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Analytical Letters
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Analysis of Melamine, Cyanuric Acid, Ammelide, and Ammeline Using MatrixAssisted Laser Desorption Ionization/Time-of-Flight Mass Spectrometry (MALDI/TOFMS)
James A. Campbell a; David S. Wunschel a; Catherine E. Petersen a a Pacific Northwest National Laboratory, Richland, Washington Online Publication Date: 01 January 2007

To cite this Article Campbell, James A., Wunschel, David S. and Petersen, Catherine E.(2007)'Analysis of Melamine, Cyanuric Acid,

Ammelide, and Ammeline Using Matrix-Assisted Laser Desorption Ionization/Time-of-Flight Mass Spectrometry (MALDI/TOFMS)',Analytical Letters,40:16,3107 3118
To link to this Article: DOI: 10.1080/00032710701646131 URL: http://dx.doi.org/10.1080/00032710701646131

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Analytical Letters, 40: 31073118, 2007 Copyright # Taylor & Francis Group, LLC ISSN 0003-2719 print/1532-236X online DOI: 10.1080/00032710701646131

ENVIRONMENTAL

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Analysis of Melamine, Cyanuric Acid, Ammelide, and Ammeline Using Matrix-Assisted Laser Desorption Ionization/Time-of-Flight Mass Spectrometry (MALDI/TOFMS)
James A. Campbell, David S. Wunschel, and Catherine E. Petersen
Pacic Northwest National Laboratory, Richland, Washington

Abstract: Melamine, cyanuric acid, two compounds connected to tainted pet food, and related analogs have been analyzed using matrix-assisted laser desorption ionization/ time-of-ight mass spectrometry. (M H) ions were observed for ammelide and ammeline under positive ion conditions with sinapinic acid as the matrix. With alpha-cyano-4-hydroxy-cinnamic acid as the matrix, a matrix-melamine complex was observed; however, no complex was observed with sinapinic acid as the matrix. (M 2 H)2 was observed for cyanuric acid with sinapinic acid as the matrix. Keywords: Melamine, matrix-assisted laser desorption ionization/time-of-ight mass spectrometry, ammelide, ammeline, cyanuric acid

INTRODUCTION Melamine (C3H6N6) (structure and molecular weight fMWg shown below in Fig. 1 and Table 1) is a chemical commonly used as a re retardant material, fertilizer component, and is also a metabolic byproduct of
Received 11 July 2007; accepted 1 September 2007 Pacic Northwest National Laboratory is operated for the Department of Energy by Battelle Memorial Institute under contract DE-AC06-76RLO. Address correspondence to Dr. James A. Campbell, Pacic Northwest National Laboratory, P.O. Box 999, MS P8-08, Richland, Washington. E-mail: james. campbell@pnl.gov 3107

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Figure 1.

Structures of melamine, ammelide, ammeline, and cyanuric acid.

cyromazine, an insect growth inhibitor (Cook and Hutter 1981; Roberts and Hutson 1998). In addition, melamine and cyanuric acid (structure and MW shown below in Fig. 1 and Table 1) have been associated with the recently reported tainted pet food and their subsequent toxic effects on animals; the exact mechanism of toxicity is still being investigated. Although largely excreted in the urine, high doses of melamine have been shown to form uroliths and carcinomas in rats (Mast et al 1983; Ogasawara et al. 1995; Cremmonezzi et al. 2004). Melamine is known to metabolize into forms sequentially replacing amino groups with the hydroxyl groups at each position to form cyanuric acid (Wackett et al. 2002). Melamine and its associated analogs have been analyzed using high performance liquid chromatography (Shelton et al. 1997; Chan et al. 2004) and gas chromatography/mass spectrometry (GC/MS) (Yokley et al. 2000). The Food and Drug Administration (FDA) is presently using silylation derivatization GC/MS for the analysis of melamine, ammelide, ammeline, and cyanuric acid in various matrices (http:/ /www.fda.gov/cvm/ MelamineAnalogs.htm). The matrix-assisted laser desorption ionization (MALDI) technique was developed by Karas and Hillenkamp (1988) and Tanaka et al. (1988) to overcome the mass range limitations of laser desorption ionization and provide a simple method for introducing high molecular weight species directly into the gas phase in both neutral and ionic forms. Matrix-assisted laser desorption ionization/time-of-ight mass spectrometry (MALDI/ TOFMS) has been primarily used to obtain spectra of very large polymers, biomolecules (Fei et al. 1996; Jackson et al.1996; Yang and Orlando 1996), and a variety of thermally labile materials (Lidgard and Duncan 1995). MALDI/TOF has also been used for the analysis of smaller molecules
Table 1. Substituent designation and MW for melamine, cyanuric acid, ammeline, and ammelide Compound Melamine Cyanuric acid Ammeline Ammelide X NH2 OH NH2 NH2 Y NH2 OH NH2 OH Z NH2 OH OH OH MW 126 129 127 128

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(e.g., , 500 mw) (Goheen et al.1997; Campbell et al. 2001; Petersen et al. 2002) even though several challenges exist when working in this mass range. As an example, the analytes of interest may have very poor ionization efciency due to the lack of high proton afnity functional groups. Also, the presence of a variety of abundant matrix-related ions in the low mass range can clutter the spectrum below 500 Da and may suppress analyte signals. In MALDI, the sample to be analyzed is mixed with a matrix, which in turn absorbs energy from irradiation with a nitrogen laser light. For example, alphacyano-4-hydroxycinnamic acid (ACHC) or sinapinic acid (SA), which are commonly, used matrices, have a carboxyl group and a benzene ring. The matrix absorbs the energy and acts as a proton donor. One of the key aspects of the typical MALDI experiment is the generation of intact molecular ions. Time-of-ight mass spectrometry (TOFMS) allows the majority of the ions generated throughout the mass range to be collected by the detector. We have analyzed melamine with ACHC and SA as matrices using MALDI/TOFMS. Ammelide, ammeline, and cyanuric acid were analyzed using SA as the matrix. A matrix-analyte complex was observed with melamine and ACHC; however, a complex was not observed with the use of SA, possibly due to steric hindrance preventing its formation. The difference in acidity between ACHC and SA may also account for this observation. (M H) ions are observed for ammelide and ammeline with SA. Cyanuric acid responds very well under negative ion conditions and (M 2 H)2 is observed with SA. This technique has the potential use as a screening tool for melamine and its analogs or contaminants. In addition, it is viable as a method for analyzing biological samples contaminated with melamine, ammelide, ammeline, or cyanuric acid.

EXPERIMENTAL Materials Melamine was obtained from MCB (Cincinnati, Ohio). Ammelide and ammeline were purchased from TCI America (Portland, Oregon). Cyanuric acid and triuoroacetic acid were obtained from Sigma (Milwaukee, Wisconsin). AldrichTriuoroacetic acid (TFA) was obtained from Sigma. Matrices were obtained from Bruker Daltonics (Billerica, Massachusetts) in the highest purity available. AnchorchipsTM were obtained from Bruker Daltonics (Billerica, Massachusetts).

Procedure for MALDI Analysis of Melamine with ACHC 3 mg of dry material was weighed out and transferred to a clean glass vial. Then 300 ml neat TFA was added to each vial. The 10 mg/ml solution was then

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vortexed. This solution was used to make a 1:1000 dilution in acetonitrile. The ACHC matrix solution was prepared as a 10 mg/ml solution in 33% acetonitrile, 33% ethanol, 32.97% water, and 0.03% TFA. Both the analyte and matrix were spotted at 0.5 ml each on a stainless steel plate and allowed to dry. AnchorchipTM Procedure for MALDI/MS Analysis of Melamine and Analogs 3 mg of dry material was weighed out and transferred to a clean glass vial. Then 300 ml neat TFA was added to each vial. The 10 mg/ml solution was then vortexed. This solution was used to make a 1:1000 dilution of each compound in acetonitrile. The SA matrix solution was prepared using 1 mg/ml SA in 90% acetonitrile with 0.1% TFA. A 0.5 ml aliquot of the diluted sample was pipetted onto a 400 mm Bruker AnchorchipTM plate without drying. Then 0.5 ml of matrix was added.

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Instrumental MALDI-TOF and MALDI-TOF-TOF analysis All the compounds were analyzed using a Bruker MALDI/TOF II (Autoex) in reectron mode. Melamine was analyzed with alpha-cyano-4-hydroxycinnamic acid in the positive ion mode. Melamine, ammelide, and ammeline were analyzed using AnchorchipsTM and SA. Cyanuric acid was analyzed with SA in the negative ion mode. The instrument was internally calibrated using matrix ions and the potassium ion to encompass the mass range. The laser was operated at 25 Hz with 300 to 500 shots collected for each spectrum. MALDI/TOFMS analysis in reector mode used a 19 kV ion source 1 (IS1) and 17.1 kV ion source 2 (IS2) accelerating voltage with a 40 ns pulsed ion extraction delay. No low mass deector was used and the reector grid 1 was set to 20 kV and grid 2 at 9.6 kV.

Figure 2. Structures of alpha-cyano-4-hydroxycinnamic acid (ACHC) and sinapinic acid (SA).

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Figure 3. MALDI spectrum of blank (top) and melamine (bottom) from m/z 300 300 with ACHC in positive ion.

Figure 4.

MS/MS spectrum of m/z 316.

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Figure 5.

Melamine-cyanuric acid complex.

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TOF/TOF Experiments were performed using the LIFTTM TOF/TOF mass spectrometer Bruker, Autoex (Billerica, Massachusetts) on the melamine-matrix complex. The mass spectrometer consists of a gridless ion source with delayed extraction electronics, a lift device for raising the potential energy of the ions, a

Figure 6. MALDI spectrum of melamine (m/z 127)and SA (top) and SA only (bottom) over mass range of 115 250 in positive ion.

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high-resolution timed ion selector, and an additional velocity focusing stage, and fast ion detectors (Suckau et al. 2003). In this system, the analyst is able to perform MS/MS experiments without an additional TOF portion or ight tube of the instrument. For the LIFT TM analysis, a parent mass window of 5 Da was used. The IS1 was set to 6 kV and IS2 to 5.2 kV with the LIFT voltages set to 19 and 4.4 kV for LIFT 1 and LIFT 2, respectively.

RESULTS AND DISCUSSION The structures of alpha-cyano-4-hydroxycinammic acid (ACHC) and sinapinic acid (SA), two MALDI matrices, are shown in Fig. 2. Melamine was analyzed using MALDI/TOF and ACHC as the matrix in the positive ion mode. The resulting MALDI spectra for melamine and matrix blank are shown in Fig. 3. The major peak of difference was observed

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Figure 7. MALDI spectra of SA only (top) and melamine with SA only (bottom) over mass range m/z 300 380 in positive ion.

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at m/z 316. MS/MS studies on m/z 316 showed a major dissociation product at m/z 127.4, and the resulting spectrum is shown in Fig. 4. This result indicates that the peak m/z 316 represents a noncovalent complex of melamine plus the matrix ion, in this instance, ACHC (MW 189). The fragment ion observed at m/ z 127.4 corresponds to the predicted mass for protonated melamine. The adduct is possibly very similar in structure to the melamine-cyanuric acid complex known to exist and shown below in Fig. 5. (Damodaran et al. 2001). Melamine was also analyzed using SA as the matrix and the resulting spectrum is illustrated in Fig. 6. The major ion formed is (M H) at m/z 127.3. It is interesting to note that there was no indication of a melamine and matrix adduct, in contrast to ACHC. Figure 7 is the MALDI mass spectrum for the mass range m/z 300 to 380 indicating no adduct ion. If an adduct were formed and detected, one would expect a peak at m/z 351

Figure 8. MALDI spectra of SA only (top) and ammeline (m/z 128) with SA (bottom) over the mass range 100 270 in positive ion.

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(225-SA 126-melamine 351). The lack of an adduct ion may be due to steric hindrance or lack of the necessary resonance form present in SA. The fact that ACHC may be a stronger acid than SA will account for this observation. Ammeline and ammelide were analyzed using SA in the positive ion mode. The ions m/z 128 and 129.6 were observed, indicating the (M H) ions for each. The spectra are shown in Figs. 8 and 9, respectively. It is important to note that for ammelide, the differential laser power necessary to ionize the matrix ions in the blank versus the very much reduced laser power necessary to see ammelide, prohibited the production of the matrix ions in the sample. This may account for the 129.6 m/z mass assignment. Additional studies are underway to understand the apparent discrepancy in mass assignment for ammelide under these conditions. Cyanuric acid was analyzed using SA acid in the negative ion mode and (M 2 H)2 was observed at m/z 128. No discernible mass spectrum was

Figure 9. MALDI spectra ammelide (m/z 129.6) and SA (top) and SA only (bottom) over the mass range 30 150 in positive ion.

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Figure 10. MALDI spectrum of cyanuric acid (m/z 129.6) with SA (top) and SA (bottom) over the ranges of 100 280 in negative ion.

observed in the positive ion mode, other than that of the matrix. The MALDI/ TOF spectrum in the negative ion mode is shown in Fig. 10.

CONCLUSIONS The growing concern over tainted food products requires exible analytical techniques to analyze not only melamine, but also the compounds that may be present as contaminants or metabolites. Importantly, the production of crystals in urine indicates that an insoluble material forms. Methods to analyze the poorly soluble material will become critical to prole the compounds that are present in pet food as well as biological matrices. MALDI/TOF has been used for the analysis of melamine, ammelide,

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ammeline, and cyanuric acid. Positive ion MALDI for melamine, ammelide, and ammeline show (M H) ions for each. The negative ion spectrum shows (M 2 H)2 for cyanuric acid. The results indicate that MALDI/TOF can be used for the analysis of melamine, ammelide, ammeline, and cyanuric acid. The technique could potentially be used as a screening tool for the analysis of these materials in biological matrices as well.

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