Application of Cone Shaped Membrane-Liquid Phase Microextraction for Analysis Nitrosodipropylamine in Salted Fish

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1 ISBN Application of Cone Shaped Membrane-Liquid Phase Microextraction for Analysis Nitrosodipropylamine in Salted Fish Yanuardi Raharjo a, Usreg Sri Handajani, Eko Aryo Wijaksono Abstract Analysis of nitrosodipropylamines as carcinogenic compounds performed as one of the development of the extraction method. Analysis of nitrosodipropylamines as carcinogenic compound in salted fish through treatment with and without the addition of KNO 3 had occurred by cone shaped membrane liquid phase microextraction technique using gas chromatographyflame ionization detector. The result were obtained analytical parameters from types of organic solvents was toluene and stirring speed was 480 rpm. It was obtained in this research that the detection limit of ppt, recovery of %, precision between 0.39% to 1.30% and true enrichment factor was times. The concentration of nitrosodipropylamines in salted fish sample without the addition of KNO 3 was not detected meanwhile in salted fish sample with the addition of KNO 3 was 0.16 ppb. Keywords: cone shaped membrane liquid phase microextraction, nitrosodiprophylamines, salted fish a Department of Chemistry, Faculty of Science and Technology, Airlangga University, Surabaya Indonesia Corresponding authors:yanuardi Raharjo, phone: ; raharjo84@gmail.com Introduction A large number of extraction techniques have been developed; including the Liquid-Liquid Extraction (LLE) is an extraction technique that is most widely used. However, LLE extraction procedure requires a long time and often uses large amounts of organic solvents that have a negative impact on the environment due to its toxic (Sanagi et al., 2007). The simpler techniques have been developed, namely the Liquid Phase microextraction (LPME). in LPME technique, carried out between a few microliters of extraction solvent (extractor or acceptor phase) and a few milliliters of the sample solution (donor phase) containing the analyte (Rasmussen and Pedersen- Bjergaard, 2004). LPME technique is one microextraction technique in the liquid phase is quite simple and has a high degree of selectivity in their usage (Psillakis and Kalogerakis, 2003). Cone Shaped Membrane technique is a sample preparation techniques recently developed by using a cone-shaped membrane. Cone-shaped membrane that acts as a protective organic solvent so that only occurs on the surface extraction solvent. This technique was developed due to have enough power high selectivity and can be injected directly into the instrument. Many advantages are obtained by using this technique, which is simple, easy, it takes quite a short time and is based green chemistry. Sanagi et al. (2007) conducted a research of existing pesticide analysis in water with CSM-LPME method. Membrane cone with nylon base material used has a pore size of 0.2 μm and thickness of 200 μm, microextraction is done with a variety of optimization parameters include an organic solvent, stirring speed and extraction time. Nitrosamines are compounds that have the potential carcinogens, mutagens, and teratogens commonly found in foods preserved by using nitrite (Scanlan, 2003). Levels of nitrosamines class of compounds that are allowed in the body ranging from 5-10 mg kg -1. Compounds nitrite can react with secondary amines in the environment and the human body as well as in processed meat to form nitrosamines compounds that can be formed due to the process of pickling or salting in high temperature (Incavo and Schafer, 2006), one of the forms of nitrosamine compounds are nitrosodipropylamine (NDPA). Nitrosamine compounds can be found in fish and processed meat products (Yurchenko and Molder, 2006). One example is when the manufacture of salted fish. The principle of preservation in the manufacture of salted fish is a combination of the addition of salt and drying. High salt concentration in the salt fish salting process will result in the death of pathogenic bacteria and spoilage are generally very sensitive to salt (Silalahi, 2006). The workings of the salt in carrying out its functions in the manufacture of salted fish is salt fish absorb body fluids so that the metabolic processes of bacteria 278 P a g e Green Chemistry Section 3: Analytical Chemistry, Yanuardi Raharjo, et al.

2 ISBN disrupted because of lack of fluids even eventually kill the bacteria. in addition to absorbing body fluids of fish, salt also absorbs liquid bacterial cells so that bacteria will experience drought and eventually die (Afrianto and Liviawaty, 2002). Methodology Equipment and Materials The materials used in this research include potassium nitrate, trichloroacetic acid, aquadem, methanol, nitrosodipropylamine standard solution (NDPA 99.99%), n-hexane, ethyl acetate, toluene, and salted fish. The tools used in this research is a set of tools GC (Gas Chromatography) type Agilent 6890 GC Plus Version A with FID detector (Flame Ionization Detector), Whatmann filter paper with the basic material of nylon membrane pore size of 0.2 μm, micro pipette, micro-pipette tube, magnetic stirrer, stir bar, a set of centrifuge and glassware commonly used in the laboratory. can be seen in Table 1 and the standard solution calibration curve obtained NDPA can be seen in Figure Parameter-Parameter Optimization Analytics a. Optimization of organic solvent. The organic solvent used in the analysis NDPA using CSM-LPME extraction includes ethyl acetate, toluene and n-hexane. The selection of these three types of organic solvents is influenced by the physical properties possessed by the organic solvent include boiling point, density and surface tension. in addition, the selectivity is also considered as the principle of like dissolve like wherein the organic solvent used has a non-polar nature of the compounds that NDPA as semi polar analytes can be extracted during the extraction process. Cone Shaped Membrane-Liquid Phase Microextraction In this research the concentration of NDPA standard solution used was 10, 20, 30, 40 and 50 ppm and optimized analytical parameters include the type of organic solvent, stirring speed and extraction time. Optimization is done by replicating as much as three times to validate the measurement and optimization of each parameter separated performed. Work steps in this research are as much as 20 ml of sample solution vial inserted in the bottle used for the extraction. Nylon membranes were used as the membrane is immersed in an organic solvent for seconds to allow the solvent to fill the pores of the membrane wall. After impregnation solvent, Cone Shaped Membrane may be positioned in a sample bottle that already contains water samples, and 80 ml of organic solvent pipetted into the membrane. Samples were stirred continuously with a magnetic stirrer at room temperature to facilitate analyte mass transfer between donor phases and acceptor phases that occur during the process of micro-extraction takes place. The extraction process is done with the optimization of organic solvent, stirring speed and extraction time, after the analyte withdrawn and transferred to Eppendorf tubes. A total of 3 ml solution is drawn into the micro syringe and injected into the instrument GC (Gas Chromatography) for further analysis. Figure 1. Set-up Cone Shaped Membrane-Liquid Phase Microextraction Table1.Dataareaof standard solutionswithoutextractionndpacsm-lpme Concentration of NDPA (ppm) (unit) Results and Discussion 1. Preparation of calibration curve without Cone Shaped Membrane LPME The data area at each concentration of standard solution NDPA without using CSM-LPME extraction Figure 2.Calibration curvewithoutcsm-lpme extraction Green Chemistry Section 3: Analytical Chemistry,Yanuardi Raharjo, et al. P a g e 279

3 (unit) ISBN Data resulting from the extraction of the three types of organic solvents can be seen in Table 2. and diagram of optimization organic solvents can be seen in Figure 3. b. Optimization of stirring speed Stirring speed optimization is done so that the NDPA compounds prone to analyte transfer to the organic solvent so that more compounds extracted NDPA (Mofazzeli, 2013). Stirring speed can also help speed up the transfer of analytes from the donor phase to the acceptor phase (Faraji and Tehrani, 2012). Data on average area obtained can be seen in Table 3, while the area to the curve of the stirring speed can be seen in Figure 4. Table2.Dataarea ofthe threetypes oforganic solvents Organic Solvent (unit) Toluene Ethyl acetate n-hexane Stirring speed (rpm) Figure 4.Curveareaonthe stirring speedoptimization 3. Preparation of the calibration curve CSM- LPME extraction NDPA standard solution measurement data using CSM-LPME extraction at optimum conditions can be seen in Table 4 and the standard calibration curve of NDPA using CSM-LPME extraction is shown in Figure Validation of Analytical Parameters Data detection limit of the analysis of compounds with and without extraction NDPA CSM-LPME can be seen in Table 5. Calculation of coefficient of variation is used to identify the reproducibility instruments used in the research of GC-FID. The coefficient of variation in this research is between 0.39 until 1.30 %. Table 4.Dataarea ofthe standard solutionndpausingcsm- LPME extraction Figure 3.Graph size of the area on the optimization of organic solvent type Table3.Dataon averageareaof thestirring speed Stirring Speed (rpm) (unit) Concentration of NDPA (ppm) (unit) Figure 5.Calibration curveof standard solutionsndpausingcsm-lpme extraction 280 P a g e Green Chemistry Section 3: Analytical Chemistry, Yanuardi Raharjo, et al.

4 ISBN Table 5. Data limit of detection and limit of quantitative NDPA with and without extraction of CSM-LPME Method Without CSM- LPME Using CSM- LPME Coefficientof Correlation (R 2 ) LOD 3.17 ppm ppt 5. Enrichment Factor Enrichment Factor describes the concentration of factors that occur during the extraction process using CSM-LPME. Analyte concentration that occurred during the extraction process using CSM- LPME can be seen in Figure 6. After calculation, the concentration factor obtained occurs during the extraction process using CSM-LPME NDPA is equal to times. While the actual or true concentration enrichment factor (EFtr) of times. Table 7. Data NDPA concentration in the sample Sample Salted Fish Salted Fish + KNO 3 Concentration of NDPA Not Detected 0.16 ppb 7. Spiking Spiking is done to determine whether there is nuisance parameters in the sample matrix used during the extraction took place. The results of the analysis for each sample spiking can be seen in Table 8. Sample Salted Fish Salted Fish + KNO3 Table 8. Data spiking samples I II III Concentration of NDPA (ppm) Recovery (%R) Figure 6.NDPA concentration curve using CSM-LPME extraction 6. Analysis of Samples The resultsof analysisofsamples ofsaltedfishwithandwithoutkno 3can be seenin Table6. Next to determine the concentration of NDPA in the sample, the extent of the area is included in the regression equation of the calibration curve generated from standard solutions NDPA using CSM-LPME extraction is y = 4,6537x-26,897 in order to obtain the concentration of NDPA in each sample are shown in Table 7. Table 6. Data analysis of samples Sample I II III Salted Fish Salted Fish + KNO From the above data recovery is quite far from 100% compared with the current standard extraction solution, it can be said that the matrix contained in the sample affect the extraction process NDPA with CSM-LPME method. The presence of organic compounds in the sample can decrease the extraction efficiency due to organic compounds contained in the sample have characteristics in common with the analyte, for example in terms of polarity, so when extracted with the extracting solvent extracted organic compound involved. The composition of the nutrient content of salted fish being a bully in the extraction matrix NDPA compounds can be seen in Table 9. It is known that the fat be one of the confounding matrix in which fat has properties extracted nonpolar so it can participate in the acceptor phase is also the nonpolar organic solvents. Table 9.Nutrient composition ofsalted fish (Singgih, 2000) Content of nutrient (%) Water Minerals Protein Lipid Conclusion Based on the research that has been done, it can be concluded as follows: Green Chemistry Section 3: Analytical Chemistry,Yanuardi Raharjo, et al. P a g e 281

5 ISBN CSM-LPME extraction technique-gc-fid can be used to analyse compounds nitrosodipropylamine (NDPA) contained in salted fish with NDPA concentration in each sample as follows, salted fish samples without the addition of KNO 3 was not detected the presence of compounds NDPA and salted fish samples with the addition of KNO 3 at 1.07 ppm. Acknowledgments The authors are grateful to Indonesia Ministry of Education for financial support, Faculty of Science and Technology and Faculty of Pharmacy, Airlangga University for the facilities. References Afrianto, E. and Liviawaty, E. (2002), Pengawetan dan Pengolahan Ikan, Kanisius, Yogyakarta Faraji, H. and Tehrani, R.M.A. (2012), Liquid phase microextraction based on the solidification of a floating organic microdrop for trace analysis of BTEX in the aqueous sample, International Journal of Industrial Chemistry, Vol.3(12): 1-8 Filho, P.J.S., Rios, A., Valcarcel, M., Zanin, K.D., Caramao, E.B. (2003), Development of a New Method for The Determination of Nitrosamines by Miceller Electrokinetic Capillary Chromatography, Water Research, Vol.37: Hashemi, M., Habibi, A., Jahanshahi, N. (2011), Determination of Cyclamate in Artificial Sweeteners and Beverages Using Headspace Single-Drop Microextraction and Gas Chromatography Flame- Ionisation Detection, Food Chemistry, Vol.124: Incavo, J, A. and Schafer, M, A. (2006), Simplified Method for The determination of N-nitrosamines in Rubber Vulcanizates, Analytica Chim. Acta, Vol. 557: Miller, J.C. dan Miller, J.N. (1988), Statistics for Analytical Chemistry Second Edition, Elllis Horwood Limited, England Mofazelli, F. (2013), Three liquid phasemicroextraction of diclofenac and ibuprofen from water Samples prior to high performance liquid chromatography, Journal Materials and Environmental Science, Vol.4(5): Psillakis, E. and Kalogerakis, N. (2003), Developments in liquid-phase microextraction, Trends Anal, Chem., Vol.22: Scanlan, R.A. (2003), Nitrosamines and Cancer, Publication Review, Vol.14: Silalahi, J. (2006), Makanan Fungsional, Kanisius, Yogyakarta Singgih, W. (2000), Industri Pemindangan Ikan, Swadaya, Jakarta Sanagi, M.M, Hong S.H., Ibrahim, W.A.W, Naim, A.A. (2007), Determination of pesticides in water by coneshaped membrane protected liquid phase microextraction prior to micro-liquid chromatography, Journal of Chromatography A, Vol.1152: Yurchenko, S. and Molder, U. (2006), Volatile N- nitrosamines in various fish products, Food Chemistry, Vol.96: P a g e Green Chemistry Section 3: Analytical Chemistry, Yanuardi Raharjo, et al.

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