sd-sample article R.A. Magbitang and others 1 SCIENCE DILIMAN (JANUARY-JUNE 2013) 25:1, 1-14 ISSN 0115-7809 Print / ISSN 2012-0818 Online Determination of Cd and Pb in Fruit Juice, Bottled Tea, Condiments and Dried Fish Samples Using ICP-MS Riza A. Magbitang1 , Melanie A. Bucsit2, Eugene S.F. T ia2, Rowena Grace Rumbaoa3, Lowela Lou M. Cervas3, Danica Angel ine Dimaya2 and Irene B. Rodriguez 1,2* 1Natural Sciences Research Institute, College of Science 2Institute of Chemistry, College of Science 3Department of Food Science and Nutrition, College of Home Economics University of the Philippines Diliman, Quezon City, 1101 Philippines ABSTRACT Metals like cadmium (Cd) and lead (Pb) are introduced in the environment through natural processes and anthropogenic activities and may end up b e i n g p r e s e n t i n f o o d , w h e r e t h e s e m e t a l s m a y p o s e h e a l t h r i s k s . A m e t h o d s u i t a b l e f o r t h e s i m u l t a n e o u s d e t e r m i n a t i o n o f C d a n d P b i n v a r i o u s m a t r i c e s o f f o o d s t u f f s w a s v a l i d a t e d a n d a p p l i e d t o d i f f e r e n t s a m p l e s i n c l u d i n g f r u i t j u i c e , b o t t l e d i c e d t e a , s e v e r a l t y p e s o f condiments, and in edible tissues of dried f ish locally produced in the Philippines. Fruit juice and bottled iced tea samples were f iltered prior t o q u a n t i f i c a t i o n o f m e t a l s u s i n g i n d u c t i v e l y co u p l e d p l a s m a m a s s s p e c t r o m e t r y ( I C P - M S ) . Co n d i m e n t s a n d d r i e d f i s h s a m p l e s w e r e m i n e r a l i z e d u s i n g m i c r o w a v e - a s s i s t e d n i t r i c a c i d d i g e s t i o n b e f o r e subsequent metal detection with ICP-MS. The method was validated using ce r tif ied reference materials DORM 3 and NIST 1643e, and evaluation o f r e c o v e r y o f s p i k e d s a m p l e s . T h e m e t h o d w a s l i n e a r i n t h e concentration range 0.01 to 500µg L -1 with correlation coeff icients of 0.999 for both analytes. The estimated detection limits were 0.060 µgL-1 and 0.186 µgL-1 for Cd and Pb, respectively. The determined levels of Cd in fruit juice were in the range 0.06 ± 0.01 to 0.67 ± 0.01 µgL -1, and Pb was detected in only one sample at 0.37 ± 0.02µgL -1. For the bottled iced tea samples, Cd was detected in only one sample (0.13 ± 0.02 µgL1) w h i l e n o n e o f t h e s a m p l e s h a d d e t e c t a b l e P b c o n c e n t r a t i o n . F o r t h e condiments, the determined Cd levels were in the range 0.83 ± 0.06 to _______________ *Corresponding Author Determination of Cd and Pb in Fruit Juice 2 306.13 ± 2.52µgL-1, whereas the determined Pb levels were in the range 2.14 ± 0.38 to 67.45 ± 7.76µgL-1. For the dried f ish samples, the Cd levels determined were in the range 2.00 ± 0.21 to 231.67 ± 5.32 µg kg-1 and that for Pb were in the range 2.38 ± 0.70 to 113.29 ± 2.25 µg kg-1. These determined levels in different foodstuffs highlight the need for routine monitoring of these contaminants. Keywords: Tr a ce m e t a l , f r u i t j u i ce s , co n d i m e n t s , d r i ed f i s h , m i c r ow a ve digestion, ICPMS INTRODUCTION Commonly consumed beverages in the Philippines include commercial fruit juices and iced tea, which are quite popular not only due to their palatable flavors but also because of their perceived nutritive value. Over the years, businesses manufacturing these beverages have continued to increase and the market has demanded for other flavors to be introduced. Fruit juices are available in various packages (tetra packs, bottled, etc.) and flavors (apple, orange, or mixtures of flavors, etc.). Aside from fruit juice, Filipinos are also consumers of condiments which are mostly fermented products such as soy sauce (from soy beans), vinegar (from sugar cane and coconut nectar), f ish sauce and f ish paste, and shrimp paste (from krill). These are often used as seasonings in main dishes, as ingredients in marinade, and as dipping sauces. Dietary guidelines worldwide increasingly recommend f ish consumption because of their nutritional benef its such as proteins, vitamins, minerals, and omega-3 polyunsaturated fatty acids (PUFAs) which exhibit protective effect against coronary heart disease and stroke (Kris-Etherton and others 2002, Joint WHO/FAO Expert Consultation 2003, Domingo 2007). However, concerns have been raised about f ish consumption due to the potential health risks associated with environmental contaminants such as presence of metals that exhibit toxicity (Domingo 2007). All of these food products may be sources of metal contaminants when these have not passed rigorous quality checks. Cadmium (Cd) and Lead (Pb) are ubiquitous in the environment and may enter the food chain due to their various applications and the improper handling of wastes. Cd is used as pigment in paints, in batteries, as stabilizers for PVC and can also be used in alloys (Jarup and others 1998). Likewise, Pb is used in batteries, alloys, pigments, cable sheathing, ammunition and as petrol additives (Garcia-Lestün and others 2010, Jarup and others 1998, ). Improper handling of wastes containing these metals and the indiscriminate use of products containing these may lead to the release of these contaminants in the environment. Consequently, they can then R.A. Magbitang and others 3 enter the food chain. The presence of these metals in food is a concern because of possible negative health effects. Adverse health effects of Cd in the body include kidney, skeletal and reproductive def iciencies (Jarup and others 1998). Pb may cause poor intellectual performance in children and may induce renal and cardiovascular diseases in adults (Fewtrell and others 2004, Garcia-Lestün and others 2010). The adverse health effects of these metals prompted the Joint FAO/WHO food standard programme Codex Committee on Contaminants in Foods (CCCF) to release, in March 2011, a new list of Maximum Levels (MLs) for contaminants and toxins. For Cd, the Committee set a Provisional Tolerable Monthly Intake (PTMI) of 25 µg kg-1 body weight. In the case of Pb, the Committee estimated that the previously established PTWI of 25 µg kg-1 body weight was no longer suff icient to prevent the effects of Pb. Consequently, a maximum level of 0.50 mg kg-1 of Pb in fruit juice was established. The Joint FAO/WHO Expert Committee on Food Additives suggested maximum levels for Cd of 2 mg kg-1 in marine bivalve mollusks and Provisional Tolerable Weekly Intake (PTWI) of 0.007 mg kg -1 body weight, and maximum levels for Pb of 0.3 mg kg-1 in f ish and PTWI of 0.025 mg kg-1 body weight (JECFA 1993). Likewise, the task group on contaminants in f ish and other seafood of the Marine Strategy Framework Directive (MSFD) of the European Union has set maximum values of these metals in muscle tissues of f ish. According to the report, Cd should not exceed 0.050 mg kg-1 based on the wet weight while Pb should not exceed 0.30 mg kg-1 wet weight (Swartenbroux and others 2010). To ensure the quality of products, especially food items, there has been a steady development in analytical techniques and instrument capabilities for monitoring metal concentration. The importance of analyzing trace contaminants has called for methodologies which are robust, able to detect trace concentrations while increasing the sample throughput and sensitivity in the analysis of varied sample matrices. Conventional techniques employed by most laboratories include the use of open- vessel acid digestion, dry ashing or a combination of both as pre-treatment steps to elemental analysis using flame atomic absorption spectroscopy (F-AAS). More advanced laboratories utilize graphite furnace AAS (GF-AAS) or plasma-based techniques such as inductively coupled plasma mass spectrometry (ICPMS) or optical emission spectroscopy (ICPOES). In this study, Cd and Pb were determined in fruit juice, bottled iced tea, condiment samples and dried f ish samples that are locally available in the Philippines after performing a suitable pre-treatment step, either microwave digestion or simple f iltration and dilution, using ICPMS. Determination of Cd and Pb in Fruit Juice 4 METHODOLOGY Chemicals and Reagents All chemicals and reagents used in this study were of analytical reagent grade unless otherwise specif ied. Nitric acid was obtained from Merck (Darmstadt, Germany). Single-element standards of Cd, Pb, indium (In) and rhenium (Re) with concentrations equivalent to 10,000 ± 30 µg mL-1 were purchased from CPI International (Santa Rosa, CA , USA). The reference material NIST 1643e (trace elements in water) used in this study was obtained from the National Institute of Standards and Technology (Maryland, USA). Another reference material used was DORM 3 (f ish protein certif ied reference material for trace metals), which was obtained from the National Research Council Canada – Institute for National Measurement Standards (Ontario, Canada). All dilutions and solution preparations were done using ultrapure water prepared using a Barnstead system (18.2 M cm resistivity, Thermo Fisher Scientif ic, Selangor Darul Ehsan, Malaysia). Instrumentation A Multiwave 3000 microwave digestion system (Anton Paar, Graz, Austria) f itted with a 16-position rotor for high digestion performance was used for the complete mineralization of the samples. An Agilent 7500cx ICPMS (Agilent Technologies, Germany) equipped with a MicroMist glass concentric nebulizer and an integrated autosampler (I-AS with type A vials, 89 x 6 mL capacity) was used as the element- selective detector. The monitored masses were m/z 111 for Cd and m/z 208 for Pb. The internal standards used were monitored at m/z equal to 115 for In and 185 for Re. The optimum conditions used for the analyses were as follows: RF power = 1550 W, carrier gas flow rate = 0.85 - 0.87 L min -1, make-up gas flow rate = 0.21 – 0.24 L min-1, nebulizer pump = 0.1 rps, sampling depth = 7.1 - 7.6 mm and the spray chamber temperature = 2.0 °C. The calibration standards were prepared in the range 0.01 µg L-1 to 500 µg L-1 which had correlation coeff icients of 0.999 or better for both analytes. The method detection limits obtained were 0.060 µg L-1 for Cd and 0.186 µg L-1 for Pb. These were estimated by getting the standard deviation of the results from the analysis of seven replicate solutions containing 1 µg L-1 of Cd and Pb, then subsequently multiplying the SD by the Student’s t value (for n = 7, t value is equal to 3.143; Ripp 1996). A drift standard, composed of 1.0 µg L-1 of both Cd and Pb, was measured repeatedly throughout the analysis sequence to determine the precision of the measurements within a day. The determined relative standard deviations of the repeated measurements of the drift standard were less than 1% and 2% for Cd and Ω R.A. Magbitang and others 5 Pb, respectively. These values indicate a stable instrument performance and good precision of measurements. Sampl ing All samples (fruit juice and condiments) included in the study were locally produced by different manufacturers and represent common products in the market. The samples were bought from a local supermarket which carries varied products. A total of 17 fruit juices and 5 bottled iced tea samples were included in the analysis. Various flavors of fruit juices were included: apple, grape, mango, orange, strawberry, calamansi, and guyabano. For the bottled iced tea samples, different brands that carry lemon flavor were included. Eighteen condiment samples were analyzed in the study, which included cane and spiced coconut vinegars, f ish and shrimp pastes, f ish sauce, soy sauce, and liquid seasoning. The dried f ish samples were collected from various wet and dry markets in Quezon City. They were identif ied to the nearest family by a research scientist at the Institute of Biology, College of Science, University of the Philippines Diliman. The identif ication of the exact species of the dried f ish samples was hampered because of the drying procedure that the samples have been subjected to, which led to the f ish samples losing most of the scales and f ins necessary for the identif ication procedure. Sample Preparation The fruit juice and iced tea samples were analyzed for the metal content after a simple f iltration and dilution process. Samples were f iltered using nylon syringe f ilters (pore size: 0.45 µm, diameter: 25 mm; obtained from Membrane Solutions LLC, Shanghai, China) immediately after opening. Three replicates were prepared for every sample. Samples were diluted 10-fold, acidif ied to 1% HNO 3 , and then analyzed using ICP-MS. Accuracy of the method was evaluated by analyzing NIST 1643e prepared in the same way and by spiking some samples with known amounts of Cd and Pb standards, which were then subjected to the same procedure. The condiment samples were mineralized by microwave-assisted acid digestion after addition of 2.0 mL of HNO 3 and 6.0 mL of water for solid samples (250 mg weighed to the nearest 0.01 mg) or 2.0 mL of HNO 3 and 5.0 mL water for liquid samples (volume of sample = 1.0 mL). The digestion step was carried out by following this optimized program (modif ied from Magbitang and Rodriguez 2012): the system was ramped to 600W for 5 min, kept at this condition for 30 min, decreased to 500 W and held at this condition for another 10 min, before allowing Determination of Cd and Pb in Fruit Juice 6 the system to cool. After digestion, the volume was adjusted to 10 mL. For the dried f ish analysis, the edible portions of the dried f ish samples were carefully scraped using acid-washed plastic knives. Triplicate aliquots (250 mg weighed to the nearest 0.1 mg) of each sample were weighed and subjected to the optimized mineralization procedure prior to ICPMS analysis. Triplicate aliquots of reference materials DORM-3 (250 mg) and NIST 1643e (1.0 mL) were digested along with the condiment samples and dried f ish samples. RESULTS AND DISCUSSION The results for the validation of the pre-treatment methods and ICPMS parameters using certif ied reference materials are summarized in Table 1. NIST reference material 1643e was used for both the validation of the f iltration step used for juice and tea samples, and the microwave-assisted digestion step used for the condiment samples. The reference material DORM 3 was also used for the validation of the microwave-assisted digestion step to simulate the complex matrix of some of the condiment samples that were derived from f ish. This was also the main reference material for the dried f ish samples. The results for NIST 1643e analyzed after simple f iltration reflected good accuracy, with 103.3% recovery for Cd and 92.4% for Pb. This was comparable to the results for NIST 1643e obtained after the microwave-assisted digestion with 104.2% recovery for Cd and 97.1% for Pb. As for the more complex matrix, the values obtained were 88.0% recovery for Cd and 84.6% recovery for Pb in DORM 3 after the complete mineralization by microwave digestion. These results suggest that the microwave digestion step would be a suff icient pre-treatment step for the condiment samples and dried f ish samples prior to ICPMS analysis. Table 1. Determined values for Cd and Pb in the certified reference materials processed using simple filtration or microwave-assisted acid d igestion prior to ICPMS analysis (expressed as mean concentration ± SD, n = 3) aPre-treatment step includes simple filtration followed by dilution. bPre-treatment step includes microwave-assisted acid digestion followed by dilution. Certified Element Certified Value Obtained Value % Recovery Reference Material NIST 1643ea 111Cd 6.568 ± 0.073 µg L-1 6.78 ± 0.16 µg L-1 103.2 208Pb 19.630 ± 0.210 µg L-1 18.14 ± 1.17 µg L-1 92.4 NIST 1643eb 111Cd 6.568 ± 0.073 µg L-1 6.85 ± 2.66 µg L-1 104.2 208Pb 19.630 ± 0.210 µg L-1 19.05 ± 2.16 µg L-1 97.1 DORM 3b 111Cd 290.000 ± 20.00 µg kg-1 255.17 ± 4.13 µg kg-1 88.0 208Pb 395.000 ± 50.00 µg kg-1 334.27 ± 17.72 µg kg-1 84.5 R.A. Magbitang and others 7 For further validation of the f iltration step used for juice and tea samples, known concentrations of the standards were spiked in some of the samples and the results are presented in Table 2. For the recovery test, juice and tea samples (one each) were spiked with 0.5, 5, and 50 µg L-1 concentrations of Cd and Pb standards. As shown above, good recovery values for the three different concentrations were obtained for Cd, which ranged from 100.3% to 106.0%. Recovery of Pb at the lowest concentration was observed to be low, which were at 57.6% and 72.6% for juice and tea samples, respectively. For the higher concentrations, 5 and 50 µg L-1, recoveries were from 95.4% to 98.9%. These recovery values are within the acceptable values as suggested by Taverniers and others (2004) and may show the influence of the matrix of the sample on the recovery of the target analytes. The good recoveries of the spiked samples indicate that the simple f iltration and dilution method used in the study is reliable in quantifying the amount of these metals in juice and bottled iced tea samples. Analysis of these samples can be done successfully without any further sample pretreatment. The results of the analysis of Cd and Pb in actual samples of fruit juice and bottled iced tea samples, as well as their respective brand codes, are summarized in Table 3. The different brands are coded from A to L because they carry various products and flavors. From the data obtained, it is evident that most of the juice samples analyzed have Pb content lower than the method detection limit while some have detectable Cd concentrations. Currently, there is no maximum limit set for Cd in fruit juices. The determined levels of Cd and Pb in these samples indicate that there is no signif icant level of the metal contaminants in these food products but the detectable levels of Cd suggest the need for routine monitoring of these contaminants in these products. Juice 0.5 0.50 ± 0.02 100.3 0.36 ± 0.22 72.6 5 5.09 ± 0.05 101.9 4.77 ± 0.15 95.4 50 51.38 ± 0.87 102.8 48.60 ± 0.22 97.2 Tea 0.5 0.53 ± 0.02 105.3 0.29 ± 0.14 57.6 5 5.16 ± 0.06 103.2 4.94 ± 0.36 98.9 50 53.01 ± 0.81 106.0 48.38 ± 0.54 96.8 Concentration of Cd and Pb (µg L-1) AddedSample Obtained value (µg L-1) Pb% RecoveryCd % Recovery Table 2. Percent recovery values for juice and tea samples spiked with known concentrations of standard Cd and Pb solutions (expressed as mean concentration ± SD, n = 3) Determination of Cd and Pb in Fruit Juice 8 Apple A < MDL 0.37 ± 0.02 B < MDL < MDL Mango A 0.25 ± 0.05 < MDL B 0.23 ± 0.05 < MDL C 0.67 ± 0.01 < MDL E < MDL < MDL Orange A < MDL < MDL B < MDL < MDL C 0.12 ± 0.01 < MDL F < MDL ND G 0.35 ± 0.02 < MDL Grape A 0.10 ± 0.02 < MDL B < MDL < MDL C 0.06 ± 0.01 < MDL Strawberry C < MDL < MDL Calamansi D < MDL < MDL Guyabano E 0.12 ± 0.03 < MDL Bottled iced tea H < MDL ND I 0.13 ± 0.02 ND J < MDL ND K < MDL ND L < MDL ND ND: No detectable value indicates that the signals from ICPMS measurements were not significantly higher than the blank. is currently a postdoctoral research fellow at the Research Center for Environmental Changes in Academia Sinica, Taiwan. Her work is centered on how trace metals interact with light to control the nitrogen cycle in marine biogeochemical systems. She is involved in research to better understand the role of trace metals in an ocean undergoing acidif ication, and how phytoplankton utilize these metals and accumulate these in the cells before eventually passing the metals up the trophic levels. Riza A. Magbitang is a University Research Associate I (URA I) at the Natural Sciences Research Institute, University of the Philippines Diliman. She is currently enrolled in the MS Chemistry program of the Institute of Chemistry of the same University, where she also obtained her BS Chemistry degree. Her research interests, among others, include environmental analytical chemistry, particularly optimization and validation of methods for the determination of environmental pollutants. Melanie A. Bucsit is currently an Instructor at the Institute of Chemistry, University of the Philippines Diliman, where she also obtained her MS Chemistry degree. Rowena Grace O. Rumbaoa is a faculty member at the Department of Food Science and Nutrition, at the College of Home Economics, University of the Philippines Diliman. She primarily handles courses in food chemistry and analysis, and food processing. She received her MS Food Science degree from the same university, where she is also currently pursuing her PhD degree. She has co-authored some works on algal polysaccharides, antioxidants, and Determination of Cd and Pb in Fruit Juice 14 proximate composition of foods. She has also completed researches on rootcrop antioxidants, processing and fortif ication of juices, modif ied atmosphere packaging and algal polysaccharides. Eugene S.F. T ia and Danica Angel ine Dimaya are graduate students at the Institute of Chemistry, College of Science, University of the Philippines Diliman. Lowela Lou M. Cervas is an Instructor at the University of the Philippines Los Baños where she handles courses on Food Analysis, Food Processing, Food Packaging and Labeling, Food Hygiene and Sanitation, and Practicum I– Pilot Scale.