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Analytica Chimica Acta 649 (2009) 91–97

Contents lists available at ScienceDirect

Analytica Chimica Acta

journa l homepage: www.e lsev ier .com/ locate /aca

etermination of melamine in milk-based products and other food and beverageroducts by ion-pair liquid chromatography–tandem mass spectrometry

aría Ibánez, Juan V. Sancho, Félix Hernández ∗

esearch Institute for Pesticides and Water, University Jaume I, E-12071, Castellón, Spain

r t i c l e i n f o

rticle history:eceived 5 May 2009eceived in revised form 3 July 2009ccepted 6 July 2009vailable online 14 July 2009

eywords:elamine

ood and beverageilk-based products

on-pair liquid chromatographyandem mass spectrometryriple quadrupole

a b s t r a c t

This paper describes a fast method for the sensitive and selective determination of melamine in awide range of food matrices, including several milk-based products. The method involves an extrac-tion with aqueous 1% trichloroacetic acid before the injection of the 10-fold diluted extract into theliquid chromatography–electrospray tandem mass spectrometry (LC–ESI-MS/MS) system, using labelledmelamine as the internal standard. As melamine is present in aqueous media in the cationic form, thechromatographic separation in reversed-phase LC requires the use of anionic ion-pair reagents, such astridecafluoroheptanoic acid (THFA). This allows a satisfactory chromatographic retention and peak shapein all the types of food samples investigated. The method has been validated in six food matrices (biscuit,dry pasta and four milk-based products) by means of recovery experiments in samples spiked at 1 and5 mg kg−1. Average recoveries (n = 5) ranged from 77% to 100%, with excellent precision (RSDs lower than5%) and limits of detection between 0.01 and 0.1 mg kg−1. In addition, accuracy and robustness of the

method was proven in different soya-based matrices by means of quality control (QC) sample analysis.QC recoveries, at 1 and 2.5 mg kg−1, were satisfactory, ranging from 79% to 110%. The method developedin this work has been applied to the determination of melamine in different types of food samples. Alldetections were confirmed by acquiring two MS/MS transitions (127 > 85 for quantification; 127 > 68 forconfirmation) and comparing their ion intensity ratio with that of reference standards. Accuracy of themethod was also assessed by applying it to a milk-based product and a baking mix material as part of an

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EU proficiency test, in wh

. Introduction

Melamine (1,3,5-triazine-2,4,6-triamine) is a nitrogen-basedndustrial chemical, used in the manufacture of durable plastics,n the production of melamine–formaldehyde resins and in theroduction of flame-retardants [1–3].

In March 2007, a pet food manufacturer alerted the US Foodnd Drug administration to animal deaths in the United States thatppeared to be linked to certain batches of their pet food. In theollowing months, consumers and veterinarians reported more ill-esses and deaths potentially associated with pet food [1]. Further

nvestigation showed that raw materials, wheat gluten and ricerotein, which were imported from China and used to manufac-ure the pet food [4] were, it appears, intentionally contaminated

ith melamine to increase their total nitrogen concentration and

onsequently the calculated protein content. Melamine in combi-ation with cyanuric acid resulted in the formation of insolubleelamine cyanurate crystals in kidneys, causing renal failure in

∗ Corresponding author. Tel.: +34 964 387366; fax: +34 964 387368.E-mail address: felix.hernandez@qfa.uji.es (F. Hernández).

003-2670/$ – see front matter © 2009 Published by Elsevier B.V.oi:10.1016/j.aca.2009.07.016

ighly satisfactory results were obtained.© 2009 Published by Elsevier B.V.

hundreds of cats and dogs in the United States who consumedadulterated feed [5]. Although initial reports suggested that con-tamination was confined to pet food, further investigations revealedthat melamine-contaminated ingredients had been also used tofeed animals intended for human consumption, such as chicken,swine and catfish [6]. Consequently, the US Food and Drug Admin-istration [2] developed a method for determination of melamineresidues in animal tissues based on liquid chromatography (LC)coupled to tandem mass spectrometry (MS/MS) using disposablestrong cation exchange solid-phase extraction (SPE) cartridges toprepare samples for LC analysis.

The need for accurate determination of melamine contentin food has led to a number of published papers where ana-lytical methods have been developed as an urgent responseto melamine incidents. Recently reported methods have useddifferent approaches, such as the use of enzyme-immunoassayLC-diode array detection (DAD) and ultra high pressure LC

(UHPLC)–tandem mass spectrometry (MS/MS) in pet food [1]; theuse of hydrophilic interaction chromatography (HILIC) columns inHPLC–MS/MS based methods applied to animal feed [7] and cat-fish [2]; HPLC–MS/MS in porcine muscle tissue [8]; or LC-DAD inrice concentrates [3]. Melamine in combination with cyanuric acid

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as also been determined by HPLC-DAD in cereal flours [9] and byC–MS/MS in meat and pet food [10-13] and in pork and fish tis-ue [14]. Most of these methods include a previous SPE step forre-concentration and/or purification of extracts.

Due to the ionic character of melamine, it is difficult to obtainufficient retention in a C18 column. Its separation can be achievedy ion-exchange or ion-pair reversed-phase LC. In general, the

on-exchange approach is not very suited to the electrospray inter-ace due to the use of buffers with high ionic strength. However,he use of ion-pair reversed-phase LC can provide satisfactoryesults, depending on the ion-pairing reagent characteristics.his approach has been used satisfactorily for the determina-ion of melamine in chard samples [15]. Other approaches tonhance the chromatographic retention of melamine are the usef zwitterionic HILIC [2,7,11] or the use of different stationaryhases, for example, CN-based phases [3] or weak anion exchange10,13].

Recently, an increased incidence of kidney stones and renal fail-re in infants was reported in China which was believed to bessociated with the ingestion of infant milk-based formula con-aminated with melamine [16]. It seemed that again nitrogen-rich

elamine was added to raw milk to boost its apparent proteinontent.

Maximum permitted concentration for melamine in food haseen set at 2.5 mg kg−1 by the European Commission [17–19]. Hongong established tolerance at 1 mg kg−1 in infant foods and Tai-anese authorities stated that melamine should not be detected in

ny food using the most sensitive instrumentation [17].Due to the urgent response needed in the face of the melamine

ncidents, analytical methodology able to accurately determine thisompound at trace levels is required to identify potential healthisks involved with consumption of contaminated foods. Becauseome of these contaminated foods could be consumed by humans,t is essential that this methodology is available as soon as possible.he goal of this paper is the development of advanced methodol-gy based on ion-pairing LC using reversed-phase C18 columns [15].andem MS with triple quadrupole analyzer is used as the detectionechnique due to its strong potential in the field of food safety. Inpite of the fact that several methods have recently been reportedor melamine in food, there is a lack of validated methodology pub-ished for milk-based products.

Only a few articles have been reported for determination ofelamine in milk or milk-based products, and some of them have

een published in Chinese journals making it difficult for the widercientific community to access the information given. In theseapers, melamine has been determined in milk and milk powder byPLC-DAD [20], thin layer chromatography-DAD [21] or HP capil-

ary electrophoresis-UV [22]. Some authors have also tried to detectelamine in milk by ultrasound-assisted extractive MS [23], by

sing low-temperature plasma (LLP) probe combined with MS/MS24] or by surface desorption atmospheric pressure chemical ion-zation (DAPCI) MS [25]. Regarding LC–MS, to our knowledge onlywo papers deal with the determination of melamine in milk. Yan etl. [26] determined melamine in raw milk and dairy products usingILIC-electrospray MS/MS, after an off-line SPE clean-up. QingQingt al. [27] developed a method based on reversed-phase LC–MS/MSsing heptafluorobutyric acid (HFBA) as the ion-pair reagent. Afterxtraction with 1% HFBA–methanol (80:20) and centrifugation, aiquid–liquid extraction with chloroform was performed. In spitef using MS/MS, the reliable identification of melamine is not suffi-iently supported in this method as only one transition is acquired

nstead of the minimum of two required for an unequivocal confir-

ation.Finally, some applications notes from instrumentation compa-

ies also describe procedures that make use of off-line SPE clean-upnd subsequent analysis by LC–MS/MS [17,28].

ca Acta 649 (2009) 91–97

For this reason, in this paper we have put special emphasis onthe analysis of milk-based products, as well as other types of foodmatrices, soya derivatives included, keeping in mind the simplifi-cation of sample handling. Thus, the SPE clean-up step has beenavoided, saving time, consumables and minimizing potential ana-lytical errors associated with this step. After extraction of sampleswith trichloroacetic acid, the food extract was diluted 10-fold withwater and directly injected into the LC–MS/MS system. The methodhas been validated in a notable number of food and beverage matri-ces, and has also been applied to two food materials within an EUMelamine Proficiency Test performed in January 2009.

2. Experimental

2.1. Reagents and chemicals

Melamine standard (95% purity) was purchased from Sigma (St.Louis, MO, USA). 2,4,6-[15N3]-triamino-1,3,5-[13C3] triazine, usedas internal standard, was supplied by Cambridge Isotope Labora-tories Inc. (Andover, MA, USA). HPLC-grade water was obtained bydistilled water passed through a MilliQ water purification system(Millipore Ltd., Bedford, MA, USA). HPLC-grade methanol (MeOH),HPLC-grade acetonitrile (ACN), reagent-grade formic acid (con-tent 98–100%) and HPLC-grade potassium dihydrogen phosphate(93%) were purchased from ScharLab (Barcelona, Spain). Tride-cafluoroheptanoic acid (TFHA, 99%) was purchased from Sigma.Analysis-grade trichloroacetic acid (TCA, 99.5%) and perchloric acid(60%) were purchased from Panreac (Barcelona, Spain). Heptafluo-robutyric acid (HFBA, 99%) was from Fluka (Buchs, Switzerland).

Stock standard solution of melamine was prepared by dissolv-ing approximately 50 mg of powder, accurately weighted, in 100 mLof acetonitrile:water 1% formic acid (50:50), obtaining a final con-centration of around 500 �g mL−1, and stored at −20 ◦C. Workingsolutions, used for LC–MS/MS analysis or for sample fortification,were obtained by diluting stock solution with methanol or water,using a maximum dilution factor of 10 in every dilution step.

The isotope-labelled melamine was purchased as 1.2 mL of100 �g mL−1 stock solution in water. A 12 �g mL−1 standard solu-tion was prepared by diluting 1.2 mL of the stock solution withwater up to 10 mL. Finally, 1.2 �g mL−1 standard working solutionwas prepared by diluting the intermediate standard solution withwater.

TFHA was prepared dissolving 0.94 g powder in 10 mL of ace-tonitrile:water (50:50) obtaining a final concentration of 250 mM.

1% TCA was prepared by dissolving 10 g TCA and diluting withwater to a final volume of 1000 mL.

2.2. Instrumentation

A HPLC system Waters Alliance 2795 (Waters, Milford, MA) wasinterfaced to a Quattro micro triple quadrupole mass spectrom-eter (Waters) using an orthogonal Z-spray–electrospray interface(ESI). The LC separation was performed injecting 20 �L and using aDiscovery C18 column (50 mm × 2.1 mm i.d., 5 �m) (Supelco, Belle-fonte, PA), at a flow rate of 300 �L min−1. The mobile phase usedwas a water 0.5 mM THFA/methanol gradient where the percent-age of methanol was changed linearly as follows: 0 min, 5%; 3 min,5%; 6 min, 50%; 7 min, 50%; 8 min, 95%; 12 min, 5%; and 17 min,5%. Drying gas, as well as nebulizing gas, was nitrogen generatedfrom pressurized air in a high-purity nitrogen generator NM30LA

230Vac Gas Station from Peak Scientific (Inchinnan, Scotland). Thedesolvation gas flow and cone gas flow were selected as 600 and60 L h−1, respectively. Infusion experiments were performed usingthe built-in syringe pump directly connected to the interface. Mass-lynx NT v 4.1 (Waters, Manchester, UK) software was used to process

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he quantitative data obtained from calibration standards and fromamples.

.3. Analytical procedure

0.5 g accurately weighed (precision 0.1 mg) of trituratedomogenised (Homogenizer K55, Dito Sama, Aubusson, France)olid samples (or 10 mL in the case of liquid samples) were trans-erred to centrifuge tubes (50 mL). Samples were extracted byhaking with 10 mL of aqueous TCA 1% on a mechanical shakerS.B.S. Instruments S.A., Barcelona, Spain) for 40 min, and thenentrifuged at 4500 rpm (3379.32 × g units) for 15 min (Centrifugeonsul, Orto-Alresa, Madrid, Spain). Afterwards, an aliquot of theupernatant was diluted 10-fold with HPLC-grade water. Then,0 �L of 250 mM TFHA solution were added to a 2 mL-vial that con-ained 1 mL of the 10-fold diluted extract and 100 �L of 1.2 mg L−1

abelled internal standard. Finally, 20 �L was injected in the LC–ESI-S/MS.

Quantification of samples was performed using calibrationurves prepared with standards in solvent that also contained thenternal standard, which provided relative response. Fortification ofolid samples for recovery experiments was performed by addingmL of the 500 �g L−1 or 2500 �g L−1 standard solution to 0.5 gomogenised blank sample in order to yield fortification levels ofand 5 mg kg−1, respectively. Fortification of liquid samples wasade by adding 1 mL of the 10 or 50 mg L−1 standard solution to

0 mL of sample yielding levels of 1 and 5 mg L−1, respectively.n both cases, fortified samples were equilibrated for 1 h prior toxtraction.

.4. Application to samples

Thirteen milk-based food and beverages products were pur-hased in different Castellon supermarkets, including milk powder,iscuits with milk-chocolate, or fruit juice and milk blends, amongstthers. As well, 20 soya-based samples were provided by the Publicealth Laboratory of Valencia (Spain).

.5. Data evaluation

In each batch of samples analyzed, a calibration curve at con-entrations between 2 and 1000 �g L−1 was injected before andfter the sample extracts. In addition, blank samples fortified withelamine at two levels were used as quality controls (QCs). QC

ecoveries in the range 70–110% were required to consider quan-ification in the sample batch satisfactory.

Confirmation of detected compounds was carried out by moni-oring both the quantification (Q) and confirmation (q) transitionsnd calculating their ion intensity ratio (Q/q). The identity of thenalyte peak was confirmed by comparison of the experimental Q/qatio in a sample with that of the reference standard. A maximumatio tolerance ±25% was accepted, as the intensity of the confir-

ative transition was 20–50% of the quantitative one (Q/q ratioetween 2 and 5) [29]. Concretely, the average Q/q ratio calculated

or melamine reference standard was approximately 4.0. The agree-ent in retention time (maximum deviation of 2.5%) for samples

nd standards was also required to confirm a positive finding.

. Results and discussion

.1. MS optimisation

Positive electrospray full-scan mass spectra and MS/MS spectraf melamine were obtained from infusion of 1 mg L−1 stan-ard solution in methanol:water (50:50, v:v) at a flow rate of0 �L min−1. The full-scan spectrum of melamine, at a cone of 40 V,

ca Acta 649 (2009) 91–97 93

showed the m/z 127.0 ion corresponding to the protonated molecule[M+H]+. The MS/MS spectrum showed fragments at m/z 84.9 (colli-sion energy 17 eV), due to the loss of H2NCN, and m/z 67.8 (collisionenergy 28 eV), due to the loss of H2NCN + NH3. The selected reactionmonitoring (SRM) transitions chosen were therefore 127.0 > 84.9and 127.0 > 67.8.

3.2. LC optimisation

Melamine is a highly polar basic compound for which it is nor-mally difficult to obtain satisfactory retention in a C18 column forseparation of this analyte from salts and polar matrix componentswhich could interfere by decreasing the MS analyte response [30].

In order to improve its retention in reversed-phase LC, ion-pairreagents can be used, although using non-volatile substances is notrecommended as they can destabilize the electrospray process andcontaminate the source, causing strong ion suppression [31]. There-fore volatile ion-pair reagents, such as perfluorinated carboxylicacids, should be used for analytes present in cationic form, likemelamine. Different perfluorinated carboxylic acids [32] added tothe mobile phase have been tested to achieve sufficient retentiontime for polar amino analytes. The acidic properties of these ion-pair reagents would reduce the peak tailing, obtaining good peakshape [33]. However, in some cases the addition of the modifier intothe mobile phase can be unsatisfactory; producing ion suppressionin LC–MS based methods. This was reported in our previous workon determination of fosetyl-Al in vegetables, where the ion-pairreagent was only added into the vial containing the final solutionto be injected into the LC–MS/MS system [34]. In our own experi-ence, when working with ion-pairing LC–MS/MS it is important tostudy the effect of adding the ion-pairing reagent into the samplevial and/or in the mobile phase as it can affect relevant parameters,such as sensitivity, reproducibility and robustness of the method[15,34–36].

In the present paper, the use of 0.5 mM HFBA as ion-pairreagent in the mobile phase did not give satisfactory retention formelamine. We also tested the effect of adding this reagent to thevial, and also in, both the vial and the mobile phase, obtaining poorresults in all cases (see Fig. 1).

However, the use of 0.5 mM TFHA in the mobile phase led to sat-isfactory chromatographic behaviour of the analyte when injectingstandard solutions, although it was insufficient for good peak shapein the samples. The addition of THFA to the vial also seemed to benecessary to obtain satisfactory chromatographic behaviour in theanalysis of samples. The optimum concentration that yielded thebest signal with enough retention time and good peak shape wasset up at 10 mM. The final, most satisfactory approach, was addingTFHA to both the vial (10 mM) and the mobile phase (0.5 mM) toensure formation of the ion-pair TFHA-melamine during the chro-matographic run, avoiding competitive processes that might occurwith matrix co-extracted compounds.

3.3. Method optimisation

Although melamine is a highly polar organic compound, quitesoluble in water, the extraction was performed with acidic sol-vent to promote its protonation which increases the extractionefficiency. Different extracting solvents such as formic acid [7],H2O:MeOH (50:50) [13], phosphoric buffer at pH 2 [15], perchloricacid 0.2 M [17] and 1% aqueous TCA [28], were tested.

First, these five extractants were tested in biscuit samples. As all

of them gave similar results, we selected the phosphoric buffer asit had been used previously in our laboratory for the determinationof melamine in chard samples [15]. However, when this extrac-tant was applied to chocolate-based samples, low recoveries (<10%)were obtained. After testing all extractants again in chocolate-based

94 M. Ibánez et al. / Analytica Chimica Acta 649 (2009) 91–97

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amples, 1% aqueous TCA was found to give satisfactory results inll matrices. This may be related to the ability of TCA to induce pro-ein precipitation. However, after direct injection of 20 �L extract,ow recoveries (around 40%) were obtained using external calibra-ion with aqueous standards, showing severe matrix suppressionn the MS instrument. Moreover, a bad peak shape was obtained.o reduce matrix effect by injecting cleaner extracts, a 10-fold dilu-ion of the raw extracts with HPLC-grade water was tested, trying tochieve similar signal response between diluted extracts and aque-us standards while maintaining satisfactory sensitivity. Despitehe 10-fold dilution, recoveries of around 60–70% were obtained.nder these conditions, matrix-matched external standard calibra-

ion using blank diluted sample extracts or the use of an adequatenternal standard was advisable to compensate for matrix effects.

As labelled melamine was commercially available, it was addedo the vial as internal standard to compensate for both the matrixffects and possible variations in the instrument signal. However,evere ionization inhibition still occurred lowering the sensitivity ofhe overall analytical procedure. Therefore, the dilution of extractsith water was assayed as a fast and simple way to minimize matrix

nterferences. According to our results, a minimum of a 10-fold dilu-ion of the sample extract was necessary for accurate quantificationith satisfactory sensitivity.

Additionally, four different extraction modes were tested:echanical shaking for 40 min, extraction with ultraturrax for

le vial on the melamine retention time and chromatographic peak shape.

5 min, and extraction in an ultrasonic bath for 15 and 30 min. Asno significant differences were observed, mechanical shaking wasselected as a simple way to automate this process.

We also checked the effect of filtering the samples. Although dif-ferent filters were tested, in all cases significant losses (up to 40%)were observed. Thus, we decided to avoid this step and centrifuga-tion was applied instead.

3.4. Method validation

The method developed was validated before application to sam-ple analysis. The validation study was carried out in six differentmatrices: milk powder, fruit juice and milk blends, biscuit, milk-chocolate biscuit, milk-chocolate egg, dry pasta (wheat) and bakingmix. The following parameters were evaluated:

- Linearity: Linearity of relative response (analyte versus internalstandard) was evaluated by injecting nine standard calibrationsolutions, in duplicate, in the range 2–1000 �g L−1. A linear cali-

bration with correlation coefficient higher than 0.99 and residualslower than 20% was obtained.

- Accuracy: This was estimated by means of recovery experiments,by analysing fortified samples in quintuplicate at two concen-tration levels (1 and 5 mg kg−1 for solid samples, and 1 and

M. Ibánez et al. / Analytica Chimi

Table 1Results obtained in the validation of milk-based products and other food samples(n = 5).

Solid samples Recovery (RSD, %) LOQ (mg kg−1) LOD (mg kg−1)

1 mg kg−1 5 mg kg−1

Milk powder 97 (3) 85 (3) 0.5 0.1Baking powder 85 (3) 80 (2) 0.3 0.1Milk-chocolate biscuit 98 (3) 81 (3) 0.4 0.1Milk-chocolate egg 94 (3) 86 (4) 0.4 0.1Biscuit 100 (5) 82 (3) 0.3 0.1Dry pasta (wheat) 82 (2) 77 (3) 0.4 0.1

Liquid samples Recovery (RSD, %) LOQ (mg L−1) LOD (mg L−1)

1 mg L−1 5 mg L−1

F

-

-

-

-3.6. Melamine proficiency test 2009

Fs

ruit juice and milk blends 92 (1) 92 (1) 0.03 0.01

5 mg L−1 for liquid samples). Satisfactory recoveries (77–100%)were obtained for all matrices, as shown in Table 1.Precision: Precision, expressed as repeatability of the method,was estimated in terms of relative standard deviation (RSD, in %)from the recovery experiments (n = 5) at each fortification level.Excellent precision (RSDs < 5%) was obtained for all matrices, asshown in Table 1.Lowest validated level (LVL) and limit of quantification (LOQ):The LVL was the lowest validated spiking level meeting themethod performance acceptability criteria (recovery in the range70–110%; with a RSD < 20%). LVL was 1 mg kg−1 for solid matricesand 1 mg L−1 for liquid samples. LOQ was estimated for a signal-to-noise of 10 from the chromatograms of samples spiked at theLVL. LOQ values are shown in Table 1.Limit of detection (LOD): This was estimated as the analyteconcentration that produced a peak signal of three times the back-ground noise (signal-to-noise of 3 from the chromatograms ofsamples spiked at the LVL). LODs were estimated in 0.1 mg kg−1

for solid samples and in 0.01 mg L−1 for liquid samples (Table 1).Specificity: This was evaluated by analysing the blank procedure,

a processed blank sample and a blank sample spiked at the LVLvalidated.

ig. 2. Illustrative LC–MS/MS chromatograms for milk powder: (a) blank milk powdertandard solution of 5 �g L−1.

ca Acta 649 (2009) 91–97 95

Typical chromatograms for a reference standard of 5 �g L−1 andmilk powder samples (blank and spiked with melamine at the LVL)are shown in Fig. 2.

3.5. Application to samples

We analyzed around 20 samples provided by the Public HealthLaboratory of Valencia. These samples corresponded to soya drink,soya sauce, black soya sauce, soya pâté, soya curd or chilli with soyasauce, among others. Each sample was fortified at two levels: 1 and2.5 mg kg−1 (mg L−1 in liquid samples) for quality control (QC). As anexample, Fig. 3 shows chromatograms for soya drink (Fig. 3a), andfor the same sample fortified at 1 mg L−1 (Fig. 3b) and 2.5 mg L−1

(Fig. 3c) together with a reference standard of 50 �g L−1(Fig. 3d).As can be seen, recoveries of 94% and 100% were obtained for thissample. Table 2 shows a summary of the results obtained in theanalysis of QC’s of soya-based products. All recoveries for QCs ana-lyzed together with each sample batch were satisfactory (between70% to 110%). Melamine was detected in one of these samples (soyadrink #013), at a concentration of 0.1 mg L−1 (Fig. 4a).

The optimised procedure was also applied to the analysis of dif-ferent milk-based food and beverages, purchased in supermarketsof the area. All analyses were performed in duplicate. One of thesamples (fruit juice and milk blends #002) was found to be con-taminated with melamine, although at a concentration lower than2.5 mg kg−1, the maximum allowed by the European Legislation(see Fig. 4b).

The two positive findings were simultaneously quantified andconfirmed by the acquisition of two SRM transitions as Fig. 4 shows.Their ion ratio was calculated and compared with that of referencestandard. In both positive samples, deviations lower than 5% wereobtained. Retention time was also used as criterion for confirma-tion, obtaining deviations lower than 0.5% between the sample andthe standard. Thus, the presence of melamine in these two sampleswas confirmed in a reliable way, avoiding false positives that couldhave been reported when acquiring only one MS/MS transition [37].

Two materials were provided by the European Commission/JointResearch Centre, Institute for Reference Materials and Measure-

sample; (b) blank milk powder sample spiked with melamine at 1 mg/kg and (c)

96 M. Ibánez et al. / Analytica Chimica Acta 649 (2009) 91–97

Fig. 3. LC–MS/MS chromatograms (quantitative transition, Q) for: (a) soya drink #011; (b) soya drink spiked at 1 mg L−1; (c) soya drink spiked at 2.5 mg L−1 and (d) 50 �g L−1

standard.

Fig. 4. LC–MS/MS chromatograms for two melamine positive samples: (a) soya drink #013, containing 6.7 �g L−1 of melamine in extract (0.13 mg L−1 in sample); (b) fruitjuice and milk blends #002, containing 4.5 �g L−1 of melamine in extract (0.09 mg L−1 in sample) (c) 10 �g L−1 standard. (Q) quantitative transition, (q) qualitative transition.

Table 2Summary of the results obtained in the analysis of quality control samples (QCs) ofsoya-based products.

Solid samplesa Liquid samplesb

1 mg kg−1 2.5 mg kg−1 1 mg L−1 2.5 mg L−1

Mean recovery (%) 96 90 103 97RSD (%) 8 6 6 6Highest value (%) 107 100 110 100L

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Table 3Results obtained in the EU Melamine proficiency test 2009.

Material A (milk powder) Material B (baking mix)

Mean value obtained(mg kg−1) ± uncertainty

10.8 ± 0.8 3.3 ± 0.1

Standard deviation 0.49 0.09

owest value (%) 79 83 94 84

a n = 13 for both levels, 1 and 2.5 mg kg−1.b n = 7 for both levels, 1 and 2.5 mg L−1.

ents/CRL Mycotoxins, within the Melamine Proficiency Test 2009.ne of them was melamine-contaminated skimmed milk powder

sample A) and the other was a baking mix containing melamine-

ontaminated milk powder (sample B)

The LC–MS/MS method described in this paper was applied tohe analysis of these two materials that were both analyzed in sex-uplicate on two different days (on January 2009). The method wasound to have excellent precision (RSD ≤ 5%) and accuracy, with

RSD (%) 5 3Z-score 0.6 0.3Theoretical value

(mg kg−1) ± uncertainty10.0 ± 0.6 3.18 ± 0.17

relative errors of 8% and 4% for the milk powder and the bak-ing mix, respectively. The results of this Proficiency Test (Table 3)together with the wide validation performed in many differentsample matrices demonstrate the reliability and robustness of themethod.

4. Conclusion

In this work, LC–ESI-MS/MS has proven to be a fast, sensitiveand selective technique for the determination of melamine in a

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ide range of food matrices in compliance with EU legislation. Dueo the high polarity of this compound, found as cationic specie inqueous media, the addition of an ion-pairing reagent, TFHA, wasequired to obtain satisfactory chromatographic retention and peakhape in reversed-phase liquid chromatography. The method devel-ped in this paper allows the rapid determination of melamineith little sample manipulation as, after the extraction step, only a

imple dilution of the sample extract with water is applied. It haseen validated at two concentration levels (1 and 5 mg kg−1) in sev-ral milk-based and other food matrices with satisfactory results.he method has been also evaluated by analyzing two materialsmilk powder and baking mix) as part of an EU proficiency test inhich excellent accuracy was obtained. Around 40 food and bever-

ge samples have been analyzed with the result that two of themere contaminated with melamine (fruit juice and milk blends,

oya drink). The identity of melamine in positive samples was con-rmed by acquiring two SRM transitions and the accomplishmentf their ion intensity ratio.

cknowledgements

The authors acknowledge the support of the Public Health Lab-ratory of Valencia (Dr. Vicente Yusa) for providing soya-basedamples and for promoting our participation in the EU Melamineroficiency Test 2009.

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