Extraction and Determination of Met and MHA

Extraction and Deter instantation of Met and MHADetermination of Methionine and Methionine Hydroxy Analogue in the Forms of Free or Metal Chelates Contained in Feed Formulations by RP-HPLCM. Salahinejad,* F .AflakiAbstractMethionine is often the first or second limiting amino sexually transmitted disease in most diets and so is most representative of amino sharps fed as nutritional supplements. It commonly supplemented as DL-methionine or as methionine hydroxy analogue. A simple and rapid method for simultaneous extraction and determination of DL-methionine and methionine hydroxy analogue in forms of exempt or in forms of metal- chelates contained in lean examines is draw. The sample extraction procedure was performed using HCl solution and heating in an sterilize or oven, which followed by the addition of EDTA and acetonitrile. Quantification and detection were carried out by reversed descriptor high performance liquid chromatography on a NovaPak C18 pillar with ultraviole t detection at 214 nm. With a expeditious phase consisted of 5% acetonitrile + 1.5% sodiumdihydrogenphosphate in water, the chromatographical run judgment of conviction were 6 min. The detection limit for DL-methionine and methionine hydroxy analogue were 2.33 and 5.46 g mL1 andMAMwith the relative standard deviation (R.S.D.) was 4.4 and 7.3% (C = 10 g mL1, n = 5) respectively. The recoveries of methionine and methionine hydroxy analogue in diet samples were 97%.Keywords Methionine hydroxy analogue, DL-methionine, Metal-chelates, Reversed phase high performance liquid chromatography (RP-HPLC)IntroductionFor optimum health and performance, the animals diets must contain adequate quantities of all nutrients needed, including amino acid. The essential amino acid furthest below the level needed to build protein is cognise as limiting amino acid. The shortage of limiting amino acid will constrain animal growth, reduce race efficiency and in constitutional cases ca design a nutri tional deficiency 1.Methionine and lysine considered the most limiting amino acids in most animal diets. Supplementation of methionine may be accomplished by the addition of DL-methionine or the hydroxyl analogue of methionine (DL-2-hydroxy-4-methylthiobutanoic acid) 2. Fig. 1 represents the structures of DL-methionine (Met) and methionine hydroxy analogue (MHA).Organic forms like metal chelates of transition metal ions in particular coat (II), Copper (II) and Manganese (II) with amino acids and peptides atomic number 18 widely used in animal feeding as they appear to induce as faster growth and better resistance to divers(a) diseases in comparison with the simple inorganic salts 3. It has been suggested that these effects are correlated with the improved metal bio-availability. The chelates are absorbed in the small intestine, possibly using transporters for amino acids small peptides 4. M either forms of metal knottyes with amino acid chelates and hydrolyzed proteins are comme rcially available, as metal amino acid chelates and complexed chelated (metal) proteinates (CCP) respectively 5-7. The methionine hydroxyl analogue largely used in animal nutrition as a source of methionine, forms stable chelates with divalent metals of grammatical construction CH3SCH2CH2CHOHCOO2 M.nH2O 8.Several methods have been used for DL-methionine determination including ion exchange chromatography in combination with pre or post column derivatization 9 and amino acid analyzer 10. These methods are not applicable to the determination of methionine hydroxy analogue because it contains -hydroxy instead of -amino group (Fig.1). Gas chromatography 10 electrophoresis 11 and high performance chromatography 12-14 were used for determination of MHA.(a) (b)Fig.1. Structures of (a) DL-methionine and (b) methionine hydroxy analogue.The use of so-called variant recipes in the production of industrial feeds causes that in practice the analyst encounters a diametriciated and unknown compo sition of the so-called matrix, i.e. the elements of a feed mixture that in m each cases made it hard to isolate and at times even make it impossible to mark MHA in the environment of a feed mixture 15. Moreover the accurate determination of methionine and methionine hydroxy analogue contained in the metallic chelates of feeds depended on complete releasing of methionine and methionine hydroxy analogue from metals. The inclination of the paper was to develop and evaluate the method of simultaneous determination of MHA and Met in forms of resign or in forms of chelates in compound feed samples.Material and Methods implementChromatographic determination were performed on a Waters Liquid Chromatograph which consisted of Waters 1525 Binary HPLC pump, Waters 2487 Dual absorbance detector, Breeze data processing system and C18 NovaPack column. An adaptable rocker shaker (Cole- Parmer 60Hz) and a feed grinder to facilitate sample preparation were used.Reagents and standardsThe stock s tandard solution of Met and MHA was prepared weekly using DL- Methionine (extra pure, Merck) and Alimet (commercial discern of the hydroxy-analogue of methionine containing 89.7% MHA in 0.1 N HCl respectively. All working solutions were prepared by diluting the stock standards as unavoidable. Deionized distilled water obtained from a Milli-Q system (Millipore, Milford, USA) was used for standard dilutions and other necessary preparations.All other chemicals such as NaH2PO4, extra pure acetonitril, isocratic grade EDTA (disodium salt) 99%, HCl 37%, orthophosphoric acid 85% and sodium hydroxyl, analytical reagent grade, were supplied by Merck. seek preparationAliquots of finely ground samples (mean particle size of 600 m) containing 0.1 gr methionine hydroxy analogues (MHA) or 0.1 gr DL-methionine (Met) in forms of free or in forms of metal-chelates were added in 20 ml of 0.1 N HCl solution and heated in autoclave in steam flow in 120 oC for 5 min or in oven with 90 oC for 20 min. A fter cooling, by adding 20 ml of EDTA solution (10% W/V) and 5 ml of acetonitrile, the samples were shacked for 10 min and then solutions were filtered using 0.45 m filter. Volume is modify to 100 ml with distilled water and a proportion of solution injected onto the HPLC column.Fig.2. Chromatogram of the extracted Met and MHA from feed.Chromatographic conditionsSeparation and quantitation of MHA and Met have been performed with reverse phase high performance liquid chromatography (RP-HPLC). The column was NovaPak C18 (150 4.6 mm, 5 m) in ambient temperature. Samples were injected in volumes ranging from 5 to 20 l using Rehodyne injector. The solvent system for separation of Met and MHA consisted of 5% acetonitrile + 1.5% NaH2PO4 in water. Using this isocratic mobile phase the chromatographic run time was 6 min. After this, a washing step was programmed to 40% acetonitrile in mobile phase so that any residual sample components would be cleaned from the column. The washing step was 5 min and column conditioned by primary mobile phase for 4 min prior the next injection. The flow rate, UV wavelength and detector attenuation used was respectively 1 ml min-1, 214 nm and 0.2 a.u.f.s. The amounts of MHA and Met contained in the samples were determined by interpolating the revalue of the tiptop area of calibration curves obtained by injecting 5, 10, 15, 20 l of mixed standard solution containing 200 mgr L-1 Met and 400 mgr L-1 MHA. The bulk standard was prepared weekly. Fig.2 shows a chromatogram which obtained by injection of the extracted sample solution.Statistical outlineIn order to verify differences of effecting factors on extraction efficiency, analysis of variance (ANOVA) was applied with the level of moment set at 0.05. The SPSS statistical program (SPSS Inc, Illinois, USA) was used to perform all statistical calculations.ResultsStudy of effective factors on extraction efficiency of Met and MHAThe effect of various parameters such as temperature, heatin g time, the presence or absence seizure of hydrochloric acid (variation of pH) and EDTA (as a strong ligand) in the convalescence of the Met and MHA in the forms of free or metal-chelates were investigated. dining table 1 shows the mean convalescence of the Met and MHA in the forms of free or metal-chelates from compounded feed at 90 oC for 20 min in 0.1 N HCl and distilled water. convalescence tests were performed by adding known amounts of different forms of Met and MHA to a compounded feed which its basic elements was maize, wheat bran, soybean ground grain, fish meal, plant oil, calcium phosphate, mineral vitamin premix. The recovery of free Met and MHA from compounded feed by distilled water was 96%, while the recovery of Met and MHA from metal-chelate was 95%.Table 1 Mean recovery of the Met and MHA from compounded feed with distilled water and 0.1 N HCl solutions at 90 oC for 20 min.a n = 4Different temperatures (25-120 oC) in different period of times (5 min -3 hours) were examined to rating of the effects of temperature and heating time in the simultaneous extraction of Met and MHA in both forms. Based on extraction efficiency of the Met and MHA in the forms of free or metal-chelates, three conditions including Autoclave (T 120 oC, t 5 min), Oven (T 90 oC, t 20 min) and Room temperature (t 3 hours) were chosen.The effect of strong ligand such as EDTA on extraction of Met and MHA in forms of metal-chelate was investigated. Table 2 represents the mean recovery of the Met and MHA in forms of metal-chelate in different heating condition (different temperature and time) in the presence or absence of EDTA as a strong ligand. The results illustrated in Table 2 reveal that the extraction of the MHA from MHA metal-chelates in feed was about 94% with heating by autoclave in 120 oC for 5 min or oven at 90 oC for 20 min. By adding the EDTA solution to the samples the recovery of MHA from MHA metal-chelates become 97%. The recovery of the Met was 96% eve n in ambient temperature and ETDA do not show a considerable effect on the Met recovery from the feed.Table 2 Mean recovery of Met and MHA (0.1 N HCl solution) in three different conditions Autoclave (T 120 oC, t 5 min), Oven (T 90 oC, t 20 min), Room temperature (T 27 oC, t 3 hours)Analytical performance of the methodQuality variables including the limit of detection (LOD) and precision, as the relative standard deviation (R.S.D.), were investigated to evaluate the analytical performance of the proposed method. According to the IUPAC realisation 16 the limit of detection (LOD, 3) of the proposed method was 2.33 and 5.46 g mL1 for Met and MHA respectively. MAMwith The R.S.D. was 4.4 and 7.3% (C = 10 g mL1, n = 5) for Met and MHA respectively. Good linear relationships exist for peak area counts versus the amount of Met and MHA (Fig. 3). The regression equation for calibration curves for Met was Y = 209551x + 296453 with a correlation coefficient (R2) of 0.9983 and for MHA was Y = 1 82603x + 294054with correlation coefficient (R2) of 0.9995 where Y is the peak area counts and x is the immersion (ppm) of analyte.Table 3 Recovery of Met or MHA from pure metal chelates complex.a n = 4Fig.3. Calibration curves for MHA and Met analysis.Method evaluationFor evaluation of the described method, the recovery of Met or MHA from pure Met or MHA metal-chelates were determined (Table 3). The results show good symmetricalness between the results of the mentioned method and the value which declared by the producers. The precision was determined by calculating the relative standard deviation of four analyses for each condition.The method also was applied for simultaneous extraction and determination of different forms of Met and MHA from compounded feed. As shown in Table 4, the obtained results prove a good agreement of the mean content of Met or MHA in mixtures with the declaration.Table 4 Simultaneous determination of different forms of Met and MHA from compounded feed.Ta ble 5 Content of Met or MHA in the analyzed industrial feed mixtures (g/Kg).a n = 4In order to evaluate the effect of typical sample matrix, numerous industrial feed samples, which their Met or MHA content declared by the producer, originating from Iran, Germany, Italy and France was qualitatively examined. The results (Table 5) show a good agreement between the obtained mean content with the declaration of free or metal-chelate form of Met or MHA in industrial feed mixtures.Basing on the above results, the usefulness of the described method for determination of the Met and MHA in form of free or in forms of metal-chelates in feed mixtures can be stated.DiscussionThe solubility of DL-methionine in aqueous solutions increases 5-fold (176.0 Vs 33.8 g L-1) when temperature is increased from 25 to 100 oC 17,18. Different temperatures (25-120 oC) in different period of times (5 min -3 hours) was examined to evaluation of the effects of temperature and heating time in simultaneous extrac tion of Met and MHA in free or metal-chelate forms. The temperature and the time of extraction have inverse effects on extraction efficiency of both analyts. When temperature increases, the time required for maximum extraction of both analyts decrease and vise versa. By performing analysis of variance (ANOVA) and student t-test between different conditions (different temperature and time) the three conditions autoclave 120 oC for 5 min, oven 90 oC for 20 min and room temperature for 3 hours had no significant differences ( p 0.05) in extraction efficiency of Met and MHA in free forms (as shown in Table 2). But extraction in room temperature significantly had lower recovery in metal-chelate form of Met and MHA. Therefore, for simultaneous extraction of Met and MHA in free or metal-chelate forms, the 90 oC for 20 min was chosen.pH can play a unique role on metalchelate formation or releasing of metal from metal-chelates 19. Experiments have shown DL- methionine extraction recoveries obtained with hydrochloric acid and with distilled water at ambient temperature are not statistically different 20. Therefore the extraction of Met and MHA in free forms could be done with distilled water at 90 oC for 20 min. The application of this procedure to be unsuitable for extraction of Met and MHA contained in metallic chelates. As shown in Table 1, the extraction recovery of Met and MHA in metal-chelate forms with distilled water is significantly lower (p EDTA is a stronger ligand than MHA therefore it can form more stable complex with metals and it must affect on recovery of MHA. Therefore by adding EDTA solution to the samples the recovery of MHA ( 97%) from MHA metal-chelates were significantly higher, but this has no significant effect on Met extraction recovery.ConclusionA simple, rapid and reliable method for simultaneous extraction and determination of Met and MHA in forms of free or in forms of metal-chelates in feed samples has been developed. This method can be us ed for analysis of free methionine or methionine hydroxy analogue as well as their metal-chelate form, from industrial feed samples without any variation. It involves a simple procedure sample preparation using 0.1 N HCl solutions and heating in autoclave or oven, which followed by addition of EDTA and acetonitrile, and quantitation by an isocratic HPLC analysis on a C18 column.References1 M. Korhonen, A.Vanhatalo, P. Huhtanen, J.Driry.Sci., 85 (2002) 1533.2 D.Hoehler, M. Rademacher, R. Mosenthin, Advances in pork production, 16 (2005) 109.4 T. L. Stanton, D. Schutz, C. Swenson, Prof. Anim. Sci. 7 (2001) 101.3 H.D. Ashmead, S.D. Ashmead, R.A. Samford, Intern.J.Appl.Res.Vet.Med.,2 (2004) 252.5 C. E. Nockels, J. DeBonis, J. Torrent, J. Anim. Sci., 71 (1993 )2539.6 H. T. Ballantine, , M. T. Socha, D. J. Tomlinson, A. B. Johnson, A. S. Fielding, J. K. Shearer, S. R.Amstel, Prof. Anim. Sci., 18 (2002) 211.77 B.L. Creech, J.W.Spears, W.L. Flowers, G.M. Hill, K.E. Lioyd, T.A. Armestrong, T .E. Engle, J.Anim.Sci., 82 (2004) 2140.8 S. Ferruzza., G. Leonardi., E.Cinti., M.Tegoni., J. Inorg. Biochem., 95 (2003) 221.9 W. Baeyens, J. Bruggeman, C. Dewaele, B. Lin, K. Imai., 5 (2005) 13.10 C. Aoyama , T. Santa *, M. Tsunoda , T. Fukushima, C. Kitada , Ka. Imai., Bio.Med.Chromatogr., 18 ( ) 630.11 A.P. Solomonova, J.S. Kamentsev, N.V. Komarova, J. Chromatogr. B Analyt Technol Biomed bearing Sci, 800 (2004) 135.12 A. Baudicheau ,J. Sci. Food Agric., 38 (1987) 1. 13 D. Wauters., J. De mol, L. Temmerman., J. Chromatogr. A,516 (1990) 375. 14 D. Balschukat, E. Kress, E. Tanner., Landwirtsch. Forsch., 41 (1988) 120.15 S. Maytyka, J. Rubaj, W. Korol, G. Bielecka, 9 (2006) 1.16 G. L. Long, J.D. Winefordner, Anal. Chem. 55, (1983) 713A-724A.17 R.C. Weast (Editor), CRC Handbook of Chemistry and Physics, CRC Press, Boca Raton, FL, 69th ed. (1988).18 Merk Index, Merck, Rahway, NJ, 10th ed. (1983).19 T.E. Brown, L.K. Zeringue, J. Dairy Sci. 77, (1994)181187.20 L. Yang, R.E. Sturgeon, S. McSheehy, Z. Mester, J. Chromatogr. A 1055, (2004) 177-184.