Sensitive determination of melamine in milk and powdered infant formula samples by high-performance liquid chromatography using dabsyl chloride derivatization followed by dispersive liquid–liquid microextraction

Accepted Manuscript Sensitive determination of melamine in milk and powdered infant formula samples by high-performance liquid chromatography using dabsyl chloride derivatization followed by dispersive liquid–liquid microextraction M. Faraji, M. Adeli PII: DOI: Reference:

S0308-8146(16)31619-3 http://dx.doi.org/10.1016/j.foodchem.2016.10.002 FOCH 19990

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Food Chemistry

Received Date: Revised Date: Accepted Date:

23 June 2016 8 September 2016 2 October 2016

Please cite this article as: Faraji, M., Adeli, M., Sensitive determination of melamine in milk and powdered infant formula samples by high-performance liquid chromatography using dabsyl chloride derivatization followed by dispersive liquid–liquid microextraction, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/j.foodchem. 2016.10.002

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1

Sensitive determination of melamine in milk and powdered infant formula

2

samples by high-performance liquid chromatography using dabsyl chloride

3

derivatization followed by dispersive liquid–liquid microextraction

4 5

M. Faraji∗'a, M. Adelib

6 7 8 9

a

Faculty of Food Industry and Agriculture, Department of Food science & Technology,

10

Standard Research Institute (SRI), Karaj P.O. Box 31745-139, Iran

11

b

12

Tehran, Iran

Knowledge Development & University Relationship Department, Iran Khodro Company,

13 14 15 16 17 18 19 20 21 22 ∗

Corresponding author. Fax: +98-26-32803870; E-mail: [email protected] 1

23

ABSTRACT

24

A new and sensitive pre-column derivatization with dabsyl chloride followed by

25

dispersive liquid-liquid microextraction was developed for the analysis of melamine (MEL)

26

in raw milk and powdered infant formula samples by high performance liquid

27

chromatography (HPLC) with visible detection. Derivatization with dabsyl chloride leads to

28

improving sensitivity and hydrophobicity of MEL. Under optimum conditions of

29

derivatization and microextraction steps, the method yielded a linear calibration curve

30

ranging from 1.0 to 500 µg L-1 with a determination coefficient (R2) of 0.9995. Limit of

31

detection and limit of quantification were 0.1 and 0.3 µg L-1, respectively. The relative

32

standard deviation (RSD%) for intra-day (repeatability) and inter-day (reproducibility) at 25

33

and 100 µg L-1 levels of MEL was less than 7.0% (n = 6). Finally, the proposed method was

34

successfully applied for the preconcentration and determination of MEL in different raw milk

35

and powdered infant formula, and satisfactory results were obtained (relative recovery ≥

36

94%).

37 38 39 40

Keywords: Melamine, Dabsyl chloride, High performance liquid chromatography, Milk, Dispersive liquid-liquid microextraction.

41 42 43 44 45 46 47 48 49

2

50

1. Introduction

51

Melamine (1,3,5-triazine-2,4,6-triamine, MEL) is an organic compound

52

often used with formaldehyde to produce MEL resin, a synthetic fire-resistant

53

and heat-tolerant polymer (Kim et al, 2008; Andersen et al., 2008). MEL is not

54

approved as an ingredient in foods, but some manufacturers illegally use it as an

55

adulterant, because of its high nitrogen level (66% by mass) and low price, to

56

increase the apparent protein content. In 2008, a large-scale MEL contamination

57

incident was made public in China and many other countries (Sun et al., 2010; Yan,

58

Zhou, Zhu, & Chen, 2009).

59

The maximum level allowed for MEL residue is regulated and set to 1.0 mg

60

kg-1 for powdered infant formula and 2.5 mg kg-1 for other foods and animal feed

61

(FAO, 2010; European Commission, 2009). Higher concentrations of MEL above the

62

safety regulation level can cause tissue injury such as acute kidney failure,

63

urolithiasis, bladder cancer, and even death (Skinner, Thomas, & Osterloh, 2010).

64

In order to detect food adulteration and evaluate food safety, several

65

analytical methods have been reported for quantitative determination of MEL in

66

different matrices (Rovina & Siddiquee, 2015), including spectrophotometry

67

(Liu et al., 2011), spectrofluorometry (Zhou, Yang, Liu, Wang, & Lu, 2010;

68

Zeng, H., Yang, R., Wang, Q., Li, J., & Qu, 2011), high performance liquid

69

chromatography with UV (HPLC–UV) or fluorescence detection (HPLC-FLD)

70

(Sun, Wang, Ai, Liang, & Wu, 2010; Venkatasami, & Sowa, 2010; Zhang et al.,

71

2014; Muniz-Valencia et al., 2008; Zheng, Yu, Li, & Dai, 2012; Filazi, Sireli,

72

Ekici, Can, & Karagoz, 2012; Gao & Jönsson, 2012), liquid chromatography–

73

tandem mass spectrometry (LC/MS/MS) (Ibanez, Sancho, & Hernandez, 2009;

74

Deng et al., 2010; Zhang et al., 2010; Wu et al., 2009; Goscinny, Hanot, 3

75

Halbardier, Michelet, & Van Loco, 2011; Chen, Zhao, Miao & Wu, 2015; Viñas,

76

Campillo, Férez-Melgarejo & Hernández-Córdoba, 2012), gas chromatography–mass

77

spectrometry (GC/MS) (Xu et al., 2009; Pan et al., 2013; Li, Qi, & Shi, 2009;

78

Li, Zhang, Meng, Wang, & Wu, 2010), gas chromatography–tandem mass

79

spectrometry (GC/MS n) (Miao et al., 2009; Tzing, & Ding, 2010), and capillary

80

zone electrophoresis (Xia et al., 2010).

81

Due to the small and polar nature of MEL, GC-based methods need a

82

further derivatization procedure. On the other hand, HPLC-based methods

83

need polar reversed-phase (RP) columns (Sun, Wang, Ai, Liang, & Wu, 2010;

84

Zheng, Yu, Li, & Dai, 2012; Ihunegbo, Tesfalidet & Jiang, 2010) or general

85

C18 and C8 RP columns with the mobile phase containing ion-pair reagent

86

(Filazi, Sireli, Ekici, Can, & Karagoz, 2012; Ibanez, Sancho, & Hernandez,

87

2009). Another challenge in MEL analysis is related to unsuitable sensitivity

88

of conventional GC and HPLC detectors. In contrast, MS and MS/MS

89

detectors introduce a high sensitivity, but instruments are expensive and the

90

running cost is high. In addition, complicated instruments and skilled

91

operators are required, which makes their popularization difficult. Therefore,

92

in order to generalize techniques and to overcome the mentioned problems,

93

development of novel sample preparation methods before injection to HPLC or

94

GC is necessary.

95

Derivatization with suitable fluorophores or chromophores can enhance

96

HPLC sensitivity and improve the chromatographic behavior of many

97

compounds (Zhang et al., 2014; Jansen, Van den Berg, Both-Miedema, &

98

Doorn, 1991). For the first time, Zhang et al. in 2014 tried derivatization of MEL

99

in order to enhance HPLC sensitivity (Zhang et al., 2014). They developed a 4

100

sensitive HPLC method with fluorescence (FL) detection for the analysis of

101

MEL by derivatizing with 10-methyl-acridone-2-sulfonyl chloride (MASC), a

102

compound with excellent fluorescence property. Their results showed that HPLC

103

sensitivity of MEL was greatly enhanced. Meanwhile, the hydrophobicity of

104

MEL was also greatly increased. According to this approach, other labeling

105

reagents containing sulphuryl chloride group could be used for quantification of

106

MEL. Amongst available reagents, dabsyl chloride is a well established UV-

107

labeling reagent that has been primarily used for covalent bonding to and

108

quantitation of amino acids (Jansen, Van den Berg, Both-Miedema, & Doorn,

109

1991), imidazole-containing compounds (Handley et al., 1998; Sormiachi, Ikeda,

110

Akimoto, & Niwa, 1995), and polyamines (Koski, Helander, Sarvas, & Vaara,

111

1987; Romero, Gazquez, Bagure, & Sanchez-Vinas, 2000). Although dabsyl

112

chloride has never been used to quantify MEL, but dabsylation method is

113

associated with some advantages. Dabsylation procedure is fast, and dabsyl

114

derivatives are very stable. Moreover, dabsyl derivatives show absorbance in the

115

range of 436–460 nm. In that way, interferences from UV-absorbing biological

116

compounds present in food extracts are mostly avoided (Aboul-Enein, 2003) in

117

comparison with MASC reagent. Further, dabsylation leads to improving the

118

hydrophobicity of the compounds (Handley et al., 1998).

119

The objective of the present work was to develop and optimize a simple

120

and fast method for determining MEL in different milk samples based on

121

derivatization

122

microextraction (DLLME) with octanol (as a compatible solvent with RP-

123

HPLC) followed by HPLC-UV-vis, for the first time. Several experimental

124

parameters of the proposed method which influence MEL derivatization and

with

dabsyl

chloride

5

and

dispersive

liquid-liquid

125

microextraction performance were investigated and optimized. Finally,

126

figures of merit of the proposed method were compared with previously

127

published methods.

128 129

2. Material and methods

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2.1. Chemicals and reagents

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HPLC-grade acetonitrile (ACN), analytical grade MEL, 1-octanol, sodium acetate,

132

triethylamine, acetone, methanol, ethanol, trichloroacetic acid, lead acetate, sodium carbonate

133

and NaCl were purchased from Merck Company (Darmstadt, Germany). 4-(4-

134

Dimethylaminophenylazo)benzenesulfonyl chloride (Dabsyl chloride) was provided from

135

Sigma-Aldrich Company (Steinheim, Germany). Dabsyl chloride reagent was dissolved in

136

ACN at concentration of 4 mg mL-1 and sonicated for 5 min. Water was purified using a

137

Milli-Q Ultrapure water purification system (Millipore, Bedford, MA, USA). Stock solution

138

of MEL in acetonitrile at 1000 mg L-1 concentration level was prepared. Fresh working

139

solutions were prepared by mixing stock solutions and diluting with water.

140

2.2. Apparatus

141

The chromatographic analysis was carried out in a EuroChrom model Knauer HPLC

142

(Berlin, Germany) consisting of a degasser, quaternary pump (model K1100), manual sample

143

injector with a 20 µL loop size, and UV-vis detector (model K2600) which it was controlled

144

by EZChrom software. The HPLC operating mode was gradient and column temperature was

145

adjusted to room temperature. The chromatography column was a Supelcosil LC-18: 25cm ×

146

4.6 mm, 5µm (Supelco, Bellefonte, PA, USA). The mobile phase used was a combination of

147

phase A (ACN: 20 mM sodium acetate, pH = 5.0, containing 0.2% triethylamine, 25:75, v/v)

148

and phase B (ACN 100%). Elution was performed as follows: from 0 to 5 min, 100%A; from

149

5 to 6 min, a linear gradient from 0 to 100%B; from 6 to 14 min, 100%B; from 14 to 15, a 6

150 151

linear gradient from 0 to 100%A; from 15 to 20 min, 100%A. The flow rate was 1.0 mL min1

. The UV-vis detector was adjusted to 460 nm. The mobile phase was filtered through a

152

0.45-µm pore size filter (Merck Millipore, Billerica, Massachusetts, USA) and degassed by

153

vacuum prior to use. A 40 kHz and 0.138 kW ultrasonic water bath with temperature control

154

(Tecno-GazSpA, Italy) was applied for ultrasonication of the samples. All of the pH

155

measurements were performed with a WTW Inolab pH meter (Weilheim, Germany). A

156

Hettich centrifuge model MIKRO 22R (Hettich Co., Kirchlengern, Germany) was used to

157

accelerate the phase separation.

158

2.3. Sample preparation

159

8.0 mL of 5% (w/v) trichloroacetic acid solution and 1.0 mL of 2.2% (w/v) lead acetate

160

solution were added to 1.0 g of each homogenized raw milk or powdered infant formula in a

161

25-mL glass beaker in order to eliminate protein and extract the analyte. The mixture was

162

placed in ultrasonic cleaner for 10 min to mix well. Then, the mixed solution was centrifuged

163

for 10 min at 10,000 rpm. For dabsyl derivatization, 200 µL of the supernatant was

164

transferred to a 10 mL conical glassware vial. Then, 50 µL of 1.5 mol L-1 sodium carbonate

165

buffer pH = 9.0 and 100 µL of 4 mg mL-1 of dabsyl chloride were added to the vial. The vial

166

was vortexed for 1 min and then allowed to react at 70 ºC for 10 min in a water bath. A

167

schematic illustration of MEL dabsylation is shown in Fig. 1. Afterward, to stop

168

derivatization reaction, appropriate cool ACN (-18 ºC) was added till the final volume of 1.0

169

mL. This solution was used as disperser solvent in the DLLME of MEL-dabsyl derivatives.

170

2.4. Dispersive liquid-liquid microextraction

171

For DLLME, 60 µL of 1-octanol (extraction solvent) was added to the vial (Section

172

2.3) and mixed. Then, 5.0 mL of deionized water was rapidly injected into the ACN phase. In

173

this step, MEL was extracted into fine droplets of 1-octanol. Subsequently, to separate the

7

174

organic phase, the mixture was centrifuged for 5 min at 4000 rpm. After this process, the

175

dispersed fine droplets of 1-octanol floated on the aqueous sample. The lower-aqueous phase

176

was separated using a syringe and 20 µL of the floated phase (30 ± 2.0 µL) was injected

177

directly into the HPLC using a microsyringe.

178 179

3. Results and Discussion

180

3.1. Optimization of melamine dabsylation conditions

181

3.1.1. Optimization of pH of derivatization

182

pH control of sample solution is very important since it greatly influences the extension

183

of derivatization reaction and, as a rule of thumb, the sample pH has to be above the pKa of

184

analyte so that it could be deprotonated. Apart from analyte, dabsylation occurs at alkaline

185

conditions usually between pH 8.5 and 9.0 (Jansen, Van den Berg, Both-Miedema, &

186

Doorn, 1991; Handley et al., 1998; Sormiachi, Ikeda, Akimoto, & Niwa, 1995;

187

Koski, Helander, Sarvas, & Vaara, 1987; Romero, Gazquez, Bagure, &

188

Sanchez-Vinas, 2000; Aboul-Enein, 2003). In order to evaluate the effect of pH on the

189

derivatization efficiency, pH of the sample solutions was adjusted in the range of 7.0–11.0

190

and the recommended procedure (Section 2.3) was followed. According to the results (Fig.

191

2a), maximum response (peak area) was obtained at pH 9.0. Maseda's research showed that

192

dabsylation did not take place when pH≤ 6.0 (Maseda, Fukui, Kimura, & Matsubara, 1983).

193

So, by increasing pH from 7.0-9.0 reponses increased.. On the other hand, higher pH values

194

(pHs> 10.0) would cause smaller responses of MEL, because a strong basic condition may

195

lead to decomposition of the analyte and the derivatizing reagent (Maseda, Fukui, Kimura, &

196

Matsubara, 1983). Thus, pH of the sample solutions for derivatization was adjusted at 9.0 by

197

using carbonate-bicarbonate buffer in subsequent experiments.

8

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3.1.2. Effect of dabsyl chloride volume

199

To guarantee the sufficient reaction of the analyte, derivatizing reagent should be

200

adequate. The effects of dabsyl chloride amount on derivatization were therefore studied in

201

the range of 50 to 150 µL of the of 4 mg mL-1 of the dabsyl chloride solution. Fig. 2b shows

202

that the response of MEL-dabsyl derivative increased obviously with the dabsyl chloride

203

amount increasing from 50 to 100 µL. Further increasing the dabsyl chloride amount beyond

204

100 µL excess had no significant effects on the response.

205

3.1.3. Effect of derivatization temperature and time

206

Reaction temperature provides the necessary activation energy to accelerate

207

derivatization reaction to completion, increasing the yield of the derivatives. Derivatization

208

using dabsyl chloride is usually carried out with medium reaction times (5–15 min) at a

209

relatively high temperature (70ºC) (Jansen, Van den Berg, Both-Miedema, & Doorn,

210

1991; Handley et al., 1998; Sormiachi, Ikeda, Akimoto, & Niwa, 1995; Koski,

211

Helander, Sarvas, & Vaara, 1987; Romero, Gazquez, Bagure, & Sanchez-Vinas,

212

2000; Aboul-Enein, 2003; Maseda, Fukui, Kimura, & Matsubara, 1983). Derivatization

213

has been shown to occur even at 25 ºC, but for adequate response an extended incubation

214

time, i.e. 30 min, has been required, and formation of by-product could be increased (Lacroix

215

& Saussereau, 2012). Therefore, in the present study, two derivatization temperatures (60 and

216

70ºC) were tested. The highest extraction efficiency for MEL was obtained at 70ºC.

217

Therefore, 70ºC was selected as optimum derivatization temperature for further experiments.

218

Also, different reaction times (5, 10, 15, 20, 30 min) were examined to find the optimum

219

condition for derivatization of MEL. According to the results, 10 min is enough for sufficient

220

dabsylation of MEL. This result is in agreement with previous studies (Jansen, Van den

221

Berg, Both-Miedema, & Doorn, 1991; Handley et al., 1998; Sormiachi, Ikeda,

222

Akimoto, & Niwa, 1995; Koski, Helander, Sarvas, & Vaara, 1987; Romero, 9

223

Gazquez, Bagure, & Sanchez-Vinas, 2000). Increasing the incubation time more than

224

15 min did show further gain in the recovery of dabsyl derivative, but led to partial hydrolysis

225

of dabsyl MEL (Maseda, Fukui, Kimura, & Matsubara, 1983).

226

An important point in derivatization is repeatability of reaction; it can be improved by

227

immediately stopping reaction at an exact time (10 min). Lacroix and Saussereau declared

228

that dabsyl derivatization reaction could be stopped by adjusting pH to below 6.0 with buffer,

229

or decreasing temperature by placing the bottom of Eppendorf vials under fresh water

230

(Lacroix & Saussereau, 2012). In this research, a new idea based on the second approach has

231

been used to improve repeatability of the derivatization, dabsylation was stopped by adding

232

proper volume of the very cold ACN (-18ºC) in to the derivatization vial (final volume = 1.0

233

mL).

234

3.1.4 Stability of melamine-dabsyl derivatives

235

One of the distinct features of dabsyl derivatives is their excellent stability (Jansen,

236

Van den Berg, Both-Miedema, & Doorn, 1991) which is very important in sample

237

analysis. Therefore, the stability of the MEL-dabsyl derivatives was investigated. The

238

derivatives at the concentration of 200 µg L-1 were repeatedly analyzed by HPLC after being

239

placed at room temperature for 0, 4, 8, 12, 24, 48, 72 h, respectively. Results indicated that

240

the responses of the MEL-dabsyl derivatives were stable with peak area deviations of less

241

than 3.6%. Thus, the stability of MEL-dabsyl derivatives was sufficient for HPLC analysis.

242

In literature, stability for at least 7 days has also been observed for dabsyl amino acid

243

derivatives (Handley et al., 1998). Furthermore, it has been demonstrated that ∆9-

244

Tetrahydrocannabinol (THC) and cannabinol (CBN), when crystallized by dabsylation, were

245

unchanged at least for one year (Maseda, Fukui, Kimura, & Matsubara, 1983).

246

10

247

3.2. Optimization of DLLME parameters

248

In this study DLLME was done for two purposes: (1) for the preconcentration of MEL-

249

dabsyl derivatives to getting further sensitivity, and (2) for omitting excess amounts of dabsyl

250

chloride reagent before injection to HPLC as result of very low extraction yield of the reagent

251

to octanol phase (the excess amount of dabsyl chloride in presence of water is converted to

252

methyl orange which is not dissolved in extraction phase (Parris & Gallelli, 1984)).

253

Therefore, in order to achieve maximum extraction efficiency, several parameters affecting

254

the DLLME of MEL-dabsyl derivatives, including the volume of extraction solvent, volume

255

of disperser solvent, and salt effect, were optimized using the one-variable-at-a-time

256

optimization method.

257

Selection of extraction solvent is very important for DLLME methods. Primary

258

requirements for an adequate extraction solvent include low solubility in water, larger density

259

than water, and high extraction efficiency for the analytes of interest (Rezaee et al., 2006;

260

Rezaee, Yamini & Faraji, 2010). Nevertheless, 1-octanol was selected because in spite of

261

being lighter than water is compatible with RP-HPLC (without need to a further

262

evaporation/reconstitution step).

263

The suitable volume of extraction solvent was investigated using 1000 µL ACN (MEL-

264

dabsyl phase) with different volumes of 1-octanol (60, 80, 100, 120 µL). As can be seen in

265

Fig. 3a, the peak area of MEL-dabsyl was decreased by increasing the extraction solvent

266

volume. This trend can be interpreted by decreasing enrichment factor due to dilution effect.

267

Consequently, 60 µL of 1-octanol was chosen for further experiments.

268

In DLLME, disperser solvent plays a crucial role, as it allows the dispersion of

269

extraction solvent into the aqueous sample where it is immiscible (Rezaee, Yamini & Faraji,

270

2010). In this study, because of using ACN as dabsyl chloride solvent and also as better

11

271

diluent, ACN was chosen as disperser solvent, and further optimization of nature of disperser

272

solvent was not done. Furthermore, the volume of ACN was optimized by varying the

273

volume between 750 and 1250 µL at a constant volume of 60 µL of 1-octanol. Extraction

274

efficiency for MEL was significantly increased by increasing the volume of ACN up to 1000

275

µL and tended to decrease after 1000 µL (Fig. 3b). It appears that at a low volume, ACN’s

276

cloudy state is not well formed, making recovery low (Rezaee, Yamini & Faraji, 2010). On

277

the other hand, solubility of extraction solvent and also MEL-dabsyl in aqueous phase

278

increased when a larger amount of the disperser solvent was used (above 1000 µL).

279

Therefore, 1000 µL of ACN was selected as the optimum volume of disperser solvent for

280

further experiments.

281

Generally, addition of salt enhances extraction of analytes, because the salting-out

282

effect can reduce the solubility of analytes in water and force more of them onto the organic

283

phase (Razmara, Daneshfar & Sahrai, 2011). On the other hand, in DLLME methods, by

284

increasing ionic strength, volume of the sediment phase increases because of the decreased

285

insolubility of the extraction solvent (Rezaee, Yamini & Faraji, 2010). To investigate the

286

effect of salt on the extraction efficiency for MEL, NaCl was added in the range of 0–15%

287

(w/v). The results revealed that salt addition had a significant effect on the extraction

288

efficiency of MEL, as the peak response was found to decrease as the ionic strength

289

increased. These results revealed that the second phenomenon is predominant and dilution of

290

extraction phase is occurred. Therefore, no salt was added in further experiments.

291

3.3. Method performance

292

The figures of merit in the proposed method, including linear dynamic range (LDR),

293

limit of detection (LOD), and limit of quantification (LOQ), and intra and inter-day

294

precisions for the extraction of MEL from matrix-matched samples (a milk sample which was

295

free from MEL) were investigated under optimum conditions. The obtained results are shown 12

296

in Table 1. Calibration curves were plotted using 8 spiking levels of MEL in concentrations

297

ranging from 1.0 to 500 µg L-1 and the good determination coefficient (R2) of 0.9952 was

298

obtained. For each level, three replicate extractions were performed under optimum

299

conditions. LOD and LOQ values based on the signal-to-noise ratio of three (LOD = 3 × S/N)

300

and signal-to-noise ratio of ten (LOQ = 10 × S/N) calculations were 0.1 µg L-1 and 0.3 µg L-1,

301

respectively. The intra-day precision of the proposed method (repeatability) was obtained 3.2

302

and 2.6 at 25 and 100 µg L-1 levels of MEL, and inter-day precision of the proposed method

303

(reproducibility) was obtained 6.7 and 5.4 at 25 and 100 µg L-1 levels of MEL, respectively.

304

3.4. Sample analysis

305

In order to evaluate the applicability of the proposed method to the analysis of MEL in

306

real samples, different raw milk and powdered infant formula samples were prepared and

307

analyzed in triplicate under optimum conditions. Moreover, in order to evaluate the accuracy

308

of the method in real sample analysis, samples were spiked at the known level of 0.5 mg Kg-

309

1

. The obtained results are presented in Table 2 based on mg MEL in Kg sample by

310

considering sample preparation steps. Good results were obtained, with average recoveries

311

ranging from 90.0 to 104.2% with RSDs% of less than 6.7%. Fig. 4 depicts the

312

chromatograms of MEL in the powdered infant formula 1 before (Fig. 4a) and after spike at

313

0.5 mg Kg-1 (Fig. 4b).

314

Evaluation of real sample analysis results showed that between tested samples MEL

315

was found in powdered infant formula 1 (0.48 mg Kg-1), powdered infant formula 5 (0.23 mg

316

Kg-1) and milk 3 (0.11 mg Kg-1) . Results demonstrated that the tested samples are in

317

agreement with the maximum level allowed for MEL residue (1.0 mg kg-1 for powdered

318

infant formula and 2.5 mg kg-1 for other foods and animal feed) (FAO, 2010; European

319

Commission, 2009).

13

320

3.5. Comparison of the applied method with other reported methods

321

The proposed method was compared with a variety of methods that had recently been

322

reported in the literature for preconcentration and determination of MEL. The distinct

323

features of the proposed method are summarized in Table 3. As can be seen from Table 3, it

324

is evident that the proposed method has a wide dynamic linear range. Moreover, LOD of the

325

method is better that some other methods which even use sensitive detection methods such as

326

LC-MS (Ibanez, Sancho, & Hernandez, 2009; Deng et al., 2010; Zhang et al.,

327

2010), GC-MS (Xu et al., 2009; Pan et al., 2013), and HPLC-FLD (Zhang et al.,

328

2014). Moreover, in regard with the running cost and complication of instrument, the

329

proposed method has a moderate running cost by using the common instrument of HPLC-

330

UV-Vis which could be applied in routine MEL analysis in food control laboratories.

331

4. Conclusions

332

In the present study, for the first time a very sensitive method was developed for the

333

analysis of MEL in powdered infant formula and raw milk samples based on dabsyl chloride

334

derivatization followed by DLLME. Dabsylation increases detection sensitivity (low

335

detection limit) and also increases hydrophobicity of polar compound of MEL, both of which

336

lead to generalization of MEL analysis with the common instrument of HPLC-UV-Vis and

337

widely used C18 columns. Meanwhile, DLLME of MEL-dabsyl derivatives resulted in

338

further preconcentration and omission of the excess amount of reagent. The proposed method

339

allows MEL determination in different powdered infant formula and milk samples with good

340

accuracy and reproducibility at levels as low as 1.0 µg L-1.

341

Acknowledgements

342

The authors are grateful for the support from the Iran National Science Foundation Fund

343

(92035384).

14

344

References

345

Aboul-Enein, H. Y. (2003). Separation techniques in clinical chemistry (1st ed.). New York:

346

Marcel Dekker (Chapter 1).

347

Andersen, W. C., Turnipseed, S. B., Karbiwnyk, C. M., Clark, S. B., Madson, M. R.,

348

Gieseker, C. M., et al. (2008). Determination and confirmation of melamine residues in

349

catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass

350

spectrometry. Journal of Agricultural and Food Chemistry, 56 4340–4347.

351

Chen D., Zhao Y., Miao H. & Wu Y. (2015) A novel dispersive micro solid phase extraction

352

using PCX as the sorbent for the determination of melamine and cyromazine in milk

353

and milk powder by UHPLC-HRMS/MS. Talanta, 134,144-152.

354

Deng, X., Guo, D., Zhao, S., Han, L., Sheng, Y., Yi, X., et al. (2010). A novel mixed-mode

355

solid phase extraction for simultaneous determination of melamine and cyanuric acid in

356

food by hydrophilic

357

chromatography. Journal of Chromatography B, 878(28), 2839-2844.

interaction

chromatography coupled

to

tandem mass

358

European Commission. 2009. Commission Regulation 1135/2009/EC of 25 November 2009

359

imposing special conditions governing the import of certain products originating in or

360

consigned from China, and repealing Commission Decision 2008/798/EC. Off. J.

361

L311:3.

362

Filazi, A., Sireli, U. T., Ekici, H., Can, H. Y. & Karagoz, A. (2012). Determination of

363

melamine in milk and dairy products by high performance liquid chromatography.

364

Journal of Dairy Science, 95, 602–608.

365

Food and Agriculture Organization (FAO), 2010. International experts limit Melamine levels 33

366

in food. The

rd Session of Codex Alimentarius Commission meeting in Geneva,

367

[accessed 08.28.16]. 15

368

Gao L. & Jönsson J. Å. (2012) Determination of melamine in fresh milk with hollow fiber

369

liquid phase microextraction based on ion-pair mechanism combined with high

370

performance liquid lhromatography. Analytical letters, 45, 2310-2323.

371

Goscinny, S., Hanot, V., Halbardier, J. F., Michelet, J. Y. & Van Loco, J. (2011). Rapid

372

analysis of melamine residue in milk, milk products, bakery goods and flour by ultra-

373

performance liquid chromatography/tandem mass spectrometry: from food crisis to

374

accreditation. Food Control, 22, 226–230.

375

Handley, M. K., Hirth, W. W., Phillips, J. G., Ali, S. M., Khan, A., Fadnis L., et al. (1998).

376

Development of a sensitive and quantitative analytical method for 1H-4-substituted

377

imidazole histamine H-receptor antagonists 3 utilizing high-performance liquid

378

chromatography and dabsyl derivatization. Journal of Chromatography B, 716, 239–

379

249.

380

Ibañez, M., Sancho, J. V., & Hernandez, F. (2009). Determination of melamine in milk-based

381

products and other food and beverage products by ion-pair liquid chromatography–

382

tandem mass spectrometry. Analytica Chimica Acta, 649, 91–97.

383

Ihunegbo F. N., Tesfalidet S. & Jiang W. (2010) Determination of melamine in milk powder

384

using zwitterionic HILIC stationary phase with UV detection. Journal of Separation

385

Science, 33, 988-995.

386

Jansen E. H. J. M., Van den Berg, R. H., Both-Miedema, R. & Doorn L-B. (1991).

387

Advantages and limitations of pre-column derivatization of amino acids with dabsyl

388

chloride. Journal of chromatography 553, 123-133.

389

Kim, B., Perkins, L. B., Bushway, R. J., Nesbit, S., Fang, T., Sheridan, R., et al. (2008).

390

Determination of melamine in pet food by enzyme immunoassay, high-performance

391

liquid chromatography with diode array detection, and ultra-performance liquid 16

392

chromatography with tandem mass spectrometry. Journal of AOAC International, 91

393

408–413.

394

Koski, P., Helander, I. M., Sarvas, M. & Vaara M. (1987). Analysis of polyamines as their

395

dabsyl derivatives by reversed-phase high-performance liquid chromatography.

396

Analytical Biochemistry, 164, 261-266.

397

Lacroix, C. & Saussereau, E. (2012). Fast liquid chromatography/tandem mass spectrometry

398

determination of cannabinoids in micro volume blood samples after dabsyl

399

derivatization. Journal of Chromatography B, 905, 85–95.

400

Li, J., Qi, H. Y. & Shi, Y. P. (2009). Determination of melamine residues in milk products by

401

zirconia hollow fiber sorptive microextraction and gas chromatography–mass

402

spectrometry. Journal of Chromatography A, 1216, 5467–5471.

403

Li, M., Zhang, L., Meng, Z., Wang, Z. & Wu, H. (2010). Molecularly-imprinted

404

microspheres for selective extraction and determination of melamine in milk and feed

405

using gas chromatography–mass spectrometry. Journal of Chromatography B, 878,

406

2333–2338.

407

Liu, Y., Deng, J., An, L., Liang, J., Chen, F., & Wang, H. (2011). Spectrophotometric

408

determination of melamine in milk by rank annihilation factor analysis based on pH

409

gradual change-UV spectral data. Food Chemistry, 126(2), 745-750.

410

Maseda, C., Fukui, Y., Kimura, K. & Matsubara, K. (1983). Chromophoric labeling of

411

cannabinoids with 4-dimethylaminoazobenzene-4'-sulfonyl chloride. Journal of

412

Forensic Science, 28, 911-921.

413

Miao, H., Fan, S., Wu, Y. N., Zhang, L., Zhou, P. P., Chen, H. J., et al. (2009). Simultaneous

414

determination of melamine, ammelide, ammeline, and cyanuric acid in milk and milk

17

415

products by gas chromatography–tandem mass spectrometry. Biomedical and

416

Environmental Science, 22, 87–94.

417

Muñiz-Valencia, R., Ceballos-Magana, S.G., Rosales-Martinez, D., Gonzalo-Lumbreras, R.,

418

Santos-Montes, A., Cubedo-Fernandez Trapiella, A., et al. (2008). Method

419

development and validation for melamine and its derivatives in rice concentrates by

420

liquid chromatography. Application to animal feed samples. Analytical and

421

Bioanalytical Chemistry, 392, 523–531.

422

Pan, X., Wu, P., Yang, D., Wang, L., Shen, X., & Zhu, C. (2013). Simultaneous

423

determination of melamine and cyanuric acid in dairy products by mixed-mode solid

424

phase extraction and GC-MS. Food Control, 30(2), 545-548.

425 426

Parris N. & Gallelli D. (1984) Dansylation of amino acids and byproduct formation. Journal of liquid chromatography, 7, 917-924.

427

Razmara, R. S., Daneshfar, A. & Sahrai, R. (2011). Determination of methylene blue and

428

sunset yellow in wastewater and food samples using salting-out assisted liquid–liquid

429

extraction. Journal of Industrial and Engineering Chemistry, 17, 533–536.

430

Rezaee, M., Assadi, Y., Milani Hosseini, M. R., Aghaee, E., Ahmadi F, Berijani S. (2006).

431

Determination of organic compounds in water using dispersive liquid-liquid

432

microextraction. Journal of Chromatography A, 1116, 1-9.

433 434

Rezaee, M., Yamini, Y. & Faraji, M. (2010). Evolution of dispersive liquid–liquid microextraction method. Journal of Chromatography A, 1217, 2342–2357.

435

Romero, R., Gazquez, D., Bagure, M. G. & Sanchez-Vinas, M. (2000). Optimization of

436

chromatographic parameters for determination of biogenic amines in wines by

18

437

reversed-phase high-performance liquid chromatography. Journal of chromatography

438

A, 871, 75-83.

439 440 441 442

Rovina K. & Siddiquee S. (2015) A review of recent advances in melamine detection techniques. Journal of Food Composition and Analysis, 43, 25-38. Skinner, C. G., Thomas, J. D. & Osterloh, J.D. (2010). Melamine Toxicity. Journal of Medical Toxicology, 6:50–55

443

Sormiachi, K., Ikeda, M., Akimoto, K. & Niwa, A. (1995). Rapid determination of dabsylated

444

hydroxyproline from cultured cells by reversed-phase high-performance liquid

445

chromatography. Journal of chromatography B, 664, 435-439.

446

Sun, F., Ma, W., Xu, L., Zhu, Y., Liu, L., Peng, C., et al. (2010). Analytical methods and

447

recent developments in the detection of melamine. Trends in Analytical Chemistry, 11,

448

1239–1249.

449

Sun, H., Wang, L., Ai, L., Liang, S., & Wu, H. (2010). A sensitive and validated method for

450

determination of melamine residue in liquid milk by reversed phase high-performance

451

liquid chromatography with solid-phase extraction. Food Control, 21(5), 686-691.

452

Tzing, S. H. & Ding, W. H. (2010). Determination of melamine and cyanuric acid in

453

powdered milk using injection-port derivatization and gas chromatography–tandem

454

mass spectrometry with furan chemical ionization. Journal of Chromatography A, 1217,

455

6267–6273.

456

Venkatasami, G., & Sowa, J. R. (2010). A rapid, acetonitrile-free, HPLC method for

457

determination of melamine in infant formula. Analytica Chimica Acta, 665(2), 227-

458

230.

459

Viñas P., Campillo N., Férez-Melgarejo G. & Hernández-Córdoba M. (2012) Determination

460

of melamine and derivatives in foods by liquid chromatography coupled to atmospheric 19

461

pressure chemical ionization mass spectrometry and diode array detection, Analytical

462

letters, 45, 2508-2518.

463

Wu, Y. T., Huang, C. M., Lin, C. C., Ho, W. A., Lin, L. C., Chiu, T.F., et al. (2009).

464

Determination of melamine in rat plasma, liver, kidney, spleen, bladder and brain by

465

liquid chromatography–tandem mass spectrometry. Journal of Chromatography A,

466

1216, 7595–7601

467

Xia, J., Zhou, N., Liu, Y., Chen, B., Wu, Y. & Yao, S. (2010). Simultaneous determination of

468

melamine and related compounds by capillary zone electrophoresis. Food Control, 21,

469

912–918.

470

Xu, X. M., Ren, Y. P., Zhu, Y., Cai, Z. X., Han, J. L., Huang, B. F., et al. (2009). Direct

471

determination of melamine in dairy products by gas chromatography/mass spectrometry

472

with coupled column separation. Analytica Chimica Acta, 650, 39–43.

473

Yan, N., Zhou, L., Zhu, Z., & Chen, X. (2009). Determination of melamine in dairy products,

474

fish feed, and fish by capillary zone electrophoresis with diode array detection. Journal

475

of Agricultural and Food Chemistry, 3, 807–811.

476

Zeng, H., Yang, R., Wang, Q., Li, J., & Qu, L. (2011). Determination of melamine by flow

477

injection analysis based on chemiluminescence system. Food Chemistry127(2), 842-

478

846.

479

Zhang, M., Li, S., Yu, C., Liu, G., Jia, J., Lu, C., et al. (2010). Determination of melamine

480

and cyanuric acid in human urine by a liquid chromatography tandem mass

481

spectrometry. Journal of Chromatography B, 878(9-10), 758-762.

482

Zhang, S., Yu, Z., Hu, N., Sun, Y., You, J. & Suo Y. (2014) Sensitive determination of

483

melamine leached from tableware by reversed phase high-performance liquid

20

484

chromatography using 10-methyl-acridone-2-sulfonyl chloride as a pre-column

485

fluorescent labeling reagent. Food Control 39, 25-29.

486 487

Zheng, X. L., Yu, B. S., Li, K. X. & Dai Y.N. (2012). Determination of melamine in dairy products by HILIC–UV with NH2 column. Food Control, 23, 245–250.

488

Zhou, Y. Y., Yang, J., Liu, M., Wang, S. F., & Lu, Q. (2010). A novel fluorometric

489

determination of melamine using cucurbit[7]uril. Journal of Luminescence, 130(5),

490

817-820.

491

21

492

Figure legends

493

Fig. 1. Derivatization scheme of dabsyl with MEL.

494

Fig. 2. Effect of pH on derivatization efficiency (Fig. 2a). Effect of dabsyl chloride volume

495 496 497 498 499

on derivatization efficiency (Fig. 2b). Fig. 3. Effect of octanol volume on extraction efficiency of DLLME method (Fig. 3a). Effect of ACN volume on extraction efficiency of DLLME method (Fig. 3b). Fig. 4. HPLC-UV-Vis chromatogram (λ =460 nm) of the powdered infant formula 1 for (a) non-spiked and (b) spike of 50 µg L-1 of MEL.

500 501 502 503 504 505 506 507 508 509 510 511 512 513 22

514

Table 1. Figures of merit of the proposed method for extraction and determination of

515

MEL.

RSD% Intra-day (n=6)

Inter-day (n=6)

25

100

25

100

(µg L-1)

(µg L-1)

(µg L-1)

(µg L-1)

3.2

2.6

6.7

5.4

LOQ

LOD

Linear Range

(µg L-1)

(µg L-1)

(µg L-1)

0.3

0.1

1.0-500

516 517 518 519 520 521 522 523 524 525 526 527 528 529 23

R2

0.9952

Table 2. Determination of MEL in different powdered infant formula and milk samples

530

a

Sample Powdered infant formula 1

Powdered infant formula 2

Powdered infant formula 3

Powdered infant formula 4

Powdered infant formula 5

Milk 1

Milk 2

Milk 3

Milk 4

Milk 5

Cadded (mg Kg-1)

Cfound (mg Kg1 )

Recovery%

-

0.48

-

1.8

0.50

1.02

104.2

2.3

-

N.Db

-

3.8

0.05

0.046

92.0

4.5

0.50

0.47

94.0

5.1

2.50

2.44

97.6

2.3

-

N.D

-

2.5

0.50

0.45

90.0

6.4

-

N.D

-

3.8

0.50

0.52

104.0

4.3

-

0.23

-

6.6

0.50

0.70

95.9

3.8

-

N.D

-

4.7

0.05

0.046

92.0

6.4

0.50

0.47

94.4

4.8

2.50

2.42

96.9

3.2

-

N.D

-

1.9

0.50

0.48

96

3.4

-

0.11

0.50

0.58

95.1

3.2

-

N.D

-

4.5

0.50

0.49

98.0

5.3

-

N.D

-

1.5

0.50

0.47

94.0

2.6

RSD (%) (n = 3)

4.8

531

a

Concentration based on based on mg MEL per Kg sample after evaluation of the sample preparation steps.

532

b

Not detected

533 534 535 536 537

24

538 539

Table 3. Comparison of the proposed method with other developed methods to determine MEL in powdered infant formula and milk samples

540

LOD (µg L-1)

LR

Running cost

Ref.

Dairy products

50

0.05-2 mg L-1

High

Xu et al. 2009

GC-MS

Dairy product

25

50-800 µg L-1

High

Pan et al. 2013

LC-MS

Milk-based products

100

Not reported

High

Ibanez et al. 2009

39.4

0-500 µg L-1

High

Deng et al. 2010

Method type

Matrix

GC-MS

and beverage products LC-MS

Different foodstuff

LC-MS

Human urine

6

10-5000 µg L-1

High

Zhang et al. 2010

HPLC-UV

Liquid milk

18

0.1-50 mg L-1

moderate

Sun et al. 2010

HPLC-UV

Infant formula

100

1.0-80 mg L-1

moderate

Venkatasami & Sowa et al. 2010

UV

Milk

12

0.4-4 mg L-1

Low

Liu et al. 2011

FL

Tainted milk

300

0.25-7.57 mg L-

Low

Zhou et al. 2011

1

FL

milk-based products

HPLC-FL

Melamine leached from

120

0.2-80 mg L-1

Low

Zeng et al. 2011

0.005-0.4

0.5-200 µg L-1

moderate

Zhang et al. 2014

0.1

1.0-500 µg L-1

moderate

This work

tableware HPLC-UV

Liquid milk, infant formula

541 542

Abbreviations: LR, linear range; LOD, limit of detection; a

Data not reported

543 544 545 546

25

547 548 549 550 551

Fig. 1

552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588

26

+

589 590 591

Fig .2a

1400000 1200000

Peak area

1000000 800000 600000 400000 200000 0 7

8

9

10

11

pH of derivatization

592 593 594 595 596

Fig .2b

1400000 1200000

Peak area

1000000 800000 600000 400000 200000 0 50

75

100

125

Dabsyl chloride volume (µL)

597 598 599 600 601

27

150

602

Fig. 3a

4000000 3500000

Peak area

3000000 2500000 2000000 1500000 1000000 500000 0 60

80 100 Octanol volume (µL)

120

603 604 605

Fig. 3b 6000000

Peak area (mAU)

5000000

4000000

3000000

2000000

1000000

0 750

1000 Disperser solvent volume (µL)

606 607 608 609

28

1250

610

Fig. 4

611

612

29

613 614

Highlights

615

•

Dabsyl derivatization followed by DLLME is used for extraction of melamine

616

•

The method provides melamine determination at trace levels by HPLC-UV-Vis.

617

•

The method is sensitive, fast, reliable, inexpensive and environmentally friendly.

618

•

A comparison with other developed methods is made.

619

•

The applicability of the procedure is evaluated with milk samples

620 621

30