxicol. 44 (3), 425–435. Ghaedi, M., Fathi, M.R., Marahel, F., Ahmadi, F., 2005. Simultaneous preconcentration and determination of copper, nickel, cobalt and lead ions content by flame atomic absorption spectrometry. Fresenius Environ. Bull. 14, 1158–1163. Ghaedi, M., Shokrollahi, A., Kianfar, A.H., Mirsadeghi, A.S., Pourfarokhi, A., Soylak, M., 2008. The determination of some heavy metals in food samples by flame atomic absorption spectrometry after their separation-preconcentration on bis salicyl aldehyde, 1, 3 propan diimine (BSPDI) loaded on activated carbon. J. Hazard. Mater. 154, 128–134. Goyer, R.A,. 1997. Toxic and essential metals interaction. Ann. Rev. Nutr. (17), 37– 50.doi: 10. 1146/annurev.nutr.17.1.37 Jalbani, N., Kazi, T.G., Jamali, M.K., Arain, B.M., Afridi, H.I., Baloch, A., 2007. Evaluation of aluminum contents in different bakery foods by electrothermal atomic absorption spectrometer. J. Food Compos. Anal. 20, 226–231. Licata, P., Trombetta, D., Cristani, M., Giofre, F., Martino, D., Calo, M., Naccari, F., 2004. Levels of ‘‘toxic” and ‘‘essential” metals in samples of bovine milk from various dairy farms in Calabria. Italy Environ. Int. 30 (1), 1–6. Mesko, M.F., De Moraes, D.P., Barin, J.S., Dressler, V.L., Knappet, G., 2006. Digestion of biological materials using the microwave-assisted sample combustion technique. Microchem. J. 82, 183–188. Mesallam, A.S., 1987. Heavy metal content of canned orange juice as determined by direct current plasma atomic emission spectrophotometry (DCPAES). Food Chem. 26 (1), 47–58. Nescentes, C.C., Arruda, M.A.Z., Nogueira, A.R.A., Nobrega, J.A., 2004. Direct determination of Cu and Zn in fruit juices and bovine milk by thermospray flame furnace atomic absorption spectrometry. Talanta. 64 (4), 912–917. Olalla, M., Gonzalez, M.C., Cabrera, C., Lopez, M.C., 2000. Optimized determination of iron in grape juice, wines, and other alcoholic beverages by. Atomic Absorption Spectrometry. 83 (1), 189–195. Onianwa, P.C., Adelota, I.G., Iwegbue, C.M.A., Ojo, M.F., Tella, O.O., 1999. Trace heavy metals composition of some Nigerian beverages and food drinks. Food Chem. 66 (3), 275–279. Onianwa, P.C., Adeyemo, A.O., Idowu, O.E., Ogabiela, E.E., 2001. Copper and zinc contents of Nigerian foods and estimates of the adult dietary intakes. Food Chem. 72 (1), 89–95. Shah, M.M.D., Griffin, I.J., ChB, M.B., Lifschitz, C.H.M.D., Abrams, S.A.M.D., 2003. Effect of orange and apple juices on iron absorption in children. Arch. Pediatr. Adolesc. Med. 157, 1232–1236. Sherman, W.C., Elvehjem Ande, C.A., Hart, B., 1934. Further studies on availability of iron in biological materials. J. Biol. Chem. 107, 383. Soylak, M., Saracoglu, S., Tuzen, M., Mendil, D., 2005. Determination of trace metals sin mushroom samples from Kayseri, Turkey. Food Chem. 92, 649–652. Underwood, E.J., 1977. Trace Elements in Human and Animal Nutrition, fourth ed. Academic Press, New York. WHO., 1973. Expert Committee on Trace Metals in Human Nutrition. World Health Organization Tech. Rep. Ser. No. 532. World Health Organization, 1996. Trace Elements in human nutrition and health WHO, Geneva. WHO, 2001, Safety Evaluation of certain food additives and contaminants; Tin. WHO Food Additives Series No. 46. Joint FAO/WHO.
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Determination of essential elements (Cu, Fe and Zn) in juices of commercially available in Pakistan Nusrat Jalbani a,*, Farooq Ahmed a,1, Tasneem Gul kazi b, Uzma Rashid a,1, Alia Bano Munshi a,1, Aftab Kandhro a,1 a b
Pakistan Council Scientific and Industrial Research Laboratories Complex, Karachi, Pakistan National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro, Pakistan
a r t i c l e
i n f o
Article history: Received 3 April 2010 Accepted 30 June 2010
Keywords: Juices Copper Iron Zinc FAAS Daily intake
a b s t r a c t In present work, the concentration of essential elements, copper (Cu), iron (Fe) and zinc (Zn) were measured in ten different juices, to evaluate whether the intakes of under study nutrients accomplished within recommended permissible levels. Flame atomic absorption spectrometry (FAAS) was used to estimate and evaluate the levels of essential elements such as Cu, Fe and Zn in ten juice samples. The elements were analyzed prior to microwave assisted acid digestion (MAD). For comparison purpose, wet acid digestion (CAD) method was used for determination of essential elements. The accuracy of proposed method was evaluated by performing addition-recovery experiments to prove the reliability of the method. All obtained analytical results by MAD and CAD showed a good agreement at a 95% confidence level. The detection limits of Cu, Fe and Zn were 22.7 lg/L, 3.85 lg/L and 3.05 lg/L. The dietary intakes of all three studied elements have also been estimated. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Juices are widely consumed by all age group due to flavor and nutritive value. The concentration of Cu and Zn in juice can vary widely depending on source, manufacturing process, and raw material (Licata et al., 2004). Copper and zinc are known to be essential nutrients for humans, stimulating the fundamental metabolic protein synthesis, but both can be toxic depending on the concentration (Underwood, 1977; Onianwa et al., 1999). Zinc constitutes about 33 lg/g of an adult body mass and it is essential as a constituent of many enzymes involved in several physiological functions, such as protein synthesis and energy metabolism (Onianwa et al., 2001). An adult human body contains about 1.5–2.0 lg/g of Cu (Nescentes et al., 2004), which is essential as a constituent of some metalloenzymes and is required in hemoglobin synthesis and in the catalysis of metabolic oxidation (Underwood, 1977). In this sense, considerable interest has been focused on Cu and Zn determination in foods and beverages (Onianwa et al., 2001). The availability of iron in foods (the proportion of the total iron content of a food which the gastro-intestinal tract and the organism are able to utilize) has added a most important point of view
* Corresponding author. Tel.: +92 3323387487; fax: +92 0213 4650785. E-mail addresses: [email protected] (N. Jalbani), farooqahmed@ yahoo.com (F. Ahmed), [email protected] (T.G. kazi), [email protected] (U. Rashid), [email protected] (A.B. Munshi), chemist_afi@yahoo.com (A. Kandhro). 1 Tel.: +92 3342231929; fax: +92 0213 4650785. 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.06.048
in the study of iron metabolism (Goyer, 1997; Sherman et al., 1934). The total iron content in the diet is of less importance than the amount of iron actually available for the use of the body in the foods employed (Olalla et al., 2000). The determination of elements in food samples traditionally been performed by digestion with acid (or acid mixtures), which is time consuming and involves some potential drawbacks such as analyte losses and sample contamined due to contaminated environment. The microwave-assisted digestion is also an adequate determinative technique, but it requires intensive sample handling, a long time to cool the reactor before opening and has a high operational cost (Soylak et al., 2005). The main advantage of microwave assisted samples pretreatment is its requirement of a small amount of mineral acids and a reduction in the production of nitrous vapors (Mesko et al., 2006; Jalbani et al., 2007). Flame atomic absorption spectrometry (FAAS) is the most extensively used for elemental determination (Ghaedi et al., 2005; Demirel et al., 2008), owing to its simple set up, low running costs, robustness, and good selectivity. For some applications FAAS is not suitable due to its relatively low sensitivity. This is mainly caused by the low efficiency of conventional pneumatic nebulizer and the short residence time of the sample in the flame (Afridi et al., 2006). The sensitivity of FAAS can be improved by increasing the efficiency of the aerosol generation/transport and by increasing the residence time of free analytic atoms in the absorption volume (Ghaedi et al., 2008). The determination of metals in foods and beverages is an important due to their essential or toxic action.
2738
N. Jalbani et al. / Food and Chemical Toxicology 48 (2010) 2737–2740
The main purpose of this work was to improve a simple and accurate method for evaluation of concentration of essential nutrients in juice. The analytical results were compared with those obtained by conventional wet acid digestion method on real samples by performing of spiking method. The accuracy of methodology was performed by standard addition method; because of the reference materials are commercially not available for juices. The duplicate samples of juices of different flavors was spiked with known amount of elements and subjected to both methods to perform a recovery test. The daily intake of the elements by people and children through different flavors of juice marketed in different cities of Pakistan.
2.3. Sampling Ten samples of juices (mango, pine apple, apple, tropical and orange) commercially available from the market of Karachi, Pakistan were collected randomly in the year of 2009. The ten collected samples were packed on different dates to observe the variation in the elemental contamination levels of the products. The results obtained for ten commercial samples of different juices are given in Table 1. According to a t-test, all determined and spiking values are in good agreement at a 95% confidence level; however the reference materials for juices are not commercially available.
2.4. Sample preparation 2. Material and methods 2.1. Reagents and glass wares All the reagents and chemicals Merck, (Darmstadt, Germany) were used of analytical grade. De-ionized water was used through out the work. Concentrated Nitric acid (65%), were spectroscopic grades Merck, (Darmstadt, Germany). Stock standard solutions (1000 lg/mL) of Cu, Fe and Zn were obtained from Fluka Kamica (Buchs, Switzerland) of corresponding metal ions. The laboratory glass wares were kept overnight in 10% (v/v HNO3) and washed with water and rinse with distilled water before use. After washing and rinsing, the glassware’s were dried in an oven at 80 °C for 1 h.
2.4.1. Microwave assisted-acid digestion method A microwave assisted-acid digestion procedure was carried out, in order to achieve a shorter digestion time and using minimum amount of acid. Duplicate of 2.0 mL of each juice samples were taken into microwave vessels but well shaken, opened and sampled by pouring directly from the original container into the microwave vessel. Added the samples to each vessels and 10 mL of a concentrated HNO3– H2O2 (2:1, v/v) and kept samples for 10 min at room temperature till the samples were homogenized, and then placed the vessels in covered PTFE container. This was then heated following a one-stage digestion programmed at 80% of total power (900 W), for 3–5 min. After cooling, the resulting solutions were evaporated to semidried mass to remove an excess acid, and then diluted upto 50.0 mL in volumetric flasks and kept as a stock sample solution.
2.2. Instrumentation A Hitachi Model 5000 Z flame atomic absorption spectrophotometer was used for determination of Cu, Fe and Zn in juice samples. The calibration curves (0– 1.2 lg/mL) for all three studied elements were established with solutions prepared from a 1000 lg/mL stock solution. The energy sources, Hollow cathode lamps (Perkin-Elmer) operating at recommended current. A milestone microwave oven (Osaka, Japan), programmable for time and microwave power from 100 to 900 W, was used for acid digestion of juice samples.
Table 1 Determination of Cu, Fe and Zn juice samples (lg/mL). S. no:
Name of Sample
Copper
Iron
Zinc
1 2 3 4 5 6 7 8 9 10
Mango Apple Pine apple Tropical Orange Frooto Classic Maza Shezan Poly
0.181 ± 0.011 0.174 ± 0.007 0.201 ± 0.011 0.222 ± 0.013 0.262 ± 0.011 0.184 ± 0.012 0.176 ± 0.009 0.203 ± 0.013 0.226 ± 0.015 0.264 ± 0.012
0.908 ± 0.051 0.413 ± 0.018 1.55 ± 0.087 1.03 ± 0.061 0.595 ± 0.031 0.912 ± 0.055 0.415 ± 0.022 1.62 ± 0.091 1.05 ± 0.064 0.598 ± 0.032
0.296 ± 0.015 0.187 ± 0.011 0.781 ± 0.049 0.407 ± 0.021 0.315 ± 0.013 0.298 ± 0.016 0.192 ± 0.011 0.784 ± 0.051 0.411 ± 0.022 0.318 ± 0.015
2.4.2. Conventional wet acid digestion method (CDM) Duplicate of 50 mL of sub samples of juices were placed into 250 mL Pyrex beaker. Added 10 mL volume of a concentrated HNO3 (65%) and 5 mL of H2O2 to each flask and solutions were heated on electric hot plate at 80 °C, for 2–3 h, till the clear transparent solutions were obtained. Finally the solutions were made up to 50 mL of volumetric flask. After making volume the prepared samples were subjected to FAAS for estimation of Cu, Fe and Zn. Blank digestions (without analyte) were also carried out. The contents of the sample solutions were evaporated, and the semidried mass and than filtered through what man No. 42 filter paper, and made up the final volume into 50 mL volumetric flasks with ultra pure water. The concentrations were obtained directly from calibration graphs after correction of the absorbance for the signal from an appropriate reagent blank. All experiments were conducted at room temperature (30 °C) following the well-established laboratory protocols. To establish the validity of the microwave assisted-acid digestion method was checked by performing standard addition method. In standard addition method, the known amount of standards (0.2, 0.4, 0.8 mg/L) were spiked with juice using replicate six samples of each, and applied both digestion methods.
2.5. Statistical analysis Statistical calculations were performed using computer program Minitab 13.2 (Minitab Inc., State College, PA) and Excel.
Table 2 The results of standard addition/recovery for Cu, Fe and Zn determination in juice (Apple) (n = 6).
a b
Added lg/mL
pffiffiffi ts= na MAD x
pffiffiffi ts= na CAD x
% Recovery
Paired t-testb Texperimental
Copper (Cu) 0.0 0.2 0.4 0.8
0.174 ± 0.007 0.368 ± 0.021 0.565 ± 0.034 0.963 ± 0.073
0.176 ± 0.009 0.372 ± 0.023 0.570 ± 0.032 0.973 ± 0.056
98.8 98.9 99.1 99.0
2.01 1.30 2.10 2.26
Iron (Fe) 0.0 0.2 0.4 0.8
0.413 ± 0.018 0.612 ± 0.037 0.812 ± 0.067 1.21 ± 0.077
0.415 ± 0.022 0.614 ± 0.035 0.813 ± 0.061 1.22 ± 0.078
99.5 99.7 99.9 99.8
2.08 2.34 1.07 2.04
Zinc (Zn) 0.0 0.4 0.6 0.8
0.187 ± 0.011 0.383 ± 0.022 0.584 ± 0.038 0.987 ± 0.082
0.190 ± 0.011 0.386 ± 0.021 0.590 ± 0.032 0.992 ± 0.078
98.4 99.2 99.0 99.5
1.65 2.08 1.87 2.03
Key = Average value ± confidence interval (P = 0.05). Paired t-test between MAD vs CAD, degree of freedom (n 1) = 5tCritical at 95% confidence limit = 2.571.
2.571
N. Jalbani et al. / Food and Chemical Toxicology 48 (2010) 2737–2740 Table 3 Daily intake of Cu, Fe and Zn by consumption of juices (mg of Cu, Fe and Zn based on 250 mL juices/person/day). Sample name
Copper
Iron
Zinc
Mango Apple Pine apple Tropical Orange Frooto Classic Maza Shezan Poly
0.045 0.044 0.050 0.056 0.066 0.046 0.044 0.051 0.057 0.066
0.227 0.103 0.388 0.259 0.149 0.228 0.104 0.405 0.264 0.150
0.074 0.047 0.195 0.102 0.079 0.075 0.048 0.196 0.103 0.080
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The spiking was performed on duplicate samples at three concentration levels 0.2, 0.4, 0.8 lg/mL for Cu, Fe and Zn, respectively. Average recovery of Cu, Fe and Zn spikes from juices matrixes as indicated in Table 2. The t-test was performed on spiked juice (Apple) samples as given in Table 2. According to t-test all the obtained values were statistically significant at 95% Confidence level. For the commercially available juice samples the concentration of studied elements were ranged as 0.181–0.262 lg/mL for Cu, 0.595–0.908 lg/mL for Fe, 0.296–0.315 lg/mL for Zn, respectively. In the Table 2 it is clearly seen that the concentration of Copper in poly was found higher, while in case of Fe and Zn the maximum concentration was observed in maza. The lower concentration was found for Fe and Zn in similar juice samples (Apple) as shown in Table 1.
2.6. Calibration and sensitivity Calibration and standard addition graphs were obtained for Cu, Fe and Zn. However, as the acid composition of both acid mixture and different matrixes of samples, the standard addition technique was used throughout the development of method.The detection and quantification limits, given by
LOD ¼ 3
s s and LOQ ¼ 10 m m
Respectively, where s is the standard deviation of ten measurements of reagent blank and m is the slope of the calibration or standard addition graph was obtained for Cu, Fe and Zn. Limit of detection (LOD) of studied three elements, Cu 22.7, Fe 3.85 and Zn 3.05 lg/L being reached, and LOQ for Cu 76.4, Fe 12.8 and for Zn 10.2 lg/L were calculated. Reproducibility of the method was assessed by using three replicates of juices (Apple) and taken from the same packages. The most reliable approach evaluating the accuracy of an analytical method requires analysis of certified reference materials with chemical composition and analytic concentration, which match those of the samples under investigation. The certified reference materials are not available, Hence the standard addition method was performed to obtain the recovery test prior to the microwave assisted-acid and conventional wet acid digestion methods. The spiking of juice was performed in duplicate at three concentration levels 0.2, 0.4, 0.8 lg/mL, respectively and in this experiment we make the volume up to 50.0 mL in volumetric flasks and were analyzed by FAAS. Average recovery of three studied essential elements spikes from juices matrixes. For recovery test the apple, was spiked with the known amount of standards of Cu, Fe, and Zn as presented in Table 2. The results of proposed method MAD showed the% recovery for Cu varied from 98.8% to 99.2%, Fe from 99.5% to 99.9% and for Zn the concentration is ranged as 98.4–99.5%. The significance of the effects was evaluated with the t-test (t of Student). A t Experimental values was calculated at degree of freedom n1 = 5, the experimental values are found lower than the critical (2.57), at a 95% of confidence level, indicate non significant difference were observed in obtained values of non essential elements by CAD and MAD method (Table 2).
3. Results The present study was carried out to determine the essential elements in ten juice samples. The concentrations of elements were determined in duplicate samples by using of FAAS after digestion by microwave closed vessels and conventional wet acid digestion method. The analyzed samples were categorized on the basis of flavors and type of fruits. The average concentration of minerals Cu 0.262 lg/mL was found higher in orange as compared to others juices (apple, pineapple, tropical and mango) as described in Tables 2. The mean concentration of Fe 1.55 lg/mL is observed maximum in pineapple as compared to other obtained juice samples. The % recoveries of understudied three elements were observed different by adding the three replicates of juice (Apple). The most reliable approach evaluating the accuracy of an analytical method requires analysis of certified reference materials with chemical composition and analytic concentration, which contest those of the samples under investigation. To the best of our knowledge, there are no any commercially available juice reference materials for understudy elements. Therefore the Standard addition method was performed by spiking of juice samples to obtain the recovery test prior to microwave assisted acid digestion method and conventional wet acid digestion method.
4. Discussion The packed fruit juices are usually consumed due to flavor and nutritive value. Fresh fruit juices are largely consumed in all age groups because of it is major source of nutrient for adult as well as older age group. Copper deficiency causes anemia, loss of weight, bone and cartilage with irregular physiological development. Abnormal ingestion causes neurological anomalies, hepatic and renal disturbances (Underwood, 1977). The iron intake of the Aberdeen poor (Franke et al., 2006; Davidson et al., 1933). Although some of the women and children were anemic, there was no correlation between the incidence of anemia and the iron content of the diet including diets affording only 6 mg. of iron daily over long periods of time. Shah et al., 2003, stated that dietary survey of various sections of the United States revealed a variation of daily iron intake from 7 mg. for the Alabama Negro to 31 mg. in the Northern lumber camps. In fruit juices, the concentration of Cu and Zn can vary from lg/ kg to mg/kg range (Fernandez et al., 2002). This aspect makes difficult the employment of conventional FAAS for direct determination of these elements. Moreover, direct aspiration of the fruit juices and milk can cause flame fluctuations and accumulation of solid deposits on the burner head (Bellido-Milla et al., 2000). The WHO (World Health Organization, WHO., 1973; World Health Organization, 1996; WHO, 2001) estimated that the daily average adult intake of copper (3.0 mg/person/day) may come from various types of foods. In case of iron, the values of daily intake is calculated as copper i.e. the major source of understudied elements are food including wheat, fruit and vegetable. The daily dietary intake of fruit is approximately (i.e. 0.334 mg/person/ day). From the study it was clearly shown that above determined elements were found under legal recommended values. The intake of above studied elements varies place to place. The variation of minerals in food is different due to the different food consumption habits and different environmental factors. The food consumption habit is depend on several socio-economic, physiological, environmental contamination where the population is living near to contaminated area whether living in non contaminated surrounding. The present study shows that the intake of Cu, Fe and Zn is different in different type of juice products in Pakistan and provides specific information on the average dietary intake of elements as shown in (Table 3). Furthermore, the present findings allowed the identification of the specific items most responsible for major and minor contribution to the intake of this nutritional essential element. The average consumption of these juices was estimated by the survey on the population of different areas of Karachi city, Pakistan. The daily intake of understudied element was calculated on the consumption of a minimum of 250 mL/person/day of the juices and compared with tolerance limits of recommended by
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WHO/FDA. Our results are consistent with reported values (Mesallam, 1987).
5. Conclusion The microwave-assisted digestion method is an inexpensive, rapid lesser amount of acid and shorter time is required for digestion of samples as compared to conventional wet acid digestion for the assessment of Cu, Fe and Zn. The juices with fruit ensure an important source of contributing the mineral with a light percentage of the DRIs of the human being. However this type of beverage is consumed by all age groups but mostly consumed by children. The Beneficial fruit juices include apple, carrots and sugar cane, grapes, cherries, grapefruit, blueberries, and lemons. Juices are not only supplies the massive amounts of nutrition to the body, but it also flushes and wash out the all organs of human body, aids in effective enzyme activity and maintain the metabolism. From the results it was clearly seen that there is no significant difference among the different type of juices. A juice does not pose a concern with regard to the prospect of iron-deficiency anemia, which remains a significant health problem in Karachi, Pakistan.
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