Gas Purge Microextraction Coupled with Stable Isotope Labeling

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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

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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

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ACS Paragon Plus Environment


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ABSTRACT

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A green, sensitive and accurate method was developed for the extraction and determination of

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bromophenols (BPs) from aquatic products by using organic solvent-free gas purge microsyringe

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extraction (GP–MSE) technique in combination with stable isotope labeling (SIL) strategy. BPs

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were extracted by NaHCO3 buffer solution with recoveries varying from 92.0% to 98.5%. The

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extracted solution was analyzed by SIL strategy during which analytes and standards were labeled

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by 10-methyl-acridone-2-sulfonyl chloride (d0-MASC) and its deuterated counterpart d3-MASC,

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respectively. The labeling reaction was finished within 10 min with good stability. The liquid

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chromatography-tandem mass spectrometry (HPLC–MS/MS) sensitivity of BPs was greatly

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enhanced due to the mass-enhancing property of MASC, while the matrix effect was effectively

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minimized by the SIL strategy. The limits of detection (LODs) were in the range of 0.10–0.30

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µg/kg, while the limits of quantitations (LOQs) were in the range of 0.32–1.0 µg/kg. The proposed

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method also showed great potential in the qualitative analysis of other bromophenols in the

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absence of standard.

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KEYWORDS: Bromophenols; GP–MSE; Stable isotope labeling; Aquatic products

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INTRODUCTION

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It was reported that about thirty-nine percent of flame retardants (FRs) is based on bromine.

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Bromophenols (BPs) are used as precursors in the synthesis of brominated flame retardants

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(BFRs). They can be formed through the photochemical degradation of BFRs in water or from the

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degradation of various product containing BFRs.1, 3 BPs themselves might also be used as FRs or

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exist as impurities in BFR technical formulations 3 Besides, the dietary components of aquatic

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animals such as marine algae, cyanobacteria and polychaetes are also BPs-producing species.4

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Possessing high lipophilicity and relatively high solubility in water, BPs are widely distributed in

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the environment and even in human body.1, 5 Due to the biological amplification effect, wild

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seafood which is preferred by consumers usually contains certain level of BPs and shows typical

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sea-like taste and flavor.6, 7 In contrast, cultivated species have a much lower BPs level and thus a

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bland flavor.8,9

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When BPs were in high level, undesirable flavor and toxicity appeared. Studies indicated that high

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concentration of BPs showed inductive effect on aromatase activity.10 Therefore, the level of BPs

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in aquatic products can be used not only as an indicator of the contamination degree of BFRs in

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marine, but also as a metric for the quality control of aquatic products.

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However, seafood flavor and quality is not always proportional to BPs content.

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European Food Safety Authority (EFSA) identified a no-observed-adverse-effect level

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(NOAEL) of 100 mg/kg b.w. per day for 2,4,6-tribromophenol (2,4,6-TBP).11 Due to the lack of

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BPs level and toxicity data, at present there is no regulation on BPs concentrations in aquatic

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product.11 However, BPs were still under supervision in many country. For example, BPs in

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aquatic products were monitored in the form of volatile phenol in China.12 The information of BPs

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level in seafood is useful for the comprehensive safety evaluation of BPs and is demanded by food

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authorities such as EFSA.11 In this study, 5 BPs which were detected in high frequency were

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chosen as analytes.

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BPs in biological samples were usually extracted by steam distillation which is materials and

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time consuming, but the recoveries of 4-bromophenol (4-BP) were usually less than 40%.4, 8 Gas

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purge microsyringe extraction (GP–MSE) distinguished itself from various solid sample extraction

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methods by high extraction efficiency and environmentally friendly property.13-15 Samples were

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placed at the bottom of a heated metal bottle. Inert gas continuously passed through and carried

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the evaporated analytes to the microsyringe barrel. Meanwhile analytes were trapped and

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concentrated by a little solvent in the microsyringe barrel. Considering the volatile property of BPs,

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it is possible to extract BPs in biological samples by the green and simple GP–MSE method.

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Matrix effect was often observed in liquid chromatography-tandem mass spectrometry

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(HPLC–MS/MS) analysis of biological samples.16, 17 Stable isotope labeling (SIL) strategy has

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been proved to be effective in overcoming matrix effect and ionization differences which often

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occur in mass spectrometry (MS) analysis.18-25 Instead of synthesizing an isotope analogy of each

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analyte, SIL strategy introduces a light isotope tag to the analyte in sample and a heavy isotope tag

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to the same analyte in standard, followed by mixing the two labeled samples for MS analysis. The

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isotopic labeled analyte pair coelute and have identical retention times in MS analysis. Therefore,

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matrix effects and ionization efficiencies between samples and standards can be expected to be the

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same. In addition, mass responses of the analytes are also enhanced through the introduction of

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permanently charged moieties or easily protonated moieties into analyte molecules.19, 26 To out

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knowledge, SIL strategy has not been applied to the MS quantitation of BPs yet.

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In this study, we aimed to establish a green and accurate method for the extraction and

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determination of BPs in biological samples by the combination of GP–MSE technique and SIL

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strategy. No organic solvent will be used in the pretreatment procedure but the recoveries will be

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effectively enhanced due to the application of GP–MSE technique. Matrix effect which was the

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common problem of MS analysis can be minimized by the SIL strategy using

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10-methyl-acridone-2-sulfonyl chloride (d0-MASC) and its deuterated counterpart d3-MASC as

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labeling reagent. Meanwhile, the MS sensitivity of BPs will also be enhanced because MASC

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can form a stable quaternary ammonium ion. Besides, the specific fragmentation character of

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MASC can be well applied in the qualitative analysis of BPs in the absence of standard. The

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proposed method provides a useful tool for the analysis and safety evaluation of BPs.

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MATERIALS AND METHODS

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Chemicals. Analytical standards of 2-bromophenol (2-BP), 4-bromophenol (4-BP),

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2,6-dibromophenol (2,6-DBP), 2,4-dibromophenol (2,4-DBP), and 2,4,6-TBP were all obtained

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from Sigma-Aldrich (USA) with purity > 99%. Methanol and acetonitrile were of HPLC grade

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and purchased from Sigma-Aldrich (USA). Pure distilled water was purchased from Watsons

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(Guangzhou, China). All other reagents used were of HPLC grade or at least of analytical grade.

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Individual stock solutions of 100 mg/L for all analytes were prepared in methanol and stored

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at 4 °C in the dark. Working solutions of all compounds and calibration concentrations were

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prepared by appropriate dilution of the stock solutions on the day of analysis. d0-MASC or

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d3-MASC were synthesized in authors’ laboratory according to the method described in our

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previous study.18 MASC solution of 5.0×10-4 mol/L was prepared by dissolving 1.5 mg MASC in

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10 mL of anhydrous acetonitrile. When not in use, all reagent solutions were stored at 4 °C in a

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refrigerator.

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Sample preparation. Prawn and fish samples were all purchased from Rizhao fishery market

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(Rizhao, Shandong province, China). Muscle tissues of aquatic products were homogenized in a

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blender.

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Sample extraction. As shown in Figure 1, the home-made GP–MSE system consists of a gas

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flow system, a 50 mL aluminum bottle (3.0 cm i.d., 7.5 cm length, Kangkede packaging company,

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Ningbo, China) and a 1.0 mL syringe (0.6×25 TWLB, Xuancheng Jiangnan Medical Instrument

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Co., Ltd. China) with 0.4 mL 0.2 mol/L NaHCO3 buffer (pH 10) solution it. The aluminum bottle

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was placed in a methyl silicone oil bath heated to 250 oC by a 100 mL SXKW lab digital heating

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mantle (Beijing ever light medical equipment Co., Ltd. China). Under this condition, the

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determined temperature inside the aluminum bottle was 240 oC. Aquatic sample of 0.5 g was

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placed into the bottom of the aluminum bottle. The evaporated analytes were carried by nitrogen

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gas at flow rate of 2.5 mL/min to the NaHCO3 extraction solvent in the syringe. After 30 min

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extraction, the extracted solution was ready for later derivatization.

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Derivatization procedure. After extraction, the extracted solution, 150 µL d0-MASC solution

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and 200 µL acetonitrile were added in a 2.0 mL screw cap vial ((1.0 cm i.d., 3.2 cm length, Agilent

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Company, USA). The vial was then sealed and allowed to react in a water bath at 65 oC for 10 min.

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Standard samples were derivatized under the same conditions with d3-MASC as labeling reagent.

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The derivatization scheme is shown in Figure 2. After the completion of the derivatization, light

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labeled sample and heavy labeled standard were mixed and diluted to 1.5 mL by acetonitrile. After

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syringe filtered using a 0.22 µm nylon filter, the diluted solution was ready for HPLC analysis.

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HPLC–MS/MS analysis. BPs were separated on an Agilent 1290 series HPLC system

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equipped with a SB C18 column (2.1×50 mm, 1.8 µm i.d., Agilent, USA). An Agilent 6460 Triple

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Quadrupole MS/MS system (Agilent, USA) was used as detector. Mobile phase A was 0.1%

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formic acid in 5% acetonitrile and B was 0.1% formic acid in acetonitrile. The flow rate was 0.3

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mL/min and the column temperature was kept at 30 °C. The elution conditions were as follows:

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40–60% B from 0 to 8 min; 60–90% B from 8 to 10 min. The HPLC–MS/MS was operated in the

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“to waste mode” for the first 3 min because the eluent was mainly composed of excess labeling

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reagent. After 3 min, the eluent was converted to MS. The injection volume was 2 µL. The mass

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spectrometer was operated in a positive ion mode for the monitoring of [M+H] + by an Agilent Jet

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Stream electrospray ionization source (ESI source). The optimal ESI source conditions were:

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capillary voltage +4.0 kV; nebulizer 40 psi; dry gas 11.0 L/min; dry temperature 300 ◦C; Sheath

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gas temperature 280 ◦C; Sheath gas flow 10 L/min. The multiple reaction monitoring (MRM)

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parameters of the target compounds are listed in Table 1.

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Method validation. The proposed method was validated by linearity, limit of detection (LOD),

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limit of quantitation (LOQ), recoveries and precision. Calibration curves were constructed by

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comparing theoretic peak area ratios of d0-/d3-MASC derivatives with the experimental peak area

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ratios. LODs and LOQs were calculated at a signal-to-noise (S/N) ratio of 3 and 10, respectively.

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Recoveries were carried out by spiking blank samples with 1.0, 5.0 and 10 µg/kg of standard

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solutions. Intra-day precision was determined by analyzing samples spiked at the same three levels

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of standards with six replicates, and inter-day precision was determined by running samples with

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spiked standards at the same levels with three replicates on three different days over a period of

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one week.

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RESULT AND DISCUSSION 7



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Extraction of BPs from biological samples by GP–MSE. Due to the volatile property of

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BPs, steam distillation extraction (SDE) was the often used extraction method. However, the SDE

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method is time and material consuming. Moreover, the recoveries of BPs with boiling points of

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higher than 200oC were not satisfying. For example, the reported recoveries of 4-BP were usually

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Gas Purge Microextraction Coupled with Stable Isotope Labeling

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