Synthesis of [15N3]melamine and[13C3]cyanuric acidYong Luo,� Liya Zhang, Weicheng Yang, Weixia Liu, Weijing Lu,and Meihua Li

This report presents an efficient syntheses of two labelled internal standards [15N3]melamine and [13C3]cyanuric acid withthe common substrates [15N]NH4Cl and [13C]urea, respectively. Both standards with excellent isotopic and chemical puritieswere prepared in good yields and could be used as internal standards without further purification.

Keywords: isotopically labelled synthesis; N-15; melamine; cyanuric acid; labelled; synthesis; internal standards

Introduction

An increased incidents of kidney stones and renal failurereported in China and the large outbreak of renal diseasesand deaths of pet animals in USA are believed to be associatedwith melamine-contaminated dairy products and pet foods inwhich melamine has been deliberately added to boost itsapparent protein content.1,2 Melamine combines with cyanuricacid to form melamine cyanurate crystal sediment whichobstructed and damaged renal tubules, leading to renalfailure.3,4 Liquid chromatography isotope diluted tandem massspectrometry (LC-IDMS/MS) methods have proved sensitive andreliable for the determination of both melamine and cyanuricacid among various analytical approaches.2,5–8

In 2002, Patil reported the preparation of [14C3]cyanuric acidfrom [14C]urea under solvent-free conditions.9 Recently,[13C3]melamine and [13C3]cyanuric acid were synthesized from[13C3]cyanuric chloride for the determination of melamine andits metabolite.10 Here we described an efficient synthesis of[15N3]melamine and [13C3]cyanuric acid starting from commer-cially available [15N]NH4Cl and [13C]urea.

Experimental

Materials and instruments

[15N]NH4Cl (99.5 atom% 15N) and [13C3]urea (99.5 atom% 13C)were provided by Shanghai Engineering Research Center ofStable Isotope. All other chemicals were of analytical grade.1H-NMR (500.13 MHz) and 13C-NMR (125.70 MHz) spectra wererecorded on Bruker DRX500 spectrometer in [D6]DMSO (TMS asinternal standard). FT-IR spectra were recorded on a NicoletFT-IR 6700 spectrometer using KBr pellets. EI-MS Spectra wereobtained with TSQ Quantum Access spectrometer.

Synthesis

2,4,6-triamino-½15N3�1,3,5- triazine, ½15N3�melamine 4

Aqueous ammonia (approximately 6.6 mol/L) which was derivedfrom [15N]NH4Cl (13.08 g, 0.24 mol) according to the procedure11

was added to a solution of cyanuric chloride (7.38 g, 0.04 mol) indioxane (40 mL) and the resulting mixture was placed in aTeflon-lined stainless steel autoclave with magnetic stirring andthen heated at 1401C for 4 h under autogenous pressure. Themixture was filtered, washed with distilled water (3–10 mL), thefiltrate was evaporated to dryness, then recrystallized in waterand dried under reduced pressure to give 5.04 g of whitepowder. [15N]NH4Cl in the filtrate could be recycled quantita-tively and the yield was better than 97.0% based on labellingsubstrate consumed. 1H NMR ( DMSO-D6): d6.606(d, J15N-1H =71.2Hz, 6H); 13C NMR( DMSO-D6):d165.367(d, J15N-13C = 78.4Hz,3C); FT-IR (KBr) cm�1: 3459, 3405, 3330, 3130, 1650, 1537, 1431;LC-MS-ESI1: [M1H] = 130.19 (100%).

2,4,6-trihydroxy-1,3,5-½13C3� triazine, ½13C3�cyanuric acid 6

[13C3]urea(5.07 g, 0.083 mol) in sulfolane(12 mL) was placed to a50 mL, three-necked flask equipped with stirrer, reflux con-denser, and vacuum source. The pressure within flask wasmaintained at 100 mm of Hg throughout the resulting run; themixture was heated until it refluxed at 2051C and kept for 2 h.After cooling to room temperature, the resulting precipitate wasfiltered, treated with 20% (wt) (20 mL) sulfuric acid at 1001C for4 h. The mixture was filtered, washed with distilled water(3–10 mL) and dried under reduced pressure to give 3.23 g ofwhite powder in 88.4% yield. 1H NMR (DMSO-D6): d11.127(s, 3H);13C NMR ( DMSO-D6): d165.367(dd, J15N-13C = 54.2 Hz,3C); FT-IR(KBr) cm�1: 3098, 3017, 2765, 1758, 1721, 1458, 1398; LC-MS-ESI�: [M-H] = 131.05(100%).

17

1

Shanghai Research Institute of Chemical Industry, Shanghai, China

*Correspondence to: Yong Luo, Shanghai Research Institute of ChemicalIndustry, Shanghai, China.E-mail: real-luoyong@hotmail.com

Research Article

Received 21 June 2010, Revised 15 August 2010, Accepted 17 August 2010 Published online 2 November 2010 in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1835

J. Label Compd. Radiopharm 2011, 54 171–172 Copyright r 2010 John Wiley & Sons, Ltd.

Results and discussion

Although the manufacture of melamine and cyanuric acid arefairly common on industrial scale, reports of the preparation ofisotopically labelled melamine and cyanuric acid are very rare.Previously Varelis described the synthesis of [13C3]melamine and[13C3]cyanuric acid in which [13C3]cyanuric chloride reacted withammonia and formic acid, respectively, to give [13C3]melamineand [13C3]cyanuric acid in moderate yields. In order to improvethe synthesis of both standards, we developed a new syntheticmethod with satisfying labeling yields with the commonsubstrates [15N]NH4Cl and [13C]urea.12

As shown in Scheme 1, multi-step ammonization of cyanuricchloride could stop at the one or two substitution stage bycontrolling the reaction temperature and the dosage ofammonia properly. In this article, [15N3]melamine was synthe-sized via a one-pot method and solvent played an importantrole in the reaction. By using dioxane as the solvent,[15N3]melamine could be synthesized with higher than 99.4%chemical purity. [15N]NH4Cl in the filtrate could be recycledquantitatively and the yield of 4 was better than 97.0% based onlabeled-substrate consumed. Other attempts using water,acetonitrile, and acetone as solvent were made and 2,4-diamine-6-chloride-1,3,5-triazine was found to be the mainproducts under the same reaction conditions.

It is known that cyanuric acid could be prepared by thepyrolysis of urea13–15 in either liquid or solid state (Scheme 2).

We found that when the reaction was carried out under reducedpressure and sulfolane as solvent, [13C3]cyanuric acid wasobtained with satisfying yield up to 88.4% based on [13C]ureaconsumed.

References

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[4] R. L. Dobson, S. Motlagh, M. Quijano, R. T. Cambron, T. R. Baker,A. M. Pullen, B. T. Regg, A. S. Bigalow-Kern, T. Vennard, A. Fix,R. Reimschuessel, G. Overmann, Y. Shan, G. P. Daston, Toxicol. Sci.2008, 106, 251–262.

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[7] L. Vaclavik, J. Rosmus, B. Popping, J. Hajslova, J. Chromatogr. A2010, 1217, 4204–4211.

[8] M. S. Filigenzi, E. R. Tor, R. H. Poppenga, L. A. Aston, B. Puschner,Rapid Commun. Mass. Sp, 2007, 21, 4027–4032.

[9] S. P. Patil, D. Padmanabhan, J. Label. Compd. Radiopharm. 2002,45, 539–542.

[10] P. Varelis, R. Jeskelis, Food. Addit. Contam. 2008. 25, 1208–1215.[11] R. Schoenheimer, S. Ratner, J. Biol. Chem. 1939, 127, 301–313.[12] W. C. Yang, Y. Luo, W. J. Lu, CN 101497587 A, Shanghai Research

Institute of Chemical Industry, 2009.[13] S. Berkowitz, H. Park, US 3563987, A1, FMC, 1971.[14] O. Marinus, P. G. Lambertus, H. Schouteten, US 3956299, A1,

Stamicarbon B. V., 1976.[15] T. D. Zhao, Y. Meng, M. A. Li, X. H. Yan, CN 101429169 A, Tianyi

Chemical Company, 2009.

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2

N N

N

Cl

ClCl

N N

N

15NH2

15NH2Cl

N N

N

Cl

15NH2Cl

N N

N

15NH2

15NH2H215N

15NH315NH3

0-5 C C30-50

15NH3

C80

1 2 3 4

Scheme 1. Synthesis of [15N3]melamine.

N N

N OHHO

OH13C3

solvent

5 6

H2N NH213C

O

Scheme 2. Synthesis of [13C3]cyanuric acid.

Y. Luo et al.

www.jlcr.org Copyright r 2010 John Wiley & Sons, Ltd. J. Label Compd. Radiopharm 2011, 54 171–172