Subscriber access provided by UNIV OF NEWCASTLE
Article
Gas Purge Microextraction Coupled with Stable Isotope Labeling-Liquid Chromatography/Mass Spectrometry for the Analysis of Bromophenols in Aquatic Products Shijuan Zhang, Qiuhui Yu, Cuncun Sheng, and Jinmao You J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04104 • Publication Date (Web): 19 Nov 2016 Downloaded from http://pubs.acs.org on November 20, 2016
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 27
Journal of Agricultural and Food Chemistry
Gas
Purge
Microextraction
Coupled
with
Stable
Isotope
Labeling-Liquid
Chromatography/Mass Spectrometry for the Analysis of Bromophenols in Aquatic Products Shijuan Zhang, *, †,‡, Qiuhui Yu,†,‡ Cuncun Sheng,†,‡ Jinmao You *, †,‡,§ †
Shandong Province Key Laboratory of Life-Organic Analysis, Qufu Normal University, Qufu, PR
China ‡
Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, Qufu
Normal University, Qufu, PR China
§
Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese
Academy of Science, Xining, PR China
Correspondence:
Dr. Shijuan Zhang and Prof. Jinmao You, Shandong Province Key
Laboratory of Life-Organic Analysis, Qufu Normal University, Qufu, PR China E-mail:
[email protected](S. Zhang);
[email protected] (J. You)
Tel.:+86 537 4456305
Fax: +86 537 4456305
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
ABSTRACT
2
A green, sensitive and accurate method was developed for the extraction and determination of
3
bromophenols (BPs) from aquatic products by using organic solvent-free gas purge microsyringe
4
extraction (GP–MSE) technique in combination with stable isotope labeling (SIL) strategy. BPs
5
were extracted by NaHCO3 buffer solution with recoveries varying from 92.0% to 98.5%. The
6
extracted solution was analyzed by SIL strategy during which analytes and standards were labeled
7
by 10-methyl-acridone-2-sulfonyl chloride (d0-MASC) and its deuterated counterpart d3-MASC,
8
respectively. The labeling reaction was finished within 10 min with good stability. The liquid
9
chromatography-tandem mass spectrometry (HPLC–MS/MS) sensitivity of BPs was greatly
10
enhanced due to the mass-enhancing property of MASC, while the matrix effect was effectively
11
minimized by the SIL strategy. The limits of detection (LODs) were in the range of 0.10–0.30
12
µg/kg, while the limits of quantitations (LOQs) were in the range of 0.32–1.0 µg/kg. The proposed
13
method also showed great potential in the qualitative analysis of other bromophenols in the
14
absence of standard.
15
KEYWORDS: Bromophenols; GP–MSE; Stable isotope labeling; Aquatic products
16 17 18 19 20 21 22 23 2
ACS Paragon Plus Environment
Page 2 of 27
Page 3 of 27
Journal of Agricultural and Food Chemistry
24
INTRODUCTION
25
It was reported that about thirty-nine percent of flame retardants (FRs) is based on bromine.
26
Bromophenols (BPs) are used as precursors in the synthesis of brominated flame retardants
27
(BFRs). They can be formed through the photochemical degradation of BFRs in water or from the
28
degradation of various product containing BFRs.1, 3 BPs themselves might also be used as FRs or
29
exist as impurities in BFR technical formulations 3 Besides, the dietary components of aquatic
30
animals such as marine algae, cyanobacteria and polychaetes are also BPs-producing species.4
31
Possessing high lipophilicity and relatively high solubility in water, BPs are widely distributed in
32
the environment and even in human body.1, 5 Due to the biological amplification effect, wild
33
seafood which is preferred by consumers usually contains certain level of BPs and shows typical
34
sea-like taste and flavor.6, 7 In contrast, cultivated species have a much lower BPs level and thus a
35
bland flavor.8,9
36
When BPs were in high level, undesirable flavor and toxicity appeared. Studies indicated that high
37
concentration of BPs showed inductive effect on aromatase activity.10 Therefore, the level of BPs
38
in aquatic products can be used not only as an indicator of the contamination degree of BFRs in
39
marine, but also as a metric for the quality control of aquatic products.
1, 2
However, seafood flavor and quality is not always proportional to BPs content.
40
European Food Safety Authority (EFSA) identified a no-observed-adverse-effect level
41
(NOAEL) of 100 mg/kg b.w. per day for 2,4,6-tribromophenol (2,4,6-TBP).11 Due to the lack of
42
BPs level and toxicity data, at present there is no regulation on BPs concentrations in aquatic
43
product.11 However, BPs were still under supervision in many country. For example, BPs in
44
aquatic products were monitored in the form of volatile phenol in China.12 The information of BPs
45
level in seafood is useful for the comprehensive safety evaluation of BPs and is demanded by food
3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
46
authorities such as EFSA.11 In this study, 5 BPs which were detected in high frequency were
47
chosen as analytes.
48
BPs in biological samples were usually extracted by steam distillation which is materials and
49
time consuming, but the recoveries of 4-bromophenol (4-BP) were usually less than 40%.4, 8 Gas
50
purge microsyringe extraction (GP–MSE) distinguished itself from various solid sample extraction
51
methods by high extraction efficiency and environmentally friendly property.13-15 Samples were
52
placed at the bottom of a heated metal bottle. Inert gas continuously passed through and carried
53
the evaporated analytes to the microsyringe barrel. Meanwhile analytes were trapped and
54
concentrated by a little solvent in the microsyringe barrel. Considering the volatile property of BPs,
55
it is possible to extract BPs in biological samples by the green and simple GP–MSE method.
56
Matrix effect was often observed in liquid chromatography-tandem mass spectrometry
57
(HPLC–MS/MS) analysis of biological samples.16, 17 Stable isotope labeling (SIL) strategy has
58
been proved to be effective in overcoming matrix effect and ionization differences which often
59
occur in mass spectrometry (MS) analysis.18-25 Instead of synthesizing an isotope analogy of each
60
analyte, SIL strategy introduces a light isotope tag to the analyte in sample and a heavy isotope tag
61
to the same analyte in standard, followed by mixing the two labeled samples for MS analysis. The
62
isotopic labeled analyte pair coelute and have identical retention times in MS analysis. Therefore,
63
matrix effects and ionization efficiencies between samples and standards can be expected to be the
64
same. In addition, mass responses of the analytes are also enhanced through the introduction of
65
permanently charged moieties or easily protonated moieties into analyte molecules.19, 26 To out
66
knowledge, SIL strategy has not been applied to the MS quantitation of BPs yet.
67
In this study, we aimed to establish a green and accurate method for the extraction and
4
ACS Paragon Plus Environment
Page 4 of 27
Page 5 of 27
Journal of Agricultural and Food Chemistry
68
determination of BPs in biological samples by the combination of GP–MSE technique and SIL
69
strategy. No organic solvent will be used in the pretreatment procedure but the recoveries will be
70
effectively enhanced due to the application of GP–MSE technique. Matrix effect which was the
71
common problem of MS analysis can be minimized by the SIL strategy using
72
10-methyl-acridone-2-sulfonyl chloride (d0-MASC) and its deuterated counterpart d3-MASC as
73
labeling reagent. Meanwhile, the MS sensitivity of BPs will also be enhanced because MASC
74
can form a stable quaternary ammonium ion. Besides, the specific fragmentation character of
75
MASC can be well applied in the qualitative analysis of BPs in the absence of standard. The
76
proposed method provides a useful tool for the analysis and safety evaluation of BPs.
77
MATERIALS AND METHODS
78
Chemicals. Analytical standards of 2-bromophenol (2-BP), 4-bromophenol (4-BP),
79
2,6-dibromophenol (2,6-DBP), 2,4-dibromophenol (2,4-DBP), and 2,4,6-TBP were all obtained
80
from Sigma-Aldrich (USA) with purity > 99%. Methanol and acetonitrile were of HPLC grade
81
and purchased from Sigma-Aldrich (USA). Pure distilled water was purchased from Watsons
82
(Guangzhou, China). All other reagents used were of HPLC grade or at least of analytical grade.
83
Individual stock solutions of 100 mg/L for all analytes were prepared in methanol and stored
84
at 4 °C in the dark. Working solutions of all compounds and calibration concentrations were
85
prepared by appropriate dilution of the stock solutions on the day of analysis. d0-MASC or
86
d3-MASC were synthesized in authors’ laboratory according to the method described in our
87
previous study.18 MASC solution of 5.0×10-4 mol/L was prepared by dissolving 1.5 mg MASC in
88
10 mL of anhydrous acetonitrile. When not in use, all reagent solutions were stored at 4 °C in a
89
refrigerator.
5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
90
Sample preparation. Prawn and fish samples were all purchased from Rizhao fishery market
91
(Rizhao, Shandong province, China). Muscle tissues of aquatic products were homogenized in a
92
blender.
93
Sample extraction. As shown in Figure 1, the home-made GP–MSE system consists of a gas
94
flow system, a 50 mL aluminum bottle (3.0 cm i.d., 7.5 cm length, Kangkede packaging company,
95
Ningbo, China) and a 1.0 mL syringe (0.6×25 TWLB, Xuancheng Jiangnan Medical Instrument
96
Co., Ltd. China) with 0.4 mL 0.2 mol/L NaHCO3 buffer (pH 10) solution it. The aluminum bottle
97
was placed in a methyl silicone oil bath heated to 250 oC by a 100 mL SXKW lab digital heating
98
mantle (Beijing ever light medical equipment Co., Ltd. China). Under this condition, the
99
determined temperature inside the aluminum bottle was 240 oC. Aquatic sample of 0.5 g was
100
placed into the bottom of the aluminum bottle. The evaporated analytes were carried by nitrogen
101
gas at flow rate of 2.5 mL/min to the NaHCO3 extraction solvent in the syringe. After 30 min
102
extraction, the extracted solution was ready for later derivatization.
103
Derivatization procedure. After extraction, the extracted solution, 150 µL d0-MASC solution
104
and 200 µL acetonitrile were added in a 2.0 mL screw cap vial ((1.0 cm i.d., 3.2 cm length, Agilent
105
Company, USA). The vial was then sealed and allowed to react in a water bath at 65 oC for 10 min.
106
Standard samples were derivatized under the same conditions with d3-MASC as labeling reagent.
107
The derivatization scheme is shown in Figure 2. After the completion of the derivatization, light
108
labeled sample and heavy labeled standard were mixed and diluted to 1.5 mL by acetonitrile. After
109
syringe filtered using a 0.22 µm nylon filter, the diluted solution was ready for HPLC analysis.
110
HPLC–MS/MS analysis. BPs were separated on an Agilent 1290 series HPLC system
6
ACS Paragon Plus Environment
Page 6 of 27
Page 7 of 27
Journal of Agricultural and Food Chemistry
111
equipped with a SB C18 column (2.1×50 mm, 1.8 µm i.d., Agilent, USA). An Agilent 6460 Triple
112
Quadrupole MS/MS system (Agilent, USA) was used as detector. Mobile phase A was 0.1%
113
formic acid in 5% acetonitrile and B was 0.1% formic acid in acetonitrile. The flow rate was 0.3
114
mL/min and the column temperature was kept at 30 °C. The elution conditions were as follows:
115
40–60% B from 0 to 8 min; 60–90% B from 8 to 10 min. The HPLC–MS/MS was operated in the
116
“to waste mode” for the first 3 min because the eluent was mainly composed of excess labeling
117
reagent. After 3 min, the eluent was converted to MS. The injection volume was 2 µL. The mass
118
spectrometer was operated in a positive ion mode for the monitoring of [M+H] + by an Agilent Jet
119
Stream electrospray ionization source (ESI source). The optimal ESI source conditions were:
120
capillary voltage +4.0 kV; nebulizer 40 psi; dry gas 11.0 L/min; dry temperature 300 ◦C; Sheath
121
gas temperature 280 ◦C; Sheath gas flow 10 L/min. The multiple reaction monitoring (MRM)
122
parameters of the target compounds are listed in Table 1.
123
Method validation. The proposed method was validated by linearity, limit of detection (LOD),
124
limit of quantitation (LOQ), recoveries and precision. Calibration curves were constructed by
125
comparing theoretic peak area ratios of d0-/d3-MASC derivatives with the experimental peak area
126
ratios. LODs and LOQs were calculated at a signal-to-noise (S/N) ratio of 3 and 10, respectively.
127
Recoveries were carried out by spiking blank samples with 1.0, 5.0 and 10 µg/kg of standard
128
solutions. Intra-day precision was determined by analyzing samples spiked at the same three levels
129
of standards with six replicates, and inter-day precision was determined by running samples with
130
spiked standards at the same levels with three replicates on three different days over a period of
131
one week.
132
RESULT AND DISCUSSION 7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
133
Extraction of BPs from biological samples by GP–MSE. Due to the volatile property of
134
BPs, steam distillation extraction (SDE) was the often used extraction method. However, the SDE
135
method is time and material consuming. Moreover, the recoveries of BPs with boiling points of
136
higher than 200oC were not satisfying. For example, the reported recoveries of 4-BP were usually
137
