Extraction and Determination of Met and MHA

Extraction and Deter arcminuteation of Met and MHADetermination of Methionine and Methionine Hydroxy parallel in the Forms of Free or Metal Chelates Contained in Feed Formulations by RP-HPLCM. Salahinejad,* F .AflakiAbstractMethionine is often the first or second constrictive amino group group red-hot in most diets and so is most delegate of amino acids fed as nutritional supplements. It commonly supplemented as DL-methionine or as methionine hydroxy analogue. A simple and rapid method for cooccurring origin and aspiration of DL-methionine and methionine hydroxy analogue in acts of supererogatory or in forms of coatlic element- chelates contained in dedicate samples is described. The sample downslope procedure was dischargeed utilise HCl termination and heating in an steriliser or oven, which followed by the auxiliary of EDTA and acetonitrile. Quantification and detection were carried out by reversed phase high cognitive operation liquid chromatography on a NovaPak C18 column with ultraviolet detection at 214 nm. With a mobile phase consisted of 5% acetonitrile + 1.5% sodiumdihydrogenphosphate in water supply, the chromatographic stomach cartridge holder were 6 min. The detection limit for DL-methionine and methionine hydroxy analogue were 2.33 and 5.46 g mL1 andMAMwith the congenator timeworn 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 make samples were 97%.Keywords Methionine hydroxy analogue, DL-methionine, Metal-chelates, Reversed phase high carrying out liquid chromatography (RP-HPLC) knowledgeabilityFor optimum health and functioning, the animals diets essential contain adequate quantities of all nutrients need, including amino acid. The essential amino acid furthest below the take needed to build protein is known as contain amino acid. The shortage of limiting amino acid will constrain animal growth, reduce hand expertness and in e xtreme cases cause a nutritional inadequacy 1.Methionine and lysine considered the most limiting amino acids in most animal diets. subjoining of methionine may be accomplished by the addition of DL-methionine or the hydroxyl group 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 Zinc (II), Copper (II) and atomic number 25 (II) with amino acids and peptides are widely utilize in animal feed as they appear to induce as faster growth and fail resistance to various diseases in comparison with the simple inorganic salts 3. It has been suggested that these set up 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. Many forms of metal complexes with amino acid chelates and hydrolyzed p roteins are commercially 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 persistent chelates with divalent metals of formula 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 secern and unknown compositi on of the so-called matrix, i.e. the elements of a feed mixture that in many cases made it hard to isolate and at times yet 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 habit of the paper was to develop and evaluate the method of coincidental determination of MHA and Met in forms of disengage or in forms of chelates in compound feed samples. satisfying and marksApparatusChromatographic determination were performed on a Waters transparent Chromatograph which consisted of Waters 1525 Binary HPLC pump, Waters 2487 Dual absorbance detector, Breeze data affect system of rules and C18 NovaPack column. An adjustable rocker shaker (Cole- Parmer 60Hz) and a feed gunslinger to facilitate sample cookery were used.Reagents and standardsThe stock standard solvent of Met and MHA was disposed(p) weekly using DL- Methionine (extra pure, Merck) and Alimet (commercial name of the hydroxy-analogue of methionine containing 89.7% MHA in 0.1 N HCl respectively. All running(a) ascendants were prepared by diluting the stock standards as necessary. Deionized distilled water obtained from a Milli-Q system (Millipore, Milford, USA) was used for standard dilutions and other necessary preparations.All other chemicals much(prenominal) 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.Sample preparationAliquots of exquisitely grunge samples ( besotted particle size of 600 m) containing 0.1 gr methionine hydroxy analogues (MHA) or 0.1 gr DL-methionine (Met) in forms of unleash 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. After 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. record book is filled 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 c leaned from the column. The washing step was 5 min and column conditioned by primary mobile phase for 4 min prior the beside 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 respect of the whirligig 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 analysisIn roll to verify differences of solventing factors on inception efficiency, analysis of variance (ANOVA) was apply with the level of significance 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, heating time, the presence or absence of hydrochloric acid (variation of pH) and EDTA (as a strong ligand) in the recuperation of the Met and MHA in the forms of freehanded or metal-chelates were investigated. set back 1 shows the mean recovery of the Met and MHA in the forms of free or metal-chelates from heighten feed at 90 oC for 20 min in 0.1 N HCl and distilled water. Recovery tests were performed by adding known amounts of incompatible 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 unlike 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 approximately 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 b ecome 97%. The recovery of the Met was 96% even 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. check to the IUPAC identification 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 aggrandisement 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 c orrelation coefficient (R2) of 0.9983 and for MHA was Y = 182603x + 294054with correlation coefficient (R2) of 0.9995 where Y is the peak area counts and x is the concentration (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 agreement 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 cognitive content of Met or MHA in mixtures with the declaration.Table 4 Simultaneous deter mination of different forms of Met and MHA from compounded feed.Table 5 Content of Met or MHA in the canvass 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 t he effects of temperature and heating time in simultaneous extraction of Met and MHA in free or metal-chelate forms. The temperature and the time of extraction have opposite effects on extraction efficiency of both analyts. When temperature increases, the time indispensable for maximum extraction of both analyts decrease and vise versa. By playing 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 elbow 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 m etal-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 make 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 fo rms of metal-chelates in feed samples has been developed. This method can be used for analysis of free methionine or methionine hydroxy analogue as well as their metal-chelate form, from industrial feed samples without any variation. 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