Determination of Melamine and Derivatives in Foods

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Determination of Melamine and Derivatives in Foods by Liquid Chromatography Coupled to Atmospheric Pressure Chemical Ionization Mass Spectrometry and Diode Array Detection a

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Pilar Viñas , Natalia Campillo , Gema Férez-Melgarejo & Manuel Hernández-Córdoba

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Department of Analytical Chemistry, Faculty of Chemistry, Regional Campus of International Excellence “Campus Mare Nostrum,” University of Murcia, Murcia, Spain Accepted author version posted online: 30 May 2012.

To cite this article: Pilar Viñas , Natalia Campillo , Gema Férez-Melgarejo & Manuel HernándezCórdoba (2012): Determination of Melamine and Derivatives in Foods by Liquid Chromatography Coupled to Atmospheric Pressure Chemical Ionization Mass Spectrometry and Diode Array Detection, Analytical Letters, 45:17, 2508-2518 To link to this article: http://dx.doi.org/10.1080/00032719.2012.694941

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Analytical Letters, 45: 2508–2518, 2012 Copyright # Taylor & Francis Group, LLC ISSN: 0003-2719 print=1532-236X online DOI: 10.1080/00032719.2012.694941

Downloaded by [Ams/Murcia Humanity], [Pilar Viñas] at 01:50 29 November 2012

Liquid Chromatography DETERMINATION OF MELAMINE AND DERIVATIVES IN FOODS BY LIQUID CHROMATOGRAPHY COUPLED TO ATMOSPHERIC PRESSURE CHEMICAL IONIZATION MASS SPECTROMETRY AND DIODE ARRAY DETECTION Pilar Vinãs, Natalia Campillo, Gema Fe´rez-Melgarejo, and Manuel Hernańdez-Co´rdoba Department of Analytical Chemistry, Faculty of Chemistry, Regional Campus of International Excellence ‘‘Campus Mare Nostrum,’’ University of Murcia, Murcia, Spain Two liquid chromatographic methods based on atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) and diode array detection (DAD) are evaluated for the rapid determination of melamine (MEL) and structurally related compounds, including ammeline (AMN), ammelide (AMD), and cyanuric acid (CA) in foods. Both procedures used ion-exchange LC and isocratic elution. Samples were extracted by homogenization with acetonitrile/water/diethylamine. Specificity was demonstrated for LC-MS by the retention characteristics and MS spectra, by comparing with commercial standards. Specificity was only demonstrated in the case of LC-DAD for MEL and AMN, considering the retention characteristics and UV spectra. The recoveries obtained for spiked samples were satisfactory for all the analytes with LC-MS. The proposed procedure, LC-APCI-MS, was successfully applied to the analysis of different baby foods, including infant formula and breakfast cereal, and samples of rice flour, potato starch, soya drink, and coconut drink. Keywords: Ammeline; Cyanuric acid; Foods; Ion exchange LC-APCI-MS; Melamine

INTRODUCTION Melamine (MEL, 1,3,5-triazine-2,4,6-triamine) is a nitrogen-rich compound commonly used for the manufacture of plastic and resins (J. Li, Qi, and Shi 2009), and its alkaline hydrolysis produces structurally related chemicals such as ammeline (AMN), ammelide (AMD), and cyanuric acid (CA). Due to the high nitrogen content, MEL has occasionally been added to some foods in order to increase the Received 26 March 2012; accepted 11 May 2012. The authors acknowledge the Comunidad Autońoma de la Regioń de Murcia (CARM, Fundacioń Seńeca, Project 15217=PI=10) and the Spanish MICINN (Project CTQ2009-08267=BQU) for financial support. G. Fe´rez acknowledges a fellowship from the Fundacioń Seńeca (CARM). Address correspondence to Prof. Manuel Hernańdez-Co´rdoba, Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, E-30071-Murcia, Spain. E-mail: [email protected] 2508


HPLC-APCI-MS FOR MELAMINE IN FOODS

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apparent level of proteins they contain. In addition of being illegal, this manipulation represents a risk for human health because of the toxicity of these compounds (Andersen et al. 2008). Indeed, none of the four chemicals should be present in foods. The safety limit for MEL in milk and milk-based products, according to the European Union, and WHO=FAO experts, is 2.5 mg kg1, while a limit of 1 mg kg1 has been stated by WHO=FAO experts for infant formula (EU Commission Decision 2008; WHO 2008). Consequently, the development of simple and effective procedures for the determination of MEL and related compounds is a subject of analytical interest. A variety of approaches has been developed for such a purpose and recently reviewed (Tittlemier 2010; Chu et al. 2010; Sun et al. 2010), mostly based on chromatographic techniques, such as gas chromatography-mass spectrometry (GC-MS) (J. Li, Qi, and Shi 2009; Miao et al. 2009; H. P. O. Tang et al. 2009; X. M. Xu et al. 2009; Xia et al. 2009; Zhao et al. 2010; M. Li et al. 2010; Tzing and Ding 2010; Lutter et al. 2011; Koh et al. 2011), liquid chromatography (LC) with ultraviolet detection or photodiode-array (DAD) (Bradley et al. 2005; Ehling, Tefera, and Ho 2007; Kim et al. 2008; Mun˜iz-Valencia et al. 2008; Zhong et al. 2011; Chao et al. 2011), and LC-MS (Andersen et al. 2008; Lutter et al. 2011; Filigenzi et al. 2008; Cheng et al. 2009; Desmarchelier et al. 2009; Ibań˜ez, Sancho, and Hernańdez 2009; Karbiwnyk et al. 2009; H. W. Tang et al. 2009; Tittlemier et al. 2009; Wu et al. 2009; Zhou et al. 2009; Y. Xu et al. 2010; Yu et al. 2010; Tran et al. 2010; Wu et al. 2010; Zhang et al. 2010; Jacob and Gamboa da Costa 2011; Han et al. 2011; Goscinny et al. 2011). Time-of-flight mass spectrometry (TOFMS) coupled to matrix assisted laser desorption ionization (MALDI) (Campbell, Wunschel, and Petersen 2007; Singh and Panchagnula 2011; Arnol et al. 2011) or to direct analysis in real time (DART) (Vaclavik et al. 2010) has been also used. However, most procedures are concerned with the determination of MEL or mixtures of MEL and CA in different matrices but do not tackle with all the mentioned species. Polar compounds are difficult to analyze by reversed-phase LC-MS, and using an ion-pair agent may be a good alternative. In such a case, care must be taken when using ion-pair reagents in combination with LC-MS due to the possibility of ion suppression, contamination of the MS source, and adduct formation. Furthermore, the ion-exchange method should preferably be compatible with the LC-MS for the analysis of polar analytes. In this paper, the application of ion-exchange LC is evaluated for determining MEL and the three derivatives. Two different detection systems have been coupled to LC, MS with APCI and DAD. The first one permitted the correct identification of the four compounds analyzed, whereas only two of the analytes studied could be specifically determined when using DAD. The finally proposed procedure, LC-MS, was validated and successfully applied to the analysis of different baby foods, as infant formula and breakfast cereal, and other samples of rice flour, potato starch, soya drink, and coconut drink.

EXPERIMENTAL Reagents Acetonitrile (ACN) and methanol (Sigma-Aldrich, Madrid, Spain) were LC grade. Doubly distilled water was purified using a Milli-Q system (Millipore,


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Bedford, MA, USA). The 10 mM ammonium acetate solution of pH 6.5 was prepared from the commercial product (Panreac, Barcelona, Spain). Other reagents were sodium hydroxide, phosphoric acid, sodium phosphate, ethanol, and diethylamine (Panreac). Stock solutions of the standards were prepared from the commercial products without previous purification. MEL (Sigma-Aldrich) and AMD (Dr Ehrenstorfer GmbH, Augsburg, Germany) were prepared by dissolving 10 mg in 25 mL of a 1:1 methanol:water mixture. AMN (Fluka, Buchs, Switzerland) was prepared by dissolving 10 mg in 25 mL of a 1:1 methanol:water mixture containing 0.05 M sodium hydroxide and sonicated for 5 min. CA (Sigma-Aldrich) was prepared by dissolving 10 mg in 50 mL of a 1:1 ethanol:water mixture containing 0.05 M sodium hydroxide and sonicated for 5 min. The aforementioned solutions were kept in dark bottles at 4 C, except AMD which was kept at 20 C. Working standard solutions were prepared by dilution with acetate buffer on the same day of use. The chemical structures of the target analytes are shown in Fig. 1. Liquid Chromatography and Detection Conditions The LC system consisted of an Agilent 1200 (Agilent, Waldbronn, Germany) binary pump (G1312A) operating at a flow-rate of 0.5 mL min1. The solvents were degassed using an on-line membrane system (Agilent G1379A). The column was maintained in a thermostated compartment at room temperature (Agilent G1316A). The diode array detector was an Agilent G1315D operating at two

Figure 1. Structures of the four target analytes.



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wavelengths of 200 nm for MEL and AMN, and 220 nm for AMD and CA. The analytical column used for the cationic exchange technique to improve resolution was a Spherisorb SCX (15 cm 4 mm, particle size 5 mm) column. The injection (40 mL) was performed using an autosampler (Agilent G1329A). The LC system was coupled to an ion-trap mass spectrometer (1036 model) equipped with an APCI interface operating in both positive and negative ion modes in selected ion monitoring (SIM) mode. The instrument parameters were: drying temperature, 350 C; APCI temperature, 400 C; drying gas flow, 6 L min1 and nebulizer gas pressure, 60 psi. Solutions were stored in 2 or 10 mL amber glass vials. An EBA 20 (Hettich, Tuttlingen, Germany) centrifuge and an ultrasonic bath (Selecta, Barcelona, Spain) were used. To filter the samples, Econofilter 25-mm diameter nylon filters (0.45 mm) were used (Agilent). Samples The samples of different types of baby food were supplied by local manufacturers: three types of infant formula (starting, follow-on, and prebiotic follow-on) and a breakfast cereal (multicereals with honey). Other samples of rice flour, glutinous rice flour, potato starch, soya drink, and coconut drink were obtained from different manufacturers. Recovery experiments were carried out using four different samples (infant formula, breakfast cereal, rice flour, and soya drink) spiked with the four analytes, when using the LC-APCI-MS method, at concentration levels between 210 ng g1 and 2.4 mg g1. The samples were left to equilibrate at room temperature for at least half an hour before starting the extraction procedure. Analytical Procedure An amount of 0.5 g of the sample was weighed into a glass tube and 5 mL of a 5:4:1 acetonitrile:water:diethylamine mixture was added (Litzau, Mercer, and Mulligan 2007). The suspension was homogenized and centrifuged at 6000 rpm for 10 min at room temperature. Then, the supernatant was recovered and filtered through a 0.45 mm nylon chromatographic filter. Aliquots of 40 mL were injected into the chromatograph with the autosampler. The mobile phase used in isocratic conditions was 10 mM ammonium acetate (pH 6.5) at 0.5 mL min1. UV–vis detection was performed in the 190–450 nm range and the diode array detector was set at wavelengths of 200 nm for MEL and AMN, and 220 nm for AMD and CA. The optimal MS response was obtained with APCI in the positive mode for MEL and AMN and in the negative mode for AMD and CA. Analyses were performed in duplicate. RESULTS AND DISCUSSION Optimization of the Chromatographic Separation Melamine is a basic and polar molecule with a pKa of 5.6 and a log P value of 1.37. The higher polarity of ionized compounds means that they are less readily retained than neutral species in reversed phase. Peak shape and reproducibility is also affected by the fact that they interact with exposed and activated silanols. To

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avoid this problem, we first used reversed phase chromatography using several C18 columns and a variety of buffers as mobile phases. Nevertheless, due to their polar nature, there was a tendency for the analytes not to be retained by the columns in any conditions and so the technique was rejected. Ion-pair chromatography (IPC) was then tested to increase the retention of melamine and its derivatives. In this case, the ionic characteristics of the compounds led to strong interactions with anionic ion-pair reagents at a given pH. Although this increased retention and resolution, column performance when using volatile buffers is extremely critical when the HPLC-MS technique is being used and IPC is discarded. We then used the cationic exchange technique with a Spherisorb SCX (15 cm 4 mm, 5 mm) column, studying the effects of pH of the mobile phase (pH 3–7) on retention times and peak shapes using buffer solutions of 50 mM phosphate at 0.5 mL min1. CA was not retained in any condition, while retention of the rest of the compounds increased at the lower pH values, especially for AMN and MEL. Mobile phases of higher volatility, in this case formate and acetate, were then tried. When 50 mM formate buffer was tested, compounds were retained by the column. Best resolution was obtained using 10 mM ammonium acetate (pH 6.5) at 0.5 mL min1, the elution order being: 1, CA (tR ¼ 2.1 min); 2, AMD (tR ¼ 2.2 min); 3, AMN (tR ¼ 3.2 min); and 4, MEL (tR ¼ 5.8 min). The method using LC-DAD was less selective and required intensive validation to ensure that there were no interferences absorbing at the low wavelengths used for detection, at around 200–220 nm. Furthermore, CA has a much weaker chromophore than the related compounds, and thus a higher detection limit. On the other hand, bands corresponding to CA and AMD were near the void time and could be not resolved. Optimization of APCI-MS Conditions A comparison of both MS ionization methods, ESI and APCI, was performed, and APCI using the heated nebulizer probe was chosen because it was more sensitive than ESI for several of these polar compounds. The optimal MS response was obtained with APCI in positive mode for MEL and AMN and in negative mode for AMD and CA. Note that AMN can be analyzed in both positive and negative mode. Accordingly, the [M þ H]þ ions were used as target ions for MEL and AMN, while the [M H] ions were used for AMD and CA (Table 1). The opposite polarities meant that polarity switching was necessary during one LC-MS run, or two different runs. The analysis was carried out in two runs to improve sensitivity. Ion suppression produced by the mobile phase buffer ions present at high concentrations is not expected to interfere with the ionization of analytes that are retained with the LC-APCI-MS method. Figure 2 depicts the extracted ion chromatograms in Table 1. LC-DAD and LC-APCI-MS conditions Compound CA AMD AMN MEL

tR (min)

Wavelength

Ionization mode

Target ion (m=z)

2.1 2.2 3.2 5.8

220 220 200 200

Negative Negative Positive Positive

128 127 128 127



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Figure 2. Extracted ion chromatograms by LC-APCI-MS in both negative and positive modes obtained using a starting infant formula spiked at 1 mg g1 of each compound.

positive and negative modes in the selected conditions. Table 1 summarizes the data referring to retention times, the UV bands, and APCI-MS data. All the compounds were identified from their mass spectra obtained by APCI-MS and from their absorption spectra in the UV-region. Sample Extraction Procedure Since baby food and animal foods are highly complex biological samples, a preparation step was necessary to simplify the matrix and signal interpretation. Precautionary steps may also be necessary to ensure sample integrity during analysis. This is critical for determining CA, which tend to precipitate. Extraction efficiency was evaluated by recovery studies involving the standard addition technique, treating the sample with acids to remove proteins. Extraction was carried out by adding 3% (m=v) trichloroacetic acid (TCA) and, after centrifugation, washing the precipitate with water. Although protein precipitation with acid, with no neutralization, is a rapid and straightforward preparation process, it involved losses in CA and MEL. However, good results were obtained when the organic solvent and alkaline medium were used together (Filigenzi et al. 2008). Thus, optimal extraction was achieved using a mixture ACN:water:diethylamine in the proportion 5:4:1 as proposed in the US FDA method (Litzau, Mercer, and Mulligan 2007). The mixture was centrifuged at 6000 rpm for 10 min and filtered through a 0.45-mm nylon filter. Validation of the Analytical Methods Used The linearity, selectivity, precision, accuracy, and detection and quantification limits of the different methods were evaluated from calibration curves obtained by


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least-squares linear regression analysis of the peak area vs. analyte concentration at six levels (ng mL1). The validation parameters, regression coefficients and range of linearity for each compound using LC-MS and external calibration are shown in Table 2. The excellent linearity obtained for the range studied is seen from the correlation coefficient values, which were greater than 0.99. Table 2 also gives the detection and quantification limits calculated on the basis of three and ten times the standard deviation of the intercept of the calibration graphs, respectively. The average relative standard deviation (RSD) for ten replicate injections of the same sample fortified at 1 mg g1 was calculated, and the resulting values (Table 2) confirm the precision of the LC-MS method for routine analysis. Matrix effects were studied in the different samples analyzed in order to evaluate to what extent signals were suppressed by co-eluting compounds from the sample matrix affecting analyte ionization. This can be attributed in MS detection to deprotonation, the presence of non-volatile components and, in complex samples, high amounts of species that compete with the ions available. We then compared the slopes of aqueous standards and standards additions calibration graphs for the different matrices. Statistical analysis pointed to significant differences between the slopes at the 95% confidence level, confirming that, to compensate any matrix effects, calibration should be carried out by means of the standard additions method. Data obtained for linearity, detection, and quantification limits by applying the standard additions method to a starting infant formula are shown in Table 2. A similar study was carried out using LC-DAD for MEL and AMN and matrix effects were also detected, proposing also the standard additions method when MEL and AMN have to be quantified by LC-DAD. The selectivity of the LC-APCI-MS method was tested by analyzing six different blank samples in order to determine the possible interference of extraneous substances. No impurity peaks were observed at the retention times corresponding to MEL and derivatives when analyzing the matrix blank and extracts of spiked samples. In the case of the LC-DAD method, a similar study was carried out to check the selectivity for MEL and AMN. To check the accuracy of the LC-APCI-MS method, a recovery study was carried out by fortifying four samples (infant formula, breakfast cereal, rice flour, and soya drink) at two concentration levels corresponding to approximately two and four times the quantitation limits. The recoveries of MEL and derivatives from spiked samples varied from 80 to 103% with an average recovery SD (n ¼ 64) of 91 5. In the case of LC-DAD, the same samples were fortified with MEL and Table 2. LC-APCI method validation data

Linear range (ng mL1) Regression coefficient Detection limit (ng mL1) Quantification limit (ng mL1) Precision (n ¼ 10), RSD%

MEL

AMN

AMD

CA

10–200 (125–2000) 0.983 2.2 (32) 7.3 (106) 9.7

10–200 (125–2000) 0.977 2.7 (35) 9.0 (116) 7.6

10–200 (150–2000) 0.978 3.0 (43) 10 (143) 6.8

100–2000 (1250–20000) 0.997 13 (180) 43 (600) 5.3

Values into brackets correspond to ng g1


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AMN at two concentration levels in the range 260–600 ng g1, and an average recovery SD (n ¼ 32) of 89 7.


Analysis of Foods Different baby foods, corresponding to three types of infant formula (starting, follow-on, and prebiotic follow-on) and a breakfast cereal (multicereals with honey), were analyzed by using the LC-APCI-MS proposed procedure. Other samples of rice flour, glutinous rice flour, potato starch, soya drink, and coconut drink were also analyzed. All samples were analyzed in triplicate. The analytes MEL, AMN, AMD, and CA were not detected in any of the samples above the detection limits. The lack of interference was seen from the chromatographic profiles. The chromatographic peaks for spiked samples were identified by comparing the retention data for the standards, the samples and the samples spiked with the standards in the same conditions. Finally, identification was confirmed using the MS spectra. CONCLUSION Cation exchange LC provides a suitable way for controlling the presence of melamine and its derivatives in foods. Although DAD allows the determination of MEL and AMN, this way of detection fails for CA and AMD. Mass spectrometric detection allows a reliable quantification of the four analytes, as the sensitivity also improved. REFERENCES Andersen, W. C., S. B. Turnipseed, C. M. Karbiwnyk, S. B. Clark, M. R. Madson, C. M. Gieseker, R. A. Miller, N. G. Rummel, and R. Reimschuessel. 2008. Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass spectrometry. J. Agric. Food Chem. 56: 4340–4347. Arnol, A., T. N. Arrey, M. Karas, and M. Persike. 2011. Fast quantitative determination of melamine and its derivatives by matrix-assisted laser desorption=ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 25: 2844–2850. Bradley, E. L., V. Boughtflower, T. L. Smith, D. R. Speck, and L. Castle. 2005. Survey of the migration of melamine and formaldehyde from melamine food contact articles available on the UK market. Food Addit. Contam. 22: 597–606. Campbell, J. A., D. S. Wunschel, and C. E. Petersen. 2007. Analysis of melamine, cyanuric acid, ammelide, and mmeline using matrix-assisted laser desorption ionization=time-offlight mass spectrometry. Anal. Lett. 40: 3107–3118. Chao, Y. Y., C. T. Lee, Y. T. Wei, H. S. Kou, and Y. L. Huang. 2011. Using an on-line microdialysis=HPLC system for the simultaneous determination of melamine and cyanuric acid in non-dairy creamer. Anal. Chim. Acta 702: 56–61. Cheng, W. C., S. K. Chen, T. J. Lin, I. J. Wang, Y. M. Kao, and D. Y. Shih. 2009. Determination of urine melamine by validated isotopic ultra-performance liquid chromatography= tandem mass spectrometry. Rapid Commun. Mass Spectrom. 23: 1776–1782. Chu, P. W. S., K. M. Chan, S. T. C. Cheung, and Y. Wong. 2010. Review of analytical techniques used in proficiency-testing programs for melamine in animal feed and milk. Trends Anal. Chem. 29: 1014–1026.


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