Highly Sensitive and High-Throughput Method for the Analysis of

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A highly sensitive and high-throughput method for the analysis of bisphenol analogues and their halogenated derivatives in breast milk Yumin Niu, Bin Wang, Yunfeng Zhao, Jing Zhang, and Bing Shao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04394 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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Journal of Agricultural and Food Chemistry

1 2

A highly sensitive and high-throughput method for the analysis of bisphenol

3

analogues and their halogenated derivatives in breast milk

4 Yumin Niu†, Bin Wang‡, Yunfeng Zhao§, Jing Zhang†, Bing Shao*, †, ∥

5 6 7

†

8

Center for Disease Prevention and Control, Beijing, 100013, China

9

‡

Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijng

College of Food Engineering and Biotechnology, Tianjin University of Science & Technology,

10

Tianjin, 300457, China

11

§

12

Safety Risk Assessment, Beijing, 100021, China

13 14

∥

Key Laboratory of Food Safety Risk Assessment, Ministry of Health and China Center for Food

Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural

University, Beijing, 100193, China

15 16 17 18 19

*Corresponding author: E-mail: [email protected]

20

Tel: +86-10-64407191

21 22

1

ACS Paragon Plus Environment


23

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ABSTRACT:

24

The structural analogs of bisphenol A (BPA) and their halogenated derivatives (together

25

referred as BPs) have been found in environment, food and even human. Limited research showed

26

that some of them exhibited similar to or even greater toxicities than BPA. Therefore, adverse

27

effects on humans with low-dose in early life exposure were expected for BPs. Breast milk is an

28

excellent matrix could reflect fetus and babies exposure to contaminants. Some of the emerging

29

BPs may present with trace or ultra-trace levels in human. However, the existing analytical

30

method in breast milk cannot quantify these BPs simultaneously with highly sensitivity using

31

small sampling weight, which is important for human biomonitoring study. In this paper, a method

32

based on Bond Elut Enhanced Matrix Removal-Lipid purification, pyridine-3-sulfonyl chloride

33

derivatization and liquid chromatography electrospray tandem mass spectrometry was developed.

34

The method required only small quantity of sample (200 µL) and allowed for the simultaneous

35

determination of 24 BPs in breast milk with ultra high sensitivity. The limit of quantifications of

36

the proposed method were 0.001-0.200 µg L-1, which were 1-6.7 times lower than the only study

37

for the simultaneous analysis of bisphenol analogs in breast milk based on 3 g sample weight. The

38

mean recoveries ranged from 86.11% to 119.05% with relative standard deviations (RSD) ≤ 19.5%

39

(n = 6). Matrix effects were within 20% with RSD < 10% for 6 different lots of samples. The

40

proposed method was successfully applied to 20 breast milk samples. BPA, bisphenol F (BPF),

41

bisphenol S (BPS) and bisphenol AF (BPAF) were detected. BPA was still the dominant BPs

42

following by BPF in the second place. This is the first time describing the occurrence of BPF and

43

BPAF in breast milk.

44 45

Key words: bisphenol analogues; Bond Elut Enhanced Matrix Removal-Lipid purification;

46

pyridine-3-sulfonyl chloride derivatization; liquid chromatography electrospray tandem mass

47

spectrometry

48

2


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INTRODUCTION Bisphenol A (BPA), one of the highest production volume chemicals worldwide, has received

50

1

51

high concerns because of its typical endocrine disrupting effects

52

2-4

53

containers 5. The tolerable daily intake (TDI) was decreased from 50 µg/kg bw/day to a temporary

54

TDI of 4 µg/kg bw/day by European Food Safety Authority (EFSA) 5 in 2015 owing to new data

55

and refined methodologies. The restrictions have led to the BPA structural analogs, such as

56

bisphenol S (BPS), bisphenol F (BPF), bisphenol B (BPB), bisphenol C (BPC), bisphenol E (BPE),

57

bisphenol M (BPM), bisphenol P (BPP), bisphenol Z (BPZ), bisphenol AF (BPAF), bisphenol AP

58

(BPAP), bisphenol BP (BPBP), bisphenol FL (BPFL) and dihydroxydiphenyl ether (DHDPE) etc.

59

gradual enter the market. These analogues have been applied in industrial and consumer products,

60

such as food container lining, canned food, thermal receipts and fluorine rubber 6. Besides the

61

analogues of BPA, halogenated derivatives of BPA, such as tetrochlorobisphenol A (TCBPA) and

62

tetrabromobisphenol A (TBBPA), were widely used as flame retardants 7. In addition, BPA could

63

be readily converted to its chlorinated derivatives during the water disinfection process 8, 9. Similar

64

transformations were observed for BPF and BPS

65

analogues and their halogenated derivatives (collectively referred to as BPs) in consumer products

66

resulted in the widespread occurrence in various environmental compartments, foods and even

67

human biological fluids

68

BPAF have been reported in the environment and human urine 6.

69

and its widespread occurrence

. Considering the uncertainty of safety, BPA has been gradually prohibited in baby food

6, 13

10-12

. The increasing application of BPA

. Elevated detection frequencies and concentrations of BPF, BPS and

An increasing number of research indicated associations between long-term at low levels of 14

70

BPA exposure in early life and neuroendocrine disorders, such as precocious puberty

71

and diabetes

72

vulnerable. Breast milk is the major or exclusive energy source for infants up to 6 months, as well

73

as a reflection of internal exposure level of contaminants in mothers and fetus. Therefore, breast

74

milk is frequently monitored for exposure assessment of BPA 5. BPA analogs and halogenated

75

derivatives have been reported similar or even greater toxic effects compared with BPA

76

However, there is little information on the occurrence of BPA related compounds in breast milk.

77

One reason is the lacking of analytical method for the simultaneous determination of BPs. The

15-17

, obesity

, as well as anxiety and hyperactivity 18. That means fetus and infants are more

6, 19-21

.

3



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78

reported methods mainly focused on the detection of BPA in breast milk. Liquid chromatography

79

tandem mass spectrometry (LC-MS) equipped with electrospray ionization (ESI) source is one of

80

the most popular analytical tool because of its higher selectivity and sensitivity compared with

81

liquid chromatography with fluorescence detector (LC-FLD) and gas chromatography mass

82

spectrometry (GC-MS)

83

(Table 1), it is infeasible to adopt these methods to other BPs directly. Till now, there are only two

84

methods reporting the simultaneous determination of bisphenol analogs in milk matrix.

85

Deceuninck et al. 23 described a sensitive analytical method for the quantification of 18 bisphenol

86

analogs (halogenated derivatives not included) in breast milk based on GC-MS after

87

N-methyl-N(trimethylsilyl)-trifluoroacetamide

88

polystyrene-divinylbenzene stationary phase cartridge and BPA molecularly imprinted polymers

89

(MIP) stationary phase cartridge were employed in the pretreatment, the method is complicated

90

and time-consuming. Cheng et al.

91

based on a quick, easy, cheap, effective, rugged and safe (QuEChERs) method. The limits of

92

quantitation (LOQs) for the method were 0.02-0.5 µg kg-1, which is limited for the determination

93

of other BPs because of their lower concentrations. Sample volume/weight is important for human

94

biomonitoring study, especially for large cohort studies. The sample weights in the two methods

95

mentioned above were 3 g and 5 g, respectively, which were too large for human biomonitoring

96

study. Therefore, the development of a low-volume sample use, high-throughput and highly

97

sensitive method is urgently needed.

22

. However, considering the varying physicochemical property of BPs

24

(MSTFA)

derivatization.

As

employed LC-MS for screening 11 BPs in dairy products

98

In this study, we aimed to establish a highly sensitive method for the simultaneous

99

determination of bisphenol analogues as well as their halogenated derivatives in breast milk using

100

small sample weight. Bond Elut Enhanced Matrix Removal-Lipid (EMR-Lipid) purification and

101

pyridine-3-sulfonyl chloride (PS-Cl) derivatization followed by LC-MS were employed.

102 103

MATERIALS AND METHODS

104

Chemical and reagents. BPA (> 98.5), BPB (> 98%), BPF (> 99%), BPS (> 98%), BPAF (>

105

98%), TCBPA (> 98%) and TBBPA (> 98%) were purchased from Tokyo Chemical Industry

106

(Tokyo, Japan). BPC (≥ 99%), BPE (≥ 98%), BPM (≥ 99%), BPP (≥ 99%), BPZ (≥ 99%), BPAP 4


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107

(≥ 99%), BPBP (≥ 98%), BPFL (≥ 99%) and DHDPE (≥ 99%) were from Sigma-Aldrich Inc. (St.

108

Louis, USA). BPA-13C12 (99%, 100 µg mL-1 in acetonitrile), BPB-13C12 (99%, 100 µg mL-1 in

109

acetonitrile), BPF-13C12 (99%, 100 µg mL-1 in acetonitrile), BPS-13C12 (98%, 100 µg mL-1 in

110

methanol), BPAF-d4 (98%), TCBPA-13C12 (99%, 50 µg mL-1 in methanol) and TBBPA-13C12 (99%,

111

50 µg mL-1 in methanol) were obtained from Cambridge Isotope Laboratories Inc. (Andover,

112

USA). Mono-chlorobisphenol A (MCBPA), dichlorobisphenol A (DCBPA, 98%) and

113

trichlorobisphenol A (TriBPA) were from Toronto Research Chemicals Int. (North York, Canada).

114

Mono-chlorobisphenol F (MCBPF), dichlorobisphenol F (DCBPF), trichlorobisphenol F(TriBPF),

115

tetrochlorobisphenol F (TCBPF), mono-chlorobisphenol S (MCBPS), dichlorobisphenol S

116

(DCBPS), trichlorobisphenol S (TriBPS), tetrochlorobisphenol S (TCBPS) were synthesized in

117

our previous study with purities all above 98% 12.

118

Derivatizing reagents, dansyl chloride (DNS-Cl, 98%) and PS-Cl (> 98%) were purchased

119

from Tokyo Chemical Industry (Tokyo, Japan). 1, 2-dimethylimidazole-4-sulfonyl chloride

120

(DMIS-4-Cl, > 95%) and 1, 2-dimethylimidazole-5-sulfonyl chloride (DMIS-5-Cl) were

121

synthesized by Life Chemicals Inc. (Niagara, Canada) and Apollo Scientific Ltd. (Stockport, UK),

122

respectively.

123

LC-MS grade methanol, acetonitrile and water were from Sigma-Aldrich (St. Louis, USA).

124

Acetone and n-hexane of HPLC grade were purchased from Dikma Technologies Inc. (Lake

125

Forest, CA). Sodium bicarbonate (ACS grade) and sodium hydroxide (98.5%) were from J&K

126

Scientific Ltd. (Beijing, China). Sodium acetate was purchased from Sinopharm Chemical

127

Reagent Co. (Beijing, China). Formic Acid (99%) and acetic acid (99%) were from Acros

128

Organics (Morris Plains, NJ). β-glucuronidase/aryl-sulfatase mixture (extracted from Helix

129

pomatia) was purchased from Roche Diagnostics GmbH (Mannhein, Germany).

130 131

Derivatization. A standard solution of BPs was evaporated to dryness under a nitrogen flow

132

and reconstituted in 200 µL of aqueous sodium bicarbonate (100 mmol L-1, pH 9.5) and 200 µL of

133

5 mg mL-1 derivating reagents dissolved in acetone. The mixtures were vortex-mixed for 1 min

134

and incubated at 60°C for 5 min. After cooled in ambiance, the reaction mixtures were extracted

135

with 400 µL of n-hexane twice. Then the n-hexane layer was evaporated to near dryness and 5



136

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reconstituted with 100 µL acetonitrile/water (50:50, v/v) before LC-MS analysis.

137 138

Breast milk sample collection. Breast milk sample were obtained from 20 healthy voluntary

139

donors living in Hunan Province, China. Sample collection was conducted from June to July in

140

2014. All samples were collected in glass bottles and frozen immediately at -20°C until analysis.

141

The study had been approved by the ethic committees of China National Center for Food Safety

142

Risk Assessment. All donors were informed of the objective of this study.

143 144

Breast milk samples and preparation. Aliquots of 200 µL of breast milk sample were

145

mixed with 50 µL of internal standard working solution. The mixtures were vortex-mixed for 1

146

min

147

β-glucuronidase/aryl-sulfatase mixture. After incubation at 37°C for 12 h, the digested samples

148

were extracted with 1.2 mL acetonitrile. The supernatants were transferred to a tube containing

149

200 mg EMR-Lipid powder and 800 µL pure water. The mixtures were vortex-mixed for 1 min

150

and centrifuged at 5 000 rpm for 10 min. Then the supernatants were transferred to another tube

151

containing 300 mg EMR-Lipid polish packing (NaCl/MgSO4 = 1:4, w/w) and vortex-mixed for 1

152

min. After centrifuged at 5 000 rpm for 10 min, the upper acetonitrile layer was evaporated to near

153

dryness under a gentle stream of nitrogen. The dried residues were derived by PS-Cl as described

154

above.

and

buffered

with

200

µL of

sodium

acetate

(pH

5.2)

and

10

µL of

155 156

LC-MS condition. LC separation was conducted using a Waters Acquity UPLC I-Class

157

system (Waters, Mildord, MA) equipped with a BEH C18 column (2.1 mm × 100 mm, 1.7 µm,

158

Waters, Mildord, MA). The mobile phase was a mixture of acetonitrile (A) and 0.1% formic acid

159

in water (B) with a flow rate of 0.3 mL min-1 under gradient conditions: 40% A increased linearly

160

to 90% in 3 min, then increased to 100% in 2 min and held for 3 min, and finally returned to the

161

initial composition in 0.1 min. The column was equilibrated for 2 min before the next injection.

162

The injection volume was 10 µL. Column oven and sampler temperature were 40°C and 10°C,

163

respectively.

164

A Waters Xevo TQ-XS (Waters, Mildord, MA) triple quadruple mass spectrometer with ESI 6


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165

ion source in positive polarity was interfaced to the LC system. Multiple reaction monitoring

166

(MRM) was employed. The capillary voltage was 2.5 kV. Nitrogen gas with purity 99.9% was

167

used as the cone gas and desolvation gas at flow rates of 150 L h-1 and 900 L h-1, respectively. The

168

source temperature and desolvation temperature were held at 150°C and 450°C.

169 170

Matrix effect. Matrix effects were evaluated by the following equation:

171

Matrix effect (%) = [1-(B/A)] × 100%

(1)

172

where A represents the peak area of the standard solution and B represents the peak area of

173

breast milk extract spiked at the same concentration of the standard before LC-MS analysis.

174

Considering the individual differences, six lots of breast milk were used to evaluate the matrix

175

effect.

176 177

Method validation. Accuracy, precision, linearity and sensitivity were validated for the

178

developed method according to Commission Decision 2002/657/EC. Accuracy and precision were

179

expressed as recovery and relative standard deviations (RSD), which were evaluated by three

180

concentration levels (LOQ, 2 × LOQ and 5 × LOQ) spiked in six replicates of blank breast milk

181

samples. The inter-day precision was evaluated over the course of five consecutive days using the

182

spiked level of 2 × LOQ. The linearity was evaluated by matrix-matched standard curve using

183

standard spiked in extracts of samples before LC-MS analysis at six different concentration levels.

184

Each concentration level was repeated in triplicate. Calibration curves for the target compounds

185

were obtained by linear regression analysis on the ratio of standard-solution areas to

186

internal-standard area versus concentration. The sensitivity of the proposed method was evaluated

187

by limit of detection (LOD) and LOQ. Since inevitable BPA in procedural blank (specified in

188

Section 3.4), ten blank breast milk sample (BPA concentration lower than procedural blank) were

189

used for the calculation of LOD and LOQ of BPA. The LOD and LOQ of BPA were 3 × standard

190

deviations (SD) and 10× SD above the mean value of the blank samples, respectively. For other

191

BPs, LOD and LOQ represented the lowest spiked concentration that can yield a signal to noise

192

ratio (S/N) greater than 3 and 10.

193 7



194 195

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RESULTS AND DISCUSSIONS Derivatization. Two established methods in our laboratory based on gel permeation 25

196

chromatography (GPC)

and Envi-Carb solid phase extraction (SPE, unpublished), respectively,

197

for the determination of some of BPs in commercial milk powder and liquid milk were tested at

198

first. However, both of the two methods displayed strong matrix effects (more than 50%) for some

199

BPs in breast milk. Besides, the two MRM transitions of some BPs, such as BPB, BPE, BPFL and

200

DHDPE et al., exhibited great responding differential (more than one order of magnitude, see

201

Figure S1 in Supporting Information). The sensitivity and accuracy were greatly decreased due to

202

the strong matrix effects and large of product ion ratio. Therefore, derivatization was taken into

203

consideration. Sulfonyl chlorides was reported to be one of the most practical derivating reagents

204

for the analysis of phenolic hydroxyl groups due to their selective reactivity and compatibility

205

with aqueous reaction solutions in previous study

206

BPA improved 180 times by the sulfonyl chlorides derivatization27, which make it possible for the

207

ultra-trace analysis of BPs with little sample volume. We compared four reported derivating agents:

208

DNS-Cl, PS-Cl, DMIS-4-Cl and DMIS-5-Cl, respectively. The MS/MS acquisition parameters for

209

DNS-Cl derivatives, PS-Cl derivatives, DMIS-4-Cl derivatives and DMIS-5-Cl derivatives of all

210

the 24 BPs were optimized in ESI positive mode by directly infusion of derivated standard

211

solution (50 µg L-1) via a syringe pump at a flow rate of 5 µL min-1 combing the mobile phase

212

(acetonile-water, 1:1, v/v) at a flow rate of 0.1 mL min-1. Diagnostic fragment ions were selected

213

and all mass-spectrometer parameters were optimized for maximum sensitivity. Take BPB for

214

example, the fragments of BPB-diDNS were m/z 235, m/z171 and m/z 156 (shown in Figure 1A),

215

which were originating from the dimethylaminonaphthalene moiety of DNS-Cl. Similarly, the

216

fragments of BPB-diDMIS-4 (m/z 159 and m/z 96, shown in Figure 1B) were resulting from the

217

dimethylimidazole moiety of DMIS-4-Cl. The collisional fragmentation of BPB-diDNS and

218

BPB-diDMIS-4 were completely retained by the derivating agents, which increasing the

219

susceptibility of detection. As an isomer of DMIS-4-Cl, the fragmentations of the derivatives of

220

DMIS-5-Cl were different. The main products were m/z 371 and m/z 96 (shown in Figure 1C). The

221

ion m/z 371 was assigned to the product ion resulting from the loss of CH2CH3 and mono-DMIS-5

222

moiety, whereas the ion m/z 96 was still from the dimethylimidazole moiety of DMIS-5-Cl. The

26

. It has been reported that the sensitivity of

8


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223

main products of BPB-diPS were m/z 354 and m/z 290 (shown in Figure 1D), which were assigned

224

to the loss of CH2CH3 and mono-PS moiety, and further loss of SO2. The two main fragmentation

225

products showed a series of intense ions that appear to have originated from the BPB moiety.

226

Therefore, PS-Cl was the best derivating agent with the highest selectivity. The fragmentations of

227

other BPs were similar with BPB, and the MS/MS fragment ions of the four derivatives were

228

displayed in Table S1 (see Supporting Information). On the other hand, the sensitivities of BPs

229

were improved 1-250 times by the derivatization of PS-Cl, which were comparable with that of

230

DNS-Cl, and much higher than DMIS-4-Cl and DMIS-5-Cl (see Supporting Information, Table

231

S2). Therefore, the optimal derivating agent was PS-Cl with excellent selectivity and outstanding

232

sensitivity for the analysis of BPs.

233

However, except for di-substituted derivatives, mono-substituted derivatives have also been 28

234

reported for the derivatization of BPs with PS-Cl

235

condition including the solvent dissolved PS-Cl, the concentration of PS-Cl, the pH of sodium

236

bicarbonate buffer, reaction time and reaction temperature. Acetone and acetonitrile were the most

237

common solvents for the derivatization of sulfonyl chloride with phenols 26-31. Therefore, acetone

238

and acetonitrile were compared at first. Almost all the reported pH for the derivatization by

239

sulfonyl chloride were 10.5

240

3.55 to 11.11 (Table 1), pH value should be reevaluated. The pH of sodium bicarbonate was 8.2.

241

Therefore, the value of pH 8.2, 9.0, 9.5, 10.0, 10.5 and 11.0 were evaluated. It has been reported

242

that the concentration of derivating agents could affect the DNS-Cl derivatives of BPA, although it

243

much more than BPA itself 32. Therefore, the concentrations of PS-Cl (1 mg mL-1, 2 mg mL-1, 3

244

mg mL-1, 4 mg mL-1 and 5 mg mL-1) were also optimized. In addition, the temperature and time of

245

the reaction were optimized. The ratios between maximum and minimum responses under each

246

condition were plotted in Figure 2A. The type of organic solvent dissolved PS-Cl has the most

247

impact on the derivatization efficacy than the other variables for all BPs. The responses of BPs

248

increased 3-43 folders when using acetone as solvent compared with acetonitrile (Figure 2B). The

249

reaction of sulfonyl chloride and phenolic conducted as the bimolecular nucleophilic substitution

250

(SN2) reaction. It is well-known that the activation barrier for the SN2 reaction is strongly affected

251

by solvent polarity

33, 34

26, 27, 29-31

. Therefore, we optimized the reaction

. However, considering the pKa values of BPs ranging from

. Reaction rates increased with less polar solvents. Therefore, acetone 9



Page 10 of 33

252

exhibited higher efficiency. However, the results were not observed for the derivatization of BPs

253

with DNS-Cl. The possible reason was due to the SN2 reaction was effected not only by solvent

254

polarity, but also by the activation energies and pre-exponential factors

255

between the reaction of BPs with PS-Cl and DNS-Cl should be further discussed. Moreover, pH is

256

another important factor effect the derivative efficiency of BPC and chloro-BPS. When pH was

257

lower than 9.0, the responses of BPC were greatly decreased, and when pH was higher than 9.5,

258

the responses of cholo-BPS were greatly decreased (Figure 2C). BPC with the highest pKa (Table

259

1), tended to form the corresponding conjugated acid under a low pH condition, which thereby

260

reduced the nucleophilic ability of BPC. However, when the pH was higher, the resulting sulfonic

261

esters of cholo-BPS who has lower pKa (ranging from 3.55-7.12, Table 1) were easily hydrolysis.

262

Therefore, cholo-BPS-monoPS products were observed in the screening of products by

263

LC-quadrupole time of flight (QTOF)-MS. The responses of mono-products increased with

264

elevating pH and the number of chlorine. Even when pH was 8.2, mono-products were still

265

observed. Besides the hydrolysis, dechlorination reaction of sulfonic esters was also observed for

266

cholo-BPS-diPS. Concentrations of PS-Cl had little influence on the efficiency of the

267

derivatization of BPs with PS-Cl, except for cholo-BPS (Figure 2A). The responses of

268

cholo-BPS-diPS increased with the concentration of PS-Cl increasing. However, hydrolysis and

269

dechlorination byproducts were still existed with PS-Cl concentration 5 mg mL-1. Reaction time

270

and temperature has little influence on the responses of all BPs-diPS. Therefore, the optimized

271

condition was 5 mg mL-1 PS in acetone and 100 mmol L-1 sodium bicarbonate (pH 9.5) incubated

272

at 60°C for 5 min. Under the condition, no mono-products were observed for all BPs except for

273

cholo-BPS. Considering the non-negligible byproducts of the derivatization of cholo-BPS with

274

PS-Cl, it is inadequate for the determination of cholo-BPS by the proposed derivatization method.

275

Therefore, in this paper, cholo-BPS were not included.

34, 35

. The distinction

276 277

Sample pretreatment. Acetonitrile was used as common extractant for biological samples 36

278

due to its excellent ability of protein precipitation

. Therefore, acetonitrile was employed to

279

extract target compounds from breast milk samples. The recoveries were between 92.3-114.2% for

280

all BPs. Lipid is another main interference in breast milk which could decrease the column 10


Page 11 of 33


281

lifetime and may bring non-negligible matrix effects. Therefore, an exhaustive clean-up of the

282

sample extract is necessary. Usually, lipids are removed by liquid partitioning based on a freezing

283

out step

284

EMR-Lipid dispersed SPE (dSPE) 42were employed for the removal of lipids from fatty samples

285

without unwanted analytes retention. Therefore, five different purification methods before

286

derivatization were compared. A: acetonitrile extracts without purification; B: acetonitrile extracts

287

were extracted by 2 mL n-hexane twice; C: acetonitrile extracts were freezen at -20°C for 1 h and

288

supernatants were used; D: acetonitrile extracts were purified by PRiME HLB cartridge (60 mg, 3

289

mL) with 1 mL extra acetonitrile eluting the cartridge; E acetonitrile extracts were purified by

290

EMR-lipid dSPE using the method mentioned above. After derived by PS-Cl, both recoveries and

291

matrix effects of the five methods were compared (Figure 3). The recoveries (Figure 3A) ranged

292

25.4%-99.0% for acetonitrile extracts without any purification (A), with relative high RSDs

293

ranging from 7.08% to 31.6%. Halogenated BPs exhibited lower recoveries and the recoveries

294

decreased with the increasing number of halogen atoms. The possible reason was due to the

295

electron-withdrawing effect of halogen in BPs reduced the nucleophilic ability to PS-Cl. The

296

lower competition ability with the binding site of PS-Cl resulted in the lower recoveries of

297

halogenated BPs. The recoveries of other BPs were slightly improved by the using of n-hexane (B)

298

or freezing method (C). However, these two methods were helpless to improve the recoveries of

299

the polyhalogenated BPs. The recoveries of all BPs were greatly improved by PRiME HLB (D)

300

and EMR-Lipid dSPE (E), ranging from 65.4% to 112.9% and from 80.4% to 118.7%, respectively.

301

On the other hand, matrix effects (Figure 3B) were also greatly improved by the using of PRiME

302

HLB (D) and EMR-Lipid dSPE (E), with matrix effects no more 26% and 18%, respectively.

303

Considering the slightly higher recoveries and lower matrix effects, EMR-Lipid dSPE was

304

preferred.

37

or n-hexane 38, GPC

39

and normal phase SPE 40. Recently, PRiME HLB

41

and Bond

305 306

Matrix effects. The composition of the mobile phase is another significant contributor to

307

matrix effects for the quantitative analysis with LC-ESI-MS, except for the sample pretreatment.

308

Therefore, a mixture of methanol-water, acetonitrile-water and the concentration of formic acid

309

were compared. First of all, responses of standard solution were compared (See Supporting 11



Page 12 of 33

310

Information, Figure S2). Interestingly, BPs with strong polarity, such as BPE, BPF, BPS, DHDPE

311

and MCBPF showed good sensitivity under the condition of a mixture of methanol and water as

312

mobile phase. However, BPs with lower polarity, such as BPC, BPM, BPZ, BPAP, BPBP, BPFL,

313

TCBPA and TBBPA showed good sensitivity under the condition of a mixture of acetonitrile and

314

water with 0.1% formic acid as mobile phase. Significant signal suppressions were observed when

315

methanol and water were used as the mobile phase for all BPs (See Supporting Information,

316

Figure S3).When 0.1% formic acid added in the water phase, signal suppressions were greatly

317

improved for BPs with relative weak retention (retention time (tR)< 2.80 min, Table 2). It was

318

probably due to the formic acid accelerated the ionization of BPs. However, BPs with strong

319

retention ability (tR> 2.80 min, Table 2) still displayed strong suppression (35.0-78.8%). Matrix

320

effects were within 20% when acetonitrile and water with 0.1% formic acid were used as mobile

321

phase (See Supporting Information, Figure S3). One possible reason was that these BPs were

322

eluted at the end of the elution gradient, where strongly retained compounds were eluted. The

323

donated protons by methanol promoted the ionization of interferences, which reduced the access

324

of the target compounds to the droplet surface. Thereby led to ionization suppression of analytes 43.

325

Considering both the responses of target compounds and matrix effects, a mixture of acetonitrile

326

and water with 0.1% formic acid was selected as the mobile phase.

327

As previously reported, the influence of mobile phase composition on ionization efficiency is

328

a well-described phenomenon in LC-MS analysis. However, the reconstitution solution also had a

329

tremendous impact on the ionization efficiency (See Supporting Information, Figure S4).

330

Methanol-water (1:1, v/v), acetonitrile-water (1:1, v/v) and a mixture of methanol/acetonitrile and

331

different concentration of formic acid in water as reconstitution solution were investigated. The

332

results showed that BPs standards dissolved in methanol-water and acetonitrile-water gave

333

comparable peak intensity. However, BPs with strong retention capability (tR> 2.80 min, Table 2)

334

showed greatly ion suppression (37.1-79.4%) when methanol-water (1:1, v/v) used as the

335

reconstitution solution. Ion suppression of BPs (no more than 17.1%) was greatly improved by

336

using acetonitrile-water (1:1, v/v) as reconstitution solution. One possible reason was due to the

337

different solubility of methanol and acetonitrile. The results were in accordance with composition

338

of mobile phase. Moreover, the addition of formic acid in the reconstitution solution has no effect 12


Page 13 of 33


339

on the matrix effect. Therefore, acetonitrile-water (1:1, v/v) was selected as the reconstitution

340

solution. The effects of reconstitution solution and mobile phase on the matrix effects need to be

341

further discussed.

342 343

Quality control. Three procedural blank (consisting of only extracting solvent) were

344

processed in the same manner as breast milk samples. BPA was detected in the procedural blank

345

with concentration 0.162 ± 0.007 µg L-1. The sensitivity of BPA was seriously affected by the high

346

background contamination. As plastics were avoided in the pretreatment, suspicions fell on the

347

materials of EMR-Lipid and EMR-Lipid polish powder. Therefore, we made an attempt on

348

washing the powders by 2 mL acetonitrilebefore using. The concentrations of BPA in procedural

349

blank were reduced to 0.042 ± 0.001 µg L-1. The background level of BPA did not decrease by

350

further washing. Therefore, the powders should be washed by 2 mL acetonitrile once before use.

351

In addition, rising levels of BPA in procedural blank were observed with the increasing storage

352

time of PS-Cl in acetone and sodium bicarbonate buffer. The reason was not clear. Therefore, the

353

derivating agent and buffer should be prepared freshly when used.

354 355

Method validation. As shown in Table 2, the LODs and LOQs of the method were

356

0.0003-0.067 µg L-1 and 0.001-0.200 µg L-1, respectively. Except for BPS and BPAF, the LOQs of

357

the proposed method were 1-6.7 times lower than the only study for the simultaneous analysis

358

bisphenol analogs in breast milk (LOQs: 0.003 to 0.1 µg kg-1) 23. The LOQs were speculated by

359

the spike level of 0.1 µg kg-1 instead of obtaining by the recovery experiments at LOQ levels.

360

Furthermore, the sample weight of the reported method was 3 g, much higher than our proposed

361

method. The linear ranges for all BPs were shown in Table 2 with the correlation coefficients (R2)

362

all above 0.99, which met the acceptance criteria. The matrix effects were within 20% with the

363

precision no more than 10%, which demonstrated the excellent suitability for the determination of

364

BPs in breast milk. The recoveries were ranging from 86.11% to 119.05% with RSD no more than

365

19.5%, which met the criteria of acceptance of Commision Decision 2002/657/EC (recovery

366

ranging from 70% to 120% with RSD no more than 20%)..

367 13



Page 14 of 33

368

Method application. The proposed method was applied for the analysis of 20 breast milk

369

samples. Table 3 listed the concentrations of BPs detected in the samples. Among the 24 BPs, only

370

BPA, BPF, BPS and BPAF were detected. BPA was the most detectable BPs as expected with

371

detection rate 85% and concentrations ranging from 0.050 µg L-1 to 0.548 µg L-1. The level of

372

BPA was consistent with recent reports in literature 23, 44, 45, which was significantly lower than the

373

research conducted in early days

374

concentrations ranging from 0.010 µg L-1 to 0.166 µg L-1. The results were accordance with the

375

findings in urine 13. BPAF was detected in three samples with concentrations ranging 0.021-0.052

376

µg L-1. BPS was detected in only one sample with concentration 0.683 µg L-1. The results alerted

377

the non-negligible levels of BPA substitutes in breast milk samples. To better understand the

378

exposure risk, BPs not only BPA should be biomonitored in the further studies. The maximum

379

residue level (MRL) of BPs in breast milk was calculated based on TDI (4 µg/kg bw/day), infant

380

ingestion data (750 mL day-1) and body weight (3.94 kg, 6 weeks), assuming that breast milk is

381

the only food source for infant. The calculated result 21.013 µg L-1 was 24 times than the highest

382

level of total BPs (No. 11, Table 3), which means it is unlikely to pose a health risk. However,

383

since the sample size was small, further biomonitoring study is still needed.

46

. BPF was following by with detection rate 60% and

384

To summarize, a highly sensitive and high-throughput method for the simultaneous

385

determination of 24 BPs in breast milk based on PS-Cl derivatization using small sample volume

386

was developed. Although the method was unable to distinguish the isomers BPM and BPP and

387

isomers DCBPA and was incapable for the determination of chloro-BPS, it can satisfy the current

388

monitoring of BPs in breast milk. BPA, BPF, BPS and BPAF were found in 20 breast milk samples.

389

BPA was still the dominant BPs in breast milk with BPF following on. This is the first study

390

reported the occurrence of BPF and BPAF in breast milk.

391 392

AUTHOR INFORMATION

393

Corresponding author

394

Bing Shao, Email: [email protected]. Tel: +86-10-64407191

395 396

Funding 14


Page 15 of 33


397

This work was supported by the Chinese National Natural Science Foundation (Grant No.

398

21607010) and Beijing Municipal Science & Technology Commission Capital Food Quality and

399

Safety Guarantee Project (Z161100000616006).

400 401

Notes:

402

The authors declare no competing financial interest.

403 404

SUPPORTING INFORMATION

405

Supplementary information for this paper is available in the online version of the paper.

406 407

Figure S1. Chromatogram of BPB (A), BPE (B), BPFL (C) and DHDPE (D) in standard solution at 5 µg L-1

408

Figure S2. Responses of BPsunder different composition of mobile phase. A: methanol-water;

409

B: methanol-water with 0.1% formic acid; C acetonitrile-water; D: acetonitrile-water with 0.1%

410

formic acid. Each bar represents the mean values ± standard deviation for three replicates.

411

Figure S3. Matrix effects of BPs under different composition of mobile phase. A:

412

methanol-water; B: methanol-water with 0.1% formic acid; C acetonitrile-water; D:

413

acetonitrile-water with 0.1% formic acid.

414 415 416 417 418

Figure S4. Matrix effects of BPs under different composition of reconstitution solution. A: methanol-water (1:1, v/v); B: acetonitrile-water (1:1, v/v). Table S1. Mass spectrometry conditions for the analysis of derivatives by different sulfonyl chlorides. Table S2. Instrumental LOQs of different derivating agent (µg L-1).

419 420

15



Page 16 of 33

421

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422

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20


Page 21 of 33

Compound Bisphenol A


Table 1 Structure and physicochemical property of BPs Acronym Structure BPA

LogKowa 3.64

pKab 10.29-10.93

4.13

10.27-10.91

4.74

10.45-11.09

3.19

9.91-10.64

3.06

9.91-10.54

6.25

10.31-10.92

6.25

10.31-10.92

2,2-Bis (4-hydroxyphenol) propane

HO

Bisphenol B

OH

BPB

2,2-Bis(4-hydroxyphenyl)butane

HO

Bisphenol C

OH

BPC

2,2-Bis(4-hydroxy-3-methylphenyl)propane HO

Bisphenol E

OH

BPE

1,1-Bis(4-Hydroxyphenyl)ethane HO

Bisphenol F

OH

BPF

4-[(4-hydroxyphenyl)methyl]phenol HO

Bisphenol M

OH

BPM

4-[2-[3-[2-(4-hydroxyphenyl)propan-2-yl]phenyl]propan-2-yl]phenol

OH HO

Bisphenol P

BPP

4-[2-[4-[2-(4-hydroxyphenyl)propan-2-yl]phenyl]propan-2-yl]phenol OH HO

21



Bisphenol S

O

BPS HO

Bis(4-hydroxyphenyl)sulphone

Page 22 of 33

S

1.65

7.464-8.23

5.00

9.91-10.51

4.47

8.74-9.38

4.86

10.22-11.11

6.08

10.01-10.91

6.08

9.58-10.19

OH

O

Bisphenol Z

BPZ

4-[1-(4-hydroxyphenyl)cyclohexyl]phenol

HO

Bisphenol AF

BPAF

OH

F

F

4-[1,1,1,3,3,3-Hexafluoro-2-(4-hydroxyphenyl)propan-2-yl]phenol F

HO

Bisphenol AP

F

F

F

OH

BPAP

4,4'-(1-Phenyl-1,1-ethandiyl)diphenol

HO

Bisphenol BP

OH

BPBP

Bis-(4-hydroxyphenyl)diphenylmethane

HO

Bisphenol FL

OH

BPFL

4,4-(9-Fluorenylidene)diphenol

HO

OH

22


Page 23 of 33


Dihydroxydiphenyl ether

O

DHDPE

3.09

9.90-10.69

4.29

9.79-10.58

-

8.98-10.54

-

9.44-10.08

-

8.93-9.73

-

8.59-9.22

-

8.50-9.14

4,4’-Dihydroxydiphenyl ether HO

Mono-chlorobisphenol A

OH

MCBPA

2-Chloro-4-[1-(4-hydroxyphenyl)-1-Methylethyl]phenol

HO

OH Cl

3,5-Dichlorobisphenol A

3,5-DCBPA

2,6-Dichloro-4-[1-(4-hydroxyphenyl)-1-methylethyl]phenol Cl

HO

OH Cl

3,3-Dichlorobisphenol A

3,3-DCBPA

2,2-Bis(3-chloro-4-hydroxyphenyl)propane Cl

HO

OH Cl

Trichlorobisphenol A

TriCBPA

2,6-Dichloro-4-[1-(3-chloro-4-hydroxyphenyl)-1-Methylethyl]phenol Cl

HO

OH Cl

Tetrochlorobisphenol A

Cl

TCBPA

2,6-dichloro-4-[2-(3,5-dichloro-4-hydroxyphenyl)propan-2-yl]phenol

Cl

Cl

HO

OH Cl

Tetrabromobisphenol A 2,6-dibromo-4-[2-(3,5-dibromo-4-hydroxyphenyl)propan-2-yl]phenol

Cl

TBBPA Br Br

HO OH Br

Br

23



Mono-chlorobisphenol F

MCBPF

Page 24 of 33

HO

OH

9.40-10.20

2-Chloro-4-[1-(4-hydroxyphenyl)-1-Methyl]phenol Cl

3,5-Dichlorobisphenol F

Cl

DCBPF

2,6-Dichloro-4-[1-(4-hydroxyphenyl)-1-methyl] phenol

8.89-10.15

HO

OH

Cl

Trichlorobisphenol F

Cl

TriCBPF

2,6-Dichloro-4-[1-(3-chloro-4-hydroxyphenyl)-1-Methyl]phenol

8.55-9.34

HO

OH

Cl

Tetrochlorobisphenol F

Cl Cl

TCBPF

2,6-dichloro-4-[(3,5-dichloro-4-hydroxyphenyl)methyl]phenol

Cl

8.20-8.84

HO

OH

Cl

Mono-chlorobisphenol S

Cl O

MCBPS HO

2-Chloro-4-[(4-hydroxyphenyl)sulfonyl]phenol

6.94-7.12

S

OH

O Cl

3,5-Dichlorobisphenol S

Cl

3,5-DCBPS

5.66-6.93

O

2,6-Dichloro-4-[(4-hydroxyphenyl)sulfonyl]phenol S

HO

OH

O Cl

3,3-Dichlorobisphenol S

Cl

3,3-DCBPS

Cl

5.21-5.99

O

Bis(3-chloro-4-hydroxyphenyl)sulphone HO

S

OH

O

Trichlorobisphenol S

Cl

TriCBPS

Cl

4.05-5.50

O

2,6-Dichloro-4-[(3-chloro-4-hydroxyphenyl)sulfonyl]phenol HO

S

OH

O

Tetrochlorobisphenol S

Cl Cl

TCBPFS

Cl

3.55-4.34

O

2,6-dichloro-4-[(3,5-dichloro-4-hydroxyphenyl)sulfonyl]phenol HO

S

OH

O Cl

Cl

24


Page 25 of 33

a b


searched from the Chemspider online database. http://www.chemspider.com/. caculated by ACDLabs/pKa dB software, v. 11.0, ACDLabs, Toronto, Canada

25



Compounds

BPA BPB BPC BPE BPF BPM/BPP BPS BPZ BPAF BPAP BPBP BPFL DHDPE MCBPA DCBPA TriCBPA TCBPA TBBPA MCBPF DCBPF TriCBPF TCBPF

Retention time (tR, min) 2.65 2.77 2.86 2.54 2.45 3.14 2.22 2.92 2.77 2.84 3.02 2.95 2.44 2.76 2.85 2.92 2.97 3.01 2.56 2.64 2.75 2.82

Table 2 Analytical performance of the LC-MS method for the determination of BPs in breast milk samples Recovery % (RSD %) Matrix effect % Linear Range LODa LOQb (RSDc %) n=6 n=6 (µgL-1) (µg L-1) (µg L-1) LOQ 2×LOQ 5 ×LOQ 0.050-50.000 0.017 0.050 -2.65 (6.24) 106.29 (3.23) 110.86 (3.92) 97.36 (6.36) 0.003-3.000 0.001 0.003 -8.35 (3.88) 94.52(2.45) 98.27 (3.62) 100.00 (5.63) 0.005-5.000 0.002 0.005 3.39 (2.16) 96.97 (5.41) 96.10 (8.71) 86.14 (6.50) 0.005-5.000 0.002 0.005 -13.21 (5.24) 118.18 (6.37) 95.61 (2.19) 94.00 (3.69) 0.005-5.000 0.002 0.005 -9.42 (4.22) 110.00 (12.9) 89.42 (6.15) 102.19 (6.73) 0.005-5.000 0.002 0.005 -4.56 (5.87) 119.05 (3.46) 105.41 (7.73) 103.67 (3.10) 0.010-10.000 0.003 0.010 8.43 (8.45) 108.24 (16.6) 109.80 (2.06) 99.33 (3.94) 0.010-10.000 0.003 0.010 -8.48 (2.71) 105.03 (1.04) 103.25 (6.65) 102.21 (1.77) 0.010-10.000 0.003 0.010 -6.70 (3.06) 102.69 (12.7) 107.99 (1.85) 97.32 (4.02) 0.005-5.000 0.002 0.005 -3.49 (4.12) 114.29 (10.8) 101.96 (2.15) 96.33 (5.33) 0.001-1.000 0.0003 0.001 -11.21 (5.74) 113.33 (10.2) 105.42 (3.55) 102.90 (1.22) 0.003-3.000 0.001 0.003 7.06 (8.29) 96.97 (5.41) 107.84 (4.92) 99.30 (0.61) 0.010-10.000 0.003 0.010 -15.64 (6.58) 95.50 (7.12) 105.03 (10.4) 97.13 (6.81) 0.003-3.000 0.001 0.003 1.43 (5.32) 113.33 (19.5) 102.23 (11.0) 105.87 (3.02) 0.005-5.000 0.002 0.005 -4.88 (4.82) 93.33 (6.19) 113.96 (3.56) 96.10 (2.30) 0.005-5.000 0.002 0.005 11.78 (2.47) 106.67 (7.53) 112.32 (4.82) 92.91 (2.38) 0.005-5.000 0.002 0.005 -9.47 (2.45) 89.68 (12.9) 101.80 (8.59) 104.6 (4.79) 0.005-5.000 0.002 0.005 -5.65 (7.29) 99.35 (16.6) 100.42 (2.06) 107.25 (6.77) 0.050-50.000 0.017 0.050 -3.87 (5.64) 96.43 (8.23) 107.78 (1.89) 98.87 (2.32) 0.200-50.000 0.067 0.200 17.04 (3.68) 91.79 (5.61) 107.39 (9.58) 101.87 (2.00) 0.200-50.000 0.067 0.200 -11.94 (4.69) 86.11 (7.27) 97.10 (5.02) 94.80 (1.20) 0.025-25.000 0.008 0.025 -6.46 (8.33) 89.12 (3.57) 87.04 (10.4) 91.39 (2.79)

Page 26 of 33

Precision % Intra-day n=5 7.25 1.79 1.93 4.85 4.57 6.21 1.84 2.40 2.24 0.59 3.14 3.84 11.34 13.49 1.97 3.47 9.00 6.62 2.20 2.11 1.98 8.84

a

LOD: limit of detection LOQ: limits of quantitation c RSD: relative standard deviations b

26


Page 27 of 33


Table 3 Concentrations of BPs in breast milk samples (µg L-1) BPA BPF BPS BPAF Sum 1 NDa 0.024 ND ND 0.024 2 0.431 ND ND ND 0.431 3 0.060 0.049 ND ND 0.109 4 0.051 0.059 ND 0.052 0.110 5 0.103 0.141 ND ND 0.244 6 ND ND ND ND ND 7 0.057 0.027 ND ND 0.084 8 0.070 0.030 ND ND 0.100 9 0.548 0.012 ND ND 0.560 10 0.050 0.166 ND ND 0.216 11 0.172 0.016 0.683 ND 0.871 12 0.077 0.018 ND ND 0.095 13 0.178 0.010 ND ND 0.188 14 0.113 ND ND ND 0.113 15 0.353 0.072 ND ND 0.425 16 ND ND ND ND ND 17 0.269 ND ND 0.021 0.290 18 0.052 ND ND 0.056 0.108 19 0.089 ND ND ND 0.089 20 0.051 ND ND ND 0.051 a ND: not detected. The concentrations were below the limit of detection (LOD). The LODs of BPA, BPF, BPS and BPAF were 0.050 µg L-1, 0.005 µg L-1, 0.010 µg L-1 and 0.010 µg L-1, respectively.

27



Page 28 of 33

Figure Caption

Figure 1 Mass spectra of product-ions and proposed fragmentation pathway of BPB-diDNS (A), BPB-diDMIS-4 (B), BPB-diDMIS-5 (C) and BPB-diPS (D).

Figure 2 Effects of different reaction condition including solvent dissolved PS-Cl, PS-Cl concentration, pH of buffer, reaction temperature and time on the responses of BPs-diPS. Ratios between maximum and minimum responses under each condition were plotted (A). Responses of a mix of BPs-diPS (0.5 µg L-1) by using acetone and acetonitrile as dissolved solvents for PS-Cl (B). Responses of BPC-diPS and chloro-BPS-diPS (0.5 µg L-1) under the reaction condition of 5 mg mL-1 PS in acetone and 100 mmol L-1 sodium bicarbonate incubated at 60°C for 5 min (C). Each bar represents the mean values ± standard deviation for three replicates.

Figure 3 Recoveries (1) and matrix effects (2) of BPs using different purification methods. A: no purification; B: purified by n-hexane; C: purified by freezing out method; D: purified by PRiME HLB; E: purified by EMR-lipid dSPE. Each bar represents the mean values ± standard deviation for three replicates.

28


Page 29 of 33


29



Page 30 of 33

Figure 1 Mass spectra of product-ions and proposed fragmentation pathway of BPB-diDNS (A), BPB-diDMIS-4 (B), BPB-diDMIS-5 (C) and BPB-diPS (D).

30


Page 31 of 33


Figure 2 Effects of different reaction condition including solvent dissolved PS-Cl, PS-Cl concentration, pH of buffer, reaction temperature and time on the responses of BPs-diPS. Ratios between maximum and minimum responses under each condition were plotted (A). Responses of a mix of BPs-diPS (0.5 µg L-1) by using acetone and acetonitrile as dissolved solvents for PS-Cl (B). Responses of BPC-diPS and chloro-BPS-diPS (0.5 µg L-1) under the reaction condition of 5 mg mL-1 PS in acetone and 100 mmol L-1 sodium bicarbonate incubated at 60°C for 5 min (C). Each bar represents the mean values ± standard deviation for three replicates.

31



Page 32 of 33

Figure 3 Recoveries (1) and matrix effects (2) of BPs using different purification methods. A: no purification; B: purified by n-hexane; C: purified by freezing out method; D: purified by PRiME HLB; E: purified by EMR-lipid dSPE. Each bar represents the mean values ± standard deviation for three replicates.

32


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TOC Graphic

33


Highly Sensitive and High-Throughput Method for the Analysis of

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