Fe3o4-Assisted Laser Desorption Ionization Mass Spectrometry For

Journal of Hazardous Materials 388 (2020) 121817

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Journal of Hazardous Materials

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Fe3O4-assisted laser desorption ionization mass spectrometry for typical T metabolite analysis and localization: Influencing factors, mechanisms, and environmental applications Wen-Wen Weia,1, Yuanhong Zhongb,1, Ting Zoua, Xiao-Fan Chena, Li Renb, Zenghua Qia, Guoguang Liua, Zhi-Feng Chena,*, Zongwei Caia,c,** a Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou, 510006, China b School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, 510006, China c State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR, China

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Editor: R. Teresa Fe3O4 has been suggested as an efficient matrix for small-molecule analysis by laser desorption ionization Keywords: mass spectrometry (LDI-MS), but thus far there has been no systematic study exploring the influencing factors

Fe3O4 of nano-Fe3O4 on the detection of typical metabolites, or the mechanism by which nano-Fe3O4 assists the Physicochemical property desorption and ionization of analytes after receiving laser energy. In this study, Fe3O4 nanoparticles with Small-molecule metabolite different physicochemical properties were synthesized and characterized. The results revealed that smaller Laser desorption ionization particle size and greater surface hydroxyl amount of nano-spherical Fe3O4 could improve the intensity and Time-of-flight mass spectrometry relative standard deviation of typical metabolites by LDI-MS. The thermally driven desorption process played

a vital role in LDI performance, but the chemical interactions between nano-Fe3O4 and analytes did not. Good

intra- or inter-spot repeatability and linearity of analytes were obtained by the optimum Fe3O4-assisted LDI- MS. Finally, the developed method was successfully used for the rapid analysis and localization of endogenous metabolites in biofluids and whole zebrafish tissue section samples. Our results not only elucidate

⁎ Corresponding author at: Guangdong University of Technology, Guangzhou, China. ⁎⁎ Corresponding author at: Hong Kong Baptist University, Hong Kong SAR, China. E-mail addresses: [email protected] (Z.-F. Chen), [email protected] (Z. Cai). 1 These authors contributed equally. https://doi.org/10.1016/j.jhazmat.2019.121817 Received 10 October 2019; Received in revised form 19 November 2019; Accepted 2 December 2019 Available online 03 December 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved. W.-W. Wei, et al. Journal of Hazardous Materials 388 (2020) 121817

the influencing factors and mechanisms of nano-Fe3O4 for the detection of typical metabolites in LDI-MS but also reveal an innovative tool for the imaging of chemicals in the regions of interest in terms of eco-toxicological research.

1. Introduction nanomaterial, nano-Fe3O4 particle size can affect the detection sensitivity of metabolites in LDI-MS (Olaitan et al., 2018), suggesting that In the 1980s, matrix-assisted laser desorption ionization (MALDI) the physicochemical properties of nano-Fe3O4 influence method sensi- was proposed as a soft ionization technique (Karas and Hillenkamp, tivity and repeatability. 1988). The first application of nanoparticles as a matrix for laser des- In this study, we attempted to use microwave-assisted and copre- orption ionization coupled with time-of-flight mass spectrometry (LDI- cipitation methods for the synthesis of nano-Fe3O4 with different phy- MS) made Koichi Tanaka a Nobel laureate (Tanaka et al., 1988). sicochemical properties (e.g., morphologies, particle sizes, and surface MALDI-MS has been successfully used to detect biomacromolecules hydroxyl amounts). Based on peak intensities and relative standard (Cornett et al., 2007; Bouslimani et al., 2015; Dallongeville et al., 2016; deviations of target analytes, an ideal matrix was selected for LDI-MS in Cravatt et al., 2007; Bruno and Ruedi, 2006), including proteins, pep- positive ionization mode. We compared the performance between as- tides, and nucleic acids, in biological samples. The remarkable ad- prepared Fe3O4-assisted LDI-MS and traditional MALDI-MS and vali- vantages of MALDI-MS include simple operation, quick analysis, high dated the repeatability and calculation curves of analytes for the de- throughput, small sample consumption, and high salt tolerance (Karas veloped method. From the results of thermal desorption calculation and et al., 1987; Min et al., 2014). Traditional organic matrices that are the UV–vis absorption spectrum, the mechanisms of the desorption and used in MALDI-MS, such as α-cyano-4-hydroxycinnamic acid (CHCA), ionization of analytes after laser irradiation were tentatively proposed. 2,5-dihydroxybenzoic acid (DHB), and sinapic acid (SA), exhibit good Finally, the developed method was successfully applied in order to performance in the analysis of biomacromolecules. However, severe identify and localize the potential metabolites in biofluids and whole background noise in the low-mass range and heterogeneous cocrys- zebrafish tissue section samples, respectively. tallization between traditional organic matrices and small-molecule analytes can limit the practicability of traditional MALDI-MS in vital 2. Experimentation endogenous metabolite analysis (Tholey and Heinzle, 2006; Weidner and Falkenhagen, 2009; Kawasaki et al., 2012; Kinumi et al., 2000; Lin 2.1. Synthesis and characterization of Fe3O4 nanoparticles et al., 2015a; Shi et al., 2017; Lopez de laorden et al., 2015). Endogenous metabolites play an essential role in the growth, de- Based on our previous work (Zhong et al., 2017), a type of nano- velopment, and reproduction of humans and other organisms (Patti spherical Fe3O4 particle (M2) was synthesized via the standard copre- et al., 2012; Shevchenko and Simons, 2010). For the accurate quanti- cipitation method, and 9 types of nano-Fe3O4 particles (M1, M3–M10) fication of metabolites with molecular weights below 1000 Da,gas with different morphologies, particle sizes, and surface hydroxyl chromatography coupled with mass spectrometry (GC–MS) (Robles amounts were synthesized using a microwave-assisted technique. M11 et al., 2017) and liquid chromatography coupled with mass spectro- was a commercial nano-Fe3O4 particle, while nano-Fe3O4 (M12) was metry (LC–MS) (Teleki and Takors, 2019) are the commonly used synthesized using a method from Yagnik et al. (Yagnik et al., 2016). The analytical instrument combinations, both of which require sample XRD patterns of the as-prepared Fe3O4 nanoparticles were obtained by preparation and extra time for chromatographic separation. In contrast an Aeris benchtop X-ray diffractometer (PANalytical B.V., Netherlands) to GC–MS and LC–MS, MALDI-MS not only enables the rapid detection with a tube voltage of 40 kV and a current of 15 mA at room tem- of analytes but also visually displays the distribution of endogenous perature. The range of angles was set from 10° to 80° (2θ) with a metabolites in tissue sections combined with an imaging technique (Liu scanning step width of 0.02° and a speed of 5°/min. The morphologies et al., 2014; Mainini et al., 2015; Wang et al., 2015; Ye et al., 2013). and particle sizes were determined from scanning electron microscope MALDI-MS imaging has shown potential for the elucidation of tox- images (SEM). SEM measurements were performed on a Hitachi icological mechanisms (Bruinen et al., 2016; Cobice et al., 2015; Liu SU8000 with 3-kV accelerating voltage and 9,400-nA emission current et al., 2017). (Hitachi Ltd., Japan). The surface hydroxyl amount of nano-Fe3O4 was In the past few years, various types of nanostructured matrices measured using a SDT Q600 simultaneous TGA-DSC thermal analyzer containing silica- and silicon-based substrates (Dupre et al., 2012; (TA Instruments, USA). The data were recorded from room temperature Zhang et al., 1999), metal nanoparticles (e.g., gold (Tang et al., 2011; to 800 °C with a heating rate of 10 °C/min and a nitrogen flow rate of Amendola et al., 2013), silver (Muller et al., 2015), and platinum(Nitta 50 mL/min. Specific heat measurements were conducted in azero et al., 2013)), metal oxides (e.g., TiO2 (Shrivas et al., 2011), Fe3O4 magnetic field with a temperature region of 271–306 K and apressure (Chiang et al., 2010; Bi et al., 2015), and ZnO (Gedda et al., 2014)), and of 9.9 × 10−6 Torr, using the specific heat option of a Quantum Design carbon-based materials (e.g., colloidal graphite (Gedda et al., 2014; Cha physical property measurement system (Quantum Design, China). The and Yeung, 2007), graphene oxide (Kim et al., 2011), and carbon dots UV–vis absorption spectrum (200−800 nm) of nano-Fe3O4 was ob- (Lin et al., 2018)) have been developed for the surface-assisted analysis tained from a MAPADA UV-3200 Spectrophotometer (Shanghai Ma- of small molecules. Due to superior optical and charge-transfer per- pada Instruments Co. Ltd., China). The detailed synthesis approaches formance, carbon-based materials (Shiea et al., 2003; Lu et al., 2016) (Text S1 and Table S1) and information (Table S2) of the nano-Fe3O4 in have attracted an enormous amount of attention. However, low solu- this study are provided in Supplementary Material. bility, poor dispersity, and carbon cluster generation hinder the utili- zation of carbon-based materials in surface-assisted laser desorption 2.2. Experimental design ionization (SALDI) (Chen et al., 2013). Yagnik et al. Yagnik et al. (2016) conducted a large-scale nanoparticle screening in order to analyze the Six metabolites with different molecular weights and categories, small molecules in LDI-MS and demonstrated the better performance of including DL-serine (SER), D-glucose (GLU), adenosine (ADE), arachidic Fe3O4-assisted LDI-MS in the positive ionization mode for sugars, amino acid (ARA), ceramide (d18:1/12:0) (CER), and triheptadecanoin (17:0/ acids, phospholipids, and glycerides. Thermally driven desorption was 17:0/17:0) (TG), were chosen as target analytes. In order to compare suggested to be a key factor for nano-Fe3O4 when compared to other the performances of different Fe3O4-assisted LDI-MS analyses with nanomaterials Yagnik et al. (2016). In addition to the type of traditional MALDI-MS, the final concentrations of each analyte in

2 W.-W. Wei, et al. Journal of Hazardous Materials 388 (2020) 121817 matrix solution were 50 mg/L. The basic information of these analytes 2.4. LDI-MS and LDI-MSI analyses is provided in Table S3. Supplier sources of chemicals and reagents can be found in Text S2. The UV–vis absorption spectrum was used to in- LDI-MS and LDI-MSI analyses were performed on an ultrafleXtreme vestigate the effects of the laser absorption capacity of3 nano-Fe O4 on II mass spectrometer (Bruker Daltonics, Germany) equipped with a 335- LDI-MS performance and to explore the affinity between matrices and nm smartbeam II laser. Mass spectra were acquired over the m/z range analytes. A modified thermal desorption model (Yagnik et al., 2016) of 100–1000 in positive ion reflector mode. Peptide Calibration was utilized to explain the performance of Fe3O4-assisted LDI-MS (Text Standard II was used for the calibration of the mass analyzer. The laser S3). In order to test method repeatability, six standard samples at parameters, including intensity and repetition, were set to 80 % and concentrations of 10 mg/L and 50 mg/L were detected 7 times in the 1000 Hz, respectively. The tissue sections were analyzed with a spatial same spot (intra-spot) and at 7 distinct spots (inter-spot). In order to test resolution of 100 μm, and 1000 laser shots per pixel were set at a full- method quantification capacity, the standard calibration curve of each scan mass spectrum. analyte was determined by analyzing the standard solution at a con- 13 centration range of 10–50 mg/L with C-Glucose (GLU 13C) as an in- 3. Results and discussion ternal standard at a concentration of 10 mg/L. For method application,

2 experiments were performed: (1) biofluid samples, including fish 3.1. Characterization of Fe3O4 nanoparticles serum, bile, and human urine, were detected by the optimum Fe3O4- assisted LDI-MS for the identification of endogenous metabolites; (2) According to the standard card of magnetite (JCPDS: 19-0629), the whole zebrafish sagittal tissue sections were analyzed using the op- XRD patterns showed that the as-prepared Fe3O4 samples had well- timum Fe3O4-assisted laser desorption ionization mass spectrometry crystallized spinel structures (Fig. S1). The SEM images revealed as- imaging (LDI-MSI) in order to map the distribution of endogenous prepared Fe3O4 with octahedral (M1, M2), spherical (M6–M11), cubic metabolites. (M3), rod-like (M4), plate-like (M5), and amorphous (M12) morphol-

ogies (Figs. 1 and S2). The mean particle sizes of as-prepared Fe3O4 2.3. Sample preparation were confirmed in the range of 19–161 nm (Table S1), indicating their nanoscale diameters. The amount of surface hydroxyl ranged from 0.18 The detailed collection procedures of the biofluid samples, in- to 7.14 % (Table S1 and Fig. S3). cluding fish serum, bile, and human urine, as well as the whole zebrafish tissue section samples, can be found in Text S4. ForLDI-MS 3.2. Optimization of physicochemical properties of nano-Fe3O4 for target analysis, Fe3O4 nanoparticles were suspended in isopropanol at the metabolite signals required concentrations. The SA, DHB, and CHCA saturated solutions (10 mg/mL) were prepared in acetonitrile/water (1:1, v/v) containing The acquired MS data demonstrated that the characteristic peaks at 0.1 % trifluoroacetic acid. Under optimum conditions, the standard m/z 128, 203, 290, 335, 504, and 871 were the sodiated adducts solution of each analyte (1 mg/mL) or biofluid sample was mixed with ([M + Na]+) of SER, GLU, ADE, ARA, CER, and TG, respectively. The the matrix solution (1:1, v/v), after which 1 μL of the mixture was pi- formation of sodiated adducts during MS ionization can be partly at- petted onto a stainless-steel sample plate, air-dried, and analyzed using tributed to the low proton affinities and high cation affinities ofana- LDI-MS. For LDI-MSI analysis, the whole zebrafish sagittal tissue section lytes (Fu et al., 2015; Han and Sunner, 2000; Ren et al., 2005). In this was sprayed with 25 mL 0.05 mg/mL as-prepared Fe3O4 solution using study, these 6 characteristic peaks were subsequently used for the a 0.2-mm nozzle airbrush (NEW-LP, China), with a nozzle-to-target analysis of target metabolites. Effects of method operations and distance of 10 cm. The final Fe3O4 material layer was approximately synthesis methods of Fe3O4 on small-molecule metabolite signals are 276.5 μg/cm2. The fish slide was dried in the open air for 15 minprior discussed in detail in Text S5. Overall, the dried-droplet preparation to LDI-MSI analysis. method, 80 % laser energy, 1 mg/mL Fe3O4, and microwave-assisted

Fig. 1. SEM images of (A) M1 and (B) M10; particle size distribution plots of (C) M1 and (D) M10; TGA-DSC plots of (E) M1 and (F) M10.

3 W.-W. Wei, et al. Journal of Hazardous Materials 388 (2020) 121817 method were selected as the optimum conditions, and used for the of analytes in LDI-MS. subsequent evaluation of the effects of nano-Fe3O4 with different phy- Nanoparticle size is closely related to the loading capacity of ana- sicochemical properties on the performance of LDI-MS (Figs. S4 and lytes. In order to assess the impact of particle size on the intensity and S5). RSD of analytes, 4 nano-spherical Fe3O4 particles with different sizes The dispersity of matrix-coating in the target plates can affect the were synthesized using a microwave-assisted method (Table S2). The performance of analytes in LDI-MS and can primarily be attributed to surface hydroxyl amounts of M6 and M8 were similar, and those of M7 the stack status of different matrix morphologies. We synthesized dif- and M10 were almost the same. It can be observed from Fig. 2A that the ferent Fe3O4 morphologies in our previous work, including nano-octa- intensities and RSDs of M6 (22 nm) and M10 (45 nm) were better than hedrons, nano-cubes, nano-rods, nano-plates, and nano-spheres. The those of M8 (45 nm) and M7 (70 nm), respectively. The small particle results revealed that the total peak intensities of analytes from Fe3O4 size increases the loading capacity of analytes, leading to increasing nano-rods (M4), nano-spheres (M6), and nano-plates (M5) were almost heat transfer from Fe3O4 to analytes during laser desorption ionization 2 or 3 times higher than those of Fe3O4 nano-cubes (M3) and nano- (Chiang et al., 2011). octahedrons (M1) (Fig. S2). There was no apparent variation among As shown in Fig. 2A, although M8, M9, and M10 have the same

M4, M5, and M6. Fe3O4 nano-plates were found to have the highest particle sizes, they have different effects on the intensity and RSDof activity of the various morphologies (Zhong et al., 2017). It is chal- analytes. These differences can be attributed to their surface hydroxyl lenging, however, to synthesize Fe3O4 nano-plates with different par- amounts. M10, with the highest surface hydroxyl amount of 4.22 %, ticle sizes and surface hydroxyl amounts. The most stable Fe3O4 mor- presented the best signal response and repeatability for LDI-MS ana- phology was reported to be the nano-sphere with the lowest energy lysis. Analyte intensity decreased with decreasing amounts of Fe3O4 (Zhong et al., 2017). As a compromise, the nano-sphere was selected as surface hydroxyl. In this study, the Fe3O4 matrix was prepared by dis- the ideal morphology of Fe3O4 for the following evaluation of the ef- solving it in isopropanol. Hydroxyl is reported to readily cover metal fects of particle size and surface hydroxyl amount on the performance oxide, which is commonly synthesized in a water medium (Blyholder

Fig. 2. (A) Effects of particle size and surface hydroxyl amount 3of nano-Fe O4 on analyte intensity. Comparison of the performances of different 3Fe O4-assisted LDI- MS analyses and traditional MALDI-MS ([Analyte] =50 mg/L); (B) mass spectra of analytes in LDI-MS with different matrices; (C) stacked column plot of analyte intensities from LDI-MS with different matrices ([Analyte] =50 mg/L); and (D) UV–vis spectra of different3 nano-Fe O4 suspensions at the same concentration of 0.1 mg/mL.

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and Richardson, 1962; Lewis and Parfitt, 1966; Yates and Lucchesi, types of nano-Fe3O4 were also used as matrices in LDI-MS (Table S2). 1961; Jr and Wickersheim, 1964). The greater the surface hydroxyl M11 was a commercial product, and M12 was synthesized by a reported amount, the better the Fe3O4 will disperse in isopropanol and on the method (Yagnik et al., 2016). The total analyte intensity in the 3 Fe3O4- LDI target plate with analytes. In addition, the surface hydroxyl may assisted LDI-MS analyses exhibited the following relative relationship: provide protons for the positive ionization of analytes and facilitate M10 > M12 > M11 (Fig. 2C). Thus, the as-obtained M10 with small ionization efficiency. Considering all of these results, M10 was selected particle size and high surface hydroxyl amount is an ideal matrix, ex- as the optimum matrix for the analysis and localization of metabolites hibiting the best overall performance for analytes in the positive ioni- in LDI-MS and LDI-MSI. zation of LDI-MS among the traditional organic matrices and nano-

Fe3O4 used in this study.

3.3. Comparison of Fe3O4-assisted LDI-MS with traditional MALDI-MS 3.4. Mechanistic studies of Fe3O4-assisted LDI-MS The background signal of M10 with no analytes in LDI-MS was determined and compared with traditional organic matrices under Once matrices receive laser irradiation from an ionization source, identical instrumental conditions (Fig. S6). CHCA, DHB, and SA pro- laser excitation will produce photo-induced electrons within them, fa- duced a large number of matrix-related peaks in the low-molecular- cilitating the ionization of nearby analytes. At the same time, these weight range of m/z 100–700. In contrast, a few small background matrices can be heated to a certain temperature, leading to the deso- signals in the low m/z range were observed in the mass spectrum of rption of nearby analytes. To investigate how laser absorption capacity

M10, for which the baseline was 3–6 times lower than those of the affects the efficiency3 ofFe O4 nanoparticle desorption and ionization, traditional organic matrices. Accordingly, M10 possesses a greater peak we assessed the full wavelength scan (200–800 nm) of 3 Fe3O4 nano- capacity to detect small-molecule analytes in biological samples. particles (M10, M11, and M12) in isopropanol solution. The results Apart from background noise, we also compared the sensitivity of revealed that M10 displayed stronger laser absorption at 355 nm than analytes obtained from traditional organic matrices with that of nano- either M11 or M12 (Fig. 2D), in agreement with the order (M10 >

Fe3O4 in LDI-MS (Figs. 2B and S7). When SA was used, most analytes M12 > M11) of total analyte intensity in Fe3O4-assisted LDI-MS could not be found in the mass spectrum. For the DHB-assisted LDI-MS (Fig. 2C). These findings indicate that laser absorption capacity ispo- measurements, the number of detected analytes increased, but the sitively related to LDI performance (Lin et al., 2015b). After laser ir- analyte intensities were far below those of the background signals. Two radiation of matrices, ionization and desorption are two essential

Fig. 3. Mechanistic study of Fe3O4-assisted LDI-MS: (A) UV–vis spectra of single analyte solution, optimum nano-Fe3O4 (M10) suspension, and analyte-M10 mixture suspension. The solid line represents the experimental data, while the dashed line represents the calculated data of the analyte-M10 mixture, which was estimated by the sum of a single analyte and M10; (B) histogram of calculated heating temperature (ΔT) and final temperature (T) produced by the optimum3 nano-Fe O4 (M10).

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processes for analytes in the ionization source. 3.5. Repeatability and calibration curves of optimum Fe3O4-assisted LDI- Laser excitation efficiently produces photo-induced free charge MS carriers or electron-hole pairs in Fe3O4 nanoparticles (Mishra et al., 2019). Good affinity at the analyte-matrix interface helps to enhance According to the results mentioned above, M10 is the optimum the rapid photo-induced electron transfer and separation capacities of matrix for LDI-MS. In order to test the method repeatability, standard photo-induced free charge carriers and electron-hole pairs (Alimpiev samples with the same concentrations, including 10 mg/L and 50 mg/L et al., 2008), facilitating the ionization of nearby analytes in Fe3O4- of each analyte, were detected 7 times in the same spot (intra-spot) and assisted LDI-MS. Analysis of the UV–vis absorption spectrum is an es- at 7 distinct spots (inter-spot). There was no obvious intensity change of sential approach for exploring structural change and complex formation any analyte on the seven replicates (Fig. 4A). The repeatability was (Dezhampanah et al., 2013; Dezhampanah and Fyzolahjani, 2013). mostly < 20 % for both intra-spot and inter-spot analyses, implying However, there was no visible red or blue shift of the maximum ab- that the developed method was robust. We conducted intra-spot ima- sorption wavelength between experimental and calculated values of ging of each analyte with different matrices. Among the traditional analyte-Fe3O4 mixtures (Fig. 3A). The analytes may be bound to the organic matrices (SA and DHB) and Fe3O4 (M10, M11, and M12), better surface of Fe3O4 by weak intermolecular interactions such as the van dispersity of M10 without larger multi-particle aggregation (Fig. 4B) der Waals force, but not strong electrical interaction (Liu et al., 2019). was found following the total intensity order of the analytes (Fig. 2C). These results demonstrate that chemical interactions between nano- These findings suggest that good dispersity of mixtures of matrices and

Fe3O4 and analytes do not play an essential role in the performance of analytes on the target plate could facilitate the performance of Fe3O4- Fe3O4-assisted LDI-MS. assisted LDI-MS. Energy transfer from matrices to analytes is considered to undergo a In order to test the potential quantification of small-molecule me- thermally driven process (Chiang et al., 2011; Alimpiev et al., 2008). A tabolites by Fe3O4-assisted LDI-MS (M10), 6 analytes at different con- modified thermal desorption model described in detail in Text S3,was centrations ranging from 10 to 50 mg/L and 1 internal standard 13 used to explain the performance of Fe3O4-assisted LDI-MS in this study (10 mg/L) were analyzed. Stable isotope labeled C-Glucose (GLU 13C) (Yagnik et al., 2016). As shown in Fig. 3B, the temperatures were was selected as the internal standard due to its similarity in chemical produced in an increasing order: M11 < M1 ≈ M4 < M12 < M10, properties to GLU as well as its commercial availability. Fig. 4C shows which was in accordance with their high LDI efficiencies (Fig. 2C). M10 the mass spectrum characteristic peaks for SER (m/z 128), GLU (m/z had the highest absorption coefficient, resulting in the highest tem- 203), ADE (m/z 290), ARA (m/z 335), CER (m/z 504), TG (m/z 871), perature (Tcal =629 K, Table S4) by laser irradiation. The thermally and GLU 13C (m/z 209). Based on the peak area ratios between the driven desorption process should be a key factor in the performance of standards and the internal standard, good linearity of the calibration 2 Fe3O4-assisted LDI-MS. curves for most analytes was obtained, with R values ranging from 0.9814 to 0.9997 (Fig. 4C), with the exception of TG, which had an R2

Fig. 4. Method performance of the optimized Fe3O4-assisted LDI-MS (M10): (A) intensities of each analyte at 10 mg/L and 50 mg/L in the intra-spots and inter-spots (n = 7); (B) intra-spot imaging of each analyte with different matrices. The intensity scale is an arbitrary relative scale ranging from red (highest) to blue (lowest);(C) calibration curves of each analyte at a concentration range of 10–50 mg/L. The lower right insets are the ion peaks of the analyte and internal standard. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

6 W.-W. Wei, et al. Journal of Hazardous Materials 388 (2020) 121817 value of 0.7785. Since the chemical structure of TG contains 3 fatty acid proposed metabolites are presented and discussed herein. chains, which bond to glycerol via ester linkages, TG is highly hydro- For the fish serum sample, the characteristic peaks at m/z 825.762 phobic and has a high molecular weight. For glucose, however, it is and 841.797 were assigned to DiMe(13,3) cholesteryl ester (CE(DiMe hydrophilic with the hydroxyl group structures. The weak linearity of (13,3))), which belongs to the class of cholesteryl esters. In a certain TG can be attributed to the improper internal standard (GLU 13C), in environment, cholesteryl esters and cholesterol can convert mutually. which the chemical properties are distinct from TG. Thus, the results Since their polarity is much less than that of free cholesterol, cholesteryl indicated the quantification potential of small-molecule metabolites by esters are used for the transport of cholesterol in plasma and the storage optimum Fe3O4-assisted LDI-MS. of cholesterol (Wishart et al., 2018). In the molecular mass range of 700–900 Da, the dominant peaks belonged to triacylglycerols (TGs). Six 3.6. Identification of endogenous metabolites in biofluid samples TGs were proposed based on their corresponding characteristic peaks (Table S5). TGs can exist in blood and become constituents of lipo- LDI-MS and LDI-MSI are effective for the rapid analysis and locali- proteins to deliver fatty acids to adipocytes. They play an essential role zation of analytes in biological samples, respectively. However, the lack in metabolism as a source of energy and as transporters of dietary fat of proper matrices is the foremost impediment in the application of LDI- (Wishart et al., 2018). In addition, D-glucose at m/z 203.092 was found, which is a principal source of energy for living organisms (Wishart MS and LDI-MSI. In this study, the optimum Fe3O4 (M10) was utilized as the matrix and then shown to successfully detect the target meta- et al., 2018). For the fish bile sample, eight characteristic peaks were bolites with different molecular weights. In order to check the prac- tentatively assigned (Table S6). It should be noted that the ion of m/z + + ticability of M10, we analyzed biofluid samples, including fish serum, 431.318 ([M+H] ) with the corresponding m/z 453.309 ([M + Na] ) bile, and human urine. In addition, we also attempted to localize me- was tentatively assigned to 7α-hydroxy-3-oxo-4-cholestenoate (7- tabolites in a whole zebrafish sagittal tissue section using optimum Hoca), which is one of the monohydroxy bile acids, alcohols, and derivatives. The presence of 7-Hoca in the fish bile proved the practic- Fe3O4-assisted LDI-MSI. Tentative assignments of characteristic peaks were made by matching measured and predicted m/z values with the ability of the optimum Fe3O4-assisted LDI-MS, since 7-Hoca is involved lowest mass error (Δppm) based on universal databases such as the in the pathway of primary bile acid biosynthesis (Wishart et al., 2018). HMDB (Wishart et al., 2018), as listed in Tables S5–S8 of the supple- Apart from the fish biological samples, human urine samples were mentary material. analyzed using the developed method. The mass range of 100–300 Da is Compared to the background baseline obtained from M10 blank, a populated by small-molecule acids, including amino acids, sulfinic large number of peaks were observed in the 3 biological samples acids, and phenylsulfates. Five proposed metabolites are listed in Table (Fig. 5A–D). A pair of characteristic peaks, in which the difference of S7. Two important amino acids were detected: homocysteine at m/z molecular weights was 22 ([M+H]+ and [M + Na]+) or 16 158.034 and 174.006, and L-tyrosine at m/z 204.063 and 220.037. ([M + Na]+ and [M + K]+), was chosen for identification. A few Homocysteine is associated with human disease. A previous study

Fig. 5. Application of the optimized Fe3O4-assisted LDI-MS and MSI. Mass spectra were acquired from (A) blank, (B) fish serum, (C) fish bile, and (D) human urine samples in the full scan mode. (E) Optical image of the whole zebrafish sagittal slice and imaging of proposed endogenous metabolites in the whole zebrafish sagittal tissue section. The intensity scale is an arbitrary relative scale ranging from color (highest) to black (lowest).

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indicated that the levels of urinary homocysteine in children with Fe3O4-assisted LDI-MS. Finally, the developed method was successfully mental retardation are higher than those of healthy children. Urinary used for the rapid analysis and localization of endogenous metabolites homocysteine levels have also been linked with premature occlusive in biofluids and whole zebrafish tissue section samples. Our results not cardiovascular disease, even in children (Wishart et al., 2018). Tyrosine only indicate the critical points of accurate synthesis of effective ma- is an essential amino acid in the human body, as well as a precursor for trices for LDI performance but also afford a promising tool for the more neurotransmitters (e.g., dopamine and adrenaline), hormones, and in-depth understanding of meaningful biological information in en- melanin. Elevated tyrosine levels in premature infants have been re- vironmental pollutant-induced toxicity. lated to decreased motor activity, lethargy, and poor feeding (Wishart et al., 2018). The results of the current study indicate that the devel- CRediT authorship contribution statement oped method could be used to identify endogenous metabolites in biofluid samples, with possible further clinical applications in thedis- Wen-Wen Wei: Writing - original draft, Formal analysis, covery of metabolites associated with human disease. In addition, as a Investigation, Methodology. Yuanhong Zhong: Writing - original draft, widely detected environmental pollutant (Chen and Ying, 2015), Funding acquisition, Formal analysis, Conceptualization, Methodology. climbazole (CBZ) and its isotope-labeled internal standard climbazole- Ting Zou: Formal analysis, Investigation. Xiao-Fan Chen: Formal

D4 (CBZ-D4) can be detected by the optimum Fe3O4-assisted LDI-MS analysis, Investigation. Li Ren: Formal analysis, Investigation. (Fig. S8). The characteristic peaks at m/z 315.137 and 319.163 were Zenghua Qi: Resources, Project administration. Guoguang Liu: + the sodiated adducts ([M + Na] ) of CBZ and CBZ-D4, respectively. Resources, Supervision. Zhi-Feng Chen: Writing - review & editing, The corresponding [M+H]+ and [M+K]+ peaks were also found, Funding acquisition, Conceptualization, Methodology, Resources, suggesting the broad application of optimum Fe3O4-assisted LDI-MS in Formal analysis, Project administration. Zongwei Cai: other compounds like environmental pollutants. Conceptualization, Resources, Funding acquisition, Supervision.

3.7. Imaging of endogenous metabolites in whole zebrafish sagittal tissue Declaration of Competing Interest sections The authors declare no competing financial interest. We have generated distribution maps of the proposed metabolites in whole zebrafish from optimum Fe3O4-assisted LDI-MSI (Fig. 5E). There Acknowledgement was a clear distinction between reproductive, digestive, and muscular tissue types. No metabolites were found in the zone of the swim bladder We would be grateful for the financial support from the National due to its absence in the zebrafish tissue section. The m/z 122.023, Natural Science Foundation of China (21507163, 91543202 and 156.981, and 258.882 ions represented the proposed metabolites L- 41602031). cysteine, phosphoglycolic acid, and PC(P-18:0/18:0), respectively. L- cysteine is a naturally occurring amino acid found in most proteins, and Appendix A. Supplementary data PC(P-18:0/18:0) is an essential component of animal fats (Wishart et al., 2018). Thus, it is reasonable to find these metabolites localized in Supplementary material related to this article can be found, in the the digestive and muscular regions of zebrafish tissue sections. The online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.121817. characteristic peak at m/z 429.299 was assigned to 25-hydroxyvitamin D3-26,23-lactone, which appeared to localize not only in the digestive References and muscular regions but also in the reproductive areas of zebrafish tissue sections. The proposed metabolite is possibly a steroid or a Alimpiev, S., Grechnikov, A., Sunner, J., Karavanskii, V., Simanovsky, Y., Zhabin, S., steroid derivative (Wishart et al., 2018). Therefore, it would be logical Nikiforov, S., 2008. On the role of defects and surface chemistry for surface-assisted laser desorption ionization from silicon. J. Chem. Phys. 128. to see this ion in gonads and blood. In addition, TG(18:4/15:0/25:5) at Amendola, V., Litti, L., Meneghetti, M., 2013. LDI-MS assisted by chemical-Free gold m/z 881.649 was found at low intensities in the areas correlating with nanoparticles: enhanced sensitivity and reduced background in the low-mass region. the zebrafish’s digestive and muscular tissues. TG(18:4/15:0/25:5) is Anal. Chem. 85, 11747–11754. Bi, C.F., Zhao, Y.R., Shen, L.J., Zhang, K., He, X.W., Chen, L.X., Zhang, K., 2015. Click the body’s self-synthesized triglyceride and primarily resides in the synthesis of hydrophilic maltose-functionalized iron oxide magnetic nanoparticles liver, followed by adipose tissue (Wishart et al., 2018). Besides, the based on dopamine anchors for highly selective enrichment of glycopeptides. ACS distribution map of other proposed endogenous metabolites, including Appl. Mater. Inter. 7, 24670–24678. small-molecule acids, ceramides, phosphatidylserines, and triacylgly- Blyholder, G., Richardson, E.A., 1962. 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