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Analysis of Melamine Cyanurate in Urine Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Ho-Wai Tang,† Kwan-Ming Ng,*,† Stephen Sin-Yin Chui,† Chi-Ming Che,*,† Ching-Wan Lam,‡ Kwok-Yung Yuen,§ Tak-Shing Siu,| Lawrence Chuen-Leung Lan,⊥ and Xiaoyan CheX Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong, Hong Kong SAR, China, Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China, Department of Microbiology, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China, Clinical Biochemistry Division, Queen Mary Hospital, Hong Kong SAR, China, Department of Surgery, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China, and Clinical Immunology Center, Southern Medical University, Guangzhou, China Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was applied to the direct analysis of melamine cyanurate (MC). The three commonly used MALDI matrixes, namely, r-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), and 2,5-dihydroxybenzoic acid (DHB), were able to desorb/ionize melamine from MC upon N2 laser irradiation, with CHCA showing the highest detection sensitivity in the positive mode. Only DHB and SA were able to desorb/ionize cyanuric acid from MC in the negative mode but with remarkably lower sensitivity. The method is able to detect melamine unambiguously from a small amount of MC (down to 12.5 µg) spiked into urine and was successfully applied for the rapid and sensitive detection of melamine in urine stones/residues of the samples collected from patients clinically confirmed of having kidney stones associated with the consumption of melamine-tainted food products. The urine matrix resulted in interfering ion peaks and suppressed the ion intensity of melamine, while a cleanup process consisting of simply washing with water eliminated such interference and enhanced the ion intensity. The merit of the method is simplicity in sample preparation. The analytical time of the method for high-throughput analysis from the time of sample treatment to analysis is less than 7 minutes per sample, with sensitive detection of the presence of melamine in the urine stones/ residues of the patient samples. Rapid and sensitive chemical analysis of body fluid/tissues is important for clinical diagnosis, as the timely collection of accurate * To whom correspondence should be addressed. Fax, (852) 2857 1586; e-mail,
[email protected] (K.-M.N.). Fax, (852) 2857 1586; e-mail,
[email protected] (C.-M.C.). † Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong. ‡ Department of Pathology, The University of Hong Kong, Queen Mary Hospital. § Department of Microbiology, The University of Hong Kong, Queen Mary Hospital. | Clinical Biochemistry Division, Queen Mary Hospital. ⊥ Department of Surgery, The University of Hong Kong, Queen Mary Hospital. X Southern Medical University.
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data can be crucial to the health or even life of an individual. Herein is described a simple and sensitive method for the chemical analysis of solid urine stones/residues containing melamine using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The outbreak of food adulteration with melamine was first discovered in dairy products, and this adulterant is now found in other types of food products such as eggs and seafood.1,2 In 2007, pet food adulteration with melamine leading to kidney toxicity in cats and dogs was reported. The incident has also affected thousands of children in China.3-9 It is believed that melamine per se is nontoxic. However, when it encounters cyanuric acid, an analogue of melamine, a stable compound (melamine cyanurate, as shown in Figure 1) with poor aqueous solubility is formed.9 This compound precipitates in renal tubules and results in the formation of kidney stones and eventually lead to renal toxicity. Public concern led to more than 24 000 clinical assessments within 3 months in Hong Kong, and more than 10 children were confirmed to be suffering from the (1) Andersen, W. C.; Turnipseed, S. B.; Karbiwnyk, C. M.; Clark, S. B.; Madson, M. R.; Gieseker, C. M.; Miller, R. A.; Rummel, N. G.; Reimschuessel, R. J. Agric. Food Chem. 2008, 56, 4340–4347. (2) Centre for Food Safety, The Government of the Hong Kong Special Administrative Region of the People’s Republic of China. Unsatisfactory Results of Testing of Melamine, December 2, 2008. Available at http:// www.cfs.gov.hk/english/whatsnew/whatsnew_fstr/files/melamine_dec/ List_of_unsatisfactory_food_samples.pdf (accessed December 17, 2008). (3) World Health Organization. Melamine-Contamination Event, China, 2008 December 8, 2008. Available at http://www.who.int/foodsafety/fs_ management/infosan_events/en/index.html (accessed December 17, 2008). (4) World Health Organization. Melamine and Cyanuric Acid: Toxicity, Preliminary Risk Assessment and Guidance on Levels in Food, October 30, 2008. Available at http://www.who.int/foodsafety/fs_management/ Melamine.pdf (accessed December 17, 2008). (5) World Health Organization. Expert Meeting to Review Toxicological Aspects of Melamine and Cyanuric Acid, Executive Summary, December 2008. Available at http://www.who.int/foodsafety/fs_management/Exec_ Summary_melamine.pdf (accessed December 17, 2008). (6) Chan, E. Y. Y.; Griffiths, S. M.; Chan, C. W. Lancet 2008, 372, 1444–1445. (7) Xin, H.; Stone, R. Science 2008, 322, 1310–1311. (8) Chiu, M. C. Hong Kong Med. J. 2008, 14, 424–426. (9) Dobson, R. L. M.; Motlagh, S.; Quijano, M.; Cambron, R. T.; Baker, T. R.; Pullen, A. M.; Regg, B. T.; Bigalow-Kern, A. S.; Vennard, T.; Fix, A.; Reimschuessel, R.; Overmann, G.; Shan, Y.; Daston, G. P. Toxicol. Sci. 2008, 106, 251–262. 10.1021/ac802752n CCC: $40.75 2009 American Chemical Society Published on Web 04/02/2009
Figure 1. Chemical structure of melamine, cyanuric acid, and melamine cyanurate.
formation of kidney stones.10,11 Considering the number of potential patients, the development of an efficient and reliable method for the detection of melamine in clinical samples is important to ease the burden on clinical laboratories. In fact, the importance of development of a quick and sensitive method for large population screening of melamine cyanurate crystals in urine has also been recognized by the World Health Organization.12 Biochemical/chemical analysis of biological samples in solid state is still a challenging task. An accurate analysis of chemical composition is important for understanding the cause of renal stone formation and determining the proper clinical treatment. Ultrasound imaging is a common method for in vivo kidney stone screening but cannot provide chemical composition information. Fourier transform infrared spectroscopy (FT-IR)13 and powder X-ray diffraction (XRD)14 are the two most common methods adopted for in vitro composition analysis of kidney stones. Both methods are relatively fast, but a large sample amount (at least the milligram level) is required. Optical and electronic microscopy have also been applied to examine the morphology of kidney stones.15,16 In fact, some of the above techniques have been successfully applied for the characterization of kidney stones in dogs associated with the consumption of melamine-containing pet food.17,18 Nevertheless, the methods mentioned above require (10) Hospital Authority, The Government of the Hong Kong Special Administrative Region of the People’s Republic of China. Hospital Authority Updates on Designated Clinics and Special Assessment Centres, December 16, 2008. Available at http://www.ha.org.hk/visitor/ha_visitor_index.asp?Content_ ID)136546&Dimension)100&Lang)ENG (accessed December 17, 2008). (11) Expert Group on Melamine Incident, Food and Health Bureau, The Government of the Hong Kong Special Administrative Region of the People’s Republic of China. Report of the Expert Group on Melamine Incident, October 20, 2008. Available at http://www.fhb.gov.hk/melamine/ doc/report20081020.pdf (accessed December, 2008). (12) World Health Organization. Expert Meeting to Review Toxicological Aspects of Melamine and Cyanuric Acid, Overall Conclusions and Recommendations, December 2008. Available at http://www.who.int/foodsafety/fs_ management/conclusions_recommendations.pdf (accessed December 17, 2008). (13) Singh, I. Int. Urol. Nephrol. 2008, 40, 595–602. (14) Bhatt, P. A.; Paul, P. J. Chem. Sci. 2008, 120, 267–273. (15) Nayir, A. Pediatr. Nephrol. 2002, 17, 425–432. (16) Sandersius, S.; Rez, P. Urol. Res. 2007, 35, 287–293. (17) Thompson, M. E.; Lewin-Smith, M. R.; Kalasinsky, V. F.; Pizzolato, K. M.; Fleetwood, M. L.; McElhaney, M. R.; Johnson, T. O. Vet. Pathol. 2008, 45, 417–426. (18) Osborne, C. A.; Lulich, J. P.; Ulrich, L. K.; Koehler, L. A.; Albasan, H.; Sauer, L.; Schubert, G. Vet. Clin. Small Anim. 2009, 39, 1–14.
reference standards, hence the analysis of substances which are not previously reported is difficult. Mass spectrometry is well-known for its molecular specificity and high sensitivity of detection. With accurate mass measurement and tandem mass spectrometric analysis, the chemical identity of analytes can be unambiguously confirmed. Liquid chromatography/mass spectrometry (LC/MS) can be applied to the chemical analysis of kidney stones in dissolved form and for the identification and quantification of target compounds.19 However, the tedious sample preparation required for minimizing the effect of complex urine matrix makes the methodology less efficient for analyzing a large number of samples. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is useful for the direct analysis of biological tissues in the solid state, including intact animal and plant tissues.20,21 In fact, MALDI-MS has been applied to the analysis of melamine and its derivatives, including ammeline, ammelide, and cyanuric acid.22 Recently, two novel mass spectrometric techniques, namely, ultrasound-assisted extractive electrospray ionization mass spectrometry (EESI-MS) and low-temperature plasma (LTP) probe tandem mass spectrometry have been developed for the rapid, sensitive and quantitative analysis of melamine in complex mixture, such as dairy samples and wheat gluten.23,24 However, melamine cyanurate solid is a giant organic crystal formed through extensive hydrogen bonding. It is insoluble in organic solvent and has very low water solubility (2 mg/mL) being 1600 times lower than that of melamine (3240 mg/mL). The significant difference in the property could create challenges to the current analytical methods for sensitive analysis of melamine cyanurate solid in a complex mixture. To the best of our knowledge, this is the first report on employing MALDI-MS method for the direct analysis of biochemical crystals containing the melamine/cyanuric acid complex in urine samples. Melamine can be desorbed/ionized upon N2 laser irradiation by simply mixing the commonly adopted MALDI matrixes with the melamine cyanurate in urine residue. The aim of our study is to apply the MALDI-MS method for the direct analysis of melamine cyanurate in urine, and to investigate the applicability of the method for analysis of melamine-containing urine stone/ residue samples collected from patients confirmed of having kidney stones associated with the consumption of melaminetainted food products. EXPERIMENTAL SECTION Chemicals and Reagents. Melamine (99%) was purchased from Alfa Aesar (Ward Hill, MA). Cyanuric acid (99%) was purchased from Lancaster (England). R-Cyano-4-hydroxycinnamic acid (99%) and sinapinic acid (>98%) were purchased from Sigma Aldrich (St. Louis, MO). 2,5-Dihydroxylbenzoic acid (AR grade) was purchased from ICN Biomedicals (Costa Mesa, CA). Methanol (HPLC grade) was purchased from Tedia (Fairfield, OH). All chemicals were used as received without further purification. (19) Perello´, J.; Sanchis, P.; Grases, F. J. Chromatogr., B 2005, 824, 175–180. (20) Reyzer, M. L.; Caprioli, R. M. Curr. Opin. Chem. Biol. 2007, 11, 29–35. (21) Ng, K.-M.; Liang, Z.; Lu, W.; Tang, H.-W.; Zhao, Z.; Che, C.-M.; Cheng, Y.-C. Anal. Chem. 2007, 79, 2745–2755. (22) Campbell, J. A.; Wunschel, D. S.; Petersen, C. E. Anal. Lett. 2007, 40, 3107–3118. (23) Huang, G.; Ouyang, Z.; Cooks, R. G. Chem. Commun. 2009, 556, 558. (24) Zhu, L.; Gamez, G.; Chen, H.; Chingin, K.; Zenobi, R. Chem. Commun. 2009, 559, 561.
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Water was purified through a Barnstead NANOpure Diamond water purification system (Thermo Fisher Scientific, Waltham, MA). Preparation of Melamine Cyanurate. Melamine cyanurate was prepared by mixing the aqueous solutions of melamine (1 × 10-3 M) and cyanuric acid (1 × 10-3 M) in equal volume (50 mL in total). The resulting white precipitates, which are melamine cyanurate (MC), were centrifuged, washed with warm water, and collected for analysis. In the method development, melamine cyanurate was also prepared in urine and is denoted as UMC, in order to mimic the real urine stones/ residues found in patients having kidney stones associated with the consumption of melamine-tainted food products. UMC was prepared according to the same procedures for the preparation of MC in water, except that the solvent was changed to urine. The urine collected from a healthy male volunteer was used as received. The UMC is light brown in color. The stock solutions of MC and UMC were prepared, respectively, by suspending the MC and UMC in water at a concentration of 0.5 mg/mL. To investigate the interference effect of urine matrix on the detection of melamine from the UMC, three urine samples spiked with UMC (containing 100 µL of UMC stock and 900 µL of control urine from the healthy individual) were prepared. The spiked samples were centrifuged for 5 min at 13 200 rpm at 8 °C, and the supernatants were removed. The residue of one spiked sample was immediately resuspended in urine (100 µL) without any other treatment, while the other two residues were washed with cold (
