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Analytical LettersPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713597227

Analysis of Melamine, Cyanuric Acid, Ammelide, and Ammeline Using Matrix-Assisted Laser Desorption Ionization/Time-of-Flight Mass Spectrometry(MALDI/TOFMS)James A. Campbell a; David S. Wunschel a; Catherine E. Petersen aa 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

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ENVIRONMENTAL

Analysis of Melamine, Cyanuric Acid,

Ammelide, and Ammeline Using

Matrix-Assisted Laser DesorptionIonization/Time-of-Flight Mass

Spectrometry (MALDI/TOFMS)

James A. Campbell, David S. Wunschel,

and Catherine E. Petersen

Pacific 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-flight 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.

(M2H)2 was observed for cyanuric acid with sinapinic acid as the matrix.

Keywords: Melamine, matrix-assisted laser desorption ionization/time-of-flight massspectrometry, 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 fire retardant

material, fertilizer component, and is also a metabolic byproduct of

Received 11 July 2007; accepted 1 September 2007

Pacific 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, Pacific Northwest National

Laboratory, P.O. Box 999, MS P8-08, Richland, Washington. E-mail: james.

[email protected]

Analytical Letters, 40: 31073118, 2007

Copyright# Taylor & Francis Group, LLCISSN 0003-2719 print/1532-236X onlineDOI: 10.1080/00032710701646131

3107


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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 recentlyreported 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; Crem-

monezzi et al. 2004). Melamine is known to metabolize into forms sequen-

tially 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 per-

formance 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 deriva-tization GC/MS for the analysis of melamine, ammelide, ammeline, andcyanuric 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-flight 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

Figure 1. Structures of melamine, ammelide, ammeline, and cyanuric acid.

Table 1. Substituent designation and MW for melamine,

cyanuric acid, ammeline, and ammelide

Compound X Y Z MW

Melamine NH2 NH2 NH2 126

Cyanuric acid OH OH OH 129

Ammeline NH2 NH2 OH 127

Ammelide NH2

OH OH 128

J. A. Campbell et al.3108


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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

efficiency due to the lack of high proton affinity 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, alpha-

cyano-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 aspectsof the typical MALDI experiment is the generation of intact molecular ions.

Time-of-flight 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 analyzedusing 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 differ-

ence 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 (M2

H)2

isobserved 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 trifluoroacetic acid were obtained from Sigma (Milwaukee,

Wisconsin). AldrichTrifluoroacetic 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

Analysis of Melamine Using MALDI/TOFMS 3109


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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% aceto-nitrile, 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 Melamineand 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 wasthen 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 thediluted sample was pipetted onto a 400 mm Bruker AnchorchipTM plate

without drying. Then 0.5 ml of matrix was added.

Instrumental

MALDI-TOF and MALDI-TOF-TOF analysis

All the compounds were analyzed using a Bruker MALDI/TOF II (Autoflex) inreflectron 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 reflector 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 deflector was used and the reflector 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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TOF/TOF

Experiments were performed using the LIFTTM TOF/TOF mass spectrometerBruker, Autoflex (Billerica, Massachusetts) on the melamine-matrix complex.

The mass spectrometer consists of a gridless ion source with delayed extrac-

tion electronics, a lift device for raising the potential energy of the ions, a

Figure 5. Melamine-cyanuric acid complex.

Figure 6. MALDI spectrum of melamine (m/z 127)and SA (top) and SA only (bot-tom) over mass range of 115250 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 flight tubeof 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) andsinapinic acid (SA), two MALDI matrices, are shown in Fig. 2.

Melamine was analyzed using MALDI/TOF and ACHC as the matrix inthe positive ion mode. The resulting MALDI spectra for melamine and

matrix blank are shown in Fig. 3. The major peak of difference was observed

Figure 7. MALDI spectra of SA only (top) and melamine with SA only (bottom)

over mass range m/z 300380 in positive ion.



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at m/z 316. MS/MS studies on m/z 316 showed a major dissociation product atm/z 127.4, and the resulting spectrum is shown in Fig. 4. This result indicatesthat the peak m/z 316 represents a noncovalent complex of melamine plus thematrix 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/z127.3. 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 massspectrum for the mass range m/z 300 to 380 indicating no adduct ion. If anadduct 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 100270 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

(M2H)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 30150 in positive ion.



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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 flexible 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 profile the

compounds that are present in pet food as well as biological matrices.

MALDI/TOF has been used for the analysis of melamine, ammelide,

Figure 10. MALDI spectrum of cyanuric acid (m/z 129.6) with SA (top) and SA(bottom) over the ranges of 100280 in negative ion.



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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 (M2H)2

for cyanuric acid.

The results indicate that MALDI/TOF can be used for the analysis ofmelamine, 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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