Effects of dietary n-3 polyunsaturated fatty acids and ... - FINS

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1 st WorkshopXIII International Feed Technology Symposiumbroilers. Development of methods of determining and mastering existing analyticalprocedures are of great importance for producers of broilers, pharmaceuticals and feedfor poultry producers. Manufacturers of broilers can get testing type and content ofcoccidiostatic in poultry feed. The existence of reliable methods of determining the drugduring manufacturing, premixes or solutions, and stability of the product. Coccidiostaticapplication is rarely applied with the most important form of containment vaccinationcoccidiosis especially in raising broilers for meat production. Since a large number ofanticoccidial preparations used in the prevention of coccidiosis we chose ionophorecoccidiostatic – salinomycin. Salinomycin, except for prophylaxis, as anticoccidialpreparations is applied as a growth promoter [4, 5]. To determine the content ofsalinomycin in samples of poultry feed before they primarily used microbiologicalmethods [9]. Spectrophotometric methods for determination salinomycin and otherionophore coccidiostatics include spectrophotometric measurement characteristics in thevisible and UV region. The visible area (colorimetry) [12] consists in the reaction ofsalinomycin solution and methanolic solution of vanilin with measuring the absorbanceof mixture at 520 nm. Thin layer chromatography is one of the most applied techniquefor separation and identification of substances due to their simplicity and selectivity indetermining [1, 7, 14]. High performance liquid chromatography is the method of choicefor determining salynomicin and other ionophore coccidiostatics in biological samples,allows for quantification with the increasing sensitivity and reducing the lower detectionlimit [2, 6, 15]. Direct performance liquid chromatography is possible only in case oflasalocid with the application of fluorescent detectors [13]. Lasalocid, alone among thecarboxylic acid ionophores, has a fluorescent chromophore. All of the HPLC-basedassays that have been developed to determine the other carboxylic acid ionophores intissue require derivatisation to introduce a suitable chromophore. Salynomicin and otherionophore coccidiostatics not have chromophore or luminophore groups, which allowUV or fluorescent detection, and due to their thermal degradability, impossible the use ofgas chromatography, resorting to post-column derivatisation (NSD-method) in order toprepare derivatives with dansylhidrazin [6] and pyridinium dichromat derivatisingreagens [8]. Frequently described is still NSD-method with vanillin reagent [2]. Sinceother methods for determination salynomicin and other ionophores shoud be notedenzyme-immuno (ELISA) method [16]. In general, the ELISA procedures developed todetect ionophore residues were significantly more sensitive compared with the earlierTLC-bioatography based sreening assays. Detection limit of this method is 0,2 ng/gsample.MATERIAL AND METHODSPreparation of samplesThe extraction efficiency and recovery of applied spectrophotometric and liquidchromatography method for the determination of salinomycin content were studied bythe standard addition method. Original extraction procedure and determinationsalinomycin content in premixes and poultry feed was developed [11]. Three grams of277
1 st WorkshopXIII International Feed Technology SymposiumSilica-gel G (for column chromatography, 70-230 mesh) was puted into the glasschromatography column. On the top of the chromatographyc column was one gram ofbroiler feed or premix with salinomycine and salinomycine was eluated with methanol infive fractions of the 1 cm 3 . Eluents were evaporated under a stream of nitrogen and theremainder was reconstituted in cm 3 of methanol. For one gram saple of feed (which isnot contained salinomycin) was added 10, 20, 40 and 60 µl primary standard solution ofsalinomycin in methanol mass concentration of 1 mg/cm 3 . In this way were obtainedfeed samples with standard addition of 10, 20, 40 and 60 µg salinomycin per 1 g of feed.Then, extraction was performed on the previously described manner.Spectrophotometric determination of salinomycine contentWe developed a spectrophotometric method for the determination of salinomycine inpremixes and broilers feed on a spectrophotometer Spekol 21 at wavelength of 520 nm.For the preparation of standard curves were prepared standard solutions of salinomycinein mass concentrations from 1 to 10 µg/cm 3 . The 1 cm 3 of each standard solution wasadded per 1 cm 3 vanilline reagent solution. Mixture is heated 25 minutes in the bathwater at 60ºC and after 10 minutes of cooling is done, colorimetric measuring at 520 nm.Identical manner done is to determine the content of salinomycin in samples of premixesand poultry feed from commercial use. After extraction, summary eluents wereevaporated under a stream of nitrogen and the remainder was reconstituted in cm 3 ofmethanol and continue to comply with the previously described analytical procedure ofspectrophotometric determination. Salinomycine concentration in investigation samplesread with a calibration curve by calculating the dilution factor.HPLC determination of salinomycine contentSince salinomycin and other ionophore coccidiostatics don’t contains chromophoregroups, don’t absorbed in the UV region, and has therefore been necessary to develop apost-column derivatisation method. The investigations were conducted on the Bio RadHPLC system with spectrophotometric detection with tungsten lamp at 520 nm. Eluateswith salinomycin were separated on a TSK ODS-120 T, 10 µm (7,8 x 300 mm) columnwith a mobile phase consisting of methanol-water-acetic acid (94:6:0.1) at a flow rate of1cm 3 /min. Post-column derivatisation was performed with vanillin-reagent solutionswhich was prepared as follows: methanol sulphate acid-vanillin (95:2:3, v/vw), protectedfrom UV light. To post-column derivatisation in the shortest possible time came todevelop color, which will alow detection salinomycin the tested samples, it is necessaryreaction chamber heated to a temperature of 90ºC. Before use, the mobile phase isdegase holding in the ultrasonic bath.RESULTS AND DISCUSSIONResults of spectrophotometric determination salinomycin in broilers feed with standardaddition method are presented in Table 1. The results show that the applied extractionmethod, based on the separation salinomycin from samples of broilers feed, on silica-gelcolumn chromatography, provides high efficiency of spectrophotometric extraction anddetermination of 83,8 ± 5,4% do 103,5 ± 6,8%. Also, determination of efficiency is evengreater if the content salinomycin, or in addition to the standard broilers feed, which278
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Effects of dietary n-3 polyunsaturated fatty acids and ... - FINS

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