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Simultaneous determination ofcyromazine, melamine and theirbiodegradation products by ion-pair high-performance liquidchromatographyHan Wangab, Lifeng Lina, Qian Suna, Qingqiang Linc, XiaofanXiongd, Kailun Wud & Chang-Ping Yua

a Key Laboratory of Urban Environment and Health, Instituteof Urban Environment, Chinese Academy of Sciences, Xiamen361021, Chinab College of Ecology and Resources Engineering, Wuyi University,Wuyishan City 354300, Chinac College of Life Science, Fujian Normal University, Fuzhou350108, Chinad College of Chemical Engineering, Huaqiao University, Xiamen361021, ChinaPublished online: 01 Apr 2014.

To cite this article: Han Wang, Lifeng Lin, Qian Sun, Qingqiang Lin, Xiaofan Xiong, KailunWu & Chang-Ping Yu (2014) Simultaneous determination of cyromazine, melamine and theirbiodegradation products by ion-pair high-performance liquid chromatography, International Journalof Environmental Analytical Chemistry, 94:12, 1173-1182, DOI: 10.1080/03067319.2014.900681

To link to this article: http://dx.doi.org/10.1080/03067319.2014.900681

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Simultaneous determination of cyromazine, melamine and theirbiodegradation products by ion-pair high-performance liquid

chromatography

Han Wanga,b, Lifeng Lina, Qian Suna, Qingqiang Linc, Xiaofan Xiongd, Kailun Wud

and Chang-Ping Yua*

aKey Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy ofSciences, Xiamen 361021, China; bCollege of Ecology and Resources Engineering, Wuyi University,Wuyishan City 354300, China; cCollege of Life Science, Fujian Normal University, Fuzhou 350108,

China; dCollege of Chemical Engineering, Huaqiao University, Xiamen 361021, China

(Received 6 August 2013; final version accepted 13 February 2014)

An ion-pair high-performance liquid chromatography with ultraviolet detection method forthe determination of cyromazine, melamine and its biodegradation products (ammeline,ammelide, cyanuric acid and biuret) was developed. C18 column was utilised to separatethe six analytes with a mobile phase consisting of perchloric acid-ammonia solution andacetonitrile, under gradient elution and variable flow rate. The detection wavelengths were205 nm for cyanuric acid and biuret and 222 nm for cyromazine, melamine, ammeline andammelide. For analysis of sediment samples, the extraction solution containing acetonitrile,ammonia and water (80:10:10 by volume) was used to extract the analytes from sedimentmatrix. Using the extraction method for the spiked sediment sample, high linearity of matrix-matched standard curve could be obtained for the six analytes. The method detection limitwas 0.1 μg g−1 for melamine and cyromazine, 0.2 μg g−1 for ammeline and ammelide,1.2 μg g−1 for cyanuric acid and 1.0 μg g−1 for biuret in sediment matrix. The recoveries ofthese compounds were 70.1–98.3% and the relative standard deviations were 0.5–4.4%.Finally, the proposed method was successfully applied to the analysis of the sediment samplenear the wastewater outlet of a melamine-producing factory.

Keywords: cyromazine; HPLC; ion-pair; melamine; sediment

1. Introduction

Melamine (2, 4, 6-triamino-1, 3, 5-s-triazine) is an important chemical, which can be manu-factured into plastics, adhesives, paints, electrical mouldings, glass-reinforced substrates orflame retardants [1–4]. Cyromazine (2-cyclopropylamino-4, 6-diamino-s-triazine), a pesticideand cyclopropyl derivative of melamine, is used to control flies in crop production and animalfeed by inhibiting insect growth. Both of them belong to s-triazine family and could be detectedin environmental samples owing to their extensive production and application [5,6].Cyromazine could be dealkylated to melamine by plants [7]. Bacteria could further degrademelamine into NH4

+ and CO2 through the metabolites of ammeline, ammelide, cyanuric acid,and biuret (Figure 1) [8]. Therefore, the two parent compounds and their biodegradationproducts may coexist in the environment. Although cyromazine or melamine may be of low

*Corresponding author. Email: cpyu@iue.ac.cn

Intern. J. Environ. Anal. Chem., 2014Vol. 94, No. 12, 1173–1182, http://dx.doi.org/10.1080/03067319.2014.900681

© 2014 Taylor & Francis

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toxicity to mammalian, the combination of melamine and cyanuric acid would induce acuterenal failure [9]. Cyromazine and melamine have stronger migration potential owing to theirhigher water-solubility. However, their transport and fate in the environment are largelyunknown and need further investigation.

Various analytical approaches have been developed for determination of cyromazine andmelamine simultaneously, including high-performance liquid chromatography with ultravio-let detection (HPLC-UV), gas chromatography-mass spectrometry (GC-MS), cation-selectiveexhaustive injection-sweeping-micellar electrokinetic chromatography, and liquid chromato-graphy coupled with tandem mass spectrometry (LC-MS/MS) [6,10–13]. HPLC-UV,LC-MS/MS and hydrophilic interaction liquid chromatography-electrospray ionisation massspectrometry have been developed for detection of melamine, ammeline, ammelide andcyanuric acid [14–16]. GC-MS also has been adopted by US Food and DrugAdministration to determine these four compounds [17]. Melamine, ammeline and ammelidealso could be monitored by high-performance cation-exchange chromatography [18]. Inaddition, surface enhanced Raman spectroscopy could be used to determine the concentra-tions of melamine and cyanuric acid in aqueous solutions [19]. A recent report showed thatLC-MS/MS could analyse cyromazine, melamine and their three metabolites [20]. However,limited methods have been developed specifically for quantifying melamine in environmen-tal samples and no method has been proposed to determine cyromazine, melamine, amme-line, ammelide, cyanuric acid and biuret (a biodegradation product after cleavage of thes-triazine ring) simultaneously. An appropriate analytical method will enhance our under-standing of their migration and transformation in the environment.

Generally, GC method requires additional preliminary derivatisation procedures. MSsystem is highly sensitive but not widely available in general laboratories because of highprice. Raman is not a common method for determining melamine, which also demandsexperienced skills. HPLC is a conventional instrument with the characteristics of beingconvenient, efficient, and cheap. However, considering the strong polarity and differentsolubility of these compounds, effective baseline separation is more difficult [21].Separation column, mobile phase and gradient procedure are important factors for effectiveseparation and simultaneous detection of the six compounds. Although conventional C18column is hard to retain these polar compounds, ion pair mobile phase may improve theirretentions.

In this study, an ion-pair HPLC method with C18 column was developed for the purpose ofsimple and simultaneous determination of cyromazine, melamine, ammeline, ammelide, cya-nuric acid and biuret, since we were unable to separate the six compounds effectively usingHPLC without ion-pair reagent. Moreover, a protocol to extract six compounds from riversediment was also established. Finally, the method was optimised and validated to detect targetcompounds in the sediment sample.

N

N

N

NH2

NH2

H2NNH

Cyromazine

N

N

N

NH2

NH2 NH

2NH

2 NH2

Melamine

N

N

N

OH

Ammeline

N

N

N

OH

OH

Ammelide

N

N

N

OH

HO OH

Cyanuric acid

O NH

NH2

O

NH2

Biuret

Figure 1. The structures of six analytes.

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2. Experimental

2.1 Chemicals and reagents

Melamine (99%) and cyanuric acid (99%) were obtained from Alfa Aesar, Germany.Cyromazine (98%), ammeline (95%) and ammelide (98%) were purchased from TokyoChemical Industry, Japan. Biuret (99.5%) was from ChemService, USA. HPLC-grade acetoni-trile was acquired from Tedia Company Inc, USA. Ammonia solution (25–28%, Guaranteedreagent), perchloric acid (Guaranteed reagent) and sodium hydroxide (Analytical reagent) werebought from Sinopharm Chemical Reagent Co. Ltd, China. Ion-pair reagent was prepared byadjusting 76.5 mM perchloric acid solution to pH 2.5 with ammonia solution.

2.2 Sample collection

Melamine-contaminated river sediment was collected from the Shaxi River near the wastewateroutlet of a melamine-producing factory located in Sanming city, Fujian, China. River sedimentobtained from 1 km upstream of the wastewater outlet was used as control. Sediment sampleswere bagged and immediately transported on ice to our laboratory in Xiamen city, Fujian,China.

2.3 Apparatus

Dionex ultimate 3000 HPLC system (USA) equipped with autosampler and rapid speed variablewavelength detector was used to analyse six target compounds. The analytical column was aHitachi lachrom C18 column (4.6 mm × 150 mm, 5 µm). Hieroglyph laborota 4000 efficientrotary evaporator (Germany) was employed to evaporate extracted solution from sedimentsamples.

2.4 Preparation of calibration standards

All stock solutions (100 µg/mL) of cyromazine, melamine, cyanuric acid and biuret weredirectly dissolved in ultra-pure water. Ammeline and ammelide were dissolved in sodiumhydroxide solution (0.05 M) owing to lower water solubility under neutral or weak acidicconditions. Melamine, ammeline and ammelide stocks were replaced every month to preventfrom hydrolysis. Series of single or mixed working standards were obtained by diluting stockswith ion-pair reagent. Working standards were prepared every time before use.

2.5 Extraction of sediment samples

The mixture of acetonitrile, ammonia and water (80:10:10 by volume) was used as an extractionsolvent. Five gram dry sediment sample was mixed with 25 mL extraction solvent, and thenshaken on a reciprocating shaker at 200 rpm. After 20 minutes the samples were centrifuged for10 minutes at 9000 rpm and the supernatant was collected. The extraction steps were carried outfour times for each sample. All 100 mL extraction solution was mixed homogeneously.

Using a rotary evaporator system at 65°C, 50 mL extraction solution was evaporated todryness. To accelerate evaporation, 10 mL n-butyl alcohol was added. Evaporation residue wasfirst redissolved with 1.25 mL sodium hydroxide solution (0.05 M) with sonication and thendiluted with 3.75 mL ion-pair reagent. The solution was filtrated with 0.45 μm cellulose acetatemembrane for HPLC analysis.

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For the spiked analysis, a series of mixed standard solution (containing six analytes) wereadded into the sediment sample. The spiked samples were treated with the same preparationprocedure before HPLC analysis as described above for calculation of recovery.

2.6 Chromatography procedure

The mobile phase consisted of acetonitrile and ion-pair reagent. The flow rate increased from0.5 to 0.7 mL/min in 0–5 min, and from 0.7 to 1.2 mL/min in 5–11 min. The flow rate decreasedto 0.5 mL/min in 11–18 min and kept 0.5 mL/min in 18–20 min. For better separation of targets,gradient elution was applied. The proportion of acetonitrile increased from 1% to 5% in0–5 min, and from 5% to 12% in 5–11 min. Acetonitrile proportion decreased gradually to1% in 11–18 min and kept at 1% in 18–20 min. The injection volume of 20 µL was used. Thedetection wavelength of 222 nm was utilised for the quantification of melamine, cyromazine,ammeline and ammelide; biuret and cyanuric acid were monitored at 205 nm. An instrumentalblank and procedural blank were also conducted for each batch.

3. Results and discussion

3.1 Selection of detection wavelengths

To choose appropriate detection wavelengths, cyromazine melamine, ammeline, ammelide,cyanuric acid and biuret were dissolved in ion-pair reagent-acetonitrile solution, respectively.These analytes in ion-pair reagent-acetonitrile solution were scanned by a UV-5200 spectro-photometer from 200 to 800 nm. The maximum absorbance wavelength was 208 and 235 nmfor melamine, 203 and 229 nm for ammeline, 222 nm for ammelide but cyanuric acid, biuretand cyromazine had no typical absorption peak. Taking their characteristic absorbance intoaccount, melamine, ammeline, ammelide and cyromazine were monitored at 222 nm. Owing todrastically declined absorption with increasing detection wavelength, 205 nm was chosen as thedetection wavelength for cyanuric acid and biuret.

3.2 Optimisation of the mobile phase

Organic phase contributes to the target elution from C18 column. Compared with cyromazine,ammeline, ammelide, cyanuric acid and biuret were easy to be eluted by the mobile phase. Twosolvents, methanol and acetonitrile, were compared with their eluting effects in this study.Acetonitrile was chosen as the eluent for improving peak shapes of the six compounds.Compared with methanol, acetonitrile also facilitated cyromazine elution. Because cyromazineretention time was distinctly delayed in C18 analytical column, gradient elution procedure wasadopted as described above in the chromatography procedure.

All six compounds are polar compounds, and melamine, ammeline, ammelide and cyanuricacid were difficult to be separated in C18 column [21]. Additionally, we found that biuret wasalso difficult to be separated from other analytes. However, perchloric acid helped analyteseparation and elution [22]. Owing to different protonation of the analytes at low pH, theresolving power of perchloric acid was effected through interaction with the positively chargedresidues of the analytes and to result in distinct hydrophobicity. For optimising the mobilephase, different perchloric acid concentrations (12–90 mM) and pH (2.5–5) of the ion-pairreagent were tested. Our results indicated that the retention times of the target compounds wereprolonged with the increase of perchloric acid content in particular for melamine and cyroma-zine (Figure 2a). There were not obvious changes in retention time when perchloric acid

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4 5 6 7 8 9 10 11 12 13 14 15 16

6

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U)

Time (min)

205 nm222 nm

0

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2 34

5 6

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2

34

5

76.5 m M perchloric acid

51 m M perchloric acid

1

2 3

4 525.5 m M perchloric acid

6

A

4 5 6 7 8 9 10 11 12 13 14 15 16

6

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205 nm222 nm

0

30

pH 2.5

pH 3.5

pH 4.5

1

2 34

56

B

Figure 2. (a) The effects of perchloric acid concentration on the separation, retention and response of theanalytes. The mobile phase (pH 2.5) was composed of perchloric acid (25.5, 51 or 76.5 mM)-ammoniasolution and acetonitrile. (b) The effects of the pH of ion-pair agent on the separation, retention andresponse of the analytes. The mobile phase (pH 2.5, 3.5 or 4.5) was composed of perchloric acid(76.5 mM)-ammonia solution and acetonitrile. Other chromatography conditions were described in thematerials and methods above. Detection wavelength: 205 and 222 nm. 1, ammelide; 2, biuret; 3, cyanuricacid; 4, ammeline; 5, melamine; 6, cyromazine.

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exceeded 76.5 mM. Most peak area of analytes increased with increasing perchloric acid contentbut did not vary distinctly after the concentration exceeded 76.5 mM. Perchloric acid alsoaffected the separation of our target compounds, especially for ammeline, biuret and cyanuricacid. The better separation was obtained when perchloric acid concentration reached 76.5 mM.Although the pH of ion-pair reagent had little effect on the absorption response of the six targetcompounds, declining pH improved analyte separation resolution (Figure 2b). When the pH wasbelow 3.5, better resolution and smooth baseline was acquired. Considering the described pH ofthe separation column (pH 2–8), pH 2.5 was selected for ion-pair reagent.

3.3 Extracting analytes from the sediment sample

Although six compounds are water-soluble, the solubility of ammeline and ammelide is lowerunder neutral pH. Furthermore, melamine and cyanuric acid will form precipitation in neutralsolution. According to our experience, the precipitation disappeared completely when solutionpH exceeded 11 or was less than 1.5. The occurrence of precipitation also depended on theconcentrations of melamine and cyanuric acid. As a result, ammonia solution, which was onecomponent of the mobile phase, was utilised as a basic modifier for extraction. The proportionof ammonia, which could dissociate analytes from sediment, influenced the effect of theextraction significantly. Acetonitrile was another important component of the extracting solu-tion. It could denature protein and precipitate a part of humus substance in the sediment.Different acetonitrile, ammonia and water volume ratios [90:5:5 (pH 11.8), 80:10:10(pH 12.4), 70:20:10 (pH 12.6), 60:30:10 (pH 12.6)] were checked for their impact on recov-eries. Our results indicated that the absolute recoveries of six analytes positively correlated withammonia concentration. Almost all recoveries of six targets increased to their maximum whenammonia proportion reached 20%. Further increase of ammonia concentration decreased therecoveries slightly (Figure 3). Considering the extracted background interferences under

Rec

ove

ry (

%)

acetonitrile:ammonia:water (90:5:5)acetonitrile:ammonia:water (80:10:10)acetonitrile:ammonia:water (70:20:10)acetonitrile:ammonia:water (60:30:10)

Cyromazine Melamine Biuret

20

40

60

80

100

0Ammeline Ammelide Cyanuric acid

Figure 3. Absolute recoveries of analytes in control sediment spiked with cyromazine 0.5 μg g−1,melamine 0.25 μg g−1, ammeline and ammelide 0.5 μg g−1, cyanuric acid 2 μg g−1 and biuret 1.5 μg g−1.

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different proportions of extraction agents (data not shown), the composition of 80:10:10 wasselected as the optimal extraction solvent.

3.4 Method validation

3.4.1 Specificity, linearity and detection limit

The analysis of real sample could be influenced by matrix effects. To consider the possibleinterference from sediment matrix, a series of mixed standard solution (containing six analytes)were spiked into the sediment control sample to obtain the matrix-matched standard curve.Spiked sediment control sample was treated as above and analysed under the optimum condi-tions. The effective baseline separation for six analytes was observed in the spiked sedimentcontrol sample. Some unknown peaks were extracted from the river sediment sample before theretention time of ammelide, but they did not interfere with the target quantification.

The calibration ranges and correlation coefficients of six target compounds are shown inTable 1. These compounds displayed high linear correlations between their concentrations andpeak areas in measured range. The method detection limits (MDLs) of melamine, cyromazine,ammeline, ammelide, cyanuric acid and biuret was 0.1, 0.1, 0.2, 0.2, 1.2 and 1.0 μg g−1

respectively in sediment matrix. The MDLs of our method were comparable to the method ofprevious study using LC-UV to quantify cyromazine and melamine residues in soil (0.1 μg g−1

for cyromazine and melamine) [6], but were higher than the method using LC-MS/MS toanalyse melamine in soil (0.020 μg g−1) [5]. Previous investigation showed that melaminecontamination in soil could be up to 41 μg g−1 depending on the sampling distance to themelamine-producing factories [5]. These results demonstrated that our method was reliable andcould be applied to quantify six analytes in environmental samples.

3.4.2 Sample analysis

The newly developed method was applied to the analysis of the river sediment sample near thewastewater outlet of a melamine-producing factory. The recovery of six analytes was evaluatedby spiking mixed standard solution to the real sample. Spiked concentrations of six compoundsranged from 1.25 to 10 times of their MDLs. After the samples were pretreated with thesuggested procedures, six analytes were determined under the optimal chromatographic condi-tion (Figure 4).

The recoveries of six compounds ranged from 70.1% to 98.3% with relative standarddeviations (RSDs) (n = 7) of 0.5–4.4% for the river sediment sample (Table 2). Melaminewas not detected in the control sediment sample 1 km upstream of the wastewater outlet.However, it was detected in the river sediment sample collected near the wastewater outlet of a

Table 1. The correlation coefficients, calibration ranges, MDLs for six analytes in sediment matrix.

Analyte Correlation coefficient Calibration range (μg g−1) MDLs (μg g−1)

Melamine 0.9991 0.1–1.0 0.1Cyromazine 0.9991 0.1–2.0 0.1Ammeline 0.9990 0.2–2.0 0.2Ammelide 0.9992 0.2–2.0 0.2Cyanuric acid 0.9545 1.2–8.0 1.2Biuret 0.9985 1.0–6.0 1.0

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melamine-producing factory with the concentration of 0.33 μg g−1 but no biodegradationproducts with concentrations higher than their MDLs was detected. Previous research hasdemonstrated that melamine was recalcitrant to biodegradation in the activated sludge andinhibitory to beneficial wastewater treatment microorganisms [23]. Our result also impliedthat melamine was not easily biodegraded in the river sediment, but research will be neededto further elucidate the fate of melamine in the sediment.

Table 2. The recoveries and their RSDs for six analytes in sediment matrix.

Analyte Content (μg g−1) Spiked (μg g−1) Found (μg g−1) Recovery (%) RSD n = 7 (%)

Melamine 0.33 0.25 0.51 72.1 2.70.33 0.125 0.42 74.8 0.5

Cyromazine – 0.25 0.22 87.3 2.5– 0.125 0.12 91.6 0.8

Ammeline – 2 1.87 93.4 2.1– 1 0.81 81 1

Ammelide – 1 0.98 98.3 3.7– 0.5 0.47 94 2.7

Cyanuric acid – 5 3.72 74.4 4.4– 2.5 1.70 68.2 0.9

Biuret – 2 1.43 71.3 1.5– 1.5 1.05 70.1 3.9

‘–’, not detected.

5 6 7 8 9 10 11 12 13 14 15

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U)

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Unspiked river sediment 205 nmUnspiked river sediment 222 nmSpiked river sediment 205 nmSpiked river sediment 222 nm

0

25

1

2

3

4

56

Figure 4. The chromatograms of the river sediment sample near a wastewater outlet of a melamine-producing factory and its spiked sample. The spiked amount of cyromazine, melamine, ammeline,ammelide, cyanuric acid and biuret was 0.25, 0.25, 2, 1, 5, 2 μg g−1, respectively. The chromatographyconditions were described in the materials and methods above. Detection wavelength: 205 and 222 nm.1, ammelide; 2, biuret; 3, cyanuric acid; 4, ammeline; 5, melamine; 6, cyromazine.

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4. Conclusions

An ion-pair HPLC-UV method was proposed to separate and determine melamine, cyromazine,ammeline, ammelide, cyanuric acid and biuret simultaneously. The method employed a C18column, gradient elution and variable flow with a mobile phase consisting of perchloric acid-ammonia solution (76.5 mM perchloric acid, pH 2.5) and acetonitrile. The optimised protocol toextract six compounds from river sediment was also established. Finally, the proposed methodwas successfully applied to analyse the river sediment sample near the wastewater outlet of amelamine-producing factory.

AcknowledgementsThis work was supported by the Science and Technology Innovation and Collaboration Team Project of theChinese Academy of Sciences, Technology Foundation for Selected Overseas Chinese Scholar ofMOHRSS, China, the Hundred Talents Program of the Chinese Academy of Sciences, Science andTechnology Planning Project of Xiamen, China (3502Z20120012), and the CAS/SAFEA InternationalPartnership Program for Creative Research Teams (KZCX2-YW-T08).

References[1] A. Kandelbauer and P. Widsten, Prog. Org. Coat. 65, 305 (2009). doi:10.1016/j.porgcoat.2008.12.003[2] W.Y. Su, S. Subyakto, T. Hata, K. Nishimiya and Y. Imamura, Ishihara S., J. Wood Sci. 44, 131–136

(1998). doi:10.1007/BF00526258[3] H. Liang, W. Shi and M. Gong, Polym. Degrad. Stabil. 90, 1 (2005). doi:10.1016/j.

polymdegradstab.2005.01.022[4] W.S. El-Sayed, A.F. El-Baz and A.M. Othman, Int. Biodeter. Biodegr. 57, 75–81 (2006).

doi:10.1016/j.ibiod.2005.11.006[5] Y. Qin, X. Lv, J. Li, G. Qi, Q. Diao, G. Liu, M. Xue, J. Wang, J. Tong, L. Zhang and K. Zhang,

Environ. Int. 36, 446 (2010). doi:10.1016/j.envint.2010.03.006[6] R.A. Yokley, L.C. Mayer, R. Rezaaiyan, M.E. Manuli and M.W. Cheung, J. Agric. Food Chem. 48,

3352 (2000). doi:10.1021/jf991231w[7] L. Lim, S.J. Scherer, K.D. Shuler and J.P. Toth, J. Agric. Food Chem. 38, 860 (1990). doi:10.1021/

jf00093a057[8] D.R. Shelton, J.S. Karns, G.W. McCarty and D.R. Durhum, Appl. Environ. Microbiol. 63, 2832

(1997).[9] B. Puschner, R.H. Poppenga, L.J. Lowenstine, M.S. Filigenzi and P.A. Pesavento, J. Vet. Diagn.

Invest. 19, 616 (2007). doi:10.1177/104063870701900602[10] X. Zhu, S. Wang, Q. Liu, Q. Xu, S. Xu and H. Chen, J. Agric. Food Chem. 57, 11075 (2009).

doi:10.1021/jf902771q[11] J.V. Sancho, M. Ibáñez, S. Grimalt, Ó.J. Pozo and F. Hernández, Anal. Chim. Acta. 530, 237 (2005).

doi:10.1016/j.aca.2004.09.038[12] H. Sun, L. Wang, N. Liu, F. Qiao and S. Liang, Chromatographia. 70, 1685 (2009). doi:10.1365/

s10337-009-1355-x[13] S. Wang, D. Li, Z. Hua and M. Zhao, Analyst. 136, 3672 (2011). doi:10.1039/c1an15086c[14] T. Sugita, H. Ishiwata, K. Yoshihira and A. Maekawa, Bull. Environ. Contam. Toxicol. 44, 567

(1990). doi:10.1007/BF01700877[15] J. Xia, N. Zhou, C. Zhou, B. Chen, Y. Wu and S. Yao, J. Sep. Sci. 33, 2688 (2010). doi:10.1002/

jssc.201000262[16] R. Muñiz-Valencia, S.G. Ceballos-Magaña, D. Rosales-Martinez, R. Gonzalo-Lumbreras, A. Santos-

Montes, A. Cubedo-Fernandez-Trapiella and R.C. Izquierdo-Hornillos, Anal. Bioanal. Chem. 392,523 (2008). doi:10.1007/s00216-008-2294-3

[17] US Food and Drug Administration, GC-MS screen for the presence of melamine, ammeline, ammelide,and cyanuric acid (2009). <http://www.fda.gov/AboutFDA/CentersOffices/OfficeofFoods/CVM/WhatWeDo/ucm134742.htm>.

[18] S. Ono, T. Funato, Y. Inoue, T. Munechika, T. Yoshimura, H. Morita, S.-I. Rengakuji and C.Shimasaki, J. Chromatogr. A. 815, 197 (1998). doi:10.1016/S0021-9673(98)00441-5

International Journal of Environmental Analytical Chemistry 1181

Dow

nloa

ded

by [

The

Uni

vers

ity o

f M

anch

este

r L

ibra

ry]

at 0

6:59

12

Nov

embe

r 20

14

[19] L. He, Y. Liu, M. Lin, J. Awika, D.R. Ledoux, H. Li and A. Mustapha, Sens. Instrum. Food Qual.Saf. 2, 66 (2008). doi:10.1007/s11694-008-9038-0

[20] H. Yu, Y. Tao, D. Chen, Y. Wang, Z. Liu, Y. Pan, L. Huang, D. Peng, M. Dai, Z. Liu and Z. Yuan,Anal. Chim. Acta. 682, 48 (2010). doi:10.1016/j.aca.2010.09.032

[21] S. Ehling, S. Tefera and I.P. Ho, Food Addit. Contam. 24, 1319 (2007). doi:10.1080/02652030701673422

[22] M. Shibue, C.T. Mant and R.S. Hodges, J. Chromatogr. A. 1080, 49 (2005). doi:10.1016/j.chroma.2005.02.063

[23] S. Xu, Y. Zhang, A. Sims, M. Bernards and Z. Hu, Water Res. 47, 2307 (2013). doi:10.1016/j.watres.2013.01.048

1182 H. Wang et al.

Dow

nloa

ded

by [

The

Uni

vers

ity o

f M

anch

este

r L

ibra

ry]

at 0

6:59

12

Nov

embe

r 20

14