Development of a QuEChERS-Based Method for Determination of

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Development of a QuEChERS-Based Method for the Determination of Carcinogenic 2-Nitrofluorene and 1-Nitropyrene in Rice Grains and Vegetables: A Comparative Study with Benzo[a]pyrene Kailin Deng, and Wan Chan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00051 • Publication Date (Web): 20 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017

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Journal of Agricultural and Food Chemistry

Development of a QuEChERS-Based Method for the Determination of Carcinogenic 2-Nitrofluorene and 1-Nitropyrene in Rice Grains and Vegetables: A Comparative Study with Benzo[a]pyrene

Kailin Deng† and Wan Chan *,†,‡

†

Environmental Science Programs and ‡ Department of Chemistry, The Hong Kong

University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

* Corresponding author (Tel: +852 2358-7370; Fax: +852-2358-1594; E-mail: [email protected])

1

ACS Paragon Plus Environment


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ABSTRACT

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Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) are ubiquitous

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environmental pollutants attracting increasing attention because of their potent

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mutagenicity to humans. Previous studies of nitro-PAHs focused on investigating

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their formation mechanisms and detecting them in atmospheric environment, however,

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few studies reported their occurrence in food samples and regulations on nitro-PAHs

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are still lacking. We report in this study the development and application of a Quick,

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Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method for the determination

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of nitro-PAHs in rice and vegetable samples. The analysis of the collected samples by

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the validated method revealed 1-nitropyrene and 2-nitrofluorene were widespread

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food contaminants. A comparative study with benzo[a]pyrene, the commonly used

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marker for PAH exposure, showed that carcinogenic nitro-PAHs existed in rice and

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vegetables at similar concentrations. Dietary exposure risk, which was estimated

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based on the surveillance data, suggested 3.28-5.03 ng/kg/day of nitro-PAHs exposure

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for Hong Kong citizens from rice grains and vegetables.

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Keywords: QuEChERS; Nitro-PAHs; Rice; Vegetables; HPLC-FLD; Dietary exposure

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INTRODUCTION

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Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) are ubiquitous

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environmental pollutants formed by multiple pathways,1 for example, during

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incomplete combustion of fossil fuels, nitro-PAHs are produced and emitted in the

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exhaust,2,3 while emerging evidence also suggested that polycyclic aromatic

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hydrocarbons (PAHs) in the atmosphere could react with N2O5, NO2, and NO3

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radicals to generate nitro-PAHs.4-6 Although present in smaller quantities than PAHs

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in the ambient environment, nitro-PAHs are still of concerns to human health due to

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their potent mutagenic and carcinogenic properties.7,8 For example, 1-nitropyrene, 1

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(Figure 1), a highly specific marker molecule for diesel exhaust, is classified by the

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International Agency for Research on Cancer as a probable cancer causing agent to

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humans.9

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Unlike PAHs which can only form mutagenic DNA adducts after hepatic oxidative

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activation,10 toxicology studies indicated that nitro-PAHs (e.g. 1-nitropyrene) can

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form covalently-bonded adducts with DNA through dual pathways;11 Reaction with

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2'-deoxyguanosine in DNA can occur through reactive intermediates generated from a

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ring oxidation or a nitro-reduction process.12 Having two pathways to produce

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covalently-bonded DNA adducts render nitro-PAHs more potent carcinogens than

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PAHs.

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Food ingestion has been proven to be the predominant pathway for PAHs exposure

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to non-smokers.13-15 However, previous studies of nitro-PAHs focused on atmospheric

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samples (gas-phase and particulate matter).4,16,17 Few studies reported on the

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quantitative determination of nitro-PAHs in food products, presumably due to their

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low concentrations and the complexity of the matrix.18,19 Thus, simple and effective

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methods capable of analyzing nitro-PAHs in food samples are urgently needed to

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obtain monitoring data.

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We recently developed a high-performance liquid chromatography method with

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fluorescence detection (HPLC-FLD) for the analysis of nitro-PAHs in meat

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products.20 The method utilizes Fe/H+-induced nitro-reduction to convert

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non-fluorescing nitro-PAHs to strongly fluorescing amino-PAHs for their sensitive

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detection using FLD. In this work, we modified and extended the application of the

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method to investigate the concentration of 1 and 2-nitrofluorene, 2, the model

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substances of carcinogenic nitro-PAHs,21,22 in jasmine rice and vegetables collected

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from supermarkets in Hong Kong and mainland China. Specifically, a Quick, Easy,

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Cheap, Effective, Rugged, and Safe (QuEChERS) based method was developed and

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used in this study to quantitate nitro-PAHs in food products. Compared to

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conventional gas chromatography-mass spectrometric-based methods that are

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time-demanding, labor-intensive, and solvent-consuming,23,24 the QuEChERS-based

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sample preparation takes only several minutes and uses merely 10 mL of acetonitrile

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for each sample,25 yet both methods have similar analytical sensitivities.18 With the

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goal of better understanding the relative concentration and dietary exposure risk of 4


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PAHs and nitro-PAHs in food products, a comparative analysis of benzo[a]pyrene, 3,

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the molecular indicator of carcinogenic PAH,26, 27 was also performed.

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MATERIALS AND METHODS Chemicals and Reagents. All chemicals and reagents used were of the highest

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purity available and were used without further purification. Iron powder, glacial acetic

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acid, 2-nitrofluorene, 1-nitropyrene, and benzo[a]pyrene were purchased from Sigma

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(St. Louis, MO). Primary and secondary amine (PSA) sorbent was obtained from

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Agela Technologies (Tianjin, China). HPLC-grade acetonitrile was acquired from

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Tedia (Fairfield, OH). Deionized water was further purified by a Milli-Q Ultrapure

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water purification system (Billerica, MA) and used in the entire study.

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Sample Collection. Food samples (vegetables n=18; rice, n=14) were obtained

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randomly from supermarkets in Hong Kong and Shenzhen (Guangdong, China). The

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samples were sealed in polypropylene bags immediately upon sampling. Vegetables

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and rice grains were kept at 0 oC and transported to the laboratory where they were

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stored in -20 oC freezer before being processed. Prior to analysis, the samples were

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washed with purified water (for vegetable samples), blot dried, homogenized in their

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raw state, and prepared by a QuEChERS-based method as described below.

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Sample Preparation. QuEChERS sample extraction. A dispersive solid phase extraction (d-SPE) based QuEChERS sample preparation method was developed and 5



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used in this study. In brief, 10 g of homogenized vegetable/rice samples were

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accurately weighed in a 50 mL Falcon tube. Acetonitrile (10 mL) was then added to

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the samples and the sample solution was ultrasonicated at room temperature for 30

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min. Sodium chloride (1 g) and anhydrous magnesium sulfate (4 g) were then added

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to the samples and mixed by vortex.

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d-SPE Clean-up. After centrifugation at 3800 rpm for 5 min, 1 mL of the

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supernatant was extracted and put into a 1.5 mL microcentrifuge tube containing PSA

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sorbent (30 mg) and anhydrous magnesium sulfate (150 mg). The samples after

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vortex mixing for 1 min were centrifuged at 13800 rpm for 10 min to settle the PSA.

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The supernatant was then transferred into 1.5 mL microcentrifuge tube and dried

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under N2 stream for Fe/H+ treatment.

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Fe/H+ Treatment. The sample extracts after QuEChERS extraction and d-SPE

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clean-up were treated with Fe/H+ to reduce the non-fluorescing nitro-PAHs to their

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corresponding amino-PAHs (Figure 2). Nitro-reduction of nitro-PAHs to amino-PAHs

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was performed essentially as described previously,20 with modification. In brief, the

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concentrated sample extracts were re-dissolved in 200 µL of 15% acetic acid in

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methanol (v/v) and iron powder (10 mg) was then added. The sample mixtures were

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vortexed for 20 min to reduce 1 and 2 to 1-aminopyrene, 4, and 2-aminofluorene, 5,

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respectively (Figure 2). The Fe/H+-treated samples were centrifuged at 13,800 rpm for

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10 min before the supernatant was transferred to an HPLC vial for HPLC-FLD

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analysis. 6


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HPLC-FLD Analysis. HPLC-FLD analysis was performed on an Agilent 1260

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Infinity LC system (Palo Alto, CA). Ten microliter of the Fe/H+-treated sample extract

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was injected on a 150 mm x 2.0 mm i.d., 5 µm, GraceSmart RP-18 column (Grace,

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Deerfield, IL) thermostated at 40 °C for chromatographic separation. The column was

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eluted with a binary solvent system, with solvent A being water, solvent B being

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acetonitrile. Gradient elution at constant flow rate of 0.4 mL/min was adopted, with

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the solvent gradient as follows: 30% B programmed linearly to 100% B in 12 min and

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held for another 5 min before re-conditioning. The HPLC was coupled to an Agilent

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1100 programmable FLD (Agilent Technologies, Palo Alto, CA) with the FLD

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time-programmed at excitation/emission wavelengths (λex/ λem) as follows: 0-7.5 min:

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λex 280 and λem 370nm; 7.5-10min: λex 240 and λem 435 nm; 10-15 min: λex 260 and

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λem 410 nm.

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Calibration and Method Validation. A stock solution mixture of 1-3 each at 1000

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mg/L was prepared in acetonitrile and stored at -20 °C until used. Working standard

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solution mixtures of individual analytes at 0.005, 0.01, 0.05, 0.1, 0.5, and 1.0 µg/mL

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were prepared by serial dilution of the stock solution with methanol. Working

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standard solution (100 µL) was dried under N2 gas stream, to which 200µL of 15%

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acetic acid in methanol (v/v) and 10 mg of iron powder were added. The standard

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solution mixtures were vortexed at room temperature for 20 min before being

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analyzed using the HPLC-FLD method described above. While calibration curves for

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nitro-PAHs (1 and 2) were established by plotting the peak areas of their 7



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corresponding amino-PAH derivative against the concentrations of nitro-PAH in the

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calibration standard solutions, that for 3 was established by plotting their

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corresponding peak areas against its concentration in the working standard solutions.

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The developed method was evaluated for sensitivity, precision, and recovery. The

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limit of detection (LOD) and limit of quantitation (LOQ) were established as the

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amount of the analyte that generated a signal 3 and 10 times that of the noise level at

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0.005µg/mL, respectively.28 The method precision was evaluated by analyzing rice,

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radish, and lettuce spiked with standard mixture at two different concentrations (0.5

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and 5.0 µg/kg), on the same day (n = 5) and over separate days in a month (n = 5).

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The method recovery was evaluated by spiking the nitro-PAHs and PAHs to samples

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(0.5 and 5.0 µg/kg), extracted by QuEChERS clean-up method, processed with Fe/H+,

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and analyzed by HPLC-FLD as described above. It is worth noting that the real

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samples may have lower extractability than spiked samples, so that after spiking the

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analytes, samples were mixed for 1 h before being processed to mimic the situation.

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Dietary Exposure Risk Assessment. Dietary exposure level (ED) was calculated

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based on standard Environmental Protection Agency (US EPA) methodologies,27,29,30

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by:

=

C × IR (Eqn.1)

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where Ci is the concentration of individual NPAH/PAH in food i (µg/kg);

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IR represents ingestion amount of food i per day (kg/day), BW represents body weight 8


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in kg. Food consumption and body weight data were obtained from the Hong Kong

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food consumption survey (2005-2007) based on 24-h food recalls.31 In this survey, the

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database covered adult and senior population (20-84 years old; n=5394,000), mean

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value of body weight was 61.25 kg for Hong Kong citizens. Regarding the monitoring

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data in this study, two different scenarios (lower [LB] and upper bound [UB]) related

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to the treatment of the non-detects (NDs) and values below the limit of quantitation

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(

Development of a QuEChERS-Based Method for Determination of

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