Synthesis And Application Of The Novel Internal Standards For The Determination Of Cyromazine And Melamine

Stable isotope-labeled(SIL) compounds have been widely used in many scientific research fields, such as life science, food safety, resources and environmrnt, new materials, etc. The stability and accuracy of detection of adulterated milk and dairy products would be improved greatly by methods of chromatogram coupled mass spectrum(LC-MS, GC-MS) with SIL intenal standard. The outbreak of "the melamine milk scandal", attracted attention of the public for dairy products, also brought confusion and challenges to Chinese dairy industry. The effective monitoring of dairy products is expected to be done to ensure the safety of its quality. No products of SIL cyromazine reagent and its derivatives are on sale. SIL cyromazine and N-methyl melamine which were to be synthesized, would be applied to the determination of cyromazine and melamine in dairy products.Three kinds of structure of stable isotope labeled internal standard, namely cyromazine-amino-15N2/15N3, cyromazine-ring-15N3, N-methyl/ethyl melamine-15N2,as well as N-methyl/ethyl melamine, were successfully designed for determitation. The novel synthetic methods of three internal standards were established by the design of organic synthesis according to literaturethe about synthesis of natural abundance cyromazine and by the experimental exploration. The optimization of process conditions were obtained by using the single-factor optimization. Natural abundance compouds were used to optimize the experimental conditions for the expensive of Isotope materials. Furthermore,stable isotope labeled syntheticexperiments were smoothly completed after the optimization process and total synthetic tests(two or three runs)were completed.(1) The excessive amounts of ammonia/cyclopropylamine in amination of natural abundance cyromazine synthesis was significantly reduced by the use of acid binding agent, which result in a large decrease amount of ammonia/cyclopropylamine and a lot of increase in the utilization of them, even with an increasing yield in synthesis of them. Such as with sodium carbonate as acid binding agent, utilization of ammonia in amination of cyanuric chloridethe increased from 59.0% to 80.0%. For the amination of Cyclopropanecarboxylic acid chloride, with triethylamine as acid binding agent, the utilization of ammonia increased from 27.2% to 50.6% and the yield of cyclopropane carboxamide increased from 37.6% to 61.1%. In the synthesis of cyromazine with sodium carbonate displace of cyclopropylamine, the utilization of it increased from 27.2% to 5.6% and amount of cyclopropylamine decrease frpm 2.2 eq to 1.2 eq with a little increase of yiled (from 72.0 to 77.3%).(2) Synthetic route of cyromazine-amino-15n2 was as follows:cyanuric chloride�'2,4-diam ino-15N2-6-chloro-1,3,5-triazine(Ⅲ-amino-15N)�'cyromazine-amino-15N2; Synthetic oute of cyromazine-amino-15N3 was as follows:cyanuric chlorid�'2,4-diamino-15N2-6-chloro-1,3.5-triazine (Ⅲ-amino-15N)(reaction with cyclopropylamine-15N)�'cyromazine-amino-15N3 (cyclopropylamine-15N ← cyclopropyl formamide-15N←cyclopropyl chloride←cyclopropanecarboxylate).(3) Cyromazine-ring-15N3 synthetic route:uret-15N2�'cyanuric-15N3�'cyanuric chloride-15N3�'2,4-diamino-6-chloro-1,3，5-triazine-15N3 (Ⅲ-ring-15N)�'cyromazine-ring-15N3 The total yield of cyromazine-ring-15N3 can reach to 19.3% (base on uret-15N2).(4) The optimized conditions, which were obtained through single-factor design, for the synthesis of 2,4-diamino-6-chloro-1,3,5-triazine (Ⅲ), were as follows:THF as solvent, n(cyanuric chloride):n (ammonia):n (sodium carbonate)=1:2.2:1,40～50℃ for 4-5 h.(5) Cyclopropylamine synthetic route was as follows:cyclopropanecarboxylate�'cyclopropyl chloride �'cyclopropyl formamide�'cyclopropanamine. A three-step process for the desired product, the total yield of cyclopropylamine can reach to 28.6% (base on cyclopropanecarboxylate).(6) In the synthesis of cyanuric chloride, Urea as a starting material, sulfolane as a high boiling solvent, between 0.013 MPa～0.019 MPa, at reflux temperature of about 205℃,1.5h to obtain cyanuric acid with yield of 83.0%. And POCl3/PCl5 as the chlorinating reagent,n(cyanuric acid):n(POCl3):n(PCl5)=1:4:4, refluxing 20 hours, the crude yield of cyanuric chloride can reach to 60.0%, the both total of yield can reach to 49.8%(base on uret).(7) In the step of cyromazine process, various systems were investigated. The proper conditions was as follows:water as solvent, sodium carbonate as acid binding agent; n(Ⅲ,2,4-diamino-6-chloro-1,3,5-triazine):n (cyclopropanamine):n (sodium carbonate)= 1:1.1:0.5, refluxing 7 h with yield 77.3%. (8) Synthetic route of N-methyl/ethyl melamine were similar to cyromazine, which as follows:cyanuric chloride�' 2,4-diamino-6-chloro-1,3,5-triazine(Ⅲ)�' N-methyl/ethyl melamine. And the synthetic process conditions were as cyromazine optimized ((4)and(7)), The total yield of N-methyl/ethyl melamine can reach to 63.0% /90.9% respectively(base on cyanuric chloride).(9) Three times of total synthesis of cyromazine amino or ring labeled were carried out with total yield of 21.0%-22.9%,18.0%-19.3% respectively.(10) The SIL synthetic process was carried out under the optimized condition. The target compound cyromazine-amino-15N2 was obtained and the total yield was 68.1%,with a chromatographic purity of 99.7%.The isotopic abundance was 98.8 atom% 15N;And total yield of cyromazine-amino-15N3 was 18.3% with a chromatographic purity of 98.2%,and the isotopic abundance of 99.3 atom% 15N; The total yield of cyromazine-ring-15N3 was17.0% with nitrogen isotopic abundance of 99.1 atom% 15N and purity of 98.0%. N-methyl/ethyl melamine-15N2 were obtained with total yield of 66.4% /95.4% respectively and purity of 99.0%/99.0%, and the isotopic abundances were 98.6atom% 15N,98.2 atom% 15N.The nitrogen isotopic abundance was not diluted essentially;which indicated that the synthetic route and process conditions were suitable for the synthesis of these five kinds of labeled compounds.(11) The target compounds and intermediates were analyzed and characterized by TLC、GC、HPLC、LC-MS and FT-IR.The results showed that their isotopic abundance and purity could meet the required specification.Furthermore, cyromazine-amino-15N2/15N3 and cyromazine-ring-15N3 were detected by external reference method and LC-MS analysis,The data showed that the isotopic abundance and purity of products were high enough for internal standard use.(12) N-methyl melamine was used as an internal standard for determination of cyromazine and melamine in milk by LC-MS/MS. In range of 50～80 mg/L. The spike recovery of melamine was 104.9～106.8% and the cyromazine was 100.8～107.8%. The melamine recovery of a matrix spike was 109.0～113.4% andthe cyromazine’s was 109.4～109.9% in the range of 5～15 mg/L..(13) Also, cyromazine-amino-15N2 was used as an internal standard for determination of cyromazine and melamineby LC-MS/MS.In the range of 50～80 mg/L, The spike recovery of melamine was 98.4～104.4%, RSD was 2.56～5.24%. And the cyromazine’s was 99.7～101.6%,RSD was 1.49～3.01%. The melamine recovery of a matrix spike was 97.7～103.9% with RSD of 0.21～2.25% and the cyromazine’s was 100.2～104.2% with RSD of 0.38～0.61% in the range of 5～15 mg/L.