Facile Preparation of Dual-Shell Novel CovalentâOrganic Framework

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Food and Beverage Chemistry/Biochemistry

Facile preparation of dual-shell novel COFs functionalized magnetic nanospheres and used for the simultaneous determination of fourteen trace Heterocyclic aromatic amines in nonsmokers and smokers of cigarettes with different tar yields based on UPLC-MS/MS Wenfen Zhang, Lan Chen, Huimin Zhang, Yanhao Zhang, Wenjing Zhang, Wuduo Zhao, Connor Johnson, Kai Hu, Fuwei Xie, and Shusheng Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06372 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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Journal of Agricultural and Food Chemistry

Facile preparation of dual-shell novel COFs functionalized magnetic nanospheres and used for the simultaneous determination of fourteen trace Heterocyclic aromatic amines in nonsmokers and smokers of cigarettes with different tar yields based on UPLC-MS/MS ＊a

Wenfen Zhang,a,b Chen Lan,a Huimin Zhang,a Yanhao Zhang,c Wenjing Zhang, ＊ Kai Hu,f Fuwei Xie,g Shusheng Zhang, a, d

Wuduo Zhao,d Connor Johnson,e

a College

of Chemistry and Molecular Engineering, Zhengzhou University, Kexue Avenue 100, Zhengzhou, Henan 450001, P. R. China. b Mechanical Engineering and Materials Science, Duke University, Durham, NC 27708, USA State c Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, 999077 d Center of Advanced Analysis and Computational Science, Key Laboratory of Molecular Sensing and Harmful Substances Detection Technology, Zhengzhou University, Kexue Avenue 100, Zhengzhou, Henan 450001, P. R. China. e Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA f Henan University of Traditional Chinese Medicine, Zhengzhou, 450008, P.R. China g Zhengzhou Tobacco Research Institute of CNTC, Fengyang Avenue, Zhengzhou, Henan 450001, P. R. China.

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Declarations of interest: none

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*Corresponding authors at:

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Zhengzhou University, 100 Kexue Avenue, Hi-tech District, Zhengzhou 450001, P.R. China, Email:

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[email protected], Telephone number: +8613513801442, Fax: +860 371 67763224, (Shusheng

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Zhang).

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Zhengzhou University, 100 Kexue Avenue, Hi-tech District, Zhengzhou 450001, P.R. China, Email:

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[email protected] , Telephone number: +8618625775683, Fax: +86 0371 67763224, (Wenjing

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Zhang).

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Abstract

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The facile preparation, characterization and application of novel dual-shell TpBD (a kind of COFs)

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coated magnetic nanospheres (TpBD) as sorbents for simple, fast and high selectivity capture of

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fourteen heterocyclic aromatic amines are reported. Quantum chemistry theory calculation was

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conducted to directly and quantifiably describe the multiple interactions, including π- π, hydrogen

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bonding, cation- π, static electricity and ion-exchange, between TpBD and heterocyclic aromatic

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amines. The excellent adsorption capacity of TpBD coated magnetic nanospheres was further

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evaluated by extraction of fourteen HAAs from non-smokers’ and smokers’ urine samples. Under

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the optimized conditions, the magnetic solid phase extraction process can be completed with high

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recovery range from 95.4% to 129.3%. After being washed with acetonitrile and water

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successively, the collected sorbents can be easily recycled and reused for five times without any

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significant difference in performance. Coupled with the ultra performance liquid chromatography-

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tandem mass spectrometer detection, the exposure level of HAAs in non-smokers and smokers

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smoking cigarettes with different tar yields were successfully explored. And it implied that the

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robust method based on the versatile TpBD coated dual-shell magnetic nanospheres sorbents

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represent a great potential application in the analysis of disease markers and body fluids.

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Keywords: Magnetic nanospheres, Heterocyclic aromatic amines, Urine, Tar yields

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1. Introduction

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Heterocyclic aromatic amines (HAAs) are the pyrolysis products of proteins and amino acids,

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which can be found in many kinds of coffee, fried food, environmental waters, smokers’ urines.

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Up to now, about thirty heterocyclic amines (HAs) have been found and several of them have been

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identified as animal carcinogens1–8. These procarcinogens are metabolically activated by N-

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hydroxylation of the exocyclic amino group, resulting in the formation of intermediates that are 2 ACS Paragon Plus Environment

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toxic and critical metabolites to DNA damage. On a daily basis, people maybe frequently exposed

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to the cigarette smoke, and faced with the healthy risk from HAs. In 1962, Poindexter and

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Carpenter first reported the existence of two HAs (carbolines and β- carbolines) in cigarette smoke.

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In 1998, D Hoffmann and I Hoffmann reported eight HAAs, which were produced by the process

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of combustion or dissociation of nitrogen- and oxygen- containing compounds when the cigarette

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burned. Among these HAs, the HAAs released from the main smoking are very low, but their

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biological toxicity is relatively high9,10. Thus, it is necessary to develop a sensitive and specific

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method for analyzing HAAs during the risk assessment of HAA exposure.

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Currently the analysis of trace HAAs in bio-fluids (urine and blood) poses challenges due to the

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existence of the matrix interferences. Sample pretreatment is always required prior to analysis of

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HAAs in real samples. For example, solid phase extraction (SPE)

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(LLE)12,13, micro-SPE14, liquid membrane extraction15, tandem solvent SPE16, microwave-assisted

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solvent extraction17, alone or in combination with one or more purification steps, were developed

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by many laboratories. Most of those pretreatments are time-consuming, labor-intensive or have

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limited diffusion and low mass transfer rates. Therefore, it is important to design an effective

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pretreatment method for the concentration of HAAs from complex urine samples. Magnetic SPE

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(MSPE), a promising modification of SPE, has attracted considerable attention and is considered

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as an excellent alternative to traditional SPE on account of its limited diffusion resistance, high

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surface area and facile surface modification. At the same time, the magnetic adsorbent can be easily

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recycled using an external magnetic field, eliminating the need for additional centrifugation or

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filtration of the sample, then greatly simplifying the SPE procedure.

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Recently, magnetic nanoparticles (MNPs) based on Fe3O4 and Fe3O4@SiO2 are most widely used

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in many application fields, such as in food safety, environmental, and biomedical analysis18–21.


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, liquid-liquid extraction


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Their suitable functionalized coating on MNPs are very significant to avoid the aggregation of the

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Fe3O4 MNPs, which can also greatly be influenced by their extraction capacity and selectivity to

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the targets. For examples, covalent-organic frameworks (COFs) with excellent thermal stability,

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high porosity, high specific surface area, and low density22–24, replacing the coordination bonds in

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metal-organic frameworks (MOFs), have demonstrated the special advantages in molecular

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adsorption and separation science25–27. However, how to obtain uniform and stable MNPs

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adsorbents with higher bonding amount and less agglomeration still remain the significant

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challenge.

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Banerjee designed and synthesized a series of COFs 28, and some of them (TpPa-1 and TpBD)

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were successfully used for the separation and concentration of various targets29,30. Among them,

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TpBD exhibits a unique porous structural framework, large conjugate system and multiple

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recognition sites, including aromatic rings (hydrophobic interaction and π-π interaction), a

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macrocyclic cavity (inclusion interaction), and partially protonated bridged nitrogen atoms (anion

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exchange interaction), which is more easily to interact with positively charged HAAs containing

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at least one heterocyclic ring, as well as at least one electron donating group amine (nitrogen-

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containing) group or methyl group (Fig. S1) to form π–π, cation-π complexes31. Herein, we try

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using Fe3O4 and SiO2 as the core and the link layer and using chemically stable amorphous COFs

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(TpBD) as the adsorptive shell to construct porous dual-shell nanospheres (TpBD-DS MNS). It is

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expected that TpBD-DS MNS can be applied to effectively extract HAAs from the urine samples.

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In this work, a novel magnetic sorbent with good dispersibility, high bonding amount and stability

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were prepared and used to extract and purify fourteen trace HAAs from smoker urine samples.

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And an accurate and reproducible ultra performance liquid chromatography- tandem mass

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spectrometer (UPLC-MS/MS) method based on TpBD-DS MNS was developed and used for the


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sensitive determination of trace HAAs in smokers’ urine samples. Based on the analysis results,

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the exposure level of HAAs in non-smokers and smokers smoking cigarettes with different tar

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yields were evaluated.

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2. Experimental

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2.1Material and instruments.

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Material. Iron (III) chloride hexahydrate (FeCl3·6H2O), ethylene glycol, polyethylene glycol,

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citric acid, acetic acid, sodium acetate (NaAc), toluene, dichloromethane, ammonium hydroxide,

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1,4-dioxane, hydrochloric acid (HCl), sodium hydroxide (NaOH), ethanol, acetone, and diatomite

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were all of analytical grade and provided by Henan Chemical Reagents Company (Henan, China).

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1,3,5-triformylphloroglucinol (TFP), tetraethyl orthosilicate (TEOS), benzidine (BD), and 3-

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aminopropyltriethoxysilane (APTES) were obtained from Sigma. A Milli-Q gradient ultrapure

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water system (Millipore, USA) was used to generate ultrapure water. HPLC-grade methanol,

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acetonitrile, formic acid were purchased from J.T. Baker (Phillips-burg, NJ, USA). Creatinine Plus

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kit purchased from the company of Roche Diagnostics (Indianapolis, IN, USA). amino-

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dipyrido[1,2-a: 3', 2'-d]imidazole (Glu-P-2), 1-methyl-9H-pyrido[3,4-b]indole (Harman), 2-

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amino-1,6-dimethylimidazo[4,5-b]-pyridine (DMIP), 9H-pyrido[3,4-b]indole (Norharman), 2-

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amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-9H-pyrido[3,4-b]indole (AαC), 2-

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amino-1,3-dimethylimidazo[4,5-f] quinoline (IQ), 2-amino-3,4-dimethyl-3H-imidazoquinoline

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(MeIQ), 2-amino-3,8-dimethyl Triazolyl Quinoxaline (MeIQx), 2-Amino-3-methyl- Imidazo [4,5-

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f] quinoxaline (IQx), amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), 3-amino-1-

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methylpyrido[4,3-b]indole (Trp-P-2), 3-amino-1,4-dimethyl-5H-pyrido[3,4-b] (Trp-P-1), amino-

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6-methyldipyrido[1,2-a: 3', 2'-d]imidazole (Glu-P-1), and all the internal standard (TriMeIQx,

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PhIP-D3, AαC-15N3, Norharman-D7, MeAαC-D3) were provided by Toronto Research Chemicals 5 ACS Paragon Plus Environment


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Inc. (North York, ON, Canada).

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Urine samples. All the smoker urine samples were obtained from the Zhengzhou Tobacco

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Research Institute of China National Tobacco Corporation (CNTC), comprising 20 non-smokers

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and 60 smokers (20 smoke high-level tar cigarettes, 20 smoke middle-level tar cigarettes, 20 smoke

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low-level tar cigarettes). The tar level was determined by the bands of the cigarettes, and this

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information was provided by Zhengzhou Tobacco Research Center. The smokers usually smoked

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10–20 cigarettes per day. All volunteers were banned from eating cooked food for 2 days before

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the sample collection. Then, urine sample of each volunteer was collected over 24 h and stored at

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-40◦C until the analysis. This study was approved by the Ethics Committee of Zhengzhou

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University, and informed consent was obtained from every subject. Non-smokers are college

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students from Zhengzhou University.

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Instrumentals. A Nicolet 6700 spectrometer (Thermo Fisher, USA) was used to record the

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Fourier-transform infrared (FT-IR) spectra. Scanning electron microscopy (SEM) and EDS

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analysis were recorded by an S-4300 SEM instrument configured with an Energy Dispersive

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Spectrometer system (Zeiss/Auriga FIB, Germany). An ARL X′TRA diffractometer with Cu Kα

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radiation (ARL, Lausanne, Switzerland) was used to collect the X-ray diffraction (XRD) spectra.

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Transmission electron microscopic (TEM) images were collected from a TalosF200S TEM

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instrument (Thermo Fisher, U.S.A.). The magnetization curves were obtained at RT on a Magnetic

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Property Measurement System called MPMS3 (LDJ Electronics, USA). An Autosorb-1 MP gas

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adsorption instrument (Quantachrome, USA) was used to perform the nitrogen sorption analysis.

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2.2 UPLC-MS/MS analysis.

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The UPLC-MS/MS analysis was conducted on an LC-30AD LC equipped with a QTRAP 6500

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Triple Quad mass spectrometer (AB SCIEX, USA). An Agilent Eclipse Plus C18 column (4.6 mm 6 ACS Paragon Plus Environment

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× 100 mm, 1.8 μm) was selected as the separation column, and a good separation was achieved at

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40℃. The flow rate and sample injection volume were 300 μL/min and 5 μL, respectively. The

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mobile phase consisted of 0.1% acetic acid solution (A) and acetonitrile (B). The gradient elution

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was carried out under the following conditions: 0-2 min, 5% B; 30 min, 30% B; 31-35 min, 100%

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B; post time, 7 min.

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The optimal mass spectrometer parameters: detection mode, multiple reaction monitoring (MRM);

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ionization mode, ESI+; dry gas (N2) temperature, 550◦C; Ion Spray Voltage, 5500 V; GS1 (N2)

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GS2 (N2), Curtain gas(N2) Voltage were 45, 50 and 30 psi respectively; The value of Collision Cell

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Exit Potential and Entrance Potential were 11 V and 10 V, respectively. The optimized parameters

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of all the HAAs in MRM analysis mode we listed in table 1.

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Matrix effect: The standard stock solution of HAAs was stepwise diluted with pure solvent or

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purified urine matrix to prepare a series of standard working solutions (1 ng/mL, 0.5 ng/mL, 0.25

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ng/mL, 0.05 ng/mL, 0.005 ng/mL). After being added with internal standard, they were analyzed

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under the optimal HPLC-MS/MS condition in quintuplicate. The slope values for standard curves

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prepared in purified urine matrix and pure solvent was compared to evaluate the matrix effect.

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2.3 Preparation of TpBD-DS MNS

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Synthesis of Fe3O4 magnetic nanospheres. A solvothermal method was used to synthesize the

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Fe3O4 nanoparticles according to a modified published method32. Briefly, FeCl3·6H2O (1.35 g, 5

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mmol) was added to ethylene glycol (50 mL), and the mixture was stirred ultrasonically to make

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the solution clear. Then NaAc (3.6 g) and polyethylene glycol (1.0 g) were added and the mixture

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was stirred vigorously for 30 min. After that, the mixture was transferred to a Teflon-lined

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stainless-steel autoclave (100 mL capacity), and heated at 200 ℃ for 12 h. The dark products were

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isolated with an external magnetic field, and then washed four times with H2O and ethanol prior 7 ACS Paragon Plus Environment


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to use.

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Synthesis of Fe3O4@SiO2 MNS. Fe3O4 (2 g wet weight) was dispersed into 0.5 M citric acid

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solution (200 ml). The mixture was stirred mechanically for 12 h at 40 °C and ultrasonicated for

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10 min. The products were separated and washed several times with deionized H2O and ethanol

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alternately. The product obtained was dispersed in aqueous ethanol (ethanol : H2O = 4:1, v/v, 160

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mL), NH4OH (4 ml) was added with sonication. Finally, TEOS (2 mL) was added with mechanical

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stirring at 45 °C. After stirring for 12 h, the products were isolated and washed twice with ethanol

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and acetone, and then with deionized H2O and dried in vacuo at 40 °C for 12 h.

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Synthesis of Fe3O4@SiO2-NH2 MNS. Fe3O4@SiO2 nanospheres (2.0 g) were dispersed in 120 ml

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of freshly distilled anhydrous toluene and sonicated for 20 min. Then APTES (4 mL) was slowly

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added dropwise with mechanical stirring under N2. The mixture was heated continuously at 115 ℃

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for 12 h with stirring. The product was extracted using a magnet and washed with toluene, acetone,

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highly deionized H2O, and ethanol and then dried in vacuo at 40 °C for 12 h.

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Preparation of Fe3O4@SiO2@TpBD dual-shell MNS (TpBD-DS MNS) adsorbent

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First, 1.53 g of Fe3O4@SiO2-NH2 MNS, 0.115 g of 1,3,5-triformylphloroglucinol (Tp) and 5mL

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3M acetic acid were added into a 250 mL round bottom pressure-resistant flask. Then, 20mL of

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anhydrous 1,4-dioxane and 1,3,5- trimethylbenzene (v:v=1:1) was added to the above solution.

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The mixture was ultrasonicated for 10 min, and then flash frozen at 77 K (liquid N2 bath) and

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degassed by a vacuum pump. Then the flask was sealed off and heated for 12h at 120 °C to form

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the monomer (Tp) functional Fe3O4@SiO2. Then, after 0.115 g 1,3,5-triformylphloroglucinol and

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0.17 g benzidine were added, the mixture was re-heated at 120 °C for 3d under a vacuum. Finally,

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the product was collected by applying a magnet, and the obtained TpBD-DS MNS adsorbent was

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washed several times with toluene, 1,4-dioxane solution, methanol, acetone, water and ethanol to 8 ACS Paragon Plus Environment

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completely remove the excess reactants. The washed magnetic sorbent was dried at 60℃ under a

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vacuum for 12 h prior to use.

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2.4 Computational Details

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The structures of HAAs and TpBD were firstly optimized by using the density functional theory

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(DFT)33,34 at the B3LYP/6-31G (d, p) level of theory. Then the molecular docking study was

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conducted to help get the rational initial guesses of the complexes of various HAAs and TpBD. In

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particular, the optimized HAAs molecules were docked into the optimized TpBD molecule by

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using the AutoDock Vina program. The grid map consists of 100 × 80 × 80 points with a spacing

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of 1.0. Choosing the best scoring representative conformation as the initial structure for each

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complex, which was further optimized at the B3LYP/6-31G (d, p) level. All geometry

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optimizations were performed using the Gaussian 09 suit of program, and the harmonic frequency

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calculations were carried out at the same level of theory to help verify that all structures have no

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imaginary frequency. Given the optimized structures of complexes, the non-covalent interaction

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(NCI) analysis was performed using the Multiwfn (version 3.5) program, and the isosurface plotted

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using the VMD (version 1.9.3) program.

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2.5 MSPE procedure

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The urine was first thawed at room temperature before the MSPE. And then simple agitation and

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hydrolysis were then performed according to the previous report with a little modification12. 100

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μL HCl solution (12 mol/L) was mixed with 2 mL urine sample and heated at 70◦C for 3 h. After

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cooling, adjust the pH of the urine to 7 with 10 mol/L NaOH. Then the mixture was transferred

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into a 100 mL beaker containing 10 mg TpBD-DS MNS adsorbent. After that, 5 μL of mixed

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internal standard (TriMeIQx, MeAαC-D3, AαC-15N3, Norharman-D7, and PhIP-D3) solution (100

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ng/mL) was added and the mixture was sonicated for 1.0 min. Then, the TpBD-DS MNSs were 9 ACS Paragon Plus Environment


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isolated from the mixture using an external magnet. The supernatant was discarded. The TpBD-

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DS MNS was first washed with 2 mL water, and then sonicated in 4 mL acetonitrile containing

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300 μL 0.1% sodium hydroxide solution for 1 min to desorb the analytes. The desorption solution

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was collected into a 10 mL centrifuge tube and evaporated under a gentle stream of nitrogen gas

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at 40 ℃. The obtained residue was redissolved in 500 μL methanol. Finally, filter the solution

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using a 0.22 μm nylon filter (Agilent, USA) prior to UPLC-MS/MS.

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Recovery Test. Recovery investigation was used to validate the developed extraction method. The

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test samples were spiked at three concentration levels (10, 100 and 1000 pg/mL) by adding

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standard solution of each HAA to non-smoker urine samples. The samples were then extracted and

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analyzed as previously described. Each concentration level was conducted three replicate

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measurements. The recovery of each HAA was calculated using the equation (Cb- Ca)/Cs, where

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Cs is the spiked concentration, and Ca and Cb are the measured concentrations before and after the

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addition of the standard solution.

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Batch-to-batch reproducibility. The batch-to-batch reproducibility was investigated by the

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recovery study. Briefly, 2 mL spiked urine sample (10 pg/mL) was concentrated and analyzed in

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triplicate as previously described using the TpBD-DS MNS from three batches. The mean

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recoveries of each HAA obtained from three batches were compared, and their RSD was calculated

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correspondingly.

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Carry-over. To evaluate the carry-over effect of the MSPE procedure, the highest concentration

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spiked sample (1000 pg/mL) and a non-spiked sample were selected as the test samples. First, the

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spiked sample was extracted and analyzed as described above. The magnetic sorbent was collected

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and washed with plenty of acetonitrile and water successively. Then, a non-spiked sample was also

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extracted and analyzed as the same procedure using the collected composites. The carry-over effect


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of each HAA was calculated using the equation (Ao- An)/Ah, where Ao and An is the peak area of

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the non-spiked sample measured using the old and new magnetic sorbent, Ah is the peak area of

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the spiked sample.

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3. Result and discussion

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3.1 The synthesis and characterization of TpBD-DS MNS

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For the preparation of MNPs adsorbents, the different methods such as embedding35, layer-by-

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layer36, encapsulation 37, physical mixing38, and chemical bonding30,32,39 were developed. In this

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work, a novel dual shell TpBD-DS MNS was synthesized by using the controllable link shell

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strategy according to the previous report with some modifications30. The bonding amount and

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stability were significantly enhanced by controlling the thickness of the link layer. In the first step,

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the influence of the TEOS amount on the distribution, morphology, and bonding thickness of SiO2

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was investigated. As shown in Fig. S2, the TEOS amount can significantly affect the thickness of

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the SiO2 shell and further regulate and control the distribution and morphology of synthesized

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MNPs. And then a monomer-mediated in situ growth strategy was applied to construct magnetic

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core–shell-shell COF nanospheres. Actually, during the process of this step, the formation of COFs

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material can be affected by the concentration of 1,3,5-triformylphloroglucinol, the pressure in the

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reaction tube, the type, ratio, and the acidity of solvent, the reaction temperature, and the time,

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such that the thicknesses can be tuned using the reaction time and the closed vacuum environment

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promote the formation of crystalline structures. In order to obtain more regular, narrow pore size,

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and good dispersion magnetic COF in high yield, the reaction was conducted at 120 °C for 3d

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under a vacuum according to the literature. Moreover, excess 1,3,5-triformylphloroglucinol was

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added during the first step to form enough reaction site which can ensure the following experiment

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runs smoothly. 11 ACS Paragon Plus Environment


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The SEM and TEM were used to investigate the structure and morphology of the synthesized

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magnetic nanospheres. As we can see from Fig. 1a, the SEM image indicated that the TpBD-DS

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MNS adsorbent possesses spherical shape at about ca. 200 nm and was relatively mono dispersed,

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which may significantly increase the adsorption sites and enhanced the adsorption ability for the

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probes due to the larger specific surface area. Moreover, the TEM images (Fig. 1b and 1c) indicated

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that the dual shell of SiO2 and TpBD were bonded on the surface of Fe3O4 with a thickness of

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approximately 40 nm and 30 nm, respectively. The plate-like structure of TpBD can easily be seen

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from the TEM images, which is consistent with the literature report. What’s more, the thickness

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of the TpBD through our method is obviously thicker than the previous report26,30, which further

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ensured the excellent adsorption ability of the prepared magnetic nanospheres. In addition to this,

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as shown in Fig.1d, 1e, the Energy dispersive X-ray (EDS) analysis further confirmed the existence

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and distribution of Fe, Si, N, C and O.

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In order to further to verify the cladding of TpBD, FT-IR spectrum was used to characterize the

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nanospheres of Fe3O4@SiO2-NH2, Fe3O4@SiO2@TpBD and TpBD (Fig. 2a). In the spectrum, the

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absorption peaks at 560 cm-1 and 1100 cm-1 were attributed to the vibration of Fe-O-Fe and Si-O-

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Si, respectively, which are attributed to providing evidence for silica coating of magnetite

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nanospheres. Both TpBD and Fe3O4@SiO2@TpBD have a strong peak at ∼1582 cm-1 and a weak

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peak at ∼3100 cm-1, which can be attributed to the stretching vibration of C=C and C=C-H,

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respectively. In addition to this, the characteristic absorption peak of the FT-IR spectrum of TpBD

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and Fe3O4 @SiO2 @TpBD at ~1276 cm-1 can be attributed to the CN functional group. All this

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result further confirms that TpBD was successfully coated on the surface of Fe3O4@SiO2.

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The XRD was used to identify the crystalline structure of Fe3O4, Fe3O4@SiO2, and

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Fe3O4@SiO2@TpBD MNPs (Fig. 2b). Diffraction peaks at 30.4°, 35.6°, 43.3°,53.8, 57.3°, and


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62.8° were observed in both Fe3O4@SiO2 and Fe3O4@SiO2@TpBD MNSs, which match well with

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the bare Fe3O4. This implied that the spinel structure of magnetite was retained in the preparation

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of these MNPs. Moreover, the first and most intense peak at ∼3.5° (2θ) and a minor peak at ∼6.1°

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demonstrate that crystallinity was achieved in both TpBD and Fe3O4@SiO2@TpBD MNS.

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However, the crystallinity of Fe3O4@SiO2@TpBD MNS was significantly decreased, which may

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be due to the embedding of Fe3O4@SiO2.

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The intensity of magnetism is essential for magnetic materials separation from the liquid medium.

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Therefore, the magnetic properties of the Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2@TpBD were

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evaluated (Fig. 2c). Their hysteresis curves exhibit that all three samples are superparamagnetic

300

with no hysteresis. The saturation intensities of magnetization were 42 emu/g for the

301

Fe3O4@SiO2@TpBD, which is obviously lower than that of Fe3O4 MNS due to the nonmagnetic

302

coating of the SiO2 and TpBD. However, the saturation intensities of magnetization are enough

303

for the separation of the magnetic nanospheres from the liquid solution.

304

Nitrogen sorption analysis was also conducted to investigate the porosity of Fe3O4@SiO2@TpBD.

305

As shown in Fig. 2d, Fe3O4@SiO2@TpBD exhibits a typically reversible isotherm (I) curve, which

306

is suggestive of a microporous structure. The BET surface area is 171.367 m2 g−1, which is lower

307

than that of the TpBD porous material (295 m2·g−1) due to the doping of Fe3O4 nanospheres.

308

Nonlocal density functional theory (NLDFT) method was used to calculate the pore width of

309

TpBD with built-in Fe3O4. As we can see from Fig. 2e, the pore size distributions for magnetic

310

TpBD consisted of two parts. One part was at about 0.5383 nm due to the Fe3O4 nanospheres being

311

embedded into the TpBD network. The other one part was at about 1.3933 nm, which can be

312

attributed to the micropore structure of the TpBD network.

313

The thermogravimetric curves (Fig. 2f) was performed to evaluate the thermal stability and the



314

bonding amount of the synthesized MNPs. As shown in Fig. 2f, the weight loss for

315

Fe3O4@SiO2@TpBD was only 3.02% in the range of 44.7-300◦C. While the weight loss was

316

approximate to 26.57% in the range of 300-800◦C, which maybe attributes to the loss of organic

317

moieties on the surface of Fe3O4@SiO2 nanospheres. Thus, we can say that TpBD was successfully

318

coated on the surface of Fe3O4@SiO2 nanospheres. Moreover, the results simultaneously proved

319

that the prepared magnetic nanospheres possess higher chemical and thermal stability.

320

3.2 Molecular interaction mechanism

321

The aromatic rings, partially protonated nitrogens and macrocyclic cavity of TpBD endowed it

322

with a unique porous structural framework, large conjugate system and the multiple recognition

323

sites. HAAs are positively changed and π-electron rich chemical compounds containing at least

324

one heterocyclic ring, as well as at least one electron donating group amine (nitrogen-containing)

325

group or methyl group. Therefore, excellent adsorption ability and high selectivity of TpBD

326

towards HAAs can be anticipated based on various possible molecular interactions, including π…π

327

interaction, LP…π interaction, N-H…O hydrogen bond, C-H…π interaction.

328

In order to further confirm our inference, 6 representational HAAs were selected as the model

329

molecular and non-covalent interaction (NCI) was performed as a theoretical strategy to visualize

330

weak interactions between TpBD and HAAs. The NCI results in Fig.3 show that the π…π

331

interaction， LP…π interaction, N-H…O hydrogen bond, and C-H…π interaction play important

332

roles in the origin of the specific selectivity. Pyridine, benzene rings and partially protonated

333

nitrogens play an important role in governing the supramolecular assembly of the complexes by

334

establishing π–π interactions and hydrogen bond interaction. Moreover, the benzene, heterocyclic

335

ring, amine group and methyl group of HAAs promote the formation of N-H…O hydrogen bond,


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336

LP…π interaction, C-H…π interaction. NCI analysis clearly indicates the excellent adsorption

337

potential of TpBD towards HAAs.

338

3.3 Optimizations of the UPLC-MS/MS parameters

339

In order to get good separation and high responses for 14 HAAs, the separation parameters were

340

optimized. Firstly, various HPLC columns and the mobile phase were evaluated and optimized.

341

Four kinds of columns were tested, including Eclipse Plus C18 (4.6 mm × 100 mm, 1.8 μm),

342

Eclipse XDB-C18 (4.6 mm × 150 mm, 5 μm), ZOBAX SB C18 (3.0 mm × 100 mm, 1.8 μm) and

343

ZOBAX SB-C18 (4.6 mm × 150 mm, 5 μm). As we can see from Fig.S3, almost all of the HAAs

344

could be completely separated, with the highest responses on the column of Eclipse Plus C18.We

345

also carried out the ultra performance liquid chromatography (UPLC) separation of HAAs with

346

different mobile phases. The best separation effects with high responses were achieved using the

347

conditions described in 2.2. The chromatograms of 14 HAAs and 5 internal standard HAAs in real

348

urine sample are shown in Fig. S4.

349

3.4The optimization of products ions of each HAA

350

The product ions of each HAA and the internal standard is correlated to the selectivity and

351

sensitivity of the built method. Therefore, a product ion scan was conducted and the obtained CID

352

spectra of all the HAAs and the internal standard are listed in Fig.4. The most sensitive transitions

353

and the secondary transitions were selected for quantification and confirmation, respectively. And

354

the optimal DP and CE value, the precursor ion (m/z) and selected product ion (m/z) and are listed

355

in Table 1.

356

3.5 Optimizations of the MSPE procedure with TpBD-DS MNS



357

In this part, the main main factors affecting MSPE, including the amount of TpBD-DS MNS used,

358

pH and volume of elution, desorption time and solvent were systematically optimized in the sample

359

pretreatment step (Fig. S5, a-e). 2 mL of standard solution sample spiked with 100 pg mL−1 of

360

HAAs were selected as the test probes. As shown in Fig. S5a, the recoveries remained almost

361

constant or even declining as the amount of TpBD-DS MNS increased from 10 mg to 50 mg. Thus,

362

10 mg was enough to achieve satisfactory concentration efficiency compared with other levels. So,

363

10 mg was selected as the optimal amount of the sorbent. The kinds and composition of elution

364

are other important factors that should be optimized. As we can see from Fig. S5b, ACN containing

365

a certain amount of NaOH gave the highest recoveries. The influence of NaOH on extraction most

366

probably occurred as a consequence of the distinct interactions among HAAs, sorbent and

367

extraction solvent. On the one hand, the high pH value can affect the ionization state of the

368

functional groups existing on the adsorbent and HAAs. Then further influence the adsorption

369

performance. On the other hand, in the alkaline solvent system, hydrogen bonding interaction and

370

the strong cation-π interaction between the sorbent and HAAs could be replaced by Na+ and OH-,

371

respectively, although many other interactions are also involved and the mechanism is complex.

372

Following, the amount of NaOH added to the elution was also investigated (Fig. S5c). In this

373

section, spiked amounts of NaOH solution (1%) ranging from 50 to 300 μL were evaluated, and it

374

was found that 300 μL was the optimal spiked amount to provide higher extraction recoveries.

375

When the desorption time was changed from 0.5 min to 10 min (Fig. S5d), the recoveries increased

376

with the desorption time from 0.5 min to 3 min and decreased from 3 min to 10 min. Therefore, 3

377

min was selected to conduct the follow-up experiment. The effects of elution volume on the MSPE

378

efficiency were also optimized. As we can see in Fig. S5e, 4 mL can meet the requirement for

379

HAAs detection. In addition to this, after being washed with plenty of acetonitrile and water


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380

successively, the collected composites can be easily recycled and reused five times (Fig. S5f)

381

without any significant loss of performance.

382

3.6 Matrix effect, Linear Regression Equation, LOD and LOQ of the Method

383

The matrix effect on the results was evaluated by comparing the response of the 14 HAAs in pure

384

solvent and purified urine matrix. Because there was no blank for the urine matrix, the standard

385

addition method was selected to conduct all the comparison experiment. The obtained results

386

showed that the matrix may suppress the response of Norharman, IQ, Glu-p-1, AaC Trp-p-2, IQx,

387

Glu-p-2, MeIQ, PHIP, and enhance the response of Glu-p-1 and DMIP. To eliminate the matrix

388

effect, the internal standards method was selected to build the calibration curves of the 14 HAAs

389

in purified urine matrix and pure solvent. The standard HAAs at five different concentration added

390

with internal standard were prepared in quintuplicate. As shown in Table 2, the squared regression

391

coefficients (R2) of all HAAs vary from 0.9989 to 0.9999 in two calibration curves, and the slopes

392

of all analytes are extremely similar. The slope ratios of the two curves ranged from 0.91 to 1.28.

393

These values were all within the acceptable limits. This may be due to the matrix effect could be

394

effectively counteracted by internal standards. For simplicity, the calibration curves of the pure

395

solvent were selected for the analysis in the ensuing experiment. The limits of detection (LOD)

396

and quantification (LOQ) were determined by a signal/noise ratio (S/N) equal to 3 and 10,

397

respectively. As shown in Table 2, the LOD range for the 14 HAAs ranged from 0.14 to 0.46

398

pg/mL, and the LOQ range for the 14 HAAs ranged from 0.41 to 1.37 pg/mL. These LOD or LOQ

399

are notably lower than the previously published works by CE-MS and HPLC-MS12,40.

400

3.7 Validation of the method

401

A spiked recovery method was used to evaluate the accuracy and precision of the developed

402

method. Non-smoker urine sample spiked with standard HAAs solution at three different 17 ACS Paragon Plus Environment


403

concentration levels were concentrated and analyzed as previously described in triplicate. As we

404

can see in table 3, the recoveries at three levels ranged from 95.4% to 129.3% with a max RSD

405

value of 7.3 %. The intra-day or inter-day precision was appraised by RSDs (below 6 %) of peak

406

areas (n = 5) at 100 pg/mL. The chromatogram of spiked sample at 10 pg/mL was shown in Fig.

407

S6. Compared with the published method12, all the results indicated that the proposed MSPE-

408

UPLC-MS/MS method based on TpBD-DS MNS possessed higher recoveries and accuracy.

409

The batch-to-batch reproducibility was confirmed by the recovery study using TpBD-DS MNS

410

obtained from different batches. The standard HAAs solution at 10 pg/mL was spiked in a non-

411

smoker urine sample, then concentrated and analyzed in triplicate under the optimum extraction

412

conditions. As shown in Table S1, the RSD value (n=3) ranged from 2.17% -12.25%, which

413

indicate the good batch-to-batch reproducibility of the synthesized magnetic sorbent. We also test

414

the TpBD shell thicknesses of six batches, the results showed that the thickness ranged from 30 to

415

32 nm with an RSD value of 3.32%。

416

3.8 Carry-over effects

417

As we all know, a disadvantage inherent in the application of SPE or MSPE is the risk of carry-

418

over from the previous sample. To investigate the carry-over effect of the MSPE procedure, the

419

highest concentration spiked sample (1000 pg/mL) was first extracted, and a subsequent non-

420

spiked sample was extracted using the collected composites, which has been washed with plenty

421

of acetonitrile and water successively. The analyte areas obtained were compared, we found carry-

422

over of 14 HAAs ranged from 0.00% to 0.06%. Therefore, it is expected there will be negligible

423

interference from carry-over in the assay.

424

3.9 Determination of HAAs in urine samples

425

To investigate the effect of cigarette tar levels on HAAs exposure levels, the urine samples from 18 ACS Paragon Plus Environment

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426

and nonsmokers (20 subjects) were compared smokers smoking cigarettes containing different tar

427

levels (60 subjects). The creatinine correction method was used to normalize the different water

428

content of the subjects selected for the project. And a commercial Creatinine Plus kit was used to

429

measure the urinary creatinine. The t-test method with the data statistics software (GraphPad Prism

430

version 5.0) was used to analysis the determination results of urinary HAAs. Based on the data

431

statistics analysis results, the relation between cigarette smoking with different tar yields and

432

urinary HAAs content was evaluated. P < 0.05 was considered statistically significant.

433

According to the results, MeAaC was only detected in several samples. While as shown in Fig. S7,

434

IQ and Trp-p-2 were detected in both smoker and non-smoker samples, and the exposure levels

435

were similar in smokers and non-smokers. On the contrary, the exposure levels of MeIQx, Glu-p-

436

1, and Glu-p-2 in most smokers samples were higher than non-smoker samples, although no

437

significant influence among smoker, non-smokers and smokers smoking cigarettes with different

438

tar level was found. However, it is worth noting that the other 6 HAAs, including Harman,

439

Norharman, DMIP, AaC, IQx, MeIQ, were detected in most urine samples and obvious relationship

440

among exposure level, smoker, non-smokers and smokers smoking cigarettes with different tar

441

level can be found. As we can see from Fig.5, almost all the P values of smokers and non-smokers

442

(

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