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Radionuclide x-ray fluorescence analysis of selected elements in agrimony tea samples with preconcentration

Radionuclide x-ray fluorescence analysis of selected elements in agrimony tea samples with... Keywords Kúcové slová: INTRODUCTION Medicinal plants can be used directly to treat various diseases: they can be used as raw material for acquiring their active constituents or for producing herbal drugs. Although the efficacy of medicinal plants for curative purposes is often assigned to their organic constituents (flavonoids, vitamins, glycosides, etc.) it has been established that there exists a relationship between the chelating of metals and some chemotherapeutic agents (Lamari et al., 2008). Trace elements play a very important role in the formation of the active chemical constituents present in medicinal plants and are responsible for their curative as well as toxic proprieties. The role of inorganic elements in plant metabolism has long been established, but the effect and influences of these elements on the administration of medicinal plants have received relatively little attention (Desideri et al., 2010). On the other hand, health research continues to implicate metals * janosova@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae as a possible cause of adverse human health effects based on the fact that they are actively involved in many biological processes (Okatch et al., 2012). Metals are non-biodegradable and are cumulative in nature rendering them as persistent pollutants. Several researchers have documented cases in which the metal content in plants is related to toxicity (Olowoyo et al., 2012). Asian traditional medicines have been reported to contain high levels of heavy metals such as arsenic, lead and mercury (Giacomino et al., 2011). Different techniques were applied to determine the elemental content in the medicinal plant materials and their infusions, e.g. neutron activation analysis (Lokhande et al., 2009), inductively coupled plasma atomic emission or mass spectrometry (Wang et al. 2012; Tokalioglu, 2012), atomic absorption spectrometry (Rego et al., 2012), and various types of X-ray fluorescence spectrometry (XRFS) (Khuder et al., 2009; Desideri et al., 2010). The advantage of XRFS involves the polycomponent character of the analysis: the method is non-destructive, fast and accurate. The reduction of detection limit can be achieved by sample preparation. This method enables the determination of total elements content without the need of knowing their chemical state (Sýkorová et al., 2009). Analysing pharmaceuticals and plant drugs with XRFS method provides qualitative and also quantitative information of the present metal ions at very low concentration levels (ppm), either single ion or a mixture of metal ions as in most cases it may be. (Krishna Murty et al., 2005). Direct measurements of metals in biological samples by XRFS without decomposition of the matrix could not be always possible because of matrix interferences and very low concentration of metal. Coupling a preconcentration step to these methods could reduce the matrix effect, increase the sensitivity, improve detection limits and provide accurate results (Jánosová et al., 2010). The usually applied preconcentration techniques are solvent extraction, cloud point extraction, solid phase extraction based on adsorption, electrodeposition, flotation, co-precipitation and ion exchange among others. When the preconcentration based on membrane filtration is used, a chelating agent is added to an aqueous sample containing the metal ion. In practice, the most frequently used chelating agents are diethyldithiocarbamate (Krasnodebska-Ostega et al., 2013), ammonium pyrrolidine dithiocarbamate (Giakisikli & Anthemidis, 2013), dithione and 8-hydroxyquinoline (Zhao et al., 2012). In this work, the preconcentration based on membrane filtration is used because of its simplicity and rapidity. After the addition of the precipitating agent to an aqueous sample containing the metal ion, the created metal precipitates are collected on the surface of the membrane by filtration under the vacuum. This filter paper with adsorbed complexes is analysed by the XRFS method. Two precipitation agents, ammonium diethyldithiocarbamate (ADDC) and thioacetamide (TAA), were investigated. Precipitates of metals were collected on membrane filters Pragopor 4, diameter 35 mm, size of the pore 3 µm, active area 4.91 cm2 (Pragochema) using filtration device constructed in the Department of Pharmaceutical Analysis and Nuclear Pharmacy (Comenius University, Bratislava, Slovak Republic). Reagents and solutions All the reagents were of analytical grade, and deionised water was used for the preparation of all aqueous solutions of TAA (Merck Darmstadt, Germany) and ADDC (Sigma Aldrich Chemie Steinheim, Switzerland). For the pH adjustment, solutions of NaOH 1.0 mol l-1 (Merck Bratislava, Slovak Republic) and HCl 35 % (Elab Nitra, Slovak Republic) were prepared. Standard solutions of Mn, Fe, Co, Ni, Cu, Zn, Hg and Pb (1 mg ml-1) were obtained from Slovak Institute of Metrology Bratislava, Slovak Republic. Plant samples for the analysis: agrimony loose tea (Agrimonia eupatoria L.) "Repíkový caj" (Fytopharma Malacky, Slovak Republic). Procedure Solid sample preparation: The agrimony tea sample of "Repíkový caj" was weighed, ground and homogenised and then pressed into tablets (20 mm diameter, 0.3000 g) with the hydraulic press under the pressure of 10 MPa. Tablets were analysed by XRFS. Infusion preparation: To 1 g of the agrimony tea sample, 250 ml of hot solution (90 °C) with optimal pH (3.5, 8.5 or 11.5) was added. After 15 min, the mixture was filtrated. To 30 ml of the infusion, 2.6 ml of TAA or 500 µl of 2 % solution of ADDC was added. Standard addition of elements: To 1 g of pulverised sample in the mortar, 30 µl (i.e. 30 µg) from stock solution of Mn, Ni, Co, Pb and Hg; 50 µl (i.e. 50 µg) from stock solution of Cu, 100 µl (i.e. 100 µg) from stock solution of Zn and 200 µl (i.e. 200 µg) from stock solution of Fe were added. The mixture was dried at room temperature and homogenised and then pressed into tablets of 0.3000 g with the hydraulic press under the pressure of 10 MPa. Tablets were analysed by XRFS. Preparation of solutions: 4 % solution of TAA and 2 % solution of ADDC were prepared. An amount of 500 ml of deionised water was adjusted to pH values 3.5, 8.5 and 11.5 by the addition of NaOH and HCl (1 mol l-1 and 0.1 mol l-1, respectively) in order to create the best pH conditions for the precipitation of the elements. Standard solutions of analysed elements were prepared from the stock solutions of each analysed elements (1 mg ml-1) where 100 µl of the standard solution corresponded to 10 µg of the element. Calibration curves for TAA: To 30 ml of the solution with pH 8.5 (optimal for the elements Fe, Zn, Ni and Hg) or with pH 11.5 (optimal for elements Mn, Co, Cu and Pb), 0, 100, 200, 300, 500 and 1000 µl of each element (corresponding to 0, 10, 20, 30, 50 and 100 µg of element) and 2.6 ml of 4 % solution of TAA were added. EXPERIMENTAL PART Instrumentation The measurement of the characteristic and L-fluorescent radiation intensity of elements Mn, Fe, Co, Cu, Zn, Pb, Ni and Hg was carried out with Si/Li semiconductor detector (ÚJV ez u Prahy, Czech Republic), thickness of beryllium window 0.25 mm, diameter of beryllium window 5 mm connected with multichannel analyzer ORTEC® and the signal was evaluated by software MAESTRO-32®. In order to excite X-rays of determined elements, radionuclide source 238Pu in the form of point disk was employed (made by Amersham, activity 880 MBq, energy 12­ 22 keV, half-life 86.4 years). All measurements were performed in noncoaxial geometrical arrangements of source, sample and detector and the acquisition time was 2000 s. The sample acidity was measured by means of pH-meter HI 9321 (Hanna Instruments Bratislava, Slovak Republic). Tablets were pressed on Carl Zeiss Jena 036-76. Calibration curves for ADDC: To 30 ml of the solution with pH 3.5 (optimal for all elements), 0, 100, 200, 300, 500 and 1000 µl of each element (corresponding to 0, 10, 20, 30, 50 and 100 µg of element) and 500 µl of 2 % solution of ADDC were added. Then the mixture was stirred in the beaker for 5 min, next it was heated at 60 °C for 5 min in water bath and cooled down (10 min in ice bath). Then the mixture was filtrated through the membrane filter Pragopor 4 using the filtration device. The filter with collected precipitates was dried at the room temperature. Dried filters were analysed by XRFS. Using the same procedure and under the same conditions, a blank solution was prepared and analysed. RESULTS AND DISCUSSION This work was aimed to determine the presence of the elements Mn, Fe, Co, Ni, Cu, Zn, Hg and Pb by XRFS in the agrimony tea ­ in original solid form and its infusion after the preconcentration of the elements from the liquid phase by precipitation with TAA and ADDC. Firstly the solid form samples were analysed. After the homogenisation, the powder was pressed into the tablets of constant parameters and these tablets were analysed with XRFS. The results are given in Table 1. The method of standard addition was applied to calculate the content of analysed elements in the sample. According to the section Procedure, the chosen amounts of each element were added to the agrimony sample and after homogenisation the pressed tablets were analysed with XRFS. Obtained counts are given in Table 2. and spectrum of the sample and the sample with added standards are presented in Fig. 1. Detection limits (LD ) for each element present in the plant matrix were calculated as a ratio of 3 ( = background noise) from the counts of non-analytical signal and counts corresponding to 1 g of element. Detection limits are presented in Table 3. Other precision parameters such as range (R), standard deviation calculated by range (SR), relative standard deviation for the area of peak in % (sr) and confidence interval (L1,2) were calculated and resulting data are given in Tables 1 and 2. The values of relative standard deviation were Table 1. Counts corresponding to selected elements after the interaction of radionuclide source 238Pu with the agrimony sample tablet element Sample No. 1. 2. 3. Average R sR sr L1,2 background 532 569 564 555 37 21.86 3.94 555±48 304 1027 1051 1054 1044 27 15.95 1.53 1044±35 500 Mn Fe Co 592 583 613 596 30 17.72 2.97 596±39 466 Ni ­1 Cu 875 835 878 863 43 25.40 2.94 863±56 523 Zn 1368 1381 1433 1394 65 38.40 2.75 1384±85 774 Hg 489 505 517 504 28 16.54 3.28 504±37 558 Pb 513 506 535 518 29 17.13 3.31 518±38 542 Counts · 2000 s for tablets of "Repíkový caj" 408 415 386 403 29 17.13 4.25 403±38 355 Table 2. Counts corresponding to selected elements after the interaction of radionuclide source 238Pu with the tablet with standard addition of elements element Sample No. 1. 2. 3. Average R sR sr L1,2 1069 1048 1042 1053 27 15.95 1.51 1053±35 4726 4831 4795 4784 105 62.03 1.30 4784±137 Mn Fe Co 1188 1268 1179 1212 89 52.58 4.34 1212±116 Ni 1139 1115 1168 1141 53 31.31 2.74 1141±69 Cu 2304 2234 2304 2281 70 41.36 1.81 2281±91 Zn 4713 4834 4609 4719 225 132.93 2.82 4719±293 Hg 1184 1212 1207 1201 28 16.54 1.38 1201±37 Pb 1245 1319 1266 1277 74 43.72 3.42 1277±96 Counts · 2000 s­1 for tablets of "Repíkový caj" solid sample sample 2000 K Zn 1500 K Fe K Ni 1000 K Mn K Co 500 K Hg K Cu K Pb standard addition Figure 1. Spectrum of a tablet of agrimony tea "Repíkový caj" and the tablet of the sample with standard addition of elements after an interaction of the radionuclide source 238Pu. less than 4.00 %, which confirms good reproducibility of the method and indicates suitability of the method for the practical evaluation of the plant preparations. The resulting contents of the selected metal elements in the agrimony tea are presented in Table 3. The content of Fe was the highest among the analysed elements. The contents of Hg and Pb were under their detection limits that confirms the good quality of the plant preparation and also negligible contamination of the processed plant Agrimonia eupatoria L. Subsequently, the liquid samples ­ infusions were analysed. Before the analysis with XRFS method, the elements were preconcentrated by precipitation with TAA and ADDC reagents. Created precipitations were filtrated through Pragopor 4 filter and the enriched filters were analysed with XRFS. Optimum pH for the multi-element analysis was chosen according to a previous study (Jánosová et al., 2010). Regarding the TAA method pH 8.5 was optimum for Fe, Ni, Zn and Hg and 11.5 for Mn, Co, Cu and Pb. When using the ADDC method, pH 3.5 was optimum for all elements. These conditions were proved to be the best for the precipitation of selected elements resulting in the lowest detection limits. First of all, calibration curves for TAA and ADDC method were constructed. The amount of added elements was in range of 0­100 µg. The measured counts corresponded to the content of metal ions collected on the membrane filter in the form of precipitations. The linear dependence of measured signals on content of element was confirmed by calculated correlation coefficients. Parameters a and b of analytical curves in the Table 3. Detection limits and content of the elements in the sample of "Repíkový caj" element LD (µg/g) Content (µg/g) Mn 3.13 15.12 Fe 3.58 29.09 Co 3.17 6.33 Ni 2.32 < 2.32 Cu 2.43 11.99 Zn 2.50 18.65 Hg 3.05 < 3.05 Pb 2.77 < 2.77 Table 4. Parameters of the calibration curve in form of y = a + bx and correlation coefficient r for selected elements for TAA and ADDC method element a b r a b r 32 Mn 60.05 -179.85 0.9971 40.28 -11.48 0.9953 Fe 111.67 -173.49 0.9982 81.12 -21.28 0.9980 Co 46.88 -63.31 0.9960 23.13 -77.88 0.9942 Ni TAA 85.66 -92.12 0.9982 ADDC 28.54 74.98 0.9854 67.717 -151.25 0.9987 102.93 -282.02 0.9981 56.50 -105.98 0.9975 66.62 -95.67 0.9984 113.36 180.67 0.9925 102.85 -254.13 0.9974 97.46 -222.91 0.9981 106.29 89.29 0.9982 Cu Zn Hg Pb form of y = a + bx and r as correlation coefficient are given in Table 4. All dependencies had linear character in chosen interval 0­100 g. Correlation coefficient was in range of 0.9925­ 0.9982 for TAA method and 0.9854­0.9987 for ADDC method. The infusions were prepared according to the section Procedure from the solution with optimum pH 3.5 for the ADDC method and optimum pH 8.5 and 11.5 for the TAA method. Also the blank test was performed. The examples of spectrum are given in Figs. 2 and 3. Calculated detection limits along with the evaluation parameters and the content of the selected elements and using TAA and ADDC method are given in Tables 5 and 6, respectively. Using the TAA method, Fe (6.25 g/g), Mn (5.25 g/g) and Cu (5.33 g/g) were determined. The contents of the other elements were under their detection limits. Using the ADDC method, Fe (6.08 g/g) and Cu (5.25 g/g) were determined. The contents of all other elements were under their detection limits. The values of relative standard deviation were under 3.00 %, which confirms good reproducibility of the method and indicates suitability of the method for the practical evaluation of the plant infusions. The detection limits of the selected elements obtained after their preconcentration with TAA were compared with the ones obtained after the preconcentration with ADDC. It was found out that TAA method combined with the XRFS analysis is the more advantageous method for the preconcentration of the selected elements Mn, Fe, Co, Ni, Cu, Hg and Pb in the plant matrices such as agrimony tea. The only exception is Zn, whose detection limits are lower in the plant matrix studied when ADDC reagent is used for the preconcentration before the XRFS analysis. However, the difference between the detection limits obtained after using the TAA method (0.62 µg/g) and the ADDC method (0.59 µg/g) was not significant. On the other hand, capability to directly determine all selected elements in one run with relatively good sensitivity is a great advantage of the ADDC method. It is far less effective when using the TAA method. In such a case, detection limits of the majority of elements obtained by the ADDC method in one run are better than the one of the TAA method. Finally, the content of the analysed elements in the solid plant sample of agrimony tea was compared with their content in the prepared infusion using the TAA and ADDC methods to estimate the percentage of the amount of the elements extracted into the infusion. As illustrated in Table 7, the content of the elements in the infusion is significantly lower than in the original solid plant sample. From the elements originally found in the solid sample ­ Mn, Fe, Co, Cu and Zn (the contents of Ni, Hg and Pb were under their detection limits) ­ only the presence of Mn, Fe and Cu were determined in the infusion. The best extracted element was Cu (44.45 %) when using TAA method. This low extraction can be explained by the different types of bounds between the metal elements and the organic compounds of the plant matrix which are not broken by this type of sample treatment (steeping in the hot water). The detection limits of the analysed elements obtained using the TAA and ADDC methods of preconcentration for XRFS analysis were significantly decreased (in one order) than those in the solid plant sample of agrimony tea. Therefore, these methods can be successfully applied in the preconcentration for XRFS analysis of medicinal plant infusions. Table 5. Content of the selected elements in the infusion using the TAA method pH element R sR sr L1,2 LD (µg/g) Content (µg/g) Fe 13 7.68 2.99 257±17 0.36 6.25 Ni 4 2.36 2.41 98±5 0.33 < 0.33 8.5 Zn 23 13.59 2.81 484±30 0.62 < 0.62 Hg 4 2.36 2.48 95±5 0.29 < 0.29 Mn 8 4.73 2.67 177±10 0.58 5.25 Co 4 2.36 2.43 97±5 0.60 < 0.60 11.5 Cu 9 5.32 2.51 212±12 0.32 5.33 Pb 5 2.96 2.55 116±7 0.30 < 0.30 Table 6. Content of the selected elements in the infusion using the ADDC method pH element R sR sr L1,2 LD (µg/g) Content (µg/g) Mn 8 4.73 2.90 163±10 0.85 < 0.85 Fe 10 6.16 2.59 238±13 0.50 6.08 Co 4 2.36 2.17 109±5 1.34 < 1.34 Ni 4 2.36 2.59 91±5 1.00 < 1.00 3.5 Cu 9 5.32 2.58 206±12 0.57 5.25 Zn 18 10.63 2.53 421±23 0.59 < 0.59 Hg 4 2.36 2.78 85±5 0.49 < 0.49 Pb 5 2.96 2.81 105±7 0.47 < 0.47 33 Table 7. Content of the elements in solid sample and in the infusion of agrimony tea "Repíkový caj" and the % of the amount of elements extracted into the infusion Element Content (µg/g) Solid sample ADDC method infusion % of extracted elements TAA method infusion % of passed elements Mn 15.12 < 0.85 5.25 34.72 Fe 29.09 6.08 20.90 6.25 21.49 Co 6.33 < 1.34 < 0.60 Ni < 2.32 < 1.00 < 0.33 Cu 11.99 5.25 43.79 5.33 44.45 Zn 18.65 < 0.59 < 0.62 Hg < 3.05 < 0.49 < 0.29 Pb < 2.77 < 0.47 < 0.30 - CONCLUSION In this work, the contents of the selected elements were studied in the samples of loose tea of agrimony "Repíkový caj" and in its infusion. The preconcentration of the elements Mn, Fe, Co, Ni, Cu, Zn, Hg and Pb by complexation with TAA and ADDC for the XRFS method was proposed. The investigated ions were collected on the cellulose nitrate membrane filter as their complexes after the reaction with TAA or ADDC and the filter was analysed by XRFS. Calibration curve proved the linear dependence of intensity of the fluorescence radiation on the content of the metal in the sample. This method was successfully applied to the determination of the selected metals in plant matrix ­ agrimony tea. Through the results obtained in this study, it is possible to conclude that the TAA and ADDC preconcentrations with precipitation, filtration on cellulose membrane and analysis of these enriched filters by XRFS significantly decrease the detection limits compared with those in solid plant samples of agrimony tea. Other benefits of the proposed TAA method are apparent from its comparison with the ADDC method. As for the detection limits, the TAA method is preferred for the majority of the elements (except for Zn, where the ADDC method was better though not significantly) when considering analysis according to the optimum pH of the analysed sample (i.e. different pH are optimum for different metals). The advantage of this procedure is the multi-element character of the analysis, speed and the lack of need to complicate pretreatment of the sample. This method can be applied in the monitoring of metals content in solid and also liquid samples of medicinal plant and other pharmaceutical plant preparations as infusions. Acknowledgement: This work was supported by the Slovak Grant Agency VEGA (project No. 1/0664/12) TAA method K Fe K Cu pH 8.5 pH 11.5 K Mn K Cu Figure 2. Spectrum of an infusion of agrimony tea "Repíkový caj" when using the TAA method at pH 8.5 and 11.5 after an interaction of the radionuclide source 238Pu. Abbreviations: TAA - thioacetamide ADDC method K Fe K Cu pH 3.5 K Fe K Cu Figure 3. Spectrum of an infusion of agrimony tea "Repíkový caj" when using the ADDC method at pH 3.5 after an interaction of the radionuclide source 238Pu. Abbreviations: ADDC - ammonium diethyldithiocarbamate REFERENCES [1] DESIDERI D, MELI MA, ROSELLI C. Determination of essential and non-essential elements in some medicinal plants by polarised X ray fluorescence spectrometer (EDPXRF). Microchem J. 2010; 95: 174-180 [2] GIACOMINO A, ABOLLINO O, MALANDRINO M, KARTHIK M, MURUGESAN V. Determination and assessment of the contents of essential and potentially toxic elements in Ayurvedic medicine formulations by inductively coupled plasma-optical emission spektrometry. Microchem J. 2011; 99: 2-6 [3] GIAKISIKLI G, ANTHEMIDIS AN. Magnetic materials as sorbents for metal/metalloid preconcentration and/or separation. A review. Anal. Chim. Acta. 2013; 789: 1-16 [4] JÁNOSOVÁ V, SÝKOROVÁ M, STROFFEKOVÁ O, HAVRÁNEK E. Determination of selected elements by radionuclide X-ray fluorescence spectrometry in liquid drug samples after the preconcentration with thioacetamide. J Anal Chem. 2010; 65(1): 56-63 [5] KHUDER A, SAWAN MKH, KARJOU J, RAZOUK AK. Determination of trace elements in Syrian medicinal plants and their infusions by energy dispersive X-ray fluorescence and total reflection X-ray fluorescence spektrometry. Spectrochim Acta B. 2009; 64: 721-725 [6] KRASNODEBSKA-OSTEGA B, SADOWSKA M, PIOTROWSKA K, WOJDA M. Thallium (III) determination in the Baltic seawater samples by ICP MS after preconcentration on SGX C18 modified with DDTC. TALANTA. 2013; 112: 73-79 [7] KRISHNA MURTY ASR, KULSHRESTA UC, NAGESWARA RAO T, KUMAR TALLURI MVN. Determination in heavy metals in selected drug substances by inductively coupled plasma ­ mass spectrometry. Indian J Chem Techn. 2005; 12: 229-231 [8] LAMARI Z, LANDSBERGER S, BRAISTED J, NEGGACHE H, LARBI R. Trace element content of medicinal plants from Algeria. J Radioanal Nucl Chem. 2008; 276 (1): 95­99 [9] LOKHANDE RS, SINGARE PU, ANDHELE ML, ACHARYA R, NAIR AGC, REDDY AVR. Study of some Ayurvedic Indian medicinal plants for the essential trace elemental contents by instrumental neutron activation analysis and atomic absorption spectroscopy techniques. Radiochim Acta. 2009; 97(6): 325-331 [10] OLOWOYO JO, OKEDEYI OO, MKOLO NM, LION GN, MDAKANE STR. Uptake and translocation of heavy metals by medicinal plants growing around a waste dump site in Pretoria, South Africa. S Afr J Bot. 2012; 78: 116-121 [11] OKATCH H, NGWENYA B, RALETAMO KM, MAROBELA KA. Determination of potentially toxic heavy metals in traditionally used medicinal plants for HIV/AIDS opportunistic infections in Ngamiland District in Northern Botswana. Anal Chim Acta. 2012; 790: 42-48 [12] REGO JF, VIRGILIO A, NOBREGA JA, NETO JAG. Determination of leadinmedicinal plants by high-resolution continuum source graphitefurnace atomic absorption spectrometry using direct solid sampling. Talanta. 2012; 100: 21-26 [13] SÝKOROVÁ M, JÁNOSOVÁ V, STROFFEKOVÁ O, HAVRÁNEK E, KOSTÁLOVÁ D, RACKOVÁ L. Determination of selected elements by XRF and total phenolics in leaves and crude methanol extract of leaves of Arctostaphylos uva ursi. Acta Facult Pharm Univ Comenianae. 2009; 56: 136-145 [14] TOKALIOGLU S. Determination of trace elements in commonly consumed medicinal herbs by ICP-MS and multivariate analysis. Food Chem. 2012; 134: 2504-2508 [15] WANG DM, FENG J, QIN FJ, MO YS. Determination of six heavy metal elements in Zanthoxylum nitidum in twelve habitats of guangxi by ICP-AES. J Chin Med Mat. 2012; 35(3): 366-368 [16] ZHAO LL, ZHONG SX, FANG KM, QIAN ZS, CHEN JR. De- termination of cadmium(II), cobalt(II), nickel(II), lead(II), zinc(II), and copper(II) in water samples using dual-cloud point extraction and inductively coupled plasma emission spektrometry. J Hazard Mat. 2012; 239: 206-212 PharmDr. Veronika Mikusová, PhD. Comenius University in Bratislava Faculty of Pharmacy Kalinciakova 8 832 32 Bratislava Slovak Republic janosova@fpharm.uniba.sk http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Pharmaceuticae Universitatis Comenianae de Gruyter

Radionuclide x-ray fluorescence analysis of selected elements in agrimony tea samples with preconcentration

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de Gruyter
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10.2478/afpuc-2013-0020
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Abstract

Keywords Kúcové slová: INTRODUCTION Medicinal plants can be used directly to treat various diseases: they can be used as raw material for acquiring their active constituents or for producing herbal drugs. Although the efficacy of medicinal plants for curative purposes is often assigned to their organic constituents (flavonoids, vitamins, glycosides, etc.) it has been established that there exists a relationship between the chelating of metals and some chemotherapeutic agents (Lamari et al., 2008). Trace elements play a very important role in the formation of the active chemical constituents present in medicinal plants and are responsible for their curative as well as toxic proprieties. The role of inorganic elements in plant metabolism has long been established, but the effect and influences of these elements on the administration of medicinal plants have received relatively little attention (Desideri et al., 2010). On the other hand, health research continues to implicate metals * janosova@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae as a possible cause of adverse human health effects based on the fact that they are actively involved in many biological processes (Okatch et al., 2012). Metals are non-biodegradable and are cumulative in nature rendering them as persistent pollutants. Several researchers have documented cases in which the metal content in plants is related to toxicity (Olowoyo et al., 2012). Asian traditional medicines have been reported to contain high levels of heavy metals such as arsenic, lead and mercury (Giacomino et al., 2011). Different techniques were applied to determine the elemental content in the medicinal plant materials and their infusions, e.g. neutron activation analysis (Lokhande et al., 2009), inductively coupled plasma atomic emission or mass spectrometry (Wang et al. 2012; Tokalioglu, 2012), atomic absorption spectrometry (Rego et al., 2012), and various types of X-ray fluorescence spectrometry (XRFS) (Khuder et al., 2009; Desideri et al., 2010). The advantage of XRFS involves the polycomponent character of the analysis: the method is non-destructive, fast and accurate. The reduction of detection limit can be achieved by sample preparation. This method enables the determination of total elements content without the need of knowing their chemical state (Sýkorová et al., 2009). Analysing pharmaceuticals and plant drugs with XRFS method provides qualitative and also quantitative information of the present metal ions at very low concentration levels (ppm), either single ion or a mixture of metal ions as in most cases it may be. (Krishna Murty et al., 2005). Direct measurements of metals in biological samples by XRFS without decomposition of the matrix could not be always possible because of matrix interferences and very low concentration of metal. Coupling a preconcentration step to these methods could reduce the matrix effect, increase the sensitivity, improve detection limits and provide accurate results (Jánosová et al., 2010). The usually applied preconcentration techniques are solvent extraction, cloud point extraction, solid phase extraction based on adsorption, electrodeposition, flotation, co-precipitation and ion exchange among others. When the preconcentration based on membrane filtration is used, a chelating agent is added to an aqueous sample containing the metal ion. In practice, the most frequently used chelating agents are diethyldithiocarbamate (Krasnodebska-Ostega et al., 2013), ammonium pyrrolidine dithiocarbamate (Giakisikli & Anthemidis, 2013), dithione and 8-hydroxyquinoline (Zhao et al., 2012). In this work, the preconcentration based on membrane filtration is used because of its simplicity and rapidity. After the addition of the precipitating agent to an aqueous sample containing the metal ion, the created metal precipitates are collected on the surface of the membrane by filtration under the vacuum. This filter paper with adsorbed complexes is analysed by the XRFS method. Two precipitation agents, ammonium diethyldithiocarbamate (ADDC) and thioacetamide (TAA), were investigated. Precipitates of metals were collected on membrane filters Pragopor 4, diameter 35 mm, size of the pore 3 µm, active area 4.91 cm2 (Pragochema) using filtration device constructed in the Department of Pharmaceutical Analysis and Nuclear Pharmacy (Comenius University, Bratislava, Slovak Republic). Reagents and solutions All the reagents were of analytical grade, and deionised water was used for the preparation of all aqueous solutions of TAA (Merck Darmstadt, Germany) and ADDC (Sigma Aldrich Chemie Steinheim, Switzerland). For the pH adjustment, solutions of NaOH 1.0 mol l-1 (Merck Bratislava, Slovak Republic) and HCl 35 % (Elab Nitra, Slovak Republic) were prepared. Standard solutions of Mn, Fe, Co, Ni, Cu, Zn, Hg and Pb (1 mg ml-1) were obtained from Slovak Institute of Metrology Bratislava, Slovak Republic. Plant samples for the analysis: agrimony loose tea (Agrimonia eupatoria L.) "Repíkový caj" (Fytopharma Malacky, Slovak Republic). Procedure Solid sample preparation: The agrimony tea sample of "Repíkový caj" was weighed, ground and homogenised and then pressed into tablets (20 mm diameter, 0.3000 g) with the hydraulic press under the pressure of 10 MPa. Tablets were analysed by XRFS. Infusion preparation: To 1 g of the agrimony tea sample, 250 ml of hot solution (90 °C) with optimal pH (3.5, 8.5 or 11.5) was added. After 15 min, the mixture was filtrated. To 30 ml of the infusion, 2.6 ml of TAA or 500 µl of 2 % solution of ADDC was added. Standard addition of elements: To 1 g of pulverised sample in the mortar, 30 µl (i.e. 30 µg) from stock solution of Mn, Ni, Co, Pb and Hg; 50 µl (i.e. 50 µg) from stock solution of Cu, 100 µl (i.e. 100 µg) from stock solution of Zn and 200 µl (i.e. 200 µg) from stock solution of Fe were added. The mixture was dried at room temperature and homogenised and then pressed into tablets of 0.3000 g with the hydraulic press under the pressure of 10 MPa. Tablets were analysed by XRFS. Preparation of solutions: 4 % solution of TAA and 2 % solution of ADDC were prepared. An amount of 500 ml of deionised water was adjusted to pH values 3.5, 8.5 and 11.5 by the addition of NaOH and HCl (1 mol l-1 and 0.1 mol l-1, respectively) in order to create the best pH conditions for the precipitation of the elements. Standard solutions of analysed elements were prepared from the stock solutions of each analysed elements (1 mg ml-1) where 100 µl of the standard solution corresponded to 10 µg of the element. Calibration curves for TAA: To 30 ml of the solution with pH 8.5 (optimal for the elements Fe, Zn, Ni and Hg) or with pH 11.5 (optimal for elements Mn, Co, Cu and Pb), 0, 100, 200, 300, 500 and 1000 µl of each element (corresponding to 0, 10, 20, 30, 50 and 100 µg of element) and 2.6 ml of 4 % solution of TAA were added. EXPERIMENTAL PART Instrumentation The measurement of the characteristic and L-fluorescent radiation intensity of elements Mn, Fe, Co, Cu, Zn, Pb, Ni and Hg was carried out with Si/Li semiconductor detector (ÚJV ez u Prahy, Czech Republic), thickness of beryllium window 0.25 mm, diameter of beryllium window 5 mm connected with multichannel analyzer ORTEC® and the signal was evaluated by software MAESTRO-32®. In order to excite X-rays of determined elements, radionuclide source 238Pu in the form of point disk was employed (made by Amersham, activity 880 MBq, energy 12­ 22 keV, half-life 86.4 years). All measurements were performed in noncoaxial geometrical arrangements of source, sample and detector and the acquisition time was 2000 s. The sample acidity was measured by means of pH-meter HI 9321 (Hanna Instruments Bratislava, Slovak Republic). Tablets were pressed on Carl Zeiss Jena 036-76. Calibration curves for ADDC: To 30 ml of the solution with pH 3.5 (optimal for all elements), 0, 100, 200, 300, 500 and 1000 µl of each element (corresponding to 0, 10, 20, 30, 50 and 100 µg of element) and 500 µl of 2 % solution of ADDC were added. Then the mixture was stirred in the beaker for 5 min, next it was heated at 60 °C for 5 min in water bath and cooled down (10 min in ice bath). Then the mixture was filtrated through the membrane filter Pragopor 4 using the filtration device. The filter with collected precipitates was dried at the room temperature. Dried filters were analysed by XRFS. Using the same procedure and under the same conditions, a blank solution was prepared and analysed. RESULTS AND DISCUSSION This work was aimed to determine the presence of the elements Mn, Fe, Co, Ni, Cu, Zn, Hg and Pb by XRFS in the agrimony tea ­ in original solid form and its infusion after the preconcentration of the elements from the liquid phase by precipitation with TAA and ADDC. Firstly the solid form samples were analysed. After the homogenisation, the powder was pressed into the tablets of constant parameters and these tablets were analysed with XRFS. The results are given in Table 1. The method of standard addition was applied to calculate the content of analysed elements in the sample. According to the section Procedure, the chosen amounts of each element were added to the agrimony sample and after homogenisation the pressed tablets were analysed with XRFS. Obtained counts are given in Table 2. and spectrum of the sample and the sample with added standards are presented in Fig. 1. Detection limits (LD ) for each element present in the plant matrix were calculated as a ratio of 3 ( = background noise) from the counts of non-analytical signal and counts corresponding to 1 g of element. Detection limits are presented in Table 3. Other precision parameters such as range (R), standard deviation calculated by range (SR), relative standard deviation for the area of peak in % (sr) and confidence interval (L1,2) were calculated and resulting data are given in Tables 1 and 2. The values of relative standard deviation were Table 1. Counts corresponding to selected elements after the interaction of radionuclide source 238Pu with the agrimony sample tablet element Sample No. 1. 2. 3. Average R sR sr L1,2 background 532 569 564 555 37 21.86 3.94 555±48 304 1027 1051 1054 1044 27 15.95 1.53 1044±35 500 Mn Fe Co 592 583 613 596 30 17.72 2.97 596±39 466 Ni ­1 Cu 875 835 878 863 43 25.40 2.94 863±56 523 Zn 1368 1381 1433 1394 65 38.40 2.75 1384±85 774 Hg 489 505 517 504 28 16.54 3.28 504±37 558 Pb 513 506 535 518 29 17.13 3.31 518±38 542 Counts · 2000 s for tablets of "Repíkový caj" 408 415 386 403 29 17.13 4.25 403±38 355 Table 2. Counts corresponding to selected elements after the interaction of radionuclide source 238Pu with the tablet with standard addition of elements element Sample No. 1. 2. 3. Average R sR sr L1,2 1069 1048 1042 1053 27 15.95 1.51 1053±35 4726 4831 4795 4784 105 62.03 1.30 4784±137 Mn Fe Co 1188 1268 1179 1212 89 52.58 4.34 1212±116 Ni 1139 1115 1168 1141 53 31.31 2.74 1141±69 Cu 2304 2234 2304 2281 70 41.36 1.81 2281±91 Zn 4713 4834 4609 4719 225 132.93 2.82 4719±293 Hg 1184 1212 1207 1201 28 16.54 1.38 1201±37 Pb 1245 1319 1266 1277 74 43.72 3.42 1277±96 Counts · 2000 s­1 for tablets of "Repíkový caj" solid sample sample 2000 K Zn 1500 K Fe K Ni 1000 K Mn K Co 500 K Hg K Cu K Pb standard addition Figure 1. Spectrum of a tablet of agrimony tea "Repíkový caj" and the tablet of the sample with standard addition of elements after an interaction of the radionuclide source 238Pu. less than 4.00 %, which confirms good reproducibility of the method and indicates suitability of the method for the practical evaluation of the plant preparations. The resulting contents of the selected metal elements in the agrimony tea are presented in Table 3. The content of Fe was the highest among the analysed elements. The contents of Hg and Pb were under their detection limits that confirms the good quality of the plant preparation and also negligible contamination of the processed plant Agrimonia eupatoria L. Subsequently, the liquid samples ­ infusions were analysed. Before the analysis with XRFS method, the elements were preconcentrated by precipitation with TAA and ADDC reagents. Created precipitations were filtrated through Pragopor 4 filter and the enriched filters were analysed with XRFS. Optimum pH for the multi-element analysis was chosen according to a previous study (Jánosová et al., 2010). Regarding the TAA method pH 8.5 was optimum for Fe, Ni, Zn and Hg and 11.5 for Mn, Co, Cu and Pb. When using the ADDC method, pH 3.5 was optimum for all elements. These conditions were proved to be the best for the precipitation of selected elements resulting in the lowest detection limits. First of all, calibration curves for TAA and ADDC method were constructed. The amount of added elements was in range of 0­100 µg. The measured counts corresponded to the content of metal ions collected on the membrane filter in the form of precipitations. The linear dependence of measured signals on content of element was confirmed by calculated correlation coefficients. Parameters a and b of analytical curves in the Table 3. Detection limits and content of the elements in the sample of "Repíkový caj" element LD (µg/g) Content (µg/g) Mn 3.13 15.12 Fe 3.58 29.09 Co 3.17 6.33 Ni 2.32 < 2.32 Cu 2.43 11.99 Zn 2.50 18.65 Hg 3.05 < 3.05 Pb 2.77 < 2.77 Table 4. Parameters of the calibration curve in form of y = a + bx and correlation coefficient r for selected elements for TAA and ADDC method element a b r a b r 32 Mn 60.05 -179.85 0.9971 40.28 -11.48 0.9953 Fe 111.67 -173.49 0.9982 81.12 -21.28 0.9980 Co 46.88 -63.31 0.9960 23.13 -77.88 0.9942 Ni TAA 85.66 -92.12 0.9982 ADDC 28.54 74.98 0.9854 67.717 -151.25 0.9987 102.93 -282.02 0.9981 56.50 -105.98 0.9975 66.62 -95.67 0.9984 113.36 180.67 0.9925 102.85 -254.13 0.9974 97.46 -222.91 0.9981 106.29 89.29 0.9982 Cu Zn Hg Pb form of y = a + bx and r as correlation coefficient are given in Table 4. All dependencies had linear character in chosen interval 0­100 g. Correlation coefficient was in range of 0.9925­ 0.9982 for TAA method and 0.9854­0.9987 for ADDC method. The infusions were prepared according to the section Procedure from the solution with optimum pH 3.5 for the ADDC method and optimum pH 8.5 and 11.5 for the TAA method. Also the blank test was performed. The examples of spectrum are given in Figs. 2 and 3. Calculated detection limits along with the evaluation parameters and the content of the selected elements and using TAA and ADDC method are given in Tables 5 and 6, respectively. Using the TAA method, Fe (6.25 g/g), Mn (5.25 g/g) and Cu (5.33 g/g) were determined. The contents of the other elements were under their detection limits. Using the ADDC method, Fe (6.08 g/g) and Cu (5.25 g/g) were determined. The contents of all other elements were under their detection limits. The values of relative standard deviation were under 3.00 %, which confirms good reproducibility of the method and indicates suitability of the method for the practical evaluation of the plant infusions. The detection limits of the selected elements obtained after their preconcentration with TAA were compared with the ones obtained after the preconcentration with ADDC. It was found out that TAA method combined with the XRFS analysis is the more advantageous method for the preconcentration of the selected elements Mn, Fe, Co, Ni, Cu, Hg and Pb in the plant matrices such as agrimony tea. The only exception is Zn, whose detection limits are lower in the plant matrix studied when ADDC reagent is used for the preconcentration before the XRFS analysis. However, the difference between the detection limits obtained after using the TAA method (0.62 µg/g) and the ADDC method (0.59 µg/g) was not significant. On the other hand, capability to directly determine all selected elements in one run with relatively good sensitivity is a great advantage of the ADDC method. It is far less effective when using the TAA method. In such a case, detection limits of the majority of elements obtained by the ADDC method in one run are better than the one of the TAA method. Finally, the content of the analysed elements in the solid plant sample of agrimony tea was compared with their content in the prepared infusion using the TAA and ADDC methods to estimate the percentage of the amount of the elements extracted into the infusion. As illustrated in Table 7, the content of the elements in the infusion is significantly lower than in the original solid plant sample. From the elements originally found in the solid sample ­ Mn, Fe, Co, Cu and Zn (the contents of Ni, Hg and Pb were under their detection limits) ­ only the presence of Mn, Fe and Cu were determined in the infusion. The best extracted element was Cu (44.45 %) when using TAA method. This low extraction can be explained by the different types of bounds between the metal elements and the organic compounds of the plant matrix which are not broken by this type of sample treatment (steeping in the hot water). The detection limits of the analysed elements obtained using the TAA and ADDC methods of preconcentration for XRFS analysis were significantly decreased (in one order) than those in the solid plant sample of agrimony tea. Therefore, these methods can be successfully applied in the preconcentration for XRFS analysis of medicinal plant infusions. Table 5. Content of the selected elements in the infusion using the TAA method pH element R sR sr L1,2 LD (µg/g) Content (µg/g) Fe 13 7.68 2.99 257±17 0.36 6.25 Ni 4 2.36 2.41 98±5 0.33 < 0.33 8.5 Zn 23 13.59 2.81 484±30 0.62 < 0.62 Hg 4 2.36 2.48 95±5 0.29 < 0.29 Mn 8 4.73 2.67 177±10 0.58 5.25 Co 4 2.36 2.43 97±5 0.60 < 0.60 11.5 Cu 9 5.32 2.51 212±12 0.32 5.33 Pb 5 2.96 2.55 116±7 0.30 < 0.30 Table 6. Content of the selected elements in the infusion using the ADDC method pH element R sR sr L1,2 LD (µg/g) Content (µg/g) Mn 8 4.73 2.90 163±10 0.85 < 0.85 Fe 10 6.16 2.59 238±13 0.50 6.08 Co 4 2.36 2.17 109±5 1.34 < 1.34 Ni 4 2.36 2.59 91±5 1.00 < 1.00 3.5 Cu 9 5.32 2.58 206±12 0.57 5.25 Zn 18 10.63 2.53 421±23 0.59 < 0.59 Hg 4 2.36 2.78 85±5 0.49 < 0.49 Pb 5 2.96 2.81 105±7 0.47 < 0.47 33 Table 7. Content of the elements in solid sample and in the infusion of agrimony tea "Repíkový caj" and the % of the amount of elements extracted into the infusion Element Content (µg/g) Solid sample ADDC method infusion % of extracted elements TAA method infusion % of passed elements Mn 15.12 < 0.85 5.25 34.72 Fe 29.09 6.08 20.90 6.25 21.49 Co 6.33 < 1.34 < 0.60 Ni < 2.32 < 1.00 < 0.33 Cu 11.99 5.25 43.79 5.33 44.45 Zn 18.65 < 0.59 < 0.62 Hg < 3.05 < 0.49 < 0.29 Pb < 2.77 < 0.47 < 0.30 - CONCLUSION In this work, the contents of the selected elements were studied in the samples of loose tea of agrimony "Repíkový caj" and in its infusion. The preconcentration of the elements Mn, Fe, Co, Ni, Cu, Zn, Hg and Pb by complexation with TAA and ADDC for the XRFS method was proposed. The investigated ions were collected on the cellulose nitrate membrane filter as their complexes after the reaction with TAA or ADDC and the filter was analysed by XRFS. Calibration curve proved the linear dependence of intensity of the fluorescence radiation on the content of the metal in the sample. This method was successfully applied to the determination of the selected metals in plant matrix ­ agrimony tea. Through the results obtained in this study, it is possible to conclude that the TAA and ADDC preconcentrations with precipitation, filtration on cellulose membrane and analysis of these enriched filters by XRFS significantly decrease the detection limits compared with those in solid plant samples of agrimony tea. Other benefits of the proposed TAA method are apparent from its comparison with the ADDC method. As for the detection limits, the TAA method is preferred for the majority of the elements (except for Zn, where the ADDC method was better though not significantly) when considering analysis according to the optimum pH of the analysed sample (i.e. different pH are optimum for different metals). The advantage of this procedure is the multi-element character of the analysis, speed and the lack of need to complicate pretreatment of the sample. This method can be applied in the monitoring of metals content in solid and also liquid samples of medicinal plant and other pharmaceutical plant preparations as infusions. Acknowledgement: This work was supported by the Slovak Grant Agency VEGA (project No. 1/0664/12) TAA method K Fe K Cu pH 8.5 pH 11.5 K Mn K Cu Figure 2. Spectrum of an infusion of agrimony tea "Repíkový caj" when using the TAA method at pH 8.5 and 11.5 after an interaction of the radionuclide source 238Pu. Abbreviations: TAA - thioacetamide ADDC method K Fe K Cu pH 3.5 K Fe K Cu Figure 3. Spectrum of an infusion of agrimony tea "Repíkový caj" when using the ADDC method at pH 3.5 after an interaction of the radionuclide source 238Pu. Abbreviations: ADDC - ammonium diethyldithiocarbamate REFERENCES [1] DESIDERI D, MELI MA, ROSELLI C. 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Determination of selected elements by XRF and total phenolics in leaves and crude methanol extract of leaves of Arctostaphylos uva ursi. Acta Facult Pharm Univ Comenianae. 2009; 56: 136-145 [14] TOKALIOGLU S. Determination of trace elements in commonly consumed medicinal herbs by ICP-MS and multivariate analysis. Food Chem. 2012; 134: 2504-2508 [15] WANG DM, FENG J, QIN FJ, MO YS. Determination of six heavy metal elements in Zanthoxylum nitidum in twelve habitats of guangxi by ICP-AES. J Chin Med Mat. 2012; 35(3): 366-368 [16] ZHAO LL, ZHONG SX, FANG KM, QIAN ZS, CHEN JR. De- termination of cadmium(II), cobalt(II), nickel(II), lead(II), zinc(II), and copper(II) in water samples using dual-cloud point extraction and inductively coupled plasma emission spektrometry. J Hazard Mat. 2012; 239: 206-212 PharmDr. Veronika Mikusová, PhD. Comenius University in Bratislava Faculty of Pharmacy Kalinciakova 8 832 32 Bratislava Slovak Republic janosova@fpharm.uniba.sk

Journal

Acta Facultatis Pharmaceuticae Universitatis Comenianaede Gruyter

Published: Dec 1, 2013

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