The Detection Of N-carbamyl-L-glutamic Acid In Feed And Synthesis Of Its Several Metal Compounds

N-Carbamyl-L-glutamic acid (NCG), is a stable, highly safe active substance,which can be used as a novel feed additive to replace or partially replace the arginine,as an animals’ catalyze metabolic activator of carbamoyl phosphate synthetase I(CPS-I), thus, it can promote the synthesis of glutamic acid and proline-citrulline, andthen promote the endogenous synthesis of arginine to produce arginine. NCG cansignificantly improve reproductive and growth performance of pig. To comply withthe safe use norms and feed additive dosage, this paper on pre-mixed feed and feedNCG established detection methods. Meanwhile, the synthesis techniques andtechnology of NCG calcium salt, sodium salt, copper salt and zinc salt were studied.Moreover, an annual output of300t NCG factory was designed on the basis of thesynthetic process of NCG. The main contents are as follows:1.It has been established a method for detecting the NCG content of pre-mixedfeed. The NCG of Pre mixed feed was extracted by ultrasonic extraction techniqueand making a comparison using methanol and water as solvents. It found that theinterference is relatively small and peak shape is better when methanol as solvent.Thus, in our experiment, methanol was employed as solvent in ultrasonic extraction,and then the results were detected by UPLC method. Chromatographicconditions: the chromatographic column was Unimicro C18(250×4.6mm,5μm); the mobile phase was methanol and phosphate water solution(pH3.0)(5:95, v/v); the flow rate was0.6mL/min; the detection wavelength was220nm; DAD detector for the detector. It indicated that NCG has a good linearrelationship in2~10mg/mL range, and the correlation coefficient was0.9999. Thequalitative detection (S/N>3) and quantitative determination (S/N>10) limit were7g/kg and20g/kg respectively; Different level of the sample substrate spikedrecoveries was97.3%-103.0%, the relative standard deviation (RSD) was less than10%.10actual pre-mix feed were detected by this method, the average content ofNCG is41.9g/kg. This method is not only simple, strong anti-interference capability, and high operability, but also high accuracy, sensitivity, to meet the pre-mixed feedNCG analytical testing requirements.2.A high performance liquid chromatography electrospray tandem massspectrometry (HPLC-ESI-MS/MS) analytical method was applied to thedetermination of NCG content in feed. Feed samples were extracted with methanol,then purified by PXA solid phase extraction column, and performed the HPLCseparation. After these treatment, the results were detected qualitatively byelectrospray ionization tandem mass spectrometry in positive ion multiple reactionmonitoring (MRM) in scanning mode, and qualitative analyzed by fragment ion m/z148.0and m/z84.0, quantitative analyzed by fragment ion m/z130.0. Thedetection limit of NCG (S/N>3) was24ug/kg, the limit of quantification (S/N>10)was80μg/kg, in mass concentration range of20~1000μ g/L, the peak area and thecontent have a good linear relationship, the correlation coefficient was0.9999. therecovery rate of NCG at three the volume level (80,200and500mg/kg) in feedwere measured, its recovery was104.0%,103.5%,95.3%, and the relative standarddeviations were7.5%,6.3%,5.8%. Five piglet feed samples and five ordinary NCGpig feed samples were tested by using the method established in this paper, it foundthat NCG was detected in the five piglet feed samples were, and the average contentof309.5mg/kg, while the ordinary pig feed samples were not detected. The methodis simple to operate, and have good purification effects, rapidity, high sensitivity andaccuracy. It meets the detection of NCG in feed samples analysis requirements.3.The synthesis of N-carbamoyl-L-glutamate with calcium, sodium, copper andzinc was carried out. In this experiment, four kinds of metal compounds wassynthesized under different conditions by reacted NCG with calcium oxide, sodiumhydroxide, copper acetate and zinc acetate. Among them, the calcium, sodium, zincsalts are white powder, NCG copper is light blue powder, all of them have a goodthermal stability. These four kinds of metal compounds were analyzed by infraredspectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy andelemental analysis identified, the results indicated that metal compounds weresuccessfully synthesized, NCG combined with calcium, copper, zinc in a molarratio of1:1, with sodium in a molar ratio of1:2. The values were measured on elemental analysis close to the theoretical content.4．An annual output of300t of N-carbamoyl-L-glutamic acid plant was designed,the total investment was13.5million yuan. The project construction land is22100m2,total construction area is11000m2. After the plant is completed, an annual incomecould reach to18million yuan, annual revenue is2.527million yuan, annual after-taxprofit is4.389million yuan, the investment profit rate is as high as32.5%and thepayback period is only3.5years. The safety rate of project management is60.7%.