Determination of Four Flavorings in Infant Formula by Solid-Phase

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Determination of Four Flavorings in Infant Formula by Solid-Phase Extraction and Gas Chromatography-Tandem Mass Spectrometry Yan Shen, Beizhen Hu, Xiangzhun Chen, Qian Miao, Chengjun Wang, Zhenou Zhu, and Chao Han J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5013083 • Publication Date (Web): 22 Oct 2014 Downloaded from http://pubs.acs.org on October 29, 2014

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Journal of Agricultural and Food Chemistry

1

Determination of Four Flavorings in Infant Formula by Solid-Phase

2

Extraction and Gas Chromatography-Tandem Mass Spectrometry

3

Yan Shen,*,† Beizhen Hu,§ Xiangzhun Chen,‡ Qian Miao,† Chengjun Wang,† Zhenou Zhu,‡ and

4

Chao Han*, ‡

5

†

College of Chemistry and Materials Engineering, Wenzhou University, Wenzhou 325035, China

6

§

Shaoxing Entry-Exit Inspection and Quarantine Bureau of P.R.C, Shaoxing 312000, China

7

‡

Wenzhou Entry-Exit Inspection and Quarantine Bureau of P.R.C, Wenzhou 325027, China

8

Title running header: Shen et al. Determination of Flavorings in infant formula by GC-MS/MS

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1

ACS Paragon Plus Environment


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ABSTRACT: Increasing attentions have been attracted to the artificial flavorings added in the

25

foods, especially those for infants and children. For the first time, a sensitive and efficient analytical

26

method based on gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed for

27

simultaneous identification and quantification of four flavoring agents (vanillin, methyl vanillin,

28

ethyl vanillin and coumarin) in infant formula samples. The flavorings in samples were extracted

29

with methanol/water (v/v, 1:1), cleaned up by solid-phase extraction, and determined by

30

GC-MS/MS in selected reaction monitoring (SRM) mode. Both isotope-labeled internal standards

31

and matrix-matched calibration solutions were used to correct the matrix effects. The limit of

32

quantification (LOQ) was calculated as 10 times of the standard deviation, and it was 10.0 µg kg -1

33

for vanillin, methyl vanillin, ethyl vanillin, and coumarin. The average recoveries were in the range

34

82.8-107.5% with relative standard deviations (RSDs) below 8.9% measured at three concentration

35

levels (10, 50 and 100 µg kg-1). The proposed method is suitable for sensitively and accurately

36

simultaneous determination of four flavoring agents in infant formula samples, and also provided

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potential use for reference in terms of real analysis of other foods.

38

KEYWORDS: flavorings, infant formula, SPE, GC-MS/MS

39 40 41 42 43 44 45 46 2


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■ INTRODUCTION

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Infant formula is a manufactured food which is deliberately designed for special dietary use

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solely as a food for infants under 12 months of age. As a complete or partial substitute for human

50

milk, infant formula provides infants all required nutrient, such as protein, lipids, carbohydrates,

51

vitamins, minerals, and so on, and it’s quality is directly related to the baby's health and growth.

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In recent years, increasing attentions have been attracted to the artificial flavorings added in the

53

foods, especially those for infants and children. Among flavoring agents usually used, vanillin and

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ethyl

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(4-hydroxy-3-methoxybenzaldehyde) is the major flavor constituent of natural vanilla extract

56

prepared from the bean of Vanilla planifolia, which is one of the highest valuble and most widely

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used flavoring aromas. In fact, artificial vanilla flavorings, which usually contains vanillin and/or

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ethyl vanillin and other related compounds synthetically prepared from low-cost raw materials, are

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often and widely used in food industry instead of expensive authentic vanilla extracts. Ethyl vanillin

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(3-Ethoxy-4-hydroxybenzaldehyde), a completely synthetic flavoring, has 3-4 times flavoring

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strength than vanillin. Vanillin and ethyl vanillin are the most commonly used flavoring agents in

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food industry, and have the largest production in the world. In addition, methyl vanillin (3,

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4-Dimethoxybenzaldehyde, or more frequently called as veratraldehyde) is another important

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vanilla-like flavoring agent been widely used as a flavorant and odorant. Noted that these three

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flavoring agents have all been listed in the “Generally Regarded as Safe” (GRAS) category by

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Flavor and Extract Manufacturers' Association（FEMA）of United States. Vanillin and ethyl vanillin

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are allowed to be used as food additives for flavoring purpose in almost all countries, but methyl

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vanillin has not been listed in the Designated Food Additive List of Japan,1 while allowed using in

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U.S.2 and China.3 These falvorings are relatively safe for common food exposure, but large amounts

vanillin

are

of

special

concern

for

their

3


widespread

use.

Vanillin


70

intake might cause nausea, headaches, emesis, and adverse effect on liver and kidney functions,4

71

especially to vulnerable infant. In order to protect the health and safety of the babies, legal

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directives to control flavors levels in baby’s foods through the maximum usage amount have been

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established in China.3 No flavorings are allowed to add in any infant formula for 0-6 months old

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infant. To follow-up formula and food (for more than 6 months old infant and child), only vanillin,

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ethyl vanillin and vanilla bean concrete (extract) are allowed to add, but must strictly comply with

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the maximum usage amount of vanillin and ethyl vanillin bellow 5 mg per 100 mL (volume of

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ready-to-use).

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For infant formula, coumarin is another serious concern. Coumarin (2H-1-benzopyran-2-one), a

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naturally occurring substance with a sweet herbaceous odor, is deemed to be hepatotoxic and

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banned for use as a food additive in the U.S. since 1956, 5–6 as well as in other countries. Moreover,

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a maximum limit of 2 mg kg

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introduced into European law in 1988.8 However, appreciable amounts of coumarin have been

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found in various foods, such as bakery products, breakfast cereals, confectionary, and so on.9–10

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Vanilla extracts adulterated with coumarin as a fragrance enhancer11 is the other potential path for

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baby’s coumarin exposure.

-1

for foods was set by the Codex alimentarius in 19857 and later

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Recently, China’s authorities and the media have reported the illegal use of flavorings in some

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infant formulas. Meanwhile, considering the low limits of the usage and complex matrix of formula

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samples, the reliable analytical method must be established for simultaneous identification and

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quantification of them. Several methods have been developed for analyzing the four above

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mentioned flavorings in various samples. High performance liquid chromatography (HPLC),12

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liquid chromatography-tandem mass spectrometry (LC-MS/MS)13 and gas chromatography-mass

92

spectrometry (GC-MS) 14 have been used for simultaneous analysis of vanillin, ethyl vanillin, and 4


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coumarin in vanilla extracts. Meanwhile, more attentions were focused on the simultaneous analysis

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of vanillin and ethyl vanillin both in vanilla extracts and foods, and various methods were

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investigated including gas chromatography (GC), 15 HPLC,16–17 capillary electrophoresis (CE),18–19

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chemiluminometry, 20 and atmospheric-pressure solids analysis probe,21 and so on. However，there

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have only HPLC method been reported for analysis of methyl vanillin in industrial waste waters

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and food.23 To the best of authors’ knowlege, there is no paper focused on the simultaneous

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determination of these four flavorings in any samples.

22

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Recently, we proposed a sufficient LC-MS/MS method for simultaneous determination of

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vanillin, ethyl vanillin and coumarin in infant formula samples,24 but methyl vanillin was unnoticed.

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Except for LC-MS/MS, Gas chromatography-tandem mass spectrometry (GC-MS/MS) has become

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one of the mostly used methods for the identification and quantification of residues in food because

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of its higher selectivity, sensitivity and the necessity for confirmation.25–27 Therefore, this study we

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developed a GC-MS/MS method for the simultaneous determination of vanillin, methyl vanillin,

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ethyl vanillin and coumarin in infant formula samples, and this is the first trail performed for this

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objective.

108 109

■ MATERIALS AND METHODS

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Chemicals. Certified standards of vanillin (CAS No. 121-33-5, 99.9% purity) and ethyl

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vanillin (CAS No. 121-32-4, 97.3% purity) were purchased from Chroma Dex (Irvine, California,

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USA). Coumarin (CAS No. 91-64-5, 98% purity) was purchased from Dr. Ehrenstorfer GmbH

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(Augsburg, Germany). Methyl vanillin (CAS No. 120-14-9, 98.0% purity), methyl vanillin-13C

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(CAS No. 1173022-44-0, 98.0% purity), Vanillin-13C6 (CAS No. 201595-58-6, 98%) and

115

coumarin-D4 (CAS No. 185056-83-1, 98%) were purchased from Toronto Research Chemicals Inc 5



116

(North York, Canada). The structures of these standards are shown in Figure 1. The water used was

117

purified with a Milli-Q water purification system from Millipore (Bedford, MA, USA). HPLC grade

118

methanol was obtained from Merck (Darmstadt, Germany).

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Preparation of Standard solutions. Individual standard stock solutions were prepared at a

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concentration of 500 mg L−1 in methanol and stored at 4 ºC in the dark. A multistandard mixture

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working solution, containing 5 mg L−1of each standard compound, was prepared by diluting each

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above stock solution with methanol and was then used for spiking blank infant formula samples and

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for preparation of matrix matched calibration solutions. Matrix matched calibration standards with

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six final concentration levels of 0.001, 0.002, 0.005, 0.01, 0.02 and 0.05 mg L-1 were prepared by

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adding to infant formula blank extracts appropriate volumes of the standard working solutions. The

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vanillin-13C6, methyl vanillin-13C and coumarin-D4 were used as internal standards and the final

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concentration was 0.01 mg L-1.

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Sample Preparation. Twenty infant formula samples for 0-12 months, 6-12 months and 6-18

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months old babies were randomly purchased from local supermarkets, and the brands covered both

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Chinese domestic brands and foreign brands. Sample preparation was based on a previously

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reported process developed by our group24 with few adaptations. 1.00 g infant formula sample was

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weighted into a 50-mL polypropylene centrifuge tube. Before added 25 mL of methanol/water (v/v,

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1:1) for extraction, 500 µL vanillin-13C6, methyl vanillin-13C and coumarin-D4 mixed internal

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standards solution (0.4 mg L-1) was first added. The sample was vortex for 1 min, and then

135

performed an ultrasonic extraction for 30 min at 30 ºC. After extraction, the suspension was

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centrifuged at 8875 × g for 15 min, and then the supernatant layer was transferred into a 50-mL

137

volumetric flask and diluted with water. This extract was then forwarded to the next clean-up

138

procedure. 6


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Solid-phase extraction. The clean-up procedure based on a solid-phase extraction was also

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referenced to our previous report.24 An Oasis HLB solid phase extraction (SPE) cartridge (60 mg, 3

141

mL, Waters, Milford, MA, USA) was used and conditioned with 5 mL methanol and then 5 mL

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water before use. 5 mL sample extracting solution was then introduced to the conditioned cartridge

143

and allowed to elute by gravity. The cartridge was washed with 5 mL of pure water, followed by 5

144

mL of methanol/water (v/v, 1:9) mixture, and then dried with vacuum for 1 min. Finally, eluted the

145

cartridge with 6 mL of methanol into a glass culture tube, and evaporated the eluate to dryness

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under vacuum at 45 ºC. The dried extract was reconstituted in 2.0 mL of methanol, vortex mixed for

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60 s, and was then filtered through a 0.22-µm nylonmembrane into a glass GC vial ready for

148

analysis.

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Blank infant formula samples were prepared in the same way as mentioned above without

150

adding the internal standard solution. The matrix matched standard solutions were prepared from

151

the blank extracts as mentioned earlier.

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GC-MS/MS analysis. A Thermo Fisher Trace 1300 GC coupled with a Thermo Fisher TSQ

153

8000 triple-quadrupole mass spectrometer and Triplus RSH autosampler (Thermo Fisher Scientific,

154

Waltham, MA, USA) was used in the GC-MS/MS system. The separation of analytes was

155

performed on a Thermo Scientific TG-SQC 30 m × 0.25 mm × 0.25 µm capillary column. A

156

splitless injection mode was used and the injection volume was 1.0 µL. The GC oven temperature

157

program was as follows: initial at 80 ºC held for 1 min, increased to 200 ºC at a rate of 30 ºC min-1,

158

then increased to 280 ºC at a rate of 15 ºC min-1 and held for 5 min. The injector temperature was

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set at 250 °C. Helium (99.9999 % purity) was used as carrier gas at a constant flow rate of 1.0 mL

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min -1, and also as collision gas in the ion trap chamber of MS/MS system. The mass spectrometer

161

was operated in electron ionization (EI) mode. The ion source and transfer line temperatures were 7



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set at 250 ºC and 280 ºC, respectively. Retention times of four flavorings were detected in full scan

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mode by recording mass range between 50 and 300 m/z. For the determination of target analytes,

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MS/MS mode was selected and performed in selected reaction monitoring (SRM) mode.

165 166

■ RESULTS AND DISCUSSION

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Optimization of MS/MS conditions. To unambiguous identification and accurate

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quantification of vanillin, methyl vanillin, ethyl vanillin and coumarin in real infant formula

169

samples at trace levels, MS/MS conditions were optimized. Firstly, individual flavoring and

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isotope-labeled standards were performed in full scan mode separately in the 50-300 m/z scan range

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to identify the optimal precursor ions. Molecular ions presented in all seven target substances

172

appeared and were selected as the parent ions beyond doubt. High masses were preferred as the

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precursor ions because it could avoid interferences from isobaric masses resulted from common

174

molecule fragments.28 Once the precursor ions were chosen, the MS/MS conditions were further

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optimized to obtain maximum resolution and sensitivity. The collision energies with different

176

values between 10 to 30 eV were applied to find the two most abundant fragments both for further

177

qualitative and quantitative intention. The dwell time was 200 ms for each compound, and the

178

optimal collision energies for each transition are listed in Table 1. Moreover, one precursor ion and

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two daughter ions were applied, which well fitted the EU criteria.29 Though the precursor ions of

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methyl vanillin and ethyl vanillin were same due to their completely same molecular formula, their

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daughter ions were different due to their different chemical structures. It permitted the separately

182

recognition of them even if encountering an unsufficient GC separation.

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A complete SRM chromatogram of a real blank infant formula sample spiked with 10 µg kg-1

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of four flavorings and three isotopic labels shown in Figure 2. It was observed that vanillin, methyl 8


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vanillin and ethyl vanillin were well separated while methyl vanillin and coumarin had almost the

186

same retention time from 6.5 to 6.6 min. But in a SRM mode, they could be unambiguous identified

187

without any question. In the MS/MS analysis mode, the target was initially identified by the

188

retention time and typical SRM transitions, and then further confirmed by the relative ion

189

abundance ratio between the qualitative ion and the quantitative ion (shown in Table 1). Meanwhile,

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a clean background was obtained in spiked sample analysis as shown in Figure 2, which indicated

191

that no chromatographic interference occurred from the sample matrix.

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The GC-MS/MS product scan spectrums of the investigated compounds were obtained and

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shown in Figure 3A-G. Moreover, based on the fragment information given by Figure 3, plausible

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interpretation of the MS/MS fragmentation was proposed and presented in Figure 4. Noted that we

195

have interpreted the MS/MS fragmentation of vanillin, vanillin-13C6, ethyl vanillin, coumarin and

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coumarin-D4 obtained in HPLC-MS/MS using a positive electrospray ionization (ESI) mode in our

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early report.24 And the obviously different cracking behavior was noticed for all compounds and

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this might be attributed to the different ionization mode. At this point, there is no related reported

199

work as reference up to now.

200

Method Validation. To investigate the applicability of the developed method for routine real

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analysis, general analytical merits including linearity, limits of quantification (LOQ), specificity,

202

precision and accuracy (recovery) were detected.

203

Matrix effects, expressing as suppression or enhancement of instrument signals, have been

204

generally recognized as an important and serious error source in quantitatively analysis of

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trace-level compound in food samples by using either GC-MS/MS or LC-MS/MS method.30–32 In

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our previous trail on analysis flavorings in infant formula by LC-MS/MS, 24 the matrix effects were

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discussed and verified by contrastive experiments. To correct matrix effects, a combination of 9



208

isotope-label internal standards and matrix-matched calibration solutions were performed and

209

satisfied results were obtained.

210

Quantification was performed based on calibration plots using the peak area of the most intense

211

MS/MS transition of the analyte which was defined as quantitative ion shown in Table 1. The

212

calibration curves with 1/x weighting were plotted for each individual target compound and the

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response data were adjusted according to the relative ratios of responses to the stable isotope

214

internal standards in the standards. Good linearity was achieved and the correlation coefficients

215

were in the range of 0.9991 to 0.9995. The limit of quantification (LOQ) was calculated as 10 times

216

of the standard deviation, and it was 10.0 µg kg-1 for vanillin, methyl vanillin, ethyl vanillin, and

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coumarin. For vanillin and ethyl vanillin permitted adding to follow-up formula (for more than 6

218

months old infant and child) in China with the maximum usage amount of 5 mg per100 mL, the

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LOQ is available without any doubt. According to the general average using instruction of infant

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formula, there are approximate 13-17 g milk powder contained in 100 mL ready-to-use liquid

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nutrition for baby, thus a vanillin or ethyl vanillin content of 294-385 mg kg-1 in powder samples

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(average 333 mg kg-1) is just comply with the regulations of China’s usage limit. For coumarin, the

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LOQ of 10.0 µg kg-1is also far below the limit of 2 mg kg-1 which set by Codex alimentarius and

224

EU. It indicated that this method is suitable for analysis of these flavoring agents in infant formula

225

samples.

226

To evaluate the specificity of the developed method, 10 different blank infant formula samples

227

were investigated. No interfering peaks from endogenous compounds of sample extracts were

228

observed at and near the retention times of all target analytes, and which indicative the specificity of

229

the method.

230

To evaluate the precision of this method, both intra-day and inter-day repeatability were 10


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examined by determination of a fortified sample at three different concentrations (low, medium and

232

high) at 10, 50 and 100 µg kg-1. Good stability and satisfied repeatability were achieved and the

233

relative standard deviations (RSDs) values were less than 3.62%.

234

The method accuracy was evaluated by analyzing series of fortified spiking samples using the

235

developed method. The appropriate amounts of the standard target compounds were spiked into the

236

“blank” infant formula samples and given at three final concentration levels of 10, 50 and 100 µg

237

kg-1. The spiked samples were treated following the same procedure as mentioned and

238

determination by GC-MS/MS. Average recoveries ranged within 82.8-107.5% (Table 2) was

239

achieved and relative standard deviations (RSDs) were less than 8.9%, indicative of the good

240

recovery and precision of the method.

241

Application to Real Samples. Twenty infant formula samples were purchased from local

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supermarkets and analyzed following the proposed method, and the blanks were periodically run

243

during the sample analysis sequence to confirm the absence of contamination. Neither ethyl vanillin

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nor coumarin was detected in all samples, while vanillin was detected at concentration levels

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ranging from 2.4 to 706.8 mg kg-1 in four samples, and these results were highly consistent with our

246

previous report by LC-MS/MS method.24 At the same time, methyl vanillin was detected at

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concentration levels ranging from 0.78 to 0.94 mg kg-1 in three samples. The detailed results were

248

summarized in Table 3, and the SRM chromatograms of a real positive sample were shown in

249

Figure 5. Vanillin was considered being added intentionally to the four vanillin-positive samples

250

for the concentration is high. Especially the sample containing 706.8 mg kg -1 vanillin, according to

251

previous analysis on Chinese vanillin usage limit, which is certainly against the China’s national

252

standard. These samples should be a matter of concern when applied to infants and even children.

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Meanwhile, the occurrence of methyl vanillin was considered being contaminated during the 11



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production process and not added intentionally, because its concentrations were so small and could

255

not effectively act as a flavoring ingredient.

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The developed method allows the simple, rapid and sensitive determination of four flavorings

257

in infant formula samples. The method showed good recoveries (82.8-107.5%) and precision (RSDs

258

below 8.9%) and could be applied to the unequivocal simultaneous identification and quantification

259

of target analytes in complex formula samples.

260 261

■ AUTHOR INFORMATION

262

Corresponding Author

263

*Phone: 86-577-88373937. Fax: 86-577-88373937. E-mail: [email protected] (Yan Shen);

264

[email protected] (Chao Han).

265 266

Funding Sources Financial support from the Science Technology Department of Zhejiang Province

267

(2013C37065), NSFC (21207102), AQSIQ (2009IK172) and Wenzhou Municipal Science and

268

Technology Bureau project (2013G0007) is gratefully acknowledged.

269 270 271 272 273 274 275 276 12


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(8) European Council. Council Directive (EEC) No. 88/388 on the approximation of the laws of

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linear ion trap mass spectrometry. J. Dairy Sci. 2014, 97, 679–686.

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and validation of a method for the determination of 159 pesticide residues in tobacco by gas

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chromatography-tandem mass spectrometry. J. Agric. Food Chem. 2013, 61, 5746–5757.

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(26) Lu, J.; Wu, J.; Stoffella, P. J.; Wilson. P. C. Analysis of bisphenol A, nonylphenol, and

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natural estrogens in vegetables and fruits using gas chromatography-tandem mass

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spectrometry. J. Agric. Food Chem. 2013, 61, 84–89.

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mass spectrometry. J. Chromatogr. A 2012, 1270, 28–40.

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(29) Commission Decision 2002/657/EC of 12 August 2002, Implementing Council Directive

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Figure Captions: There are five figures in this manuscript.

367

Figure 1. Chemical structure of vanillin (A), methyl vanillin (B), ethyl vanillin (C), coumarin (D),

368

vanillin-13C6 (E), methyl vanillin-13C (F) and coumarin-D4 (E).

369

Figure 2. GC-MS/MS SRM chromatograms of a blank infant formula sample spiked 10 µg kg-1

370

four flavorings. Vanillin (A), vanillin-13C6 (B), ethyl vanillin (C), methyl vanillin (D), methyl

371

vanillin-13C (E), coumarin (F), and coumarin-D4 (G).

372

Figure 3.

373

methyl vanillin (D), methyl vanillin-13C (E) coumarin (F), and coumarin-D4 (G).

374

Collision energy (eV): A: 20; B: 20; C: 15; D: 15; E: 15; F: 10; G: 10.

375

Figure 4. Proposed fragmentation pattern with tentative structures for the product ions of vanillin

376

(A), vanillin-13C6 (B), ethyl vanillin (C), methyl vanillin (D), methyl vanillin-13C (E) coumarin (F),

377

and coumarin-D4 (G).

378

Figure 5. The SRM chromatograms of a real positive sample. vanillin (A), methyl vanillin (B).

GC-MS/MS product scan spectrum of vanillin (A), vanillin-13C6 (B), ethyl vanillin (C),

17



Page 18 of 25

Figures

CHO

CHO

CHO O

OCH 3

OCH 3

O

O

OH

OCH 3

OH

(A)

(B)

(C)

(D) D

13

CHO 13

H 13C H 13C 13

C

13

CH

13

C

C

CHO

D

O

D OCH 3

OCH3

OH

OCH3

(E)

(F)

Figure 1


D

(G)

O

Page 19 of 25


Figure 2




Page 20 of 25

Page 21 of 25




Figure 3


Page 22 of 25

Page 23 of 25


CHO

CHO

H13C

C+

H13C

-CHO -29

-CHO -29

13C

13C + 13CH

H13C H13C

13C

OCH3

13C

OCH3

OH

Mr 129.0

-14

-H -1

-CH 2

-14

-1

-H

-CH2

Mr 123.0

+

+ CO

CO C+ H13C H13C

OH

OCH3 OH

CH

Mr 158.0 Vanillin-13C6

OH

OH Mr 152.0 Vanillin

13C

13

OH

OCH3

OCH3

13C

Mr 151.0

OH

13C

13C

13

CH

H 13C

13C

OCH 3 Mr 157.0

CHO C+ -CHO -29 O

O OH

OH

Mr 166.0

-14-14

-CH2- CH2

Ethyl vanillin

Mr 137.0

C+

OH (C)

OH


Mr 109.0

13C + 13CH 13 13C

OH

OH

Mr 109.0

(A)

H 13C

(B)

C OH Mr 115.0


+ + CO

CHO

CHO -H -1

OCH3 OCH3 Mr 165.0

-15 OCH 3 OCH3 Mr 166.0 Methyl vanillin

-H -1

OCH 3 OCH3 Mr 166.0

OOCH3 Mr 151.0

H+ C

-CO-CH 2

-28-14 OCH 3 OCH3 (D) Mr 137.0

13CHO

-CH3 -15 OCH3 OOCH 3 Mr 167.0 OCH 3 Mr 152.0 Methyl vanillin-13C - 13CHO -30

-CHO

-29 C

+

13CHO

CO

-CH 3

Page 24 of 25

C+

Mr 95.0 OCH3

-CO-CH

Mr 96.0 -28-13 OCH 3 OCH3 OCH 3 (E) Mr 137.0

D

D

O

O

D

O

-CO -28

O

O

-28 D

Mr 118.0

O

-CO

D

Mr 146.0

D Mr 122.0

D

Mr 150.0

-CO -28

Coumarin-D4

-CO -28

Coumarin

D

D

C+

D

CH 2+ (F)

C+

D

Mr 90.0

Figure 4


(G)

D

CH2+ Mr 94.0

Page 25 of 25


Figure 5


Determination of Four Flavorings in Infant Formula by Solid-Phase

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