Ultra-High-Throughput Analytical Strategy Based on UHPLC-DAD in

Subscriber access provided by READING UNIV

Article

Ultra-High-Throughput Analytical Strategy Based on UHPLC-DAD in Combination with Syringe Filtration for the Quantification of 9 Synthetic Colorants in Beverages: Impacts of Syringe Membrane Types and Sample pH on Recovery Je Young Shin, and Mun Yhung Jung J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03882 • Publication Date (Web): 30 Oct 2017 Downloaded from http://pubs.acs.org on October 30, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

Journal of Agricultural and Food Chemistry 1

1 2

Ultra-High-Throughput Analytical Strategy Based on UHPLC-DAD in Combination with

3

Syringe Filtration for the Quantitation of 9 Synthetic Colorants in Beverages: Impacts of

4

Syringe Membrane Types and Sample pH on Recovery

5 6 7

Je Young Shin and Mun Yhung Jung*

8 9 10 11

Department of Food Science and Biotechnology, Graduate School, Woosuk University,

12

Samnye-ro 443, Samnye-eup, Wanju-gun, Jeonbuk Province 565-701, Republic of Korea. Tel)

13

82-63-290-1438 Fax) 82-63-291-9312, e-mail) [email protected]

14 15

*To whom correspondence should be addressed.

16 17 18 19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 29 2

20

ABSTRACT

21

An ultra-high-throughput approach based on UHPLC-DAD in combination with simple

22

syringe filtration was successfully developed and validated for the quantitation of 9 synthetic

23

colorants in beverages. The recoveries of the colorants from the beverages were found to be

24

dramatically affected by the syringe filter membrane types and pH of the sample solution.

25

The high recoveries of the 9 colorants (92.7 - 105.9%) were achieved by the syringe filtration

26

with PVDF membrane following the pH adjustment of sample solution at pH 7.0. The sample

27

treatment procedure was very simple and took only 1 min. The fast chromatographic

28

separation (1 min) of the nine synthetic colorants was achieved by UHPLC-DAD using a

29

C18-core-shell column. This analytical approach (UHPLC-DAD combined with syringe

30

filtration) took only approximately 3 min. The established method was ultrafast, sensitive,

31

precise, accurate and reliable. The method was successfully applied to rapidly determine the

32

9 colorants in 17 beverages.

33 34

Keywords: Synthetic colorant, syringe filter, beverages, UHPLC–DAD, method validation

35 36 37 38 39 40


Page 3 of 29


41

INTRODUCTION

42

Food colorants are commonly used for increasing the commercial values, acceptability and

43

appetite of various foods including beverages. Colored beverages are popular in the market

44

due to the visual preference of consumers.1 Synthetic colorants are preferred in beverages due

45

to their low price and high stability against light, temperature, and oxygen, as compared to

46

natural colorants.2 However, synthetic colorants have been reported to increase various

47

human health risks including allergy and asthmatic reaction, DNA damage, hyperactivity, and

48

carcinogenesis.3-8 The use of synthetic colorants in foods and beverages is strictly regulated

49

by legislation throughout the world.9 Thus, the contents of synthetic colorants in beverages

50

should be continuously monitored.

51 52

Many analytical techniques including thin-layer chromatography, liquid chromatography,

53

adsorptive voltammetry, spectrophotometric methods, and differential pulse polarography

54

have been developed for the determination of synthetic food colorants.10-16 Most popular

55

methods for the determination of synthetic food colorants are liquid chromatographic

56

methods in combination with diode array detectors (DADs), ultraviolet (UV), or mass

57

spectrometer (MS).12-16 However, high throughput analytical methods for the determination

58

of synthetic colorants in the food products are still required, especially in the laboratories

59

facing the challenges of saving the analytical time and treating the large amount of samples in

60

a short period. UHPLC with a core-shell column may provide the dramatic increase in

61

analytical speed.17,18 However, the UHPLC in combination with core shell column has never

62

been previously applied for the fast separation of synthetic colorants. In addition, for the fast

63

analysis of colorants, not only the fast chromatographic separation but also rapid sample



Page 4 of 29 4

64

preparation techniques are required. However, time consuming sample preparation techniques

65

(extraction and purification) have been generally practiced for the determination of the

66

synthetic colorants. Simple dilution and direct syringe filtration has been previously

67

introduced for the chromatographic analysis of synthetic colorants in beverages.14,19-22 In our

68

preliminary study, however, it was found that considerable amounts of colorants were

69

retained in the filter membranes of certain types of syringe filters, resulting in the significant

70

losses of the target colorants during syringe filtration. We also found that the retention

71

behavior of the synthetic colorants was greatly dependent on the syringe membrane types and

72

pH of the sample solution. To date, however, the impact of the types of syringe filter

73

membrane on the recovery of synthetic colorants in beverages has never been previously

74

studied. 14,19-22 Furthermore, the impact of pH of the sample solution for the recovery of the

75

colorants during syringe filtration has not been also reported.

76 77

The main objective of this research was to develop and validate an ultrafast analytical method

78

based on UHPLC-DAD combined with syringe filtration for the determination of nine

79

synthetic colorants (Indigo Carmine, Erythrosine B, Tartrazine, Sunset Yellow FCF, Fast

80

Green FCF, Brilliant Blue FCF, Allura Red AC, Amaranth, and Ponceau 4R) in beverages. To

81

achieve this, the optimum sample preparation conditions such as the impact of syringe types

82

(cellulose, nylon, PES, and PVDF) and pH of the sample solution on the recovery of the

83

colorants were studied. UHPLC-DAD using a core-shell column was also established for the

84

ultrafast separation and quantification of the synthetic colorants in beverages. The chemical

85

structures of nine synthetic colorants are shown in Figure 1.

86


Page 5 of 29


87 88

MATERIALS AND METHODS

89

Reagents and samples

90

Synthetic colorant standards such as Tartrazine (E102, 95% purity), Sunset Yellow FCF (E

91

110, 90% purity), Indigo carmine (E 132, 95% purity), Ponceau 4R (E 124, 82 % purity),

92

Amaranth (E123, 90.0% purity). Erythrozine B (E127, 95.0% purity), Fast Green FCF (E143,

93

85.0% purity), Allura red AC (E 129, 80% purity) and Brilliant Blue FCF (E 133, 95 %

94

purity) were obtained from Tokyo Chemical Industry (Tokyo, Japan). Ammonium acetate

95

and HPLC-grade methanol were purchased from Riedel-de Haen (Seelze, Germany) and JT

96

Baker Chemical (Phillipsburg, NJ), respectively. Acetonitrile and water (HPLC-grade) were

97

obtained from Fisher Scientific (Fair Lawn. NJ). The syringe filters of different membrane

98

types (pore size, 0.45 µm; PVDF, Nylon, Cellulose, and PES) were purchased from Agela

99

Technologies (Bonna-Agela Technologies Inc., Wilmington, DE, USA). Colored beverages

100

(n=17) with synthetic colorant additive were purchased from several local markets.

101 102

Preparation of standard solutions

103

Individual standard stock solutions containing each colorant (500 mg/L) were prepared with

104

distilled deionized water. Then, working standard solutions (0.05–50.0 mg/L) were prepared

105

by a serial dilution of the stock standard solutions with deionized water. The standards

106

solutions were stored in a refrigerator (4-6 ºC) until used. It was found that the stability of the

107

standard solutions were stable at least for 53 days in the refrigerator.

108 109

Sample preparation



Page 6 of 29 6

110

Carbonated beverages were transferred into 50 mL-capacity polypropylene conical tubes

111

(SPL Lifesciences Co. Ltd, Pocheonsi, Republic of Korea) and placed in a sonication bath for

112

10 min at room temperature to remove the CO2 gas. Noncarbonated beverages were not

113

treated in the sonication bath. The impact of filter types (Nylon, PVDF, Cellulose, and PES)

114

on the recovery of the colorants was studied by determining the recovered colorant contents

115

after the filtration of the standard solution (10.0 mg/L) with the syringe filters. To study the

116

impact of pH of the sample solution on the recovery of the colorants, the beverage samples

117

spiked with the colorants at three different concentrations (1.0, 4.0, and 10.0 mg/L) were

118

prepared. Then, the pH of the spiked beverage samples (20 mL) was adjusted to pH 3.5, 4.5,

119

5.0, 5.5, 6.0, 6.5, 7.0, and 7.5 with 10% sodium hydroxide solution and then brought to 25

120

mL with distilled water. Then, the sample solutions were filtered with a syringe filter (PVDF).

121

UHPLC-DAD was conducted for the calculation of the recovery rates.

122 123

UHPLC-DAD analysis

124

UHPLC-DAD method was conducted with a Nexera UHPLC system (Shimadzu, Tokyo,

125

Japan) consisting of dual-pump (LC-30 AD), auto-sampler (SIL-30AC), semi-micro

126

photodiode array detector (SPD-M20A), and controller (CTO-20AC). The column used was a

127

2.1 x 100 mm x 2.1 mm i.d., 2.6 µm, core-shell Kinetex C18 (Phenomenex, Torrance, CA,

128

USA). The mobile phase system was composed of 0.02M ammonium acetate in water (A)

129

and methanol/ acetonitrile (80:20, v/v) (B). The mobile phase gradient program for the

130

UHPLC was as following: gradient program of 0-0.10 min, 10% B; 0.10-0.20 min 45% B;

131

0.20-0.45 min 50% B; 0.45-0.55 min 70% B; 0.55-0.75 min 90% B; 0.75-1.20 min, 100% B;

132

1.20-1.60 min, 100% B; 1.60-1.70 min, 10% B; 1.80-2.00 min. 10% B. The flow rate of


Page 7 of 29


133

mobile phase was 1.0 mL/min. The injection volume was 5 µL and the column temperature

134

was maintained at 40 o C. Quantification of the colorants was performed by the DAD detector

135

at the appropriate absorbance wavelengths (Table 1). Identifications of the chromatographic

136

peaks were performed by matching with the retention times of the authentic colorants. The

137

identities of colorants were further confirmed by comparing their scanning absorption spectra

138

with those of the standard colorants.

139 140

Method validations

141

The working standard solutions (0.05, 0.10, 0.25, 0.50, 1.0, 5.0, 10.0, and 50.0 mg/L) were

142

analyzed by the UHPLC-DAD and UHPLC-MS/MS methods to draw the standard calibration

143

curves for the colorants. Then, the linearities of the curves, limit of detection (LOD), and

144

limit of quantification (LOQ) were obtained. The LOD and LOQ were calculated by the

145

formulas: LOD = 3.0 x standard error of y-intercept/slope, and LOQ = 10 x standard error of

146

y-intercept/slope. Intra-day precision was studied by the 10 times repeated analysis of the

147

standard solutions at the concentrations of 0.25, 2.0, and 5.0 mg/L. Inter-day precision of the

148

method was also studied by the repeated analysis of the nine common synthetic colorants at

149

the concentrations of 0.25, 2.0, and 5.0 mg/L for 6 different dates in triplicate analysis per

150

sample for each day. Accuracy of the method was tested by analyzing the sample beverage

151

spiked at the concentrations of 1.0, 4.0, and 10.0 mg/L.

152 153

Statistical analysis

154

A SPSS statistical analysis program (SPSS 14.0 K, SPSS) was used for conducting the

155

Tukey’s multiple-range test to ascertain the statistical differences in the obtained quantitative



Page 8 of 29 8

156

data at α = 0.05 by using.

157 158

RESULTS AND DISCUSSION

159

UHPLC-DAD method optimization

160

The UHPL-DAD method was performed with a sub-3 µm core-shell C18 column, which has

161

been known to provide high separation efficiency similar to a sub-2 µm fully porous columns,

162

while maintaining lower back pressure. A series of experiments was conducted to obtain the

163

optimal separation condition. Due to the lower column back pressure with the core-shell

164

column, high flow rate (1 mL/min) of mobile phases could be applied. The mobile phase

165

system was composed of 0.02M ammonium acetate in water and methanol: acetonitrile

166

(80:20 v/v). The mobile phase gradient program was optimized to obtain the efficient

167

separation of the nine colorants. In this study, a satisfactory ultrafast chromatographic

168

separation (less than 1 min) was achieved for all the 9 synthetic colorants including the

169

critical pair of Fast Green FCF and Brilliant Blue FCF (Figure 2). The resolution (R) of the

170

critical pair of Fast Green FCF and Brilliant Blue FCF was calculated to be 0.93, which was

171

acceptable for the quantification purpose (Figure 2). A fast HPLC-DAD method, which took

172

13 min chromatographic running time for the analysis of 3 synthetic colorants (Amaranth,

173

Sunset Yellow, and Tartrazine), has been previously reported.14 Chemometrics-assisted

174

HPLC-DAD has been reported to take 6 min for the separation of six synthetic colorants

175

(Allura Red, Amaranth, Brilliant Blue, Carmine, Sunset Yellow, and Tartrazine).19 A

176

previously reported UHPLC-DAD-MS/MS method took 3 min for the separation of 10

177

synthetic colorants (Allura Red AC, Amaranth, Brilliant Blue FCF, Erythrosine B,

178

Carmoisine, Indigo Carmine, Paten Blue V, Ponceau 4R, Sunset Yellow FCF, and


Page 9 of 29


179

Tartrazine).23 However, these previous chromatographic methods could not be directly

180

compared with the present technique due to the different target colorants for the analysis. A

181

previous HPLC-DAD method,24 took 18 min for the separation of the same type of nine

182

synthetic colorants. Korea Food and Drug Administration official method (KFDA official

183

method 2.4.1) includes the 20 min of chromatographic running time for the same nine

184

synthetic colorants. Our present UHPLC-DAD method took only 1 min for the

185

chromatographic separation of the same nine synthetic colorants.

186 187

Standard curves, linearity, LOD and LOQ, and repeatability

188

The established UHPLC-DAD was conducted to obtain the calibration curves, linearity, LOD

189

and LOQ of authentic standard solutions. Table 1 shows the linearity and equations of the

190

standard curves, LOD and LOQ for the nine synthetic colorant as obtained by the UHPLC-

191

DAD. The UHPLC-DAD provided excellent linearity of the standard calibration curves in the

192

tested concentration range. The coefficients of the determination (r2) of the curves were

193

higher than 0.999. UHPLC-DAD method provided low LOD and LOQ. The LODs of the

194

UHPLC-DAD for the synthetic colorants were in the ranges of 0.04 – 0.15 mg/L. The intra-

195

day repeatability of the UHPLC-DAD method was obtained by 10 repeated injection of the

196

standard solutions at the concentrations of 0.25, 2.0, and 5.0 mg/L. The relative standard

197

deviation (% RSD) was in the range of 0.01 – 1.07% for the intra-day repeated analysis,

198

showing excellent intra-day precision. The inter-day repeatability with standard solutions at

199

the three different concentrations was also conducted by the repeated analysis for six

200

different dates. The relative standard deviation (% RSD) was in the range of 0.04 – 0.67% for

201

the inter-day analysis of the colorants in standard solutions. The results clearly indicated that



Page 10 of 29 10

202

the UHPLC-DAD method had excellent intra- and inter day-precisions for the colorants.

203 204

Sample preparation

205

Fast analysis of colorants requires not only the fast chromatographic separation but also rapid

206

sample preparation technique. Table 2 shows the effects of syringe filter membrane type on

207

the recovery rates of the colorants in the standard solution (standards dissolved in distilled

208

water, 10.0 mg/L). The result clearly showed that the recovery rate was greatly different with

209

the types of filter membrane and colorant. PVDF showed the highest recovery rates of

210

colorants, ranging from 98.5 to 102.1 %. Cellulose type syringe filter showed also acceptable

211

recovery rates for all the tested colorants. Nylon type syringe filter provide the poor recovery

212

rates, ranging from 0 to 61.7%. The high retention of synthetic food colorants in nylon

213

membrane syringe filter may be due to the amide groups in its chemical structure. The amide

214

groups in nylon membrane may exist in a hydride of two resonance structures (ne as a dipole),

215

which could form ion-dipole interaction and hydrogen bonding with polar colorants. The

216

strong binding capacity of nylon membrane with certain analytes including acetaminophen,

217

ibuprofen, and loratadine has been previously reported.25,26 However, this is the first report on

218

the high retention of the synthetic colorants in nylon syringe filter. PES syringe filter showed

219

selectively low recovery rate for Erythrosine B. The recovery rates of Erythrosine B was

220

24.3%. Direct syringe filtration for the HPLC analysis of synthetic colorants in beverages has

221

been previously reported.13,14,20-22 However, our result was not consistent with the those of the

222

previous reports. The recovery rates of Tartrazine, Amaranth, Ponceau 4R and Sunset Yellow

223

FCF using nylon syringe filter for the sample treatment from soft drink have been reported to

224

be in the range of 95.5 – 102.1%.22 However, our data showed that the nylon syringe filter


Page 11 of 29


225

showed the least recovery rates among the tested syringe filters, resulting in the recovery of

226

61.7, 46.7, 42.5 and 49.8% for Tartrazine, Amaranth, Ponceau 4R and Sunset Yellow FCF,

227

respectively. It has been previously reported that the recovery of three synthetic colorants

228

(Tartrazine, Amaranth, and Sunset Yellow FCF) by direct syringe filter of beverages were in

229

the range of 97 - 105%.14 Unfortunately, the authors did not specify the syringe filter type

230

used for the sample preparation. Our results showed that these colorants (Tartrazine,

231

Amaranth, and Sunset Yellow FCF) were fully recovered by the PVDF, cellulose and PES

232

type syringe filters, but nylon syringe filter provided the low recovery of the colorants (Table

233

2). In a previous report, the recovery of Carmoisine from fruit flavored drink by direct

234

syringe filter has been reported to be 97.9%.21 However, the authors did not specify the type

235

of syringe they used. Furthermore, the study on the recovery of other colorants from the

236

beverages has not been conducted. Our data clearly showed the recovery of colorants was

237

greatly dependent on the types of syringe filter. We conducted experiments to check the

238

recovery of the colorants from the beverage sample with PVDF syringe filter, which showed

239

excellent recovery of all the tested colorants in the standard solutions. However, the recovery

240

rate of the colorants in the real beverage sample was unexpectedly greatly lower than those in

241

pure standard solutions. We assumed that these low recovery rates was due to the pH

242

difference between the real sample and standard solution. The pH values of standard solution

243

and the sport drink were pH 7.0 and pH 3.5, respectively. Thus we studied the effects of pH

244

of the beverage on the recovery rates of colorants with PVDF syringe filter (Table 3). The

245

results showed that the pH of the sample solution greatly affected the recovery of the

246

colorants from the beverage. For example, Erythrosine B was not recovered at all at pH 3.5 of

247

the sample solution with PVDF syringe filter. However, the recovery rate of Erythrosine B



Page 12 of 29 12

248

increased dramatically as pH increased, reaching full recovery rate (approximately 100 %) at

249

pH 7.0. There was no significant difference in recovery rates of colorants between pH 7 and

250

7.5, so we selected pH 7.0 as an extraction condition for the extraction of the colorants from

251

beverages. This pretreatment process is very simple and efficient for the analysis of synthetic

252

colorants from the beverages. The results suggested that the sample preparation technique

253

with the PVDF syringe at pH 7.0 of the sample solution gives high accuracy of the method as

254

assessed by studying the recovery of spiked beverage with different concentrations of

255

synthetic colorant. The simple syringe filtration technique provided high accuracy and

256

rapidity for the sample preparation. To our knowledge, the pH dependent recovery of

257

colorants by syringe filter has never been previously reported.

258 259

Application of the method

260

The established analytical approach was applied to determine the synthetic colorant in 17

261

beverages available in Korean market. Figure 3 showed exemplary chromatograms of the

262

synthetic colorant in commercial beverages. No interference was found in the UHPLC-DAD

263

chromatograms of all the tested beverage samples. The contents of synthetic colorants in the

264

beverages are shown in Table 4. There was a great variation on the type and contents of

265

colorants in the samples depending on the products. Single colorant was found in 9 beverage

266

products. Mixtures of two different colorants were found in nine out of 17 beverages tested.

267

Mixtures of three different colorants were found in one beverage product. Only four colorants

268

(Tartrazine, 0.73 - 10.81 mg/L; Sunset Yellow FCF, 1.17 - 57.98 mg/L; Allura Red AC, 1.00

269

- 56.23 mg/L; and Brilliant Blue FCF, 1.14 - 6.92 mg/L) were found in the beverages. Allura

270

Red AC and Sunset Yellow FCF were the most widely employed colorants in the beverages


Page 13 of 29


271

tested, followed by Tartrazine, Brilliant Blue FCF, and Ponceau 4R in a decreasing order.

272

The concentrations of synthetic colorants in the beverages were in the range of 0.73 – 57.98

273

mg/L. The contents of synthetic colorant in all the tested beverages were lower than the legal

274

maximum limits set by Korean Food & Drug Administration. Our present data were within

275

the range of the previously reported values of the synthetic colorants in beverages. The

276

contents of synthetic colorants in 28 soft drinks in Korean market has been previously

277

reported.24 The authors reported that the mean concentration of colorants in the soft drinks

278

was 16.68 mg kg-1 with the ranges of 27.77 - 69.45 mg/kg of Sunset Yellow FCF, 0.89 -

279

16.50 mg/kg of Tartrazine, 1.61 - 33.60 mg/kg of Allura Red AC, 2.79 - 56.19 mg/kg of

280

Amaranth, and 0.21 - 7.20 mg/kg of Brilliant Blue FCF.24 The contents of synthetic colorants

281

in four commercial beverages in Korean market has been also previously reported.27 The

282

results showed that the mean contents of total synthetic colorants in the 4 beverages was 0.22

283

mg kg-1 with the ranges of 0.00 - 2.83 mg/kg of Tartrazine, 0.00 - 0.40 mg/kg of Sunset

284

Yellow FCF, 0.00 - 0.58 mg/kg of Brilliant Blue FCF, and 0.00 - 02.13 mg/kg of Allura Red

285

AC.27 In a previous report,15 the contents of synthetic colorants in 13 soft drinks in Italian

286

market were reported to be in the range of 0.23 - 473.21 mg/L. The contents of synthetic

287

colorants in 4 fruit juices in China were reported to be in the range of 0.99 - 11.55 mg/L.28

288

The contents of synthetic colorants in a carbonated drink and a fruit-flavored drink in China

289

has been reported to be 9.07 and 1.58 mg/kg, respectively.29

290 291

In summary, an analytical approach based on a syringe filtration combined with UHPLC-

292

DAD was successfully developed and validated for the ultrafast quantification of nine

293

synthetic colorants in beverages. It was found, for the first time, that syringe types and pH of



Page 14 of 29 14

294

the sample solution dramatically affected the recovery of the colorants from the samples. The

295

filtration with PVDF membrane syringe filter at pH 7.0 of the sample solution provided the

296

highest recovery (92.7-105.9%) of all the 9 synthetic colorants tested. The sample preparation

297

procedure was very simple and took only approximately 1 min. The UHPLC-DAD using core

298

shell C18 column was developed for the ultrafast satisfactory separation of the nine colorants.

299

The established method provided high linearity of standard calibration curves, low LOD and

300

LOQ, and high precision and accuracy for the determination of nine synthetic colorants. The

301

method was successfully applied to analyze the synthetic colorants in 17 beverages in Korea

302

Market. The total analytical time including the sample preparation and chromatographic

303

operation for the quantification of nine synthetic colorants in beverages was approximately 3

304

min. We believe that this analytical method would be an efficient tool for the rapid analysis of

305

synthetic colorants in beverages.

306 307

ASSOCIATED CONTENT

308

The supporting information:

309

This material is available free of charge via the Internet at http://pubs.acs.org.

310

Table S1. Effect of pH of the Sample Solution on the Recovery Rates of Authentic Colorant

311

from the Spiked Non-Colored Sport Drink (1, 4, and 10 mg/L Spiked) with PVDF Syringe

312

Filter.

313

Table S2. Intra-day Repeatability of Contents of Authentic Colorants in Standard Solutions

314

Obtained from 10 Repeated Analysis by the UHPLC-DAD Method

315

Table S3. Inter-day Repeatability of Contents of Authentic Colorants Obtained from Six


Page 15 of 29


316

Different Day Analysis

317

Figure S1. UV/Vis scanning spectra of 9 synthetic tar colorant standards obtained by

318

UHPLC-DAD

319 320

AUTHOR INFORMATION

321

Corresponding Author

322

*Phone: +82-63-290-1438. Fax: +82-63-290-1435. E-mail: [email protected]

323 324

Notes

325

The authors declare no competing financial interest.

326 327

REFRENCES

328

(1) Shen, Y.; Zhang, X.; Prinyawiwatkul, W.; Xu, Z. Simultaneous determination of red and

329

yellow artificial food colourants and carotenoid pigments in food products. Food Chem. 2014.

330

157, 553-558.

331

.

332

(2) Olgun, F.A.O.; Ozturk, B.D.; Apak, R. Determination of synthetic food colorants in

333

water-soluble beverages individually by HPLC and totally by Ce (IV)-oxidative

334

spectrophotometry. Food Anal. Methods. 2012, 5, 1335-1341.

335



Page 16 of 29 16

336

(3) Amchova, P.; Kotolova H.; Ruda-Kucerova J. Health safety issues of synthetic food

337

colorants. Regul. Toxicol. Pharm. 2015, 914-922.

338 339

(4) Mpountoukas, P.; Pantazaki, A.; Kostareli, E.; Christodoulou, P.; Kareli, D.; Poliliou, S.;

340

Mourelatos, C.; Lambropoulou, V.; Lialiaris, T. Cytogenetic evaluation and DNA interaction

341

studies of the food colorants amaranth, erythrosine and tartrazine. Food Chem. Toxicol. 2010,

342

48, 2934-2944.

343 344

(5) Sasaki, Y. F.; Kawaguchi, S.; Kamaya, A.; Ohshita, M.; Kabasawa, K.; Iwama, K.; K

345

Taniguchi, K.; Tsuda, S. The comet assay with 8 mouse organs: results with 39 currently used

346

food additives. Mutat. Res. 2002, 519, 103–119.

347 348

(6) McCann, D.; Barrett, A.; Cooper, A.; Crumpler, D.; Dalen, L.; Grimshaw, K.; Kitchin, E.;

349

Lok, K.; Porteous, L.; Prince, E. Food additives and hyperactive behaviour in 3-year-old and

350

8/9-year-old children in the community: A randomised, doubleblinded, placebocontrolled

351

trial. Lancet 2007, 370, 1560–1567.

352 353

(7) Rowe, K. S.; Rowe, K. J. Synthetic food coloring and behavior: A dose response effect in

354

a double-blind, placebo controlled, repeated-measures study. J. Pediatr. 1994, 125, 691–698.

355 356

(8) JECFA. Amaranth. In WHO food additives series (vol. 8). 1975, Geneva: World Health


Page 17 of 29


357

Organization.

358 359

(9) European Parliament and Council Directive 94/36/EC of 30 June 1994 on colours for use

360

in foodstuffs. Off. J. Eur. Commun. 1994, L237, 13-29.

361 362

(10) de Andrade, F.I.; Guedes, M.I.F.; Vieira, Í. G. P.; Mendes, F.N.P.; Rodrigues P.A.S;

363

Maia, C.S.C.; Ávila, M.M.M.; Ribeiro, L.M. Determination of synthetic food dyes in

364

commercial soft drinks by TLC and ion-pair HPLC. Food Chem. 2014, 157, 193-198.

365 366

(11) Combeau, S.; Chatelut, M.; Vittori, O. Identification and simultaneous determination of

367

azorubin, allura red AC and ponceau 4R by differential pulse polarography: application to

368

soft drinks. Talanta 2002, 56, 115–122.

369 370

(12) Chai, W.; HuijuanWang, H.; Zhang, Y.; Ding, G. Preparation of polydopamine-coated

371

magnetic nanoparticles for dispersive solid-phase extraction of water-soluble synthetic

372

colorants in beverage samples with HPLC analysis. Talanta 2016, 149, 13-20

373

(13) Serdar, M.; Knezvic, z. Simultaneous LC analysis of food dyes in soft drinks.

374

Chromatographia 2009, 70, 1519-1521.

375 376

(14) Culzoni, M.J.; Schenone, A.V.; Llamas, N. E.; Garrido, M.; Di Nezio, M.S.; Band, B.S.F.;

377

Goicoechea, H.C. Fast chromatographic method for the determination of dyes in beverages



Page 18 of 29 18

378

by using high performance liquid chromatography-diode array detection data and second

379

order algorithms. J. Chromatogra. A. 2009, 1216, 7063-7070.

380 381

(15) Gianotti, V.; Angioi, S.; Gosetti, F.; Marengo, E.; Gennaro, M. C. Chemometrically

382

asssisted development of IP‐RP‐HPLC and spectrophotometric methods for the identification

383

and determination of synthetic dyes in commercial soft drinks. J. Liq. Chromatogra. R. T.

384

2005, 28, 923-937

385 386

(16) Tsai, C.F.; Kuo, C.H.; Shih, D.Y.C. Determination of 20 synthetic dyes in chili powders

387

and syrup-preserved fruits by liquid chromatography/tandem mass spectrometry. J. Food

388

Drug Anal. 2015, 23,453-462.

389 390

(17) Park, S.Y.; Jung, M.Y. UHPLC- ESI- MS/MS for the quantification of eight major

391

gingerols and shogaols in ginger products: effects of ionization polarity and mobile phase

392

modifier on the sensitivity. J. Food Sci. 2016, 81, C2457-C2465.

393 394

(18) Park, H.J.; Jung, M.Y. One step salting-out assisted liquid-liquid extraction followed by

395

UHPLC-ESI-MS/MS for the analysis of isoflavones in soy milk. Food Chem. 2017, 229,

396

797-804.

397 398

(19) Yin, X.L.; Wu,, H.L.; Gu, H.W.; Hu,Y.; Wang, L.; Xia, H.; Xiang, S.X.; Yu, R.Q.


Page 19 of 29


399

Chemometrics-assisted high performance liquid chromatography-diode array detection

400

strategy to solve varying interfering patterns from different chromatographic columns and

401

sample matrices for beverage analysis. J. Chromatogra. A. 2016, 1435, 75-84

402 403

(20) Gosetti, F.; Chiuminatto, U.; Mazzucco, E.; Calabrese,G.; MC Gennaro. M.C.; Marengo.

404

E. Non-target screening of allura red AC photodegradation products in a beverage through

405

ultrahigh performance liquid chromatography coupled with hybrid triple quadrupole/linear

406

ion trap mass spectrometry. Food Chem. 2013, 136, 617-623.

407 408

(21) Minioti, K.S.; Sakellariou, C.F.; Thomaidis, N.S. Determination of 13 synthetic food

409

colorants in water-soluble foods by reversed-phase high-performance liquid chromatography

410

coupled with diode-array detector. Anal. Chim. Acta. 2007, 583,103–110

411 412

(22) Ma, M.; Luo, X.; Chen, B.; Su, S.; Yao, S. Simultaneous determination of water-soluble

413

and fat-soluble synthetic colorants in foodstuff by high-performance liquid chromatography–

414

diode array detection–electrospray mass spectrometry. J. Chromatogra. A. 2006, 1103, 170–

415

176

416 417

(23) Ji, C.; Feng. F.; Chen. Z.; Chu. X. Highly sensitive determination of 10 dyes in food with

418

complex matrices using SPE followed by UPLC-DAD-tandem mass spectrometry. J. Liq.

419

Chromatogra. R. T. 2001, 34, 93-105.

420



Page 20 of 29 20

421

(24) Kim, H.Y.; Nam, H.S.; Jung, Y.H.; Lee, J.H.; Ha, S.C. Tar colors in foods distributed

422

throughout the Gyeong-In region; Monitoring favorite food items of children near elementary

423

schools. Korean J. Food Sci. Technol. 2008, 40, 243-250.

424 425

(25) Lindenberg, M.; Wiegand, C.; Dressman, J. B. Comparison of the adsorption of several

426

drugs to typical filter materials. Dissolut. Technol. 2005, 12, 22–25.

427 428

(26) Joshi, V.: Blodgett, J.: George, J.: Brinker, J. Impact of sample preparation on dissolution

429

testing: drug binding and extractable impurities and their effect on dissolution data. Dissolut.

430

Technol. 2008, 15, 20–27.

431 432

(27) Lee, Y.M.; Na, B.J.; Lee, Y.S.; Kim, S.C.; Lee, D.H.; Lee, D.H.; Seo, I.I.W.; Choi, S.H.;

433

Ha, S.D. Monitoring of tar color content in children's snack and its exposure assessment. J.

434

Food Hyg. Saf.2011, 26, 57-63.

435 436

(28) Liu, F.J.; Liu, C.T.; Li, W.; Tang, A, N. Dispersive solid-phase microextraction and

437

capillary electrophoresis separation of food colorants in beverages using diamino moiety

438

functionalized silica nanoparticles as both extractant and pseudostationary phase. Talanta.

439

2015, 132, 166-372.

440 441

(29) Wu, H.; Guo, J.; Du, L.; Tian, H.; Hao, C.; Wang, Z.; Wang , J. A rapid shaking-based

442

ionic liquid dispersive liquid phase microextraction for the simultaneous determination of six


Page 21 of 29


443

synthetic food colourants in soft drinks, sugar- and gelatin-based confectionery by high-

444

performance liquid chromatography. Food Chem. 2013, 141, 182-186.

445 446

447

Figure 1. Chemical structures of nine synthetic colorant standards.

448 449

Figure 2. UHPLC-DAD chromatograms of mixed nine colorant standards.

450 451

Figure 3. UHPLC-DAD chromatograms of the synthetic colorants in selected beverages

452



Page 22 of 29 22

453 454 455

Table 1. Linear Equations of Standard Curves, Retention Time (tR), Detection Wavelength (nm), LOD, and LOQ of Nine Authentic Colorants by the UHPLC-DAD Method

456 Colorant

tR

Detection Wavelength (nm)

Standard curve

LOD (mg/L)

LOQ (mg/L)

Tartrazine

0.345

420

y = 19857x-1212.1

0.14

0.46

Amaranth

0.423

520

y =11168x -1930.7

0.05

0.17

Indigo Carmine

0.486

600

y = 15313x -2.74

0.06

0.19

Ponceau 4R

0.514

520

y =10662x -369.75

0.04

0.13

Sunset Yellow FCF

0.564

480

y = 11085x -318.48

0.06

0.21

Allura Red AC

0.599

520

y = 13551x - 418.13

0.08

0.25

Fast Green FCF

0.701

600

y =43092x+ 917.70

0.15

0.50

Brilliant Blue FCF

0.734

600

y = 22422x - 997.35

0.04

0.14

Erythrosine B

0.905

520

y = 19174x +543.96

0.12

0.40

457 458 459 460 461 462 463 464 465 466 467 468 469


Page 23 of 29


470 471

Table 2. Effects of Syringe Filter Membrane Type on the Recovery Rates of Colorants in the Standard Solutions. Recovery rate (%)1) Compound

472

Nylon

PVDF

Cellulose

PES

Tartrazine

61.7±0.3c

99.2±0.5ab

98.7±0.4b

100.1±0.6a

Amaranth

46.7±0.9b

101.9±0.2a

101.2±0.3a

100.5±0.7a

Indigo Carmine

55.8±1.0d

102.1±0.7a

95.3±0.5c

99.7±1.0b

Ponceau 4R

42.5±0.6b

99.2±0.3a

99.8±0.4a

100.2±0.2a

Sunset Yellow FCF

49.8±0.5c

99.3±0.9a

96.9±0.9b

98.4±0.9ab

Allura Red AC

32.1±0.4d

100.9±0.7a

98.7±0.4b

94.6±0.8c

Fast Green FCF

57.5±0.9c

97.9±0.1a

90.0±1.0b

95.0±0.6a

Brilliant Blue FCF

61.2±0.4d

98.5±0.9b

90.2±0.8c

104.3±0.7a

Erythrosine B

0

99.6±0.6a

98.6±0.5a

24.3±0.7b

1)

The data with same superscript within the same row are not statistically different at α=0.05.

473

474

475

476

477



Page 24 of 29 24

478 479

Table 3. Contents of Synthetic Colorants in Beverages Containing Synthetic Colorants as Determined by the Established UHPLC Following Simple Syringe Filtration Mean content Sample

Colorants

RSD(%) (mg/L)

B-1

Tartrazine

10.81

0.13

Sunset Yellow FCF

15.18

0.30

Allura Red AC

1.00

4.68

Tartrazine

3.36

0.23

Sunset Yellow FCF

1.17

0.41

Tartrazine

5.02

0.50

Brilliant Bule FCF

1.43

2.57

B-4

Tartrazine

1.58

0.09

B-5

Tartrazine

1.56

0.48

Allura Red AC

3.36

1.30

Tartrazine

1.63

1.29

Allura Red AC

10.56

0.82

Tartrazine

0.73

5.05

Brilliant Bule FCF

2.73

0.28

B-8

Sunset Yellow FCF

13.72

0.46

B-9

Sunset Yellow FCF

57.98

0.20

B-10

Allura Red AC

29.27

0.54

Brilliant Bule FCF

6.92

2.90

Allura Red AC

56.23

0.79

B-2

B-3

B-6

B-7

B-11


Page 25 of 29


B-12

Brilliant Bule FCF

3.51

0.97

B-13

Brilliant Bule FCF

3.74

0.65

B-14

Allura Red AC

17.11

1.16

Brilliant Bule FCF

1.14

0.16

Brilliant Bule FCF

6.07

0.40

Allura Red AC

31.97

0.36

B-16

Brilliant Bule FCF

3.38

0.51

B-17

Allura Red AC

0.93

5.01

B-15

480



Page 26 of 29

Fig 1

Allura Red AC

Amaranth

Erythrosine B

Fast Green FCF

Ponceau

Sunset Yellow FCF


Brilliant Blue FCF

Indigo Carmine

Tartazine

Page 27 of 29


Fig 2

Dataf ile Name:151023)9mix_10ppm.lcd mAU 300 Ch1-520nm,4nm Ch2-420nm,4nm Ch3-480nm,4nm Ch4-600nm,4nm 200

Sunset Yellow FCF

Tartrazine

Ponceau 4R Indigo Carmine

Fast Green FCF

Allura Red AC

Erythrosine B

Brilliant Blue FCF

Amaranth

100

0 0.0

0.1

0.2

0.3

0.4

0.5

0.6


0.7

0.8

0.9

min


Fig 3

Sunset yellow FCF Tartrazine B-1 Allura Red FCF

Allura Red AC

Peak intensity

B-10

Brilliant Blue FCF

Allura Red AC B-11

ACS Paragontime Plus(min) Environment Retention

Page 28 of 29

Page 29 of 29


Ultrafast Analysis of Synthetic Colorants in Beverages Simple Treatment for High Recovery

DatafUltrafast ile Name:151023)9mix_10ppm.lcd Separation mAU 300 Ch1-520nm,4nm Ch2-420nm,4nm Ch3-480nm,4nm Ch4-600nm,4nm 200

100

1 min pH 7.0 adjustment

0

PVDF Syringe filter

0.0

0.1

0.2

0.3

0.4

0.5

0.6

UHPLC-DAD

0.7

0.8

0.9

min

0.94 min


Ultra-High-Throughput Analytical Strategy Based on UHPLC-DAD in

Recommend Documents