Development of a Sensitive HILIC-MS/MS Method for Quantification of Melamine, Derivatives, and Potential Precursors in Various Water Matrices
Abstract
1. Introduction
2. Materials and Methods
2.1. Reagents and Stock and Standard Solutions
2.2. Instrumentation
2.3. Method Optimization
2.4. Determination of Limits of Detection and Quantification
2.5. Method Validation
2.6. Stability Test
2.7. Proof of Concept
3. Results
3.1. Sample Preparation
3.2. Optimum Chromatographic and MS Conditions
3.3. Limits of Detection and Quantification
3.4. Method Validation
3.4.1. Recovery of Evaporation
3.4.2. Calibration Linearity
3.4.3. Matrix Effects
3.4.4. Repeatability
3.5. Stability Test
3.6. Proof of Concept
4. Discussion
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
Abbreviations
AMD | Ammeline |
AMN | Ammelide |
CE | Collision energy |
CXP | Collision cell exit potential |
CYA | Cyanuric acid |
CYRO | Cyromazine |
DAD | Diode array detector |
DP | Declustering potential |
ECHA | European Chemicals Agency |
GC | Gas chromatography |
EP | Entrance potential |
HMMM | Hexa(methoxymethyl)melamine |
HILIC | Hydrophilic interaction liquid chromatography |
HPLC | High-performance liquid chromatography |
IS | Isotope-labeled internal standards |
LOD | Limit of detection |
LOQ | Limit of quantification |
MEL | Melamine |
MS | Mass spectrometry |
MS/MS | Tandem mass spectrometry |
PE | Polyethylene |
PS | Polystyrene |
REACH | Registration, evaluation, authorization, and restriction of chemicals |
S/N | Signal-to-noise ratio |
SPE | Solid-phase extraction |
UV | Ultraviolet detector |
WWTP | Wastewater treatment plant |
References
- Wu, Y.; Zhang, Y. Analytical chemistry, toxicology, epidemiology and health impact assessment of melamine in infant formula: Recent progress and developments. Food. Chem. Toxicol. 2013, 56, 325–335. [Google Scholar] [CrossRef] [PubMed]
- ECHA (European Chemicals Agency). Substance Infocard Melamine. Available online: https://echa.europa.eu/substance-information/-/substanceinfo/100.003.288 (accessed on 15 April 2025).
- Charron, I.; Magueresse-Battistoni, B.L.; Habert, R.; Canivenc-Lavier, M.C.; Mhaouty-Kodja, S.; Michel-Caillet, C. Melamine regulatory assessment for endocrine disruption. Environ. Int. 2024, 194, 109188. [Google Scholar] [CrossRef] [PubMed]
- Ministère de la Transition Écologique et de la Cohésion des Territoires. Regulatory Management Option Analysis Conclusion Document. 2024. Available online: https://echa.europa.eu/documents/10162/1f5fde3d-96ae-340a-78bd-61a650ab3fb4 (accessed on 12 June 2025).
- Ono, S.; Funato, T.; Inoue, Y.; Munechika, T.; Yoshimura, T.; Morita, H.; Rengakuji, S.-I.; Shimasaki, C. Determination of melamine derivatives, melame, meleme, ammeline and ammelide by high-performance cation-exchange chromatography. J. Chromatogr. A 1998, 815, 197–204. [Google Scholar] [CrossRef]
- Shelton, D.R.; Karns, J.S.; McCarty, G.W.; Durham, D.R. Metabolism of melamine by Klebsiella terragena. Appl. Environ. Microbiol. 1997, 63, 2832–2835. [Google Scholar] [CrossRef]
- 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.; et al. Identification and characterization of toxicity of contaminants in pet food leading to an outbreak of renal toxicity in cats and dogs. Toxicol. Sci. 2008, 106, 251–262. [Google Scholar] [CrossRef]
- Pote, D.H.; Daniel, T.C.; Edwards, D.R.; Mattice, J.D.; Wickliff, D.B. Effect of drying and rainfall intensity on cyromazine loss from surface-applied caged-layer manure. J. Environ. Qual. 1994, 23, 101–104. [Google Scholar] [CrossRef]
- Rauert, C.; Kaserzon, S.L.; Veal, C.; Yeh, R.Y.; Mueller, J.F.; Thomas, K.V. The first environmental assessment of hexa(methoxymethyl)melamine and co-occurring cyclic amines in Australian waterways. Sci. Total Environ. 2020, 743, 140834. [Google Scholar] [CrossRef]
- Alhelou, R.; Seiwert, B.; Reemtsma, T. Hexamethoxymethylmelamine—A precursor of persistent and mobile contaminants in municipal wastewater and the water cycle. Water Res. 2019, 165, 114973. [Google Scholar] [CrossRef]
- Tyan, Y.-C.; Yang, M.-H.; Jong, S.-B.; Wang, C.-K.; Shiea, J. Melamine contamination. Anal. Bioanal. Chem. 2009, 395, 729–735. [Google Scholar] [CrossRef]
- Tittlemier, S.A. Methods for the analysis of melamine and related compounds in foods: A review. Food Addit. Contam. Part A 2010, 27, 129–145. [Google Scholar] [CrossRef]
- Puschner, B.; Reimschuessel, R. Toxicosis caused by melamine and cyanuric acid in dogs and cats: Uncovering the mystery and subsequent global implications. Clin. Lab. Med. 2011, 31, 181–199. [Google Scholar] [CrossRef] [PubMed]
- Fan, Y.; Ma, X.; Li, Z.; Chen, M. Fast derivatization followed by gas chromatography–mass spectrometry for simultaneous detection of melamine, ammeline, ammelide, and cyanuric acid in fish and shrimp. Food Anal. Methods 2016, 9, 16–22. [Google Scholar] [CrossRef]
- Wong, Y.-L.; Mok, C.-S. A single analytical procedure for the simultaneous and confirmatory determination of melamine and related compounds in various food matrices by isotope dilution gas chromatography-mass spectrometry (ID-GC-MS). Anal. Methods 2013, 5, 2305–2314. [Google Scholar] [CrossRef]
- Miao, H.; Fan, S.; Zhou, P.P.; Zhang, L.; Zhao, Y.F.; Wu, Y.N. Determination of melamine and its analogues in egg by gas chromatography-tandem mass spectrometry using an isotope dilution technique. Food Addit. Contam. 2010, 27, 1497–1506. [Google Scholar] [CrossRef]
- Tzing, S.-H.; Ding, W.-H. Determination of melamine and cyanuric acid in powdered milk using injection-port derivatization and gas chromatography–tandem mass spectrometry with furan chemical ionization. J. Chromatogr. A 2010, 1217, 6267–6273. [Google Scholar] [CrossRef]
- Wang, H.; Lin, L.; Sun, Q.; Lin, Q.; Xiong, X.; Wu, K.; Yu, C.-P. Simultaneous determination of cyromazine, melamine and their biodegradation products by ion-pair high-performance liquid chromatography. Int. J. Environ. Anal. Chem. 2014, 94, 1173–1182. [Google Scholar] [CrossRef]
- Sun, H.; Qin, X.; Ge, X.; Wang, L. Effective separation and sensitive determination of cyanuric acid, melamine and cyromazine in environmental water by reversed phase high-performance liquid chromatography. Enivron. Technol. 2011, 32, 317–323. [Google Scholar] [CrossRef]
- Ehling, S.; Tefera, S.; Ho, I.P. High-performance liquid chromatographic method for the simultaneous detection of the adulteration of cereal flours with melamine and related triazine by-products ammeline, ammelide, and cyanuric acid. Food Addit. Contam. 2007, 24, 1319–1325. [Google Scholar] [CrossRef]
- Vinas, P.; Campillo, N.; Férez-Melgarejo, G.; Hernández-Córdoba, M. Determination of melamine and derivatives in foods by liquid chromatography coupled to atmospheric pressure chemical ionisation mass spectrometry and diode array detection. Anal. Lett. 2012, 45, 2508–2518. [Google Scholar] [CrossRef]
- Draher, J.; Ehling, S.; Cellar, N.; Reddy, T.; Henion, J.; Sousou, N. Determination of emerging nitrogenous economic adulterants in milk proteins by high-performance liquid chromatography/compact mass spectrometry. Rapid Commun. Mass Spectrom. 2016, 30, 1265–1272. [Google Scholar] [CrossRef] [PubMed]
- Kolkman, A.; Vughs, D.; Sjerps, R.; Kooij, P.J.F.; van der Kooi, M.; Baken, K.; Louisse, J.; de Voogt, P. Assessment of highly polar chemicals in Dutch and Flemish drinking water and its sources: Presence and potential risks. Environ. Sci. Technol. Water 2021, 1, 928–937. [Google Scholar] [CrossRef]
- Zhu, H.; Kannan, K. Melamine and cyanuric acid in foodstuffs from the United States and their implications for human exposure. Environ. Int. 2019, 130, 104950. [Google Scholar] [CrossRef] [PubMed]
- García-Miguel, E.; Meza-Márquez, O.G.; Osorio-Revilla, G.; Téllez-Medina, D.I.; Jiménez-Martínez, C.; Cornejo-Mazón, M.; Hernández-Martínez, D.M.; Gallardo-Velazquez, T. Detection of cyanuric acid and melamine in infant formula powders bei mid-FTIR spectroscopy and multivariate analysis. J. Food Qual. 2018, 2018, 7926768. [Google Scholar] [CrossRef]
- Braekevelt, E.; Lau, B.P.-Y.; Feng, S.; Ménard, C.; Tittlemier, S.A. Determination of melamine, ammeline, ammelide and cyanuric acid in infant formula purchased in Canada by liquid chromatography-tandem mass spectrometry. Food Addit. Contam. 2011, 28, 698–704. [Google Scholar] [CrossRef] [PubMed]
- Lütjens, L.H.; Pawlowski, S.; Silvani, M.; Blumenstein, U.; Richter, I. Melamine in the environment: A critical review of available information. Environ. Sci. Eur. 2023, 35, 2. [Google Scholar] [CrossRef]
- Warner, W.; Licha, T. Melamine—A PMT/vPvM substance as a generic indicator for anthropogenic activity and urbanisation? An explorative study on melamine in the water cycle and soil. Chemosphere 2025, 370, 143918. [Google Scholar] [CrossRef]
- Zhu, H.; Kannan, K. Occurrence and distribution of melamine and its derivatives in surface water, drinking water, precipitation, wastewater, and swimming pool water. Environ. Pollut. 2019, 258, 113743. [Google Scholar] [CrossRef]
- Liu, S.-S.; Cai, Q.-S.; Li, C.; Cheng, S.; Wang, Z.; Yang, Y.; Ying, G.-G.; Sweetman, A.J.; Chen, C.-E. In situ measurement of an emerging persistent, mobile and toxic (PMT) substance—Melamine and related triazines in waters by diffusive gradient in thin-films. Water Res. 2021, 206, 117752. [Google Scholar] [CrossRef]
- Neuwald, I.J.; Hübner, D.; Wiegand, H.L.; Valkov, V.; Borchers, U.; Nödler, K.; Scheurer, M.; Hale, S.E.; Arp, H.P.H.; Zahn, D. Occurence, distribution, and environmental behavior of persistent, mobile, and toxic (PMT) and very persistent and very mobile (vPvM) substances in the sources of German drinking water. Environ. Sci. Technol. 2022, 56, 10857–10867. [Google Scholar] [CrossRef]
- Schulze, S.; Zahn, D.; Montes, R.; Rodil, R.; Quintana, J.B.; Knepper, T.P.; Reemtsma, T.; Berger, U. Occurrence of emerging persistent and mobile organic contaminants in European water samples. Water Res. 2019, 153, 80–90. [Google Scholar] [CrossRef]
- He, L.; Su, Y.; Shen, X.; Zheng, Y.; Guo, H.; Zeng, Z. Solid-phase extraction of melamine from aqueous samples using water-compatible molecularly imprinted polymers. J. Sep. Sci. 2009, 32, 3310–3318. [Google Scholar] [CrossRef]
- Johannessen, C.; Helm, P.; Metcalfe, C.D. Detection of selected tire wear compounds in urban receiving waters. Environ. Pollut. 2021, 287, 117659. [Google Scholar] [CrossRef]
- GTFCH (Gesellschaft für Toxikologische und Forensische Chemie). Requirements for the Validation of Analytical Methods. 2009. Available online: https://www.gtfch.org/cms/images/stories/files/Appendix%20B%20GTFCh%2020090601.pdf (accessed on 12 June 2025).
- Gong, H.; Tang, S.; Zhang, T. Catalytic hydrolysis of waste residue from the melamine process and the kinetics of melamine hydrolysis in NaOH solution. React. Kinet. Mech. Catal. 2016, 118, 377–391. [Google Scholar] [CrossRef]
- Johannessen, C.; Parnis, J.M. Environmental modelling of hexamethoxymethylmelamine, its transformation products, and precursor compounds: An emerging family of contaminants from tire wear. Chemosphere 2021, 280, 130914. [Google Scholar] [CrossRef] [PubMed]
- Dsikowitzky, L.; Schwarzbauer, J. Hexa(methoxymethyl) melamine: An emerging contaminant in German rivers. Water Environ. Res. 2015, 87, 461–469. [Google Scholar] [CrossRef] [PubMed]
- Johannessen, C.; Helm, P.; Metcalfe, C.D. Runoff of the tire-wear compound hexamethoxymethyl-melamine into urban watersheds. Archives Environ. Contam. Toxicol. 2022, 82, 162–170. [Google Scholar] [CrossRef]
- Zhu, H.; Kannan, K. Determination of melamine and its derivatives in textiles and infant clothing purchased in the United States. Sci. Total Environ. 2020, 710, 136396. [Google Scholar] [CrossRef]
- Judzentiene, A.; Zdaniauskiene, A.; Ignatjev, I.; Druteikiene, R. Evaluation of physico-chemical characteristics of cement superplasticizer based on polymelamine sulphonate. Materials 2024, 17, 1940. [Google Scholar] [CrossRef]
- Wang, H.; Yang, X.; Xiong, W.; Liu, X.; Zhang, Z. Synthesis and the effects of new melamine superplasticizer on the properties of concrete. Int. Sch. Res. Notices 2013, 1, 708063. [Google Scholar] [CrossRef]
- Seitz, W.; Winzenbacher, R. A survey on trace organic chemicals in a German water protection area and the proposal of relevant indicators for anthropogenic influences. Environ. Monit. Assess. 2017, 189, 244. [Google Scholar] [CrossRef]
- An, H.; Li, X.; Yang, Q.; Wang, D.; Xie, T.; Zhao, J.; Xu, Q.; Chen, F.; Zhong, Y.; Yuan, Y.; et al. The behavior of melamine in biological wastewater treatment system. J. Hazard. Mater. 2017, 322, 445–453. [Google Scholar] [CrossRef] [PubMed]
- Jutzi, K.; Cook, A.M.; Hütter, R. The degradative pathway of the s-triazine melamine. The steps to ring cleavage. Biochem. J. 1982, 208, 679–684. [Google Scholar] [CrossRef]
- Takagi, K.; Fujii, K.; Yamazaki, K.; Harada, N.; Iwasaki, A. Biodegradation of melamine and its hydroxy derivatives by a bacterial consortium containing a novel Nocardioides species. Appl. Microbiol. Biot. 2012, 94, 1647–1656. [Google Scholar] [CrossRef] [PubMed]
- Chen, Z.; Su, Y.; Chen, J.; Li, Z.; Wang, T. Study on the health risk of cyanuric acid in swimming pool water and its prevention and control measures. Front. Public Health 2024, 11, 1294842. [Google Scholar] [CrossRef] [PubMed]
Direct injection | ||||||||
Sample volume | 1 mL | |||||||
Sample pH | Original | |||||||
Internal standards | MEL-13C3, AMN-13C3, AMD-13C3, CYA-13C3, CYRO-d4, 20 µL of a 0.1 mg/L solution in methanol | |||||||
Sample dilution | Dilution of 100 µL with 900 µL acetonitrile | |||||||
Pre-concentration by evaporation | ||||||||
Sample volume | 10 mL | |||||||
Sample pH | Original | |||||||
Internal standards | MEL-13C3, AMN-13C3, AMD-13C3, CYA-13C3, CYRO-d4, 5 µL of a 0.1 mg/L solution in methanol | |||||||
Pre-concentration program | Start | 1 | 2 | 3 | 4 | 5 | 6 | |
Time (h:m) | 00:03 | 04:00 | 00:05 | 04:00 | 00:05 | 04:00 | ||
Temperature (°C) | 30 | 40 | 40 | 35 | 35 | 30 | 30 | |
Vacuum (mbar) | 10 | 10 | 10 | 10 | 10 | 10 | ||
Rotational speed (min−1) | 900 | 900 | 1200 | 1200 | 1200 | 1200 | ||
Reconstitution of the dry residue | In 225 µL acetonitrile + 25 µL ultrapure water |
Analyte | Ionization Mode | Retention Time (min) | Q1 | Q3 | DP (V) | EP (V) | CE (V) | CXP (V) |
---|---|---|---|---|---|---|---|---|
MEL | Positive | 3.1 | 127.0 | 68.0 * | 106 | 10 | 37 | 10 |
85.0 | 25 | 12 | ||||||
AMN | Positive | 3.4 | 128.0 | 86.0 * | 96 | 10 | 23 | 12 |
43.1 | 55 | 6 | ||||||
AMD | Negative | 2.4 | 127.0 | 83.9 * | −50 | −10 | −16 | −7 |
42.0 | −28 | −5 | ||||||
CYA | Negative | 1.5 | 127.9 | 42.1 * | −45 | −10 | −26 | −5 |
85.0 | −14 | −7 | ||||||
CYRO | Positive | 1.9 | 167.0 | 68.0 * | 91 | 10 | 43 | 10 |
85.0 | 25 | 12 | ||||||
HMMM | Positive | 1.2 | 391.1 | 177.1 * | 31 | 10 | 39 | 12 |
359.2 | 11 | 26 | ||||||
IS-MEL-13C3 | Positive | 3.1 | 130.0 | 87.1 * | 120 | 10 | 25 | 6 |
IS-AMN-13C3 | Positive | 3.4 | 131.0 | 88.0 * | 131 | 10 | 23 | 10 |
71.0 | 37 | 12 | ||||||
IS-AMD-13C3 | Negative | 2.4 | 130.0 | 86 * | −55 | −10 | −16 | −5 |
43.0 | −36 | −5 | ||||||
IS-CYA-13C3 | Negative | 1.5 | 130.9 | 43.1 * | −45 | −10 | −38 | −9 |
IS-CYRO-d4 | Positive | 1.9 | 171.0 | 86.0 * | 71 | 10 | 27 | 10 |
Direct Injection (µg/L) | Evaporation (µg/L) | |||||||
---|---|---|---|---|---|---|---|---|
Analyte | Drinking Water | Surface Water | WWTP Effluent | WWTP Influent | Drinking Water | Surface Water | WWTP Effluent | WWTP Influent |
MEL | 0.10 | 0.10 | 0.10 | 0.10 | 0.01 | 0.01 | 0.05 | 0.10 |
AMN | 0.20 | 0.20 | 0.20 | 0.20 | 0.01 | 0.01 | 0.05 | 0.10 |
AMD | 0.10 | 0.10 | 0.10 | 0.10 | 0.01 | 0.01 | 0.05 | 0.10 |
CYA | 1.00 | 1.00 | 2.00 | 2.00 | 0.05 | 0.05 | 0.25 | 0.50 |
CYRO | 0.10 | 0.10 | 0.10 | 0.10 | 0.01 | 0.01 | 0.05 | 0.10 |
HMMM | 0.10 | 0.10 | 0.10 | 0.10 | 0.01 | 0.01 | 0.05 | 0.10 |
Analyte | Recovery of Evaporation Procedure (%) |
---|---|
MEL | 103.2 |
AMN | 100.9 |
AMD | 91.8 |
CYA | 81.5 |
CYRO | 96.9 |
HMMM | 76.1 |
Sample | Matrix | MEL (µg/L) | AMN (µg/L) | AMD (µg/L) | CYA (µg/L) | CYRO (µg/L) | HMMM (µg/L) |
---|---|---|---|---|---|---|---|
1 | Drinking water | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ |
2 | Groundwater | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ |
3 | Groundwater | 0.015 | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ |
4 | Surface water | 0.69 | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ |
5 | Surface water | 1.2 | <LOQ | <LOQ | <LOQ | <LOQ | 1.6 |
6 | Road run-off | 0.71 | <LOQ | 1.3 | 12.0 | <LOQ | 0.74 |
7 | Road run-off | 0.49 | <LOQ | 2.1 | 5.9 | <LOQ | 0.77 |
8 | WWTP effluent | 3.0 | <LOQ | <LOQ | 1.0 | <LOQ | 0.29 |
9 | WWTP effluent | 1.3 | <LOQ | <LOQ | 1.1 | <LOQ | 0.18 |
10 | WWTP influent | 6.9 | <LOQ | <LOQ | <LOQ | <LOQ | 0.23 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Käberich, M.; Nemetz, L.; Sacher, F. Development of a Sensitive HILIC-MS/MS Method for Quantification of Melamine, Derivatives, and Potential Precursors in Various Water Matrices. Analytica 2025, 6, 27. https://doi.org/10.3390/analytica6030027
Käberich M, Nemetz L, Sacher F. Development of a Sensitive HILIC-MS/MS Method for Quantification of Melamine, Derivatives, and Potential Precursors in Various Water Matrices. Analytica. 2025; 6(3):27. https://doi.org/10.3390/analytica6030027
Chicago/Turabian StyleKäberich, Merle, Lisann Nemetz, and Frank Sacher. 2025. "Development of a Sensitive HILIC-MS/MS Method for Quantification of Melamine, Derivatives, and Potential Precursors in Various Water Matrices" Analytica 6, no. 3: 27. https://doi.org/10.3390/analytica6030027
APA StyleKäberich, M., Nemetz, L., & Sacher, F. (2025). Development of a Sensitive HILIC-MS/MS Method for Quantification of Melamine, Derivatives, and Potential Precursors in Various Water Matrices. Analytica, 6(3), 27. https://doi.org/10.3390/analytica6030027