- Email: [email protected]
Accelerator-based trace element analysis of foods and agriculture products
Available online at www.sciencedirect.com
NIM B Beam Interactions with Materials & Atoms
Nuclear Instruments and Methods in Physics Research B 266 (2008) 2391–2395 www.elsevier.com/locate/nimb
Accelerator-based trace element analysis of foods and agriculture products Manuel C. Lagunas-Solar a,*, Cecilia Pin˜a U a, Corina Solı´s b, Alibech Mireles b a
Crocker Nuclear Laboratory, 1 Shields Avenue, University of California, Davis, CA 95616-8569, USA b Physics Institute, National Autonomous University of Me´xico, Me´xico, DF, Mexico Available online 6 March 2008
Abstract An accelerator-based analytical method for measuring trace elements in foods and agricultural products was developed, optimized, validated and compared using reference standards. The method’s initial phase is a new, rapid and effective digestion process of a small mass analyte in an aqueous media containing H2O2. Digestion is initiated by radicals formed in water with pulsed UV (PUV) induced (laser) photolysis, which rapidly react with organic matter. After digestion, trace metals are pre-concentrated as carbamates and deposited as thin targets onto Teflon filters. Conventional particle induced X-ray emission (PIXE) or X-ray fluorescence (XRF) methods are then used to analyze elements in the sample. When foods and other agricultural commodities (i.e., soils, feeds) are analyzed, the combined method named pulsed UV (PUV)/PIXE results in enhanced detection of trace elements such as Fe, Co, Ni, Cu, Zn and Pb at 1 mg/kg (1 ppm) levels, without lengthy, acid-based digestions. It provides improvements in digestion kinetics and processing time enhancing analytical sensitivity and element recovery. Precision and recovery yields were confirmed with food reference standards. The analysis of edible foods from contaminated agricultural areas is also reported. Ó 2008 Elsevier B.V. All rights reserved. PACS: 29.20. c; 29.20.Hm; 82.80. d; 82.80.Ej; 78.70.En Keywords: Accelerators; PIXE; XRF; Laser-assisted digestion; Elemental analyses; Foods
1. Introduction The analysis of trace elements in foods and agricultural materials is essential to assure that adequate standards are set to minimize risks to human and environmental health. For centuries, industrial and agricultural practices have released metals to soil, water resources and atmosphere. Past and current practices are not adequate to prevent the spread of toxic elements and many are being reported in foods and in agricultural and urban areas worldwide [1–3]. As a result, new public and environmental health standards are being set prompting the need for improved analytical techniques to facilitate trace element analysis in foods and in the natural environs. *
Corresponding author. Tel.: +1 530 752 7439; fax: +1 530 754 8246. E-mail address: [email protected] (M.C. Lagunas-Solar).
0168-583X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2008.03.027
Accelerator-based PIXE and XRF techniques are effective to detect a wide range of elements with adequate analytical sensitivity. However, faster methods of sample preparation are desirable to optimize their use. A combined process including a fast, complete PUV digestion followed by a selective sample pre-concentration step using carbamates was then developed and tested. Results of this combined approach are reported for various types of food samples. Previously, other studies dealing with other aspects have been published [4–6,7]. 2. Materials and methods Fresh produce from markets and agricultural areas in California and Tla´huac, near Me´xico City, were used. Samples were washed, dried (70 °C, 72 h) and macerated into a fine (120–200 mesh) powder (Micro mill; Bel-Art Products,
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M.C. Lagunas-Solar et al. / Nucl. Instr. and Meth. in Phys. Res. B 266 (2008) 2391–2395
New Jersey, NJ) and filtered (80–200 mesh sieves) (Fisher Scientific, Pittsburg, PA). For method validation, Spinach leaves [NIST 1570a], Peach leaves [NIST 1547a], Pd solution (Inductively Coupled Plasma (ICP) Standard 1000 mg/kg, VWR International, Brisbane, CA) and ICP Standard Solution VIII (100 mg/kg, Merck, Germany) were used. Multiple samples (50–100 mg each, with 3–6 mL of suprapure H2O2 (30%) and 200 lL Antifoam A-5758 [Sigma, San Luis, MO] in 10 mL quartz tubes (1 cm OD), were digested simultaneously using a 12-sample rotating (adjustable) holder and exposed to PUV from an excimer laser (Lambda Physik LPX 200, Go¨ttingen, Germany) (KrF mode; 400 mJ/pulse; 30–60 pps) [5]. UV exposure per sample was measured with Joulemeters (Molectron EPM 1000 Laser Energy Power Meter, Molectron Inc., Portland, OR). Digested samples were analyzed directly by PIXE (with 0.5 lL Pd as internal standard), or submitted to one of two pre-concentration steps. In either case, a solution (3 mL) spiked with 5 mg/kg of ICP Standard Solution VIII, was previously adjusted to a pH 3.5–4 using standardized HCl and NH4OH. Deionized water was added to complete 50 mL sample volumes. Pre-concentration of trace metals (as carbamates) in the sample were achieved by: Method 1: 1 mL of a 10% (w/w) solution of ammonium pyrrolidinedithiocarbamate (APDC) (Sigma, San Luis, MO) and 10% (w/w) of sodium diethyldithiocarbamate (DDTC) (Sigma, San Luis, MO), or Method 2: 5 mL of a 30 mg APDC, 300 mg of L-ascorbic acid, 20 mg Cupferron, 15 mg of bisulfite (Sodium Hidrogensulfite ACS) solution dissolved in 4 mL deionized water and 30 mg of 8-hidroxiquinoline (oxine) dissolved in 1 mL of 2-propanol. A 100 lL Pd internal standard solution and co-precipitating agent was also added and the solution was filtered (0.45 lm Nucleopore). Digested samples (3 mL) and blanks were prepared with identical procedures and stored in membrane filters in a desiccator at 35 °C until PIXE analyses. The pulsed UV photolysis digestion process was evaluated by varying the mass sample/H2O2 ratio using 30% of suprapure H2O2, irradiation time (5–10 min) and sample
temperatures (30 °C and 90 °C) by multiple experimental replicates of each factor. 3. Results and discussion The development of PUV/PIXE method focused on improving digestion procedures and enhancing analytical sensitivity. Method validation and several applications of this approach are given in this work and are also detailed by Mireles [7]. 3.1. Optimization of pulsed UV assisted digestion 3.1.1. Mass reduction Sample mass reduction was found to be independent of analyte concentration (data not shown). Results of mass reduction experiments with Spinach and Plum reference standards are shown in Fig. 1(A) and (B), respectively. The optimum mass reduction was 75% for Spinach (10 min irradiation at 30 °C) and 90% for Plum (5 min irradiation at 90 °C), with 3–6% variation coefficients and were obtained with 3 mL of 30% H2O2. The laser beam must cover the entire sample area as radicals formed react quickly having insufficient time to diffuse to unirradiated volumes. Other concentrations of H2O2 (50%) were not explored, as suprapure purity is not available, raising concerns of potential cross contamination. As shown in Fig. 2 with samples from various plants and plant sections, mass reduction was also matrix dependent. 3.1.2. Recovery factors after pulsed UV digestion With digestions at 30 °C and 90 °C, recovery of elements such as Fe, Co, Ni, Cu, Zn, Se, Sr and Pb varied from 92% to 100% (data not shown). However, due to the formation of volatile compounds, elements such as Cr and Mn had lower recoveries (60% and 80%, respectively). Overall, recovery was independent from the sample matrix and the element’s initial concentration. When digestion was conducted at 90 °C, only Se and Sr showed a minor temperature effect, as recoveries were lowered.
Fig. 1. Mass reduction (%) for (A) spinach and (B) plum as a function of time of digestion, temperature and volume of H2O2.
M.C. Lagunas-Solar et al. / Nucl. Instr. and Meth. in Phys. Res. B 266 (2008) 2391–2395
Fig. 2. Results for mass reduction (%) in several commodities, including edible and non-edible parts, treated with PUV at 30 °C and 90 °C.
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ing self-absorption effects on the sample. This approach enhances overall detection of trace elements. For most of the selected elements (Fe, Co, Ni, Cu, Zn, Se, Tl, Pb and Bi), Method 2 (APDC only) yielded better overall results than Method 1 (APDC and DDTC) (data not shown) [7]. Recovery of Cr, Mn, Sr and Ba were low for either method, as they do not form carbamates due to low pH (3.5–4), have non-suitable oxidation states, or because they compete with the formation of hydroxides. Table 1 gives the recovery (%) with variation coefficients (CV) for elements of interest in Spinach + ICP Standard solution VIII samples after PUV digestion (30 and 90 °C) and concentrated as carbamates using Method 2. Except for Se, recovery levels was not affected by matrix, irradiation time or digestion temperature and remained similar in all cases. Selenium is apparently oxidized by radicals formed in the PUV photolysis and does not form carbamates. 3.2.2. Optimized PUV/PIXE method An optimized PUV/PIXE method shall consist of: (1) dried, powder sample (50–100 mg) is added to 3 mL of suprapure H2O2 (30%) with 200 lL of Antifoam; (2) PUV digestion for 5 min at 90 °C using a high peak power (80 MW/cm2) pulsed UV photon (248 nm, 5 eV/photon) beam (1 3 cm2) from a KrF excimer laser; (3) solution is filtered (0.45 lm Nucleopore; pre-weighed) to remove the non-digested organic material; (4) multiple samples may be pooled to improve detection and quantification of trace elements; (5) pre-concentration with carbamates (Method 2 is preferred) and filtration onto 0.45 lm Nucleopore membrane filter and (6) sample filters are then analyzed by PIXE (or XRF).
Fig. 3. Mass reduction (%) of agricultural commodities collected in Tla´huac, Me´xico. Comparison between conventional dry ashing (150– 550 °C) and PUV digestion method.
3.1.2. Comparison with dry ashing Results shown in Fig. 3, indicated that PUV digestion (5–10 min) provides similar mass reduction efficiencies as conventional dry ashing (5 h; 150–550 °C) but allows considerable savings in time. However, PUV digestion requires a complex infrastructure (lasers) and trained personnel, factors that may somehow diminish its advantages. 3.2. Enhanced analytical sensitivity 3.2.1. Pre-concentration with carbamates Trace elements were concentrated using carbamates as a coordinating and complexating agent. This step allowed a smaller mass (and area) of the metal-carbamate complexes to be used in preparing appropriate samples onto filters for PIXE or XRF analyses. Using carbamates allows the elimination of macronutrients, such as Na, K, Ca, Mg and P and significantly minimizes X-ray background while reduc-
3.2.3. Validation of PUV/PIXE method Linearity, accuracy, repeatability, analytical stability, robustness, precision and analytical sensitivity were determined using established criteria [8]. Validation was based on six replicated samples using the optimized PUV/PIXE method and Pd as internal standard. The optimized method was lineal in the 0.025–20 lg/cm2 range (r2 > 98%). Results were accurate and repeatable. The sample’s analytical stability was evaluated by varying the amount of time elapsed between the addition of carbamates and the filtration steps. Samples were stable after 0.5 h (tested up to 24 h), although no stability concerns exists with longer storage times. The robustness of the method was evaluated by studying the effect of small variations in the sample pH (from 3 to 4.5), before adding carbamates. In this pH interval, the method proved robust. Precision was determined with replicate analysis of Fe, Co, Ni, Cu, Zn and Pb in the 0.025–5 lg/cm2 range, which is a representative range for elemental content in fruits and vegetables [9]. The statistical evaluation showed variations <10% for 0.025 lg/cm2 samples (<0.002 SD) reaching a variation of <1.5% (<0.04 SD) for 5 lg/cm2 samples. Finally, the analytical sensitivity expressed as a limit of detection (LOD) and a limit of quantification (LOQ) was
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Table 1 Comparison of element recoveries (%) with coefficients of variation (CV) for spinach digested with PUV (30 °C and 90 °C) and pre-concentrated with Method 2 Recovery (%) for spinach 30 °C
Element
Recovery (%) for spinach 90 °C
Pd internal standard and co-precipitating agent (lg)
Cr Mn Fe Co Ni Cu Zn Se Sr Pb
10
20
100
200
10
20
100
200
2 (2) 0.2 (0.1) 91 (4) 94 (3) 99 (3) 98 (3) 96.2 (0.6) n.d 0.2 (0.4) 97 (3)
1 (5) n.d 92 (2) 98 (4) 102 (3) 97 (4) 98 (3) 0.4 (0.4) 0.5 (0.1) 94 (4)
1 (1) n.d 98 (2) 99 (2) 96.1 (0.5) 96 (4) 93 (5) 1.9 (0.6) 0.1 (0.1) 93 (1)
1.5 (0.3) n.d 99 (3) 98 (2) 94 (1) 98 (1) 96 (2) n.d 0.3 (0.1) 93 (2)
4 (2) 0.1 (0.1) 93 (4) 100 (3) 102 (3) 100 (2) 98.2 (0.7) 0.3 (0.5) 0.2 (0.4) 94 (3)
0.8 (0.3) n.d 101 (2) 96 (3) 94 (3) 98 (4) 99 (2) 0.4 (0.4) n.d 94 (4)
0.8 (0.1) n.d 92 (2) 98 (2) 96.0 (0.5) 98 (4) 95 (5) 6.0 (0.6) 0.3 (0.1) 98 (1)
0.5 (0.3) n.d 97 (3) 100 (1) 96 (1) 99 (1) 98 (3) 3.6 (0.5) 0.1 (0.1) 98 (2)
n.d = not detected.
Table 2 Limit of detection (LOD) and quantification (LOQ) attainable with PUV/ PIXE method Element
Detection limit (mg/kg)
Quantification limit (mg/kg)
Fe Co Ni Cu Zn Pb
0.16 0.18 0.2 0.2 0.28 0.22
0.64 0.72 0.74 0.76 1.02 0.8
increased approximately five times, with detection limits of 0.2 mg/kg in 50 mg of digested samples, in comparison with 1 mg/kg for biological samples as reported in the literature [10]. Results of LOD and LOQ are shown in Table 2. Application of PUV/PIXE to a field study: The validated PUV/PIXE method was applied to a field study to determine the uptake of toxic metals in a variety of edible plants (fruits and vegetables) cultivated in Tla´huac, Me´xico and irrigated with wastewater from Me´xico City. Results are given in Table 3, along with soil and phytotoxic levels [2]. Average values for plants and sections (n = 3) and for soils (n = 10)
Table 3 Concentrations of Fe, Co, Ni, Cu, Zn and Pb (mg/kg) with standard deviations (SD) determined with PUV/PIXE method in edible plants cultivated in Tla´huac, Me´xicoa Plant
Quelite Tomatillo Tomato Zucchini Prickly Pear Nopal Quince Rosemary Swiss Chard Pear Hot Pepper Purslane Cauliflower Spinach Beet Roman Lettuce Lettuce Radish
Element
Fe
Co
Ni
Cu
Zn
Pb
Soil level (mg/kg)
18,000 (1500)
63 (6)
25 (1)
30 (3)
62 (5)
35 (2)
Phytotoxic levels (mg/kg)
–
15–50
10–100
20–100
100–400
30–300
Sections
Measured concentrations (mg/kg)
Leaves Fruit Ripe fruit Unripe fruit Flower Fruit Leaves Fruit Leaves Leaves Fruit Fruit Leaves Heart Leaves Tubercle Heart Leaves Leaves Tubercle
210 (10) 127 (6) 330 (20) 330 (20) 270 (10) 33 (1) 48 (2) 33 (1) 63 (3) 130 (6) 77 (3) 34 (1) 152 (7) 116 (5) 240 (10) 320 (20) 47 (2) 220 (10) 158 (7) 480 (20)
1.43 (0.07) 4.2 (0.2) 4.1 (0.2) 1.77 (0.09) 6.1 (0.3) 1.60 (0.08) 0.59 (0.03) 0.39 (0.02) 3.2 (0.2) 2.7 (0.1) 1.2 (0.1) 0.96 (0.05) 3.8 (0.2) 1.00 (0.05) 1.12 (0.06) 6.7 (0.3) 0.33 (0.02) 0.59 (0.03) 1.13 (0.06) 0.94 (0.05)
13.8 (0.7) 18 (1) 16 (1) 14.8 (0.7) 18 (1) 3.8 (0.2) 12.1 (0.6) 2.2 (0.1) 15 (1) 48 (2) 16 (1) 4.4 (0.2) 10.5 (0.5) 12.4 (0.6) 5.5 (0.3) 20 (1) 23 (1) 29 (2) 3.8 (0.2) 11.4 (0.6)
31 (2) 19 (1) 22 (1) 24 (1) 24 (1) 16.2 (0.8) 31 (2) 2.5 (0.1) 24 (1) 55 (3) 29 (1) 20 (1) 26 (1) 17 (1) 25 (1) 59 (3) 21 (1) 25 (1) 17 (1) 39 (2)
0.57 (0.03) 0.44 (0.02) 0.54 (0.03) n.d 1.18 (0.06) 0.050 (0.003) 0.77 (0.04) 0.13 (0.01) 0.33 (0.02) n.d 1.47 (0.07) 0.12 (0.01) 0.60 (0.03) 0.21 (0.01) n.d 6.3 (0.3) 0.15 (0.01) 0.80 (0.04) 0.46 (0.02) 5.1 (0.3)
n.d 0.69 (0.03) 0.91 (0.05) 0.20 (0.01) 1.03 (0.05) 0.11 (0.01) 0.76 (0.04) 0.04 (0.002) n.d 2.4 (0.1) 1.34 (0.07) 0.04 (0.002) 1.22 (0.06) n.d n.d 2.7 (0.1) n.d n.d 0.16 (0.01) 1.16 (0.06)
n.d = not detected, Purslane: Portuclaca oleracea, Quelite: Chenopodium sp. a Numbers in bold exceed the top limit (0.2 mg/kg) recommended by FAO/WHO for human consumption of produce.
M.C. Lagunas-Solar et al. / Nucl. Instr. and Meth. in Phys. Res. B 266 (2008) 2391–2395
are given. In most vegetables, tubercles and fruits concentrations are below levels considered as phytotoxic for plants [2] or safe for human consumption [3]. However, in most foods, Pb concentrations exceeded 0.2 mg/kg a top level recommended by FAO/WHO [9]. Lead concentrations in beetroots and radish were high, as tubercles are in direct contact with soil. Other elements (Fe, Co, Ni, Cu, Zn) do not exceed recommended levels [1,3]. 4. Conclusions With PUV digestion, matrix dissolution of a variety of fruits and vegetables is nearly complete. Mass reduction is comparable to dry ashing, but PUV digestion provides a significant reduction in time. Although direct PIXE analysis allows detection of a higher number of elements, with carbamates, a higher analytical sensitivity is reached (LOD from 1 to 0.2 mg/kg) with 50 mg of original sample. The PUV/ PIXE method proved to have high precision and reproducibility with NIST standards and was shown to compare favorably with NAA and AA analyses [5]. It was also shown as reliable for measuring trace concentrations of metals in different plant materials. Finally, the PUV/PIXE method may provide advantages to study trace elements in foods and to address health and environmental concerns. So far, analytical limitations and cost have limited the execution of statistically sound studies of this kind, hindering the availability of critical information needed to develop technologies for precise and sustainable agriculture.
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Acknowledgements Support by UC MEXUS-CONACYT and DGAPAUNAM (Grant No. IN228603-3) is recognized. Alibech Mireles received a fellowship from DGEP, UNAM. References [1] National Academy of Science (Commitee on Food Protection), Toxicants Occurring Naturally in Foods, second ed., National Academy of Science, Washington, DC, 1973. [2] A. Kabata-Pendias, H. Pendias, Trace Elements in Soils and Plants, CRC Press, Boca Raton, FL, 2000. [3] WHO, Trace Elements in Human Health and Nutrition, WHO Publications, Geneva, Switzerland, 1996. [4] R.G. Flocchini, M. Lagunas-Solar, B.P. Perley, Int. J. PIXE 6 (1–2) (1996) 375–394. [5] M. Lagunas-Solar, C. Solı´s, A. Mireles, C. Pin˜a, R.G. Flocchini, Int. J. PIXE 15 (3–4) (2005) 309–316. [6] C. Solı´s, M. Lagunas-Solar, B.P. Perley, C. Pin˜a, L.F. Aguilar, R.G. Flocchini, Nucl. Instr. and Meth. B 189 (2002) 77–80. [7] A. Mireles, Development and optimization of sample preparation and quantitative analysis of trace elements in fruits and vegetables with PIXE, Ph.D. Thesis, National Autonomous University of Mexico, 2004. (in Spanish) [8] Joint FAO/IAEA Expert Consultation, Validation of Analytical Methods for Food Control, Vienna, 2–4 December 1997. [9] FAO/WHO CODEX STAN 210-2001, Rev. 1, Maximum Levels for Lead, 2003. [10] S.A.E. Johansson, J.L. Campbell, PIXE: A Novel Technique For Elemental Analysis, Wiley & Sons, New York, 1988; See also C.L. Keen, T. Jue, C.D. Tran, J. Vogel, R. Gregory Downing, V. Yyengar, R.B. Rucker, J. Nutr. 133 (2003) 1574S.
NIM B Beam Interactions with Materials & Atoms
Nuclear Instruments and Methods in Physics Research B 266 (2008) 2391–2395 www.elsevier.com/locate/nimb
Accelerator-based trace element analysis of foods and agriculture products Manuel C. Lagunas-Solar a,*, Cecilia Pin˜a U a, Corina Solı´s b, Alibech Mireles b a
Crocker Nuclear Laboratory, 1 Shields Avenue, University of California, Davis, CA 95616-8569, USA b Physics Institute, National Autonomous University of Me´xico, Me´xico, DF, Mexico Available online 6 March 2008
Abstract An accelerator-based analytical method for measuring trace elements in foods and agricultural products was developed, optimized, validated and compared using reference standards. The method’s initial phase is a new, rapid and effective digestion process of a small mass analyte in an aqueous media containing H2O2. Digestion is initiated by radicals formed in water with pulsed UV (PUV) induced (laser) photolysis, which rapidly react with organic matter. After digestion, trace metals are pre-concentrated as carbamates and deposited as thin targets onto Teflon filters. Conventional particle induced X-ray emission (PIXE) or X-ray fluorescence (XRF) methods are then used to analyze elements in the sample. When foods and other agricultural commodities (i.e., soils, feeds) are analyzed, the combined method named pulsed UV (PUV)/PIXE results in enhanced detection of trace elements such as Fe, Co, Ni, Cu, Zn and Pb at 1 mg/kg (1 ppm) levels, without lengthy, acid-based digestions. It provides improvements in digestion kinetics and processing time enhancing analytical sensitivity and element recovery. Precision and recovery yields were confirmed with food reference standards. The analysis of edible foods from contaminated agricultural areas is also reported. Ó 2008 Elsevier B.V. All rights reserved. PACS: 29.20. c; 29.20.Hm; 82.80. d; 82.80.Ej; 78.70.En Keywords: Accelerators; PIXE; XRF; Laser-assisted digestion; Elemental analyses; Foods
1. Introduction The analysis of trace elements in foods and agricultural materials is essential to assure that adequate standards are set to minimize risks to human and environmental health. For centuries, industrial and agricultural practices have released metals to soil, water resources and atmosphere. Past and current practices are not adequate to prevent the spread of toxic elements and many are being reported in foods and in agricultural and urban areas worldwide [1–3]. As a result, new public and environmental health standards are being set prompting the need for improved analytical techniques to facilitate trace element analysis in foods and in the natural environs. *
Corresponding author. Tel.: +1 530 752 7439; fax: +1 530 754 8246. E-mail address: [email protected] (M.C. Lagunas-Solar).
0168-583X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2008.03.027
Accelerator-based PIXE and XRF techniques are effective to detect a wide range of elements with adequate analytical sensitivity. However, faster methods of sample preparation are desirable to optimize their use. A combined process including a fast, complete PUV digestion followed by a selective sample pre-concentration step using carbamates was then developed and tested. Results of this combined approach are reported for various types of food samples. Previously, other studies dealing with other aspects have been published [4–6,7]. 2. Materials and methods Fresh produce from markets and agricultural areas in California and Tla´huac, near Me´xico City, were used. Samples were washed, dried (70 °C, 72 h) and macerated into a fine (120–200 mesh) powder (Micro mill; Bel-Art Products,
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M.C. Lagunas-Solar et al. / Nucl. Instr. and Meth. in Phys. Res. B 266 (2008) 2391–2395
New Jersey, NJ) and filtered (80–200 mesh sieves) (Fisher Scientific, Pittsburg, PA). For method validation, Spinach leaves [NIST 1570a], Peach leaves [NIST 1547a], Pd solution (Inductively Coupled Plasma (ICP) Standard 1000 mg/kg, VWR International, Brisbane, CA) and ICP Standard Solution VIII (100 mg/kg, Merck, Germany) were used. Multiple samples (50–100 mg each, with 3–6 mL of suprapure H2O2 (30%) and 200 lL Antifoam A-5758 [Sigma, San Luis, MO] in 10 mL quartz tubes (1 cm OD), were digested simultaneously using a 12-sample rotating (adjustable) holder and exposed to PUV from an excimer laser (Lambda Physik LPX 200, Go¨ttingen, Germany) (KrF mode; 400 mJ/pulse; 30–60 pps) [5]. UV exposure per sample was measured with Joulemeters (Molectron EPM 1000 Laser Energy Power Meter, Molectron Inc., Portland, OR). Digested samples were analyzed directly by PIXE (with 0.5 lL Pd as internal standard), or submitted to one of two pre-concentration steps. In either case, a solution (3 mL) spiked with 5 mg/kg of ICP Standard Solution VIII, was previously adjusted to a pH 3.5–4 using standardized HCl and NH4OH. Deionized water was added to complete 50 mL sample volumes. Pre-concentration of trace metals (as carbamates) in the sample were achieved by: Method 1: 1 mL of a 10% (w/w) solution of ammonium pyrrolidinedithiocarbamate (APDC) (Sigma, San Luis, MO) and 10% (w/w) of sodium diethyldithiocarbamate (DDTC) (Sigma, San Luis, MO), or Method 2: 5 mL of a 30 mg APDC, 300 mg of L-ascorbic acid, 20 mg Cupferron, 15 mg of bisulfite (Sodium Hidrogensulfite ACS) solution dissolved in 4 mL deionized water and 30 mg of 8-hidroxiquinoline (oxine) dissolved in 1 mL of 2-propanol. A 100 lL Pd internal standard solution and co-precipitating agent was also added and the solution was filtered (0.45 lm Nucleopore). Digested samples (3 mL) and blanks were prepared with identical procedures and stored in membrane filters in a desiccator at 35 °C until PIXE analyses. The pulsed UV photolysis digestion process was evaluated by varying the mass sample/H2O2 ratio using 30% of suprapure H2O2, irradiation time (5–10 min) and sample
temperatures (30 °C and 90 °C) by multiple experimental replicates of each factor. 3. Results and discussion The development of PUV/PIXE method focused on improving digestion procedures and enhancing analytical sensitivity. Method validation and several applications of this approach are given in this work and are also detailed by Mireles [7]. 3.1. Optimization of pulsed UV assisted digestion 3.1.1. Mass reduction Sample mass reduction was found to be independent of analyte concentration (data not shown). Results of mass reduction experiments with Spinach and Plum reference standards are shown in Fig. 1(A) and (B), respectively. The optimum mass reduction was 75% for Spinach (10 min irradiation at 30 °C) and 90% for Plum (5 min irradiation at 90 °C), with 3–6% variation coefficients and were obtained with 3 mL of 30% H2O2. The laser beam must cover the entire sample area as radicals formed react quickly having insufficient time to diffuse to unirradiated volumes. Other concentrations of H2O2 (50%) were not explored, as suprapure purity is not available, raising concerns of potential cross contamination. As shown in Fig. 2 with samples from various plants and plant sections, mass reduction was also matrix dependent. 3.1.2. Recovery factors after pulsed UV digestion With digestions at 30 °C and 90 °C, recovery of elements such as Fe, Co, Ni, Cu, Zn, Se, Sr and Pb varied from 92% to 100% (data not shown). However, due to the formation of volatile compounds, elements such as Cr and Mn had lower recoveries (60% and 80%, respectively). Overall, recovery was independent from the sample matrix and the element’s initial concentration. When digestion was conducted at 90 °C, only Se and Sr showed a minor temperature effect, as recoveries were lowered.
Fig. 1. Mass reduction (%) for (A) spinach and (B) plum as a function of time of digestion, temperature and volume of H2O2.
M.C. Lagunas-Solar et al. / Nucl. Instr. and Meth. in Phys. Res. B 266 (2008) 2391–2395
Fig. 2. Results for mass reduction (%) in several commodities, including edible and non-edible parts, treated with PUV at 30 °C and 90 °C.
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ing self-absorption effects on the sample. This approach enhances overall detection of trace elements. For most of the selected elements (Fe, Co, Ni, Cu, Zn, Se, Tl, Pb and Bi), Method 2 (APDC only) yielded better overall results than Method 1 (APDC and DDTC) (data not shown) [7]. Recovery of Cr, Mn, Sr and Ba were low for either method, as they do not form carbamates due to low pH (3.5–4), have non-suitable oxidation states, or because they compete with the formation of hydroxides. Table 1 gives the recovery (%) with variation coefficients (CV) for elements of interest in Spinach + ICP Standard solution VIII samples after PUV digestion (30 and 90 °C) and concentrated as carbamates using Method 2. Except for Se, recovery levels was not affected by matrix, irradiation time or digestion temperature and remained similar in all cases. Selenium is apparently oxidized by radicals formed in the PUV photolysis and does not form carbamates. 3.2.2. Optimized PUV/PIXE method An optimized PUV/PIXE method shall consist of: (1) dried, powder sample (50–100 mg) is added to 3 mL of suprapure H2O2 (30%) with 200 lL of Antifoam; (2) PUV digestion for 5 min at 90 °C using a high peak power (80 MW/cm2) pulsed UV photon (248 nm, 5 eV/photon) beam (1 3 cm2) from a KrF excimer laser; (3) solution is filtered (0.45 lm Nucleopore; pre-weighed) to remove the non-digested organic material; (4) multiple samples may be pooled to improve detection and quantification of trace elements; (5) pre-concentration with carbamates (Method 2 is preferred) and filtration onto 0.45 lm Nucleopore membrane filter and (6) sample filters are then analyzed by PIXE (or XRF).
Fig. 3. Mass reduction (%) of agricultural commodities collected in Tla´huac, Me´xico. Comparison between conventional dry ashing (150– 550 °C) and PUV digestion method.
3.1.2. Comparison with dry ashing Results shown in Fig. 3, indicated that PUV digestion (5–10 min) provides similar mass reduction efficiencies as conventional dry ashing (5 h; 150–550 °C) but allows considerable savings in time. However, PUV digestion requires a complex infrastructure (lasers) and trained personnel, factors that may somehow diminish its advantages. 3.2. Enhanced analytical sensitivity 3.2.1. Pre-concentration with carbamates Trace elements were concentrated using carbamates as a coordinating and complexating agent. This step allowed a smaller mass (and area) of the metal-carbamate complexes to be used in preparing appropriate samples onto filters for PIXE or XRF analyses. Using carbamates allows the elimination of macronutrients, such as Na, K, Ca, Mg and P and significantly minimizes X-ray background while reduc-
3.2.3. Validation of PUV/PIXE method Linearity, accuracy, repeatability, analytical stability, robustness, precision and analytical sensitivity were determined using established criteria [8]. Validation was based on six replicated samples using the optimized PUV/PIXE method and Pd as internal standard. The optimized method was lineal in the 0.025–20 lg/cm2 range (r2 > 98%). Results were accurate and repeatable. The sample’s analytical stability was evaluated by varying the amount of time elapsed between the addition of carbamates and the filtration steps. Samples were stable after 0.5 h (tested up to 24 h), although no stability concerns exists with longer storage times. The robustness of the method was evaluated by studying the effect of small variations in the sample pH (from 3 to 4.5), before adding carbamates. In this pH interval, the method proved robust. Precision was determined with replicate analysis of Fe, Co, Ni, Cu, Zn and Pb in the 0.025–5 lg/cm2 range, which is a representative range for elemental content in fruits and vegetables [9]. The statistical evaluation showed variations <10% for 0.025 lg/cm2 samples (<0.002 SD) reaching a variation of <1.5% (<0.04 SD) for 5 lg/cm2 samples. Finally, the analytical sensitivity expressed as a limit of detection (LOD) and a limit of quantification (LOQ) was
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Table 1 Comparison of element recoveries (%) with coefficients of variation (CV) for spinach digested with PUV (30 °C and 90 °C) and pre-concentrated with Method 2 Recovery (%) for spinach 30 °C
Element
Recovery (%) for spinach 90 °C
Pd internal standard and co-precipitating agent (lg)
Cr Mn Fe Co Ni Cu Zn Se Sr Pb
10
20
100
200
10
20
100
200
2 (2) 0.2 (0.1) 91 (4) 94 (3) 99 (3) 98 (3) 96.2 (0.6) n.d 0.2 (0.4) 97 (3)
1 (5) n.d 92 (2) 98 (4) 102 (3) 97 (4) 98 (3) 0.4 (0.4) 0.5 (0.1) 94 (4)
1 (1) n.d 98 (2) 99 (2) 96.1 (0.5) 96 (4) 93 (5) 1.9 (0.6) 0.1 (0.1) 93 (1)
1.5 (0.3) n.d 99 (3) 98 (2) 94 (1) 98 (1) 96 (2) n.d 0.3 (0.1) 93 (2)
4 (2) 0.1 (0.1) 93 (4) 100 (3) 102 (3) 100 (2) 98.2 (0.7) 0.3 (0.5) 0.2 (0.4) 94 (3)
0.8 (0.3) n.d 101 (2) 96 (3) 94 (3) 98 (4) 99 (2) 0.4 (0.4) n.d 94 (4)
0.8 (0.1) n.d 92 (2) 98 (2) 96.0 (0.5) 98 (4) 95 (5) 6.0 (0.6) 0.3 (0.1) 98 (1)
0.5 (0.3) n.d 97 (3) 100 (1) 96 (1) 99 (1) 98 (3) 3.6 (0.5) 0.1 (0.1) 98 (2)
n.d = not detected.
Table 2 Limit of detection (LOD) and quantification (LOQ) attainable with PUV/ PIXE method Element
Detection limit (mg/kg)
Quantification limit (mg/kg)
Fe Co Ni Cu Zn Pb
0.16 0.18 0.2 0.2 0.28 0.22
0.64 0.72 0.74 0.76 1.02 0.8
increased approximately five times, with detection limits of 0.2 mg/kg in 50 mg of digested samples, in comparison with 1 mg/kg for biological samples as reported in the literature [10]. Results of LOD and LOQ are shown in Table 2. Application of PUV/PIXE to a field study: The validated PUV/PIXE method was applied to a field study to determine the uptake of toxic metals in a variety of edible plants (fruits and vegetables) cultivated in Tla´huac, Me´xico and irrigated with wastewater from Me´xico City. Results are given in Table 3, along with soil and phytotoxic levels [2]. Average values for plants and sections (n = 3) and for soils (n = 10)
Table 3 Concentrations of Fe, Co, Ni, Cu, Zn and Pb (mg/kg) with standard deviations (SD) determined with PUV/PIXE method in edible plants cultivated in Tla´huac, Me´xicoa Plant
Quelite Tomatillo Tomato Zucchini Prickly Pear Nopal Quince Rosemary Swiss Chard Pear Hot Pepper Purslane Cauliflower Spinach Beet Roman Lettuce Lettuce Radish
Element
Fe
Co
Ni
Cu
Zn
Pb
Soil level (mg/kg)
18,000 (1500)
63 (6)
25 (1)
30 (3)
62 (5)
35 (2)
Phytotoxic levels (mg/kg)
–
15–50
10–100
20–100
100–400
30–300
Sections
Measured concentrations (mg/kg)
Leaves Fruit Ripe fruit Unripe fruit Flower Fruit Leaves Fruit Leaves Leaves Fruit Fruit Leaves Heart Leaves Tubercle Heart Leaves Leaves Tubercle
210 (10) 127 (6) 330 (20) 330 (20) 270 (10) 33 (1) 48 (2) 33 (1) 63 (3) 130 (6) 77 (3) 34 (1) 152 (7) 116 (5) 240 (10) 320 (20) 47 (2) 220 (10) 158 (7) 480 (20)
1.43 (0.07) 4.2 (0.2) 4.1 (0.2) 1.77 (0.09) 6.1 (0.3) 1.60 (0.08) 0.59 (0.03) 0.39 (0.02) 3.2 (0.2) 2.7 (0.1) 1.2 (0.1) 0.96 (0.05) 3.8 (0.2) 1.00 (0.05) 1.12 (0.06) 6.7 (0.3) 0.33 (0.02) 0.59 (0.03) 1.13 (0.06) 0.94 (0.05)
13.8 (0.7) 18 (1) 16 (1) 14.8 (0.7) 18 (1) 3.8 (0.2) 12.1 (0.6) 2.2 (0.1) 15 (1) 48 (2) 16 (1) 4.4 (0.2) 10.5 (0.5) 12.4 (0.6) 5.5 (0.3) 20 (1) 23 (1) 29 (2) 3.8 (0.2) 11.4 (0.6)
31 (2) 19 (1) 22 (1) 24 (1) 24 (1) 16.2 (0.8) 31 (2) 2.5 (0.1) 24 (1) 55 (3) 29 (1) 20 (1) 26 (1) 17 (1) 25 (1) 59 (3) 21 (1) 25 (1) 17 (1) 39 (2)
0.57 (0.03) 0.44 (0.02) 0.54 (0.03) n.d 1.18 (0.06) 0.050 (0.003) 0.77 (0.04) 0.13 (0.01) 0.33 (0.02) n.d 1.47 (0.07) 0.12 (0.01) 0.60 (0.03) 0.21 (0.01) n.d 6.3 (0.3) 0.15 (0.01) 0.80 (0.04) 0.46 (0.02) 5.1 (0.3)
n.d 0.69 (0.03) 0.91 (0.05) 0.20 (0.01) 1.03 (0.05) 0.11 (0.01) 0.76 (0.04) 0.04 (0.002) n.d 2.4 (0.1) 1.34 (0.07) 0.04 (0.002) 1.22 (0.06) n.d n.d 2.7 (0.1) n.d n.d 0.16 (0.01) 1.16 (0.06)
n.d = not detected, Purslane: Portuclaca oleracea, Quelite: Chenopodium sp. a Numbers in bold exceed the top limit (0.2 mg/kg) recommended by FAO/WHO for human consumption of produce.
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are given. In most vegetables, tubercles and fruits concentrations are below levels considered as phytotoxic for plants [2] or safe for human consumption [3]. However, in most foods, Pb concentrations exceeded 0.2 mg/kg a top level recommended by FAO/WHO [9]. Lead concentrations in beetroots and radish were high, as tubercles are in direct contact with soil. Other elements (Fe, Co, Ni, Cu, Zn) do not exceed recommended levels [1,3]. 4. Conclusions With PUV digestion, matrix dissolution of a variety of fruits and vegetables is nearly complete. Mass reduction is comparable to dry ashing, but PUV digestion provides a significant reduction in time. Although direct PIXE analysis allows detection of a higher number of elements, with carbamates, a higher analytical sensitivity is reached (LOD from 1 to 0.2 mg/kg) with 50 mg of original sample. The PUV/ PIXE method proved to have high precision and reproducibility with NIST standards and was shown to compare favorably with NAA and AA analyses [5]. It was also shown as reliable for measuring trace concentrations of metals in different plant materials. Finally, the PUV/PIXE method may provide advantages to study trace elements in foods and to address health and environmental concerns. So far, analytical limitations and cost have limited the execution of statistically sound studies of this kind, hindering the availability of critical information needed to develop technologies for precise and sustainable agriculture.
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Acknowledgements Support by UC MEXUS-CONACYT and DGAPAUNAM (Grant No. IN228603-3) is recognized. Alibech Mireles received a fellowship from DGEP, UNAM. References [1] National Academy of Science (Commitee on Food Protection), Toxicants Occurring Naturally in Foods, second ed., National Academy of Science, Washington, DC, 1973. [2] A. Kabata-Pendias, H. Pendias, Trace Elements in Soils and Plants, CRC Press, Boca Raton, FL, 2000. [3] WHO, Trace Elements in Human Health and Nutrition, WHO Publications, Geneva, Switzerland, 1996. [4] R.G. Flocchini, M. Lagunas-Solar, B.P. Perley, Int. J. PIXE 6 (1–2) (1996) 375–394. [5] M. Lagunas-Solar, C. Solı´s, A. Mireles, C. Pin˜a, R.G. Flocchini, Int. J. PIXE 15 (3–4) (2005) 309–316. [6] C. Solı´s, M. Lagunas-Solar, B.P. Perley, C. Pin˜a, L.F. Aguilar, R.G. Flocchini, Nucl. Instr. and Meth. B 189 (2002) 77–80. [7] A. Mireles, Development and optimization of sample preparation and quantitative analysis of trace elements in fruits and vegetables with PIXE, Ph.D. Thesis, National Autonomous University of Mexico, 2004. (in Spanish) [8] Joint FAO/IAEA Expert Consultation, Validation of Analytical Methods for Food Control, Vienna, 2–4 December 1997. [9] FAO/WHO CODEX STAN 210-2001, Rev. 1, Maximum Levels for Lead, 2003. [10] S.A.E. Johansson, J.L. Campbell, PIXE: A Novel Technique For Elemental Analysis, Wiley & Sons, New York, 1988; See also C.L. Keen, T. Jue, C.D. Tran, J. Vogel, R. Gregory Downing, V. Yyengar, R.B. Rucker, J. Nutr. 133 (2003) 1574S.