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Development and Validation of a Microwave Digestion – FAAS Procedure for Cu, Mn and Zn Determination in Liver ARABELA UNTEA1,2, RODICA DIANA CRISTE2, LUMINITA VLADESCU1* 1 University of Bucharest, Department of Analytical Chemistry, Faculty of Chemistry, 2-14 Blv. Regina Elisabeta, 030018, Bucharest, Romania. 2 National Research and Development Institute for Biology and Animal Nutrition (INCDBNA) 1, Calea Bucuresti, 077015, Balotesti, Ilfov, Romania

In this paper the evaluation of the dry, wet and microwave digestion methods applied on certified bovine liver (CRM 12-02-01) has been performed. The microwave digestion method was selected and validated for the copper, manganese and zinc determination in liver samples by FAAS, because it is simple, rapid (digestion was done in 2.5 hours, with reduced chemical use) and reliable. Furthermore, microwave digestion method is characterized by a recovery between 98.13 and 99.09% and standard deviations between 0.22% and 4.13% for liver samples preparation and gave a precision close to 100%. The correlation coefficients, R2 of the linear regression equations exceeded 0.997 values, demonstrating a good linearity of the proposed method. The relative standard deviation for repeatability and reproducibility ranged from 0.80% to 1.96%. Liver trace elements analysis procedure using microwave digestion and FAAS determination, was applied for the Cu, Mn, and Zn determination in liver samples of recently weaned piglets. Linear correlation between the values determined by FAAS and those obtained by GF-AAS, both applied on the same samples proves the correctness of the proposed microwave digestion method used in the preparation of the piglet liver samples. Keywords: pig liver, digestion methods, FAAS, microelements determination

Elements such as iron, copper, manganese, zinc and selenium are essential nutrients with multiple biochemical functions in all living organisms and therefore they are the object of complex mineral feeding studies [1]. The need to determine the trace elements in biological samples required the development of many analytical methods to evaluate the mineralization procedures with certified reference materials (CRM) having a similar biological composition as the analysed sample [1, 2]. Evaluation of various mineralization methods in samples preparation for FAAS, GF-AAS or ICP-MS trace element analysis of vegetables and fruits [1], mushrooms [3], wheat [4, 5], human food [6 – 10], spices [11], tea [12], marine invertebrates [13], sardines [14], bovine liver [2], as well as of wool and human hair [15], samples has been performed. Some of the classical indicators of dietary minerals bioavailability are their concentrations in the liver, the main storage place of the organism [16, 17]. Hence, the exact determination of the trace elements content in the liver is of a major importance in the studies of mineral nutrition [17]. The mineralization is an essential step in the sampling for the liver sample analysis. Due to the complex organic matrix interferences may occur in the digestion procedures, mainly because of volatilization [18, 19] and contamination from reagents or laboratory glassware [6]. Few analytical data for cadmium, copper, lead and zinc concentrations determination by both ICP-MS and FAAS in the digests of the reference material samples (CRM 12-02-01, SRM 1577b) were reported [2, 20]. In this paper the evaluation of the dry, wet and microwave digestion methods applied on certified bovine liver (CRM – BCR 185R) has been performed. The developed microwave digestion method was selected and validated for the copper, manganese and zinc content determination in liver samples by FAAS. The validated microwave

digestion – FAAS determination procedure was applied to evaluate the Cu, Zn and Mn concentrations in liver of recently weaned piglets. Linear correlation between the values determined by FAAS and those obtained by GF-AAS, both applied on the same samples proves the correctness of the proposed microwave digestion method used in the preparation of the piglet liver samples. Experimental part Chemicals All the reagents used were supplied by Merck and were of analytical purity. Concentrated aqueous solutions 36% HCl (d = 1.19 kg . L-1), 65% HNO3 (d = 1.39 kg . L-1) and 30% H2O2 were used. The reference materials for AAS were CertiPUR® (Merck). Stock solutions Traceable to SRM from NIST: Zn(NO3)2, Mn(NO3)2 and Cu(NO3)2 are prepared in HNO3 0.5 mol . L-1 and contain 1,000 mg . L-1 ion of interest. For calibration of the atomic absorption spectrometer standard solutions were prepared using stock solutions and distilled water (Milli-Q Ultrapure, 18.2 MΩ/cm). The certified reference material Bovine liver (CRM – BCR 185R) was used. Equipment Instruments used were the following: - Atomic absorption spectrometer Thermo Electron – SOLAAR M6 Dual Zeeman Comfort (Cambridge, UK), with deuterium lamp for background correction and airacetylene flame has been used (Metrological certificate: 0170549 / 21.11.2007). The working parameters are listed in table 1. - Atomic absorption spectrometer Thermo Electron – SOLAAR M6 Dual Zeeman Comfort (Cambridge, UK), with Zeeman background correction and graphite furnace has been used (Metrological certificate: 0170549 / 21.11.2007). The working parameters are listed in table 1.

* email: [email protected] REV. CHIM. (Bucharest) ♦ 63 ♦ No. 4 ♦ 2012

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Table 1 WORKING PARAMETERS FOR Cu, Mn AND Zn DETERMINATION BY FAAS AND GF-AAS

- Digestion system FOSS Tecator Digestor Auto (Hilleroed, Denmark). - Microwave digestion system, remote temperature measurement, BERGHOF, Speedwave MWS-2 Comfort (Eningen, Germany). - Analytical scale Sartorius balance (Gottingen, Germany). - Stove BMT ECOCELL Blueline Comfort (Neuremberg, Germany). - Sand bath, Falc Instruments (Treviglio, Italy). - Water distiller Milli-Q Ultrapure Water Purification System, Millipore (Billerica, USA). - Calcination oven NABERTHERM Labotherm L15/11/ P320 Comfort (Bremen, Germany). Class A glassware was used for transvasation, dilution and storage. Digestion Methods Dry digestion – ten CRM liver samples of 0.5 ± 2 . 10–4 g each were weighed in porcelain crucibles, charred on a hot plate with stepwise increasing temperature up to 350°C for 4 h and then burned in the oven at 550oC for 16 h until a white ash. The ash treated with 2 mL 36% HCl and 5 mL 18% HCl in the porcelain crucibles were heated until dry. Each residue was dissolved in boiling deionized water, filtered through filter paper and passed quantitatively with deionized water into a 50 mL volumetric flasks filled to the mark with deionized water. A blank control was carried out in the same way using the reagent solutions alone. Wet digestion – ten CRM liver samples of 0.5 g each were weighed with ± 2 . 10–4 g accuracy. Each sample was quantitatively transferred with 16 mL digestion acid mixture 65% HNO3 : 30% H2O2 (3:1, v/v) in a 250 mL FOSS digester; the digesters were introduced in the digestor at 1300C for 30 min. After cooling at room temperature, 16 mL mixture of HNO3 : H2O2 (3:1, v/v) were again added and then the digesters reintroduced in the digestor for 30 min at 1300C. After full cooling each solution was filtered through filter paper in a 50 mL volumetric flask using deionized water and the flasks were filled to the mark with deionized water. A blank was carried out in the same way. Microwave digestion – ten CRM liver samples of 0.5 ± 2 . 10–4 g each were weighed. Each sample was quantitatively transferred with 7 mL mixture of 65% HNO3 : 30% H2O2 (5:2, v/v) into a 60 mL Teflon DAP – 60K vessels used for digestion. The vessels closed with safety lids and then with screw-in lids, were introduced inside the oven. Digestion conditions: 8 min at 1300C, 80% energy; 5 min at 1550C, 80% energy; 12 min at 170 0C, 80% energy. Maximal microwave oven power was 1000 W. After full cooling at room temperature the solutions were filtered through filter paper in a 50 mL volumetric flasks using boiling deionized water. After cooling under water flow, the flasks were filled 342

to the mark with deionized water. A blank digest was carried out in the same way. Validation of the microwave digestion – FAAS determination method The linearity, working area and sensitivity of the determination method have been evaluated using ten standard solutions with concentrations ranging between 0.4 - 4 ppm for copper, 0.2 - 2 ppm for manganese and 0.1 - 1 ppm for zinc. The detection limits, LOD and quantification limits, LOQ have been determined experimentally using series of 5 solutions, by diluting the standard solutions having the lowest concentration used to plot the calibration curves, 40 times for Cu (the lowest standard being 0.4 ppm), 20 times for Mn (the lowest standard being 0.2 ppm) and 10 times for Zn (the lowest standard being 0.1 ppm). LOD and LOQ were calculated by adding 3 x standard deviation, and 10 x standard deviation, respectively, with the average determined concentrations. The selectivity of the digestion method was evaluated by applying it on a certified reference material Bovine liver (CRM – BCR 185R) samples, with complex matrix and comparing the results with the reference values. The microwave digestion method above described in Digestion Methods section was used. In order to check the method accuracy, eight liver CRM reference samples with element content of 277 mg . L-1 for Cu, 138.6 mg . L-1 for Zn and 11.07 mg . L-1 for Mn have been made, each one being the object of 3 determinations. The accuracy was calculated with the equation: (X / μ) x 100, where X is the average of the eight determinations and μ is the reference value. The bias interval was calculated with the formula: [(X - μ) / μ] x 100. The precision of the microwave digestion method expressed by the repeatability (intraday assay) was determined using five repeated determinations for three samples containing: 0.2001 g, 0.3002 g and 0.4002 g CRM each respectively, by a single analyst, at short time intervals, using the same equipment and the same working technique. The precision of the microwave digestion method expressed by the reproducibility (interdays assay) was determined using eight repeated determinations for two samples containing 0.3002 g CRM each. The determinations were performed on 5 different days by two analysts, using different glassware, different reagents and the same working technique. Precision was calculated with the formula: (standard deviation/mean) x 100. The recovery was evaluated using eight CRM samples and another eight samples to which 1 mg . L-1 of Cu(II), 1.5 mg . L-1 of Mn(II) and 0.4 mg . L-1 of Zn(II) were added. The percentage of analyte recovery was calculated with the


REV. CHIM. (Bucharest) ♦ 63 ♦ No. 4 ♦2012

Table 2 VALIDATION OF THE TRACE ELEMENTS ANALISYS PROCEDURE BY MICROWAVE DIGESTION AND FAAS DETERMINATION USING CERTIFIED REFERENCE MATERIAL BOVINE LIVER (CRM – BCR 185R)

formula: (X1 - X) x 100 / X1, where X is the average of the eight sample concentrations without added analyte and X1 is the average of the eight sample concentrations with added analyte. Piglet liver samples for analytical procedure application A 3 weeks experiment on 8 piglets (Landrace × Large White), 13.5 ± 2.8 kg, males, castrated, half-brother piglets was conducted. The animals were housed in individual metabolic cages located in an experimental house of the Laboratory for animal physiology of the National Research and Development Institute for Biology and Animal Nutrition (INCDBNA), Romania, provided with natural light regime, 24°C temperature and optimal conditions of hygiene. No antibiotics (for prophylactic or therapeutic reasons) were given to the animals at either facility. Commencing at weaning piglets were fed ad libitum on compound feeds produced by INCDBNA according to the norms for the specific category of weaned piglets (10-30 kg) based on corn and soybean [21]. The weaning age of 28 days was considered as day 0. Four piglets were slaughtered at day 0 (28 days age), the others four at the day 22 (50 days age). From each piglet the liver samples were collected, dried at 65°C and ground. Results and discussions Selection of the method for digestion of liver sample In order to choose the optimal digestion method for microelements determination in piglet liver samples by FAAS, the results of the three digestion methods: dry digestion, wet digestion and microwave digestion, applied on certified bovine liver (CRM – BCR 185R) were compared. REV. CHIM. (Bucharest) ♦ 63 ♦ No. 4 ♦ 2012

The obtained results recommended the uses of microwave digestion method characterized by a recovery of 99.09, 98.74 and 98.18% for Cu, Mn and Zn, respectively, and by a standard deviation of 1.50% for Cu, 2.02% for Mn and 2.04% for Zn, respectively. For an economical evaluation of the three digestion methods the following parameters: quantities of reagents (expressed as volume of concentrated acids HCl, HNO3) used for sample treatment, time needed for preparation of samples and accuracy of the method were taken into account. To compare the time, only actual working time of the devices was considered (i.e., in dry digestion – roasting in the oven and dissolved on a sand bath; in wet digestion – hob heating time to reach boiling temperature and then cooling times, in each of the two phases of the method; in microwave digestion – crucibles heating in the oven and crucibles cooling). Based on results shown in figure 1 microwave method was chosen as optimal digestion method for microelements content determination in piglets liver samples by FAAS, because it required the shortest working time, an average reagent amount and ensure a good accuracy. Therefore, microwave method was validated and after that it was applied on piglet liver samples collected from mineral balance experiments. Validation of the liver trace elements analysis procedure using microwave digestion and FAAS determination For the validation of the liver trace elements analysis procedure using microwave digestion and FAAS determination, several parameters to characterize the


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Fig. 1 Evaluation of three digestion methods according with following parameters: volumes of concentrated reagents used for sample treatment, time needed for preparation of samples and accuracy of the method.

determination method (linearity, working domain, sensitivity, limit of detection and limit of quantification) and several validation parameters of the sample preparation method (selectivity, accuracy, precision, repeatability, reproducibility and recovery) were considered according to the international rules [22, 23]. The results are presented in table 2. The required performance criteria are: accuracy within the bias interval of -2 – 2%; maximal RSD value is set according to the analyte concentration, according to Horwitz equation: RSD = 2(1-0.5lgC); RSD =10.72 % for a concentration of 1 mg . L-1; the recovery values in the interval 80 – 120%. The obtained results were evaluated using the Student’s t test: the value given in the table [24] is t = 2.26 for p ≤ 0.05 and 9 degree of freedom and it is in all cases greater than the calculated ones (between 1.30 – 1.73), showing that the data belong to the same population of values as the reference value.

The relative standard deviation for repeatability and reproducibility ranged from 1.18 to 1.92%. The linearity of the method was checked in the range 0.4 – 4.4 mg . L-1 for Cu (II), 0.1 – 1.0 mg . L-1 for Zn (II) and 0.2 – 2.0 mg . L-1 for Mn (II). The correlation coefficients, R2 of the linear regression equations exceeded 0.997 values, demonstrating a good linearity of the proposed method. The results presented in table 2 show that the method is validated for precision and accuracy parameters. Applications The gastro-intestinal problems that appear in the piglets during weaning (such as diarrhoea) prompt the use of antibiotics and trace elements (i.e. copper, zinc) as growth promoters [25]. Knowing the mineral bioavailability of a diet for recently weaned piglets, in such a critical period is necessary. Trace elements analysis procedure using

Table 3 RESULTS OF Cu, Mn AND Zn CONCENTRATIONS DETERMINATION IN RECENTLY WEANED PIGLETS SAMPLES USING MICROWAVE DIGESTION COUPLED WITH FAAS AND GF-AAS DETERMINATION METHOD, RESPECTIVELY

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Fig. 2 Correlation plot of the copper concentrations (mg . kg-1) determined by FAAS and GF-AAS in microwave digests of the liver piglet samples: Day 0 (Y = -6.81 + 1.506 X; R2 = 0.8989); Day 22 (Y = -2.487 + 1.413 X; R2 = 0.8931).

Fig. 3 Correlation plot of the manganese concentrations (mg . kg-1) determined by FAAS and GF-AAS in microwave digests of the liver piglet samples: Day 0 (Y = 3.79 + 1.008 X; R2 = 0.970); Day 22 (Y = -30.286 + 3.998 X; R2 = 0.9562).

Fig. 4 Correlation plot of the zinc concentrations (mg . Kg-1) determined by FAAS and GF-AAS in microwave digests of the liver piglet samples: Day 0 (Y = -162.62 + 1.896 X; R2 = 0.8367); Day 22 (Y = -24.872 + 1.1725 X; R2 = 0.6790)

microwave digestion and FAAS determination, was applied for the Cu, Mn and Zn determination in liver samples of recently wanted piglets. Determination of the Cu, Mn and Zn concentrations in the liver of piglets sacrificed at the beginning of a nutrition experiment (day 0; 28 days age) and at the end of the experiment (day 22; 50 days age) were made. The obtained results are presented in table 3 and figures 2 – 4. As shown in figures 2 – 4, there is a good correlation between the values determined by the proposed method and those obtained by GF-AAS, which proves the correctness of the microwave digestion method used in the liver samples preparation. Comparing the data in figures 2 – 4 it is noted that after 22 days of weaning only the manganese content in the piglet’s liver increased, while copper and zinc concentrations decreased. REV. CHIM. (Bucharest) ♦ 63 ♦ No. 4 ♦ 2012

Conclusions Three preparation methods of the liver sample: dry, wet and microwave digestion methods were applied on certified bovine liver (CRM – BCR 185R). The results of analysis were compared in order to select the best one to be used for copper, manganese and zinc determination in liver samples by FAAS. The most efficient is the microwave method because it is a closed system with considerably reduced potential hazards and chemical use (7 mL concentrated acid solutions compared to 12 mL in wet digestion). It is also economic, efficient and rapid (digestion was done in 2.5 h, compared to 18 h for dry digestion and 6 h for wet digestion) and it is reliable. Furthermore, microwave digestion method proposed was characterized by recovery, standard deviations and precision. For the caracterization of the trace elements analysis method using microwave digestion and FAAS determination, several


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parameters for the determination method (linearity, working domain, sensitivity, limit of detection and limit of quantification) and several validation parameters of the sample preparation method (selectivity, accuracy, precision, repeatability, reproducibility and recovery) were determined according to the international rules. The correlation coefficients, R2 exceeded 0.997 values, demonstrating a good linearity of proposed method. The relative standard deviation for repeatability and reproducibility ranged from 1.18 to 1.92%. Trace elements analysis procedure using microwave digestion and FAAS determination, was applied for the Cu, Mn, and Zn content determination in liver samples of recently weaned piglets. Linear correlation between the values determined by FAAS and those obtained by GF-AAS, both applied on the same samples proves the correctness of the proposed microwave digestion method used in the preparation of the piglet liver samples. References 1. DEMIREL, S., TUZEN, M., SARACOGLU, S., SOYLAK, M., J. Hazard. Mater., 152, 2008, p. 1020. 2. GERBER, N., BROGIOLI, R., HATTENDORF, B., SCHEEDER, M. R. L., WENK, C., GUNTHER, D., Animal, 3, 2009, p. 166. 3. KUCAK, A., BLANUSA, M., Arh. Hig. Rada Toksikol., 49, 1998, p. 335. 4. ADAMS, M. L., CHAUDRI, A. M., ROUSSEAU, I., MCGRATH, S. P., Int. J. Environ. Anal. Chem., 83, 2003, p. 307. 5. DONER, G., AKMAN, S., Anal. Lett., 33, 2000, p. 3333. 6. ZUDOWSKA, J., BIZIUK, M., J. Food Sci., 73, 2008, p. 21. 7. SOYLAK, M., TUZEN, M., NARIN, I., and SARI, H., J. Food Drug Anal., 12, 2004, p. 254. 8. DA-COL, J. A., DOMENE, S. M. A., PEREIRA-FILH, E. R., Food Anal. Methods., 2, 2009, p. 110.

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