Facile in-situ polymerization of polyaniline-functionalized melamine sponge preparation for mass spectrometric monitoring of perfluorooctanoic acid and perfluorooctane sulfonate from biological samples

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Facile in-situ polymerization of polyaniline-functionalized melamine sponge preparation for mass spectrometric monitoring of perfluorooctanoic acid and perfluorooctane sulfonate from biological samples Liang Qi , Jicheng Gong PII: DOI: Reference:

S0021-9673(19)31225-7 https://doi.org/10.1016/j.chroma.2019.460777 CHROMA 460777

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

24 September 2019 3 December 2019 7 December 2019

Please cite this article as: Liang Qi , Jicheng Gong , Facile in-situ polymerization of polyanilinefunctionalized melamine sponge preparation for mass spectrometric monitoring of perfluorooctanoic acid and perfluorooctane sulfonate from biological samples, Journal of Chromatography A (2019), doi: https://doi.org/10.1016/j.chroma.2019.460777

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Highlights 

A novel kind of polyaniline-functionalized melamine sponge (PMs) was prepared;



PMs was used for extraction of two representative perfluorinated chemicals (PFCs);



The proposed method was used to measure PFC concentrations in biological samples.

Facile in-situ polymerization of polyaniline-functionalized melamine sponge preparation for mass spectrometric monitoring of perfluorooctanoic acid and perfluorooctane sulfonate from biological samples Liang Qi and Jicheng Gong* Beijing Innovation Center for Engineering Science and Advanced Technology, State Key Joint Laboratory for Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, and Center for Environment and Health, Peking University, Beijing 100871, P. R. China *Correspondence to Jicheng Gong, 5 Yiheyuan Road, Haidian Beijing, 100871, China; contact: 86-10-62753229, email: [email protected]

Abstract In this present work, a novel polyaniline-functionalized melamine sponge (PMs) was successfully prepared using a simple unstirred in-situ polymerization process. The PMs was characterized using a scanning electron microscope and contact angle measurements. Its adsorption performance was initially determined via dye adsorption assays, and the conditions affecting the synthesis including polymerization time, acidity, molar ratio, and number and sizes of raw melamine sponge were optimized. The PMs was then used as an efficient adsorbent for the development of a novel, low-cost method for the detection of two representative perfluorinated chemicals, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), using ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QqQ-MS/MS) with the internal standard method. To achieve the best extraction efficiency with this method, several variables were optimized, including adsorption time, pH value, the number of PMs, and desorption conditions. Calibration graphs showed a good linear degree at concentration ranging from 0.1 to 50 μg L−1 for PFOA and 0.01-10 μg L−1 for PFOS, with a coefficient of detection R2 = 0.998. The intra-day and inter-day relative standard deviations were found to range from 5.9% to 8.2% for PFOA, and 5.5% to 7.7% for PFOS. Under these optimized conditions, the method was successfully used to measure PFOA and PFOS content in real human serum and urine samples, with average spiked recoveries ranging from 79% and 91% for PFOA, and 5.5% to 7.7% for PFOS.

Keywords: Polyaniline-functionalized melamine sponge; in-situ polymerization; perfluorooctanoic acid and perfluorooctane sulfonate; UPLC-QqQ-MS/MS detection; human serum and urine samples

1. Introduction In

recent

decades,

conducting

polymers

(e.g.,

polyaniline,

polypyrrole,

polythiophene and their derivatives) have been extensively investigated due to their curious electronic properties and potential electrochemical applications [1-4]. In particular, polyaniline (PANi) is a unique conjugated polymer that has aroused considerable interest due to unique properties, excellent environmental stability and potential application in electronic devices [5]. As PANi has 4 different states, it can be tailored for specific applications in other fields through a non-redox acid/base doping process [6]; thus, PANi has also become an attractive material for use in sensor [7-9] and separation applications [10, 11]. The preparation process of PANi is facile, and as a kind of potential nano-adsorbent, coating PANi onto those separable templates (e.g., magnetic nanoparticles and a stir bar) [10, 11] will increase its applicability in separation sciences. It is well known that microporous melamine sponge, consisting of a formaldehyde-melamine-sodium bisulfite copolymer, has open-cell structures and excellent hydrophilicity, as well as a negligible cost [12, 13]. The open-cell structure indicates that porous melamine sponge has a very large specific surface area to attach various nano-adsorbents, and it’s easy to be separated from sample solution. Thus, melamine sponges are indeed excellent templates for nano-adsorbents. Until now, graphene functionalized melamine sponges have been well developed, and a novel kind of urea-formaldehyde co-oligomers functionalized sponge has also been reported [19]. These functionalized sponges have been widely used in adsorption studies [12, 14-17], as well as for lesser analytical purposes [13, 18]. From this perspective, functionalizing a melamine sponge with PANi could also be a good strategy for developing a novel kind of adsorbent. The typical synthetic methods of PANi are certain to offer important reference for the preparation of PANi-functionalized melamine sponge (PMs). In most cases, PANi is polymerized or synthesized to form various nano-composites under normal stirring conditions, a synthesis method that usually produces large PANi particles (~100 μm) [5, 11]. In general, mechanical agitation is a common method for disrupting aggregates, as it

can ensure sufficient contact between aniline monomers and templates, especially when its nanocomposites are synthesized. Li et al. [19] investigated the effect of mechanical agitation on the aggregation of PANi nanoparticles during synthesis, and found that the resulting particles are usually highly aggregated if the reaction is stirred during the polymerization, while the absence of stirring favors the formation of high-quality polyaniline nanofibers. This can be explained in terms of a new aggregation mechanism in which aggregation is triggered by heterogeneous nucleation. Therefore, stirring could be an important factor influencing the preparation of PMs. Perfluorinated chemicals (PFCs) are commonly used in industrial and consumer products, including surfactants, food packaging, lubricants, paper and textile coatings, and polishes [20]. Due to the existence of strong C-F bonds, PFCs possess good chemical and thermal stability. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are the two most frequently used and detected PFCs and are abundant in the environment, and both United States Environmental Protection Agency and Health Canada have specified the maximum acceptable PFOA and PFOS concentrations in drinking water in 2016. The wide use of PFOA and PFOS is leading to potential human exposure through food and drinking water. For example, data gathered from animals indicate that PFOA can cause several types of tumors and neonatal death, and may have toxic effects on the immune, liver, and endocrine systems [21]. Cumulative serum PFOA was found to be positively associated with liver injury biomarkers [22]. Furthermore, a recent

cross-sectional

analysis

provided

epidemiological

evidence

that

environment-related levels of serum PFOA may be positively associated with the prevalence of diabetes in men and with total cholesterol in adults [23]. In addition, PFCs have displayed the inhibitory ability on the activity of UDP-glucuronosyltransferase isoforms [24]. Various issues (e.g., detection or water purification) in the domain of environment have gained a huge attention to develop easy-to-make systems and to achieve good

performances [25, 26]. For PFCs, an effective analytical method could also assist in the evaluation of the risks associated with exposure to them, allowing for an in-depth investigation of the molecular mechanisms of the subsequent health effects. The solid-phase extraction (SPE) methods have been widely utilized to determine PFC levels in

environmental

and

human

biological

samples

[20,

27-29].

In

addition,

electrochemiluminescence [30] and fluorescence [31-33] biosensors have been developed for PFC assays. Compared with SPE, the presence of a matrix effect makes it more challenging when using the biosensor for the analysis of real samples; however, commercial SPE methods are time consuming, expensive and cumbersome to perform [34, 35]. Therefore, to overcome these drawbacks, a low-cost, easy-to-use and reliable strategy for the accurate detection of PFCs is needed. To decrease the difficulty involved in post-washing and considering the suspendability of melamine sponges in water, a facile unstirred in-situ polymerization method was developed for the fabrication of a novel kind of PMs. The PMs was further used for the extraction of two model PFCs, PFOA and PFOS, and as a novel detection method for PFCs with ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QqQ-MS/MS) instrumentation. The validated method was further used to analyze PFOA and PFOS in human urine and serum samples (Fig. 1). (Fig. 1) 2. Experimental Section 2.1. Chemicals and materials Melamine sponges were purchased from an online vendor. Analytical grade aniline and ammonium persulfate (APS) were obtained from Macklin Biochemical Co., Ltd (Shanghai, China). PFOA and PFOS standards were purchased from Macklin Biochemical Co., Ltd (Shanghai, China) and a methanolic solution of 13C8-labeled PFOA and PFOS that served as the internal standards were purchased from Cambridge Isotope Laboratories (CIL), Inc. (Andover, MA, USA). Methylene blue (MB) and methyl orange

(OG) standards were obtained from Energy Chemical Co., Ltd (Shanghai, China) and Macklin Biochemical Co., Ltd (Shanghai, China), respectively. K2Cr2O7 was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. LC-MS grade methanol and water were purchased from Thermo Fisher Scientific Inc. (Waltham, USA) and Merck KGaA (Darmstadt, Germany), respectively. All other chemicals used were of highest purity and without further purification. 2.2. Instrumentation Chromatographic separation and analysis were conducted with a UPLC system (Agilent) coupled to a triple quadrupole mass spectrometer (Agilent 6470), which was controlled with its MassHunter software. The UPLC system included two pumps (1290 Infinity II Series). The column used for separation was a reversed-phase C18 column (ZORBAX EclipsePlus C18, 2.1 × 50 mm, 1.8 µm; Agilent, USA), kept at 30 °C. The mobile phase consisted of water containing 2 mM ammonium acetate (A) and methanol (B). The injection volume was maintained at 20 μL using an autosampler (1290 Infinity II Series). Isocratic elution was performed for 0 to 2 min, maintaining 25% (v/v) A with a flow rate of 0.4 mL min-1. The Agilent Jet Stream (AJS) ESI parameters were set at a Nebulizer of 35.0 psi, sheath gas temperature at 400 °C, gas temperature at 350 °C, and N2 sheath gas/gas glow both at 12.0 L min-1, respectively. Quantitative analysis was performed in the multiple reaction monitoring (MRM) and negative ionization modes. For PFOA (m/z > m/z: 413 > 169), the fragmentor and collision energy were optimized to 102 V and 17 V, respectively; The fragmentor and collision energy were optimized to160 V and 32V for PFOS (m/z > m/z: 499 > 80). The 169 (m/z) transition for PFOA corresponds to the C3F7- fragment, and the 99 (m/z) transition for PFOS corresponds to the FSO3- fragment. The PMs were prepared with metal spraying for scanning electron microscope (SEM) characterization, and images were obtained with a SEM (S-4800, Hitachi, Japan) under vacuum conditions. Contact angle measurements were gathered using an optical contact

angle and contour analysis device (OCA 20, DataPhysics Instruments, Germany). The weights of the PMs were measured with an analytical balance (AND, Japan). 2.3. PMs synthesis The raw melamine sponge was cut into 30 × 15 × 3 mm pieces (~20 mg each), rinsed with water and ethanol, and left to dry. The strategy for preparing PMs was developed based on that for preparing PANi nanoparticles. A total of 1.2 mL of aniline monomer and 0.75 g of APS were dissolved in two glass beakers containing 40 mL of 1 M HCl. Two pieces of melamine sponges were initially dipped beforehand into the aniline solution completely. Then, the two solutions were both kept at 4 °C for 30 min. The APS solution was rapidly poured into the aniline solution and shaken vigorously for ~30 s. The above reaction mixtures, containing aniline and APS with the addition of melamine sponges, were left to stand at 4 °C for 4.5 h. The products were washed thoroughly with deionized water and ethanol and were subsequently dried at 40 °C for 10 h. 2.4. Dye adsorption assay MB and OG adsorption assays were conducted by adding 10 mg of PMs into an MB or OG water solution with different concentration and volume. The mixture was shaken for 90 min at 170 rpm using a thermostatic water bath oscillator. The UV-vis spectra of MB and OG before and after adsorption were both measured using a spectrometer (NanoDrop OneC, Thermo Fiseher Scientific, USA). 2.5. Sample extraction 2.5.1 Human urine Precipitation of trace proteins (0.5 mL of urine) was induced by mixing the samples with acetonitrile (1:1). After centrifugation at 4000 rpm for 10 min, 0.5 mL of the supernatant was diluted with 9.5 mL of Tris-HCL buffer (10 mM, pH 7.0) to reach a final volume of 10 mL. Two milligrams of PMs were added to each sample. After shaking for 90 min at 170 rpm using a thermostatic water bath oscillator, the aforementioned PMs

were gently rinsed with pure water and subsequently dipped into 2 mL methanol to desorb the target analyte. 2.5.2 Human serum Again, 100 μL of acetonitrile was added to the serum sample (0.1 mL) for protein precipitation. After centrifugation at 13000 rpm for 20 min, 0.12 mL of the serum supernatant was diluted with 9.88 mL of Tris-HCL buffer (10 mM, pH 7.0). After the addition of 2 mg of PMs, each sample was shaken for 90 min at 170 rpm, and the aforementioned PMs were gently rinsed with pure water and subsequently dipped into 2 mL methanol to desorb the target analyte. The eluent was evaporated to dryness and the residue was reconstituted in 100 μL of MeOH/2 mM ammonium acetate (3:1, v/v), shaken and ultrasonicated for 5 min followed by injection into the UPLC system. The urine and serum samples were spiked with different concentrations of the standard solution, respectively, in order to measure and calculate recoveries. The serum and urine matrix effects were examined and calculated using 10 μg L−1 of PFOA and PFOS. 2.6. Method validation PFOA and PFOS were quantified with the internal standard and the isotopically labeled internal standard for the sample was added before final detection. An individual stock solution of PFOA and PFOS (200 μg L−1) were prepared by accurately weighing and dissolving PFOA and PFOS in LC-MS grade methanol. PFOA and PFOS working solutions were prepared by appropriate dilution of the stock solutions in MeOH/2 mM ammonium acetate (3:1, v/v). A 200 μg L−1 working solution of internal standard containing

13

C8-PFOA and

13

C8-PFOS was prepared by diluting the stock solution with

MeOH/2 mM ammonium acetate (3:1, v/v). The serum and urine matrix effects were examined and calculated using 10 μg L−1 of PFOA and PFOS. The carryover effect was calculated from the ratio of the signal response at the retention time of PFOA and PFOS in pure water (blank control) to the

highest-concentration calibration sample (20 μg L−1). 3. Results and discussion 3.1 Preparation of PMs and dye adsorption assay We first prepared a PANi-functionalized melamine sponge (PMs) while stirring, but found that completely removing the unattached and granular PANi particles embedded inside the sponge required significant effort, and this result could confirm Li’s previous report about aggregated particles [19]. It is well known that washing is always necessary for the purification of newly-prepared nanomaterials or nanocomposites since it can help remove non-reacted reactants and reactive solvents, as well as the unattached nanoparticles. However, the presence of aggregated PANi particles partially hampered post-synthetic washing when a melamine sponge was used as the template. Thus, we further examined an unstirred in-situ polymerization process for the preparation of PMs. During PMs preparation, the adsorption behavior of anionic OG (adsorption time: 0.5 h) in the PMs was initially evaluated, and the adsorption efficiency was used as the criterion to investigate the optimized synthesis conditions, including polymerization time, acidity, and molar ratio. All the experiments were conducted at a low temperature (4 °C) because it has been reported that both the molecular weight of PANi and its crystallinity increase as the reaction temperature decreases [36]. 3.1.1 Polymerization time The efficiency of PMs preparation can be improved by optimizing the polymerization time. We examined three different polymerization time (2, 4.5 and 12 h) using the PMs prepared from the condition of 1 M HCL and an aniline:APS molar ratio of 4:1. The results show that the adsorbency of OG (20 μM, 15 mL) on PMs was 79±4.2% (n=3) when the polymerization time was 2 h, reaching 90±4.9% when the polymerization time was extended to 4.5 h. However, when the polymerization time (12 h) was more than doubled, no significant increases were observed in adsorbency (94±3.5%). That could be due to the low reaction temperature reducing the rate at which the unreacted

aniline monomers and APS entered the sponge, thus the increase in PANi yield coating the skeletons is limited. Finally, 4.5 h was selected as the polymerization time. 3.1.2 Acidity and molar ratio After determining the optimal polymerization time, another two synthesis conditions, HCL concentration (0.1 M and 1 M) and aniline:APS molar ratio (4:1 and 4:4), were investigated. We found it difficult to form emeraldine PMs at a low acidity (0.1 M HCL) when small amounts of APS were used (aniline:APS = 4:1). PMs can be successfully prepared under higher oxidant and acid dosages, and the adsorbance of OG solution (20 μM, 15 mL) before and after the addition of each PMs (10 mg) is listed in Table S1 (Supplementary data). The results showed that PMs (1 M HCL, aniline:APS = 4:1) exhibited the highest adsorption efficiency of OG (~90±4.9%, n=3), and that PMs synthesized from other acids and oxidant dosages exhibited a weaker adsorbency (38±2.2% and 46±3.5%). It has been reported that PANi chains are more likely to be produced under more acidic conditions [36, 37], and another study showed that the quantity of granular particles starts to increase as the concentration of HCL is lowered [38], thus, improving the acidity may increase the dopants and the amount of nanofibers, while excess oxidants may go against the conjugate regularity of PANi particles. Therefore, a high acid dosage of 1 M HCL and an aniline:APS molar ratio of 4:1 were selected as the optimized acidity and aniline:APS molar ratio. Fig. S1A and B (Supplementary data) shows the UV-vis adsorption spectra of OG and MB respectively, in the absence and presence of PMs under the synthesis conditions of 1 M HCL and molar ratio of aniline:APS = 4:1. After nearly all of the OG molecules were removed after adding 10 mg of PMs, only ~35% of MB molecules were adsorbed. In addition, an adsorption efficiency of ~85% can be achieved when PMs was used to adsorb Cr2O72- at the same condition (Fig. S1C). These results show that PMs exhibited a superior adsorption capability towards anionic OG and Cr2O72-, which is in accordance with the cationic property of PMs. The weights of the melamine sponge before and after

loading PANi was also recorded (n=4), and the results show that the mass loading of PANi was about 75±4 mg g-1 of raw sponges for the PMs (1 M HCL, aniline:APS = 4:1). 3.1.3 Number of raw melamine sponge The production of a greater amount of PMs per batch is a great advantage in terms of time and cost [18]. For this reason, 1 and 2 pieces of PMs (~20 mg per raw sponge) at a time were synthesized respectively under the synthesis conditions of 1M HCL and a molar ratio of aniline:APS = 4:1. Several small cuboids (~2 mg each) cut from one piece of raw sponge were also utilized to prepare the PMs directly. After the synthesis, we cut several cuboids (2 mg, 6 × 7.5 × 3 mm) from the 1-piece and 2-piece PMs respectively, and examined the adsorbance of the OG solution (20 μM, 10 mL) to compare it with that of the small cuboids prepared directly. The results are listed in Table S1, which show that there was almost no difference in their adsorption efficiencies, indicating that the size of the raw sponge does not affect the adsorption performance of the PMs. Considering the volume of the reaction mixture and the operation flexibility when used as the adsorption assay for different analytes, 2 pieces of PMs (~20 mg each) were synthesized at a time and cut into pieces as needed. 3.2. Characterization of PMs The APS solution was rapidly poured into an aniline solution containing melamine sponges and shaken vigorously. Subsequently, after several minutes of polymerization, pale green nanoparticles began to appear, until the entire solution was dark green in color. At the end of the reaction, the white melamine sponges also turned green. The color of the PMs was consistent with that of doped emeraldine PANi nanoparticles, and visible color variations from white to green indicated the formation of PANi nanoparticles inside the sponge. The typical morphology of PMs was first characterized using an SEM. As can be seen from Fig. 2A, the raw sponge possesses an inner structure composed of a smooth skeleton. Moreover, relatively dense nanoparticles accumulate on the skeleton of the

melamine sponge, which were found to roughen the surface, as shown in Fig. 2B and C. However, the visible amount of PANi nanoparticles only slightly increased after the synthesis time was extended to 12 h, as shown in Fig. 2C. (Fig. 2) Afterwards, the contact angle measurements of the melamine sponge and PMs were determined, and these corresponding data are shown in Fig. 3. Fig. 3A shows that the contact angle could not be detected when a water droplet was placed on the surface of the hydrophilic melamine sponge. Conversely, due to the hydrophobicity of PANi, Fig. 3B shows that the PMs had a contact angle of ~128° when the water droplet was placed on the surface of the PMs, illustrating its increased hydrophobicity. (Fig. 3) 3.3. Optimization of the extraction procedure To achieve the best extraction efficiency when applying the PMs to the analysis of PFOA and PFOS, several parameters, including adsorption time, pH value, number of PMs and desorption conditions were optimized. 3.3.1. Adsorption time An optimum adsorption time always contributes to an increased the amount of adsorbed analytes. The buffer solution (10 mM, pH=7, 40 mL) was spiked with 2 μg of PFOA and PFOS, and 4 mg of PMs was used to determine the best adsorption time within a range of 10 to 120 min (Fig. 4A). The results show that the adsorption reaction was very rapid during the initial stage (i.e., from 0 to 30 min for PFOA and 0 to 40 min for PFOS), which may be due to the fact that most of the adsorption sites were unbound. An adsorption equilibrium was reached at approximately 90 min for both PFOA and PFOS, and the adsorption rate subsequently slowed as the number of available adsorption sites decreased further. Considering both the adsorption efficiency and the time consumption, 90 min was selected as the optimum adsorption time. Based on the above results, the adsorption kinetics of PFOA and PFOS onto PMs

were further investigated by a pseudo-first-order model and a pseudo-second-order model (Supplementary data), respectively. It can be seen from Table S2 that when the pseudo-second-order model was applied, the experimental values of qe (the amounts of adsorbed PFCs at equilibrium) were more consistent with the calculated ones for both PFOA and PFOS. Thus, the pseudo-second-order model is better than the pseudo-first-order model to describe the adsorption of PFOA and PFOS by PMs. 3.3.2. Effect of pH pH values ranging from 2 to 9 were investigated, and the buffer solutions spiked with 2 μg of PFOA and PFOS (40 mL) were selected to study the optimum pH value. Fig. 4B shows that the PMs exhibited a strong adsorption of PFOA and PFOS in acidic or neutral conditions, and that the adsorption quantity clearly decreased in alkaline conditions. In this assay, PANi nanoparticles were synthesized in acidic condition with APS as the oxidant, and doping resulted in PANi with a certain number of positive sites. On the other hand, different ionized forms of PFOA and PFOS are expected to exist in solutions with various pH values. PFOA contains a carboxy group and PFOS contains a sulfonic group, which can release a proton into the solution in weakly acidic or neutral conditions. More negative ions can form in the neutral solution as the pH increased from 2 to 9. Nevertheless, the doping level in PANi gradually decreased at higher pH ranges, which may explain why PMs began to lose their adsorbency for PFOA and PFOS in the alkaline solution. In general, these model PFCs contain acidic groups, thus it is believed that PMs can adsorb PFOA and PFOS molecules primarily through electrostatic interactions, which could increase definite selectivity of the proposed method. Comparatively speaking, the commercial SPE columns (e.g., HLB or C8 and C18) adsorb PFOA and PFOS mainly through hydrophobic interaction [39], which typically lacks selectivity. According to these results, PMs exhibited good adsorbency for both PFOA and PFOS at pH 7. Thus, this was finally selected as the optimum pH value for use in the subsequent experiments. The use of a buffer solution ensures a consistent extraction pH

from batch to batch, hereby allowing for the good repeatability of the extraction efficiency, especially when very complex samples are examined. 3.3.3. Amount of PMs After determining the optimized adsorption time and pH, the amount of PMs (1-5 mg) was investigated. A neutral buffer solution containing 50 ng of PFOA and PFOS (10 mL) was used, to which different amounts of PMs were added to determine the optimum amount of PMs. As shown in Fig. 4C, for PFOA, the adsorption efficiency exceeded 90% when 1 mg of PMs was added with subsequent shaking for 90 min, and nearly 100% of the PFOA was adsorbed when 5 mg of PMs was added; For PFOS, an adsorption efficiency of over 90% was reached when 2 mg of PMs was added. Similar to PFOA, 5 mg of PMs could also adsorb nearly 100% of the PFOS. (Fig. 4) The extraction efficiency results obtained from PMs are shown in Fig. 5, and the branched peak of PFOS was similar to that reported in a previous study [30]. Finally, 2 mg of PMs was chosen as the optimal amount of PMs for the subsequent assays considering its slightly large-volume. Compared with PMs, raw melamine sponges are able to adsorb less than 8% of PFOA and PFOS under the same conditions. (Fig. 5) Under the above optimized extraction conditions, the reproducibility of the extraction using 2 mg of PMs cut from big pieces was further examined by performing 3 replicate analyses every 2 h during one day and 3 replicate analyses over 3 consecutive days. The values of batch to batch reproducibility were less than 5%. Thus, the good reproducibility of extraction may contribute to the evaluation of the elution conditions, discussed in the following section. 3.3.4. Elution conditions As a very good solvent for PFOA and PFOS, methanol is commonly used for the elution of the 2 PFC molecules in several aforementioned SPE-based methods [40].

Therefore, we first investigated the elution efficiency of methanol in this extraction assay and found it to be a good elution solvent for desorbing PFOA and PFOS from the PMs in this assay. Different volumes of methanol (1000 to 2200 μL) were investigated, whereby 1800 μL of methanol was found to be adequate for the elution of PFOA from the PMs. Likewise, 2000 μL of methanol was enough to elute PFOS. In other words, nearly all of the analytes could be desorbed from the sorbent (Fig. 4D). Therefore, methanol (2000 μL) was chosen as the elution solvent for the assay. 3.3.5 Analytical performance The analytical parameters for quantitative analysis of PFOA and PFOS were investigated under the optimized conditions and the results are summarized in Table S3. The response was found to be linear at concentration ranging from 0.1 to 50 μg L−1 for PFOA and 0.01-10 μg L−1 for PFOS, with high coefficient of determination (R2 = 0.999). The intra-day precision was tested by performing 5 replicate analyses every 2 h during one day, whereas inter-day precision was evaluated by performing 6 replicate analyses over 6 consecutive days. The values ranged from 5.9% to 8.2% for PFOA, and 5.5 to 7.7% for PFOS respectively. Analyte recovery was determined using human urine and serum, and they were found to range from 79% to 91% for PFOA, and 81 to 87% for PFOS. Acceptable matrix effects of serum and urine were obtained, as shown in Table S3. Considering the matrix effect, these recoveries should be acceptable. In addition, the results from 3 replicate tests show a negligible carryover effect of 0.3% for PFOA and 0.7% for PFOS. Reusability is also an important factor for evaluating the performance of the adsorbent. Our results indicate that small decreases (~6.9%) in the recoveries can be observed after 4 extraction/elution cycles. In general, the cost of PMs is much lower compared than that of a commercial SPE column. As such, the negligible cost and effort taken to synthesize it make its reuse not very necessary [13]. 3.4 Application to real samples

The performance of the method was further evaluated by analyzing the PFOA and PFOS concentrations in two different types of biological samples, human urine and serum. PFOA and PFOS were extracted from urine and serum as described above, which subsequently detected using UPLC-QqQ-MS/MS. Four urine and 4 serum samples were selected randomly. The concentration and content of PFOA in the urine (250 μL) and serum (60 μL) are listed in Table S4. Two representative chromatograms of PFOA and PFOS from urine (no. 4) and serum (no. A) are shown in Fig. 6. The results show that the both the concentrations of PFOA and PFOS in urine were lower than that in the serum. The ranges of PFOA and PFOS concentrations are consistent with previous results [27, 41]. (Fig. 6) 3.5 Comparison with published methods The comparison between this proposed method and previous methods is shown in Table S5. The recovery efficiency and detection limit are both comparable to those previously reported; Larger volume of blood or urine samples were needed when most of the SPE methods were developed; Owing to the large specific surface area and selective adsorption of PMs, a much smaller amount of adsorbents was required for the extraction of PFOA and PFOS. Thus, this proposed method based on PMs shows several unique advantages when used for PFCs analysis. 4. Conclusions In this study, a novel PMs was successfully prepared through a simple unstirred in-situ polymerization process. Compared with melamine sponges, the PMs exhibited good hydrophobicity and adsorbency, especially for anionic dyes or analytes (e.g., methyl orange and K2Cr2O7). The proposed PMs-extraction protocol combined with UPLC-QqQ-MS/MS detection offers a valuable, low-cost approach for measurement of PFOA and PFOS in human serum and urine samples. The chemical structures of PFOA and PFOS are similar to those of other PFCs, and it can be concluded that PMs can be

used for the extraction of other PFCs through similar mechanism. Futhermore, our findings demonstrate that PMs have great potential for use in the analysis of other target analytes in real samples. The authors declare no conflict of interests. Author contribution Dr. Liang Qi is responsible for the assay design and operation, and Dr. Jicheng Gong is responsible for the design of whole assay and the text revision, et al.

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Fig. 1. (A) Synthesis of PMs under low temperature and static conditions; (B) Analytical procedure of quantifying PFOA and PFOS in serum and urine based on PM extraction and UPLC-QqQ-MS/MS detection.

Fig. 2. Characterization of melamine sponges and PMs (1 M HCL, aniline:APS molar ratio = 4:1). TEM images of (A) melamine sponges, (B) PMs (4.5 h), and (C) PMs (12 h); (D) partial magnification from areas in B) and C), respectively.

Fig. 3. Contact angle measurements on (A) melamine sponges and (B) PMs. The insets show 2 digital photographs of the corresponding sponges and PMs showed a contact angle of ~128°.

Fig. 4. Effect of (A) adsorption time, (B) pH, (C) amount of adsorbent and (D) eluent solvent volume on extraction efficiency (n=3).

Fig. 5. Chromatograms from standard solutions (Control: 10 μg L−1, 10 mL) of PFOA and PFOS extracted with PMs (2 mg). The standard sloutions were prepared in the buffer solution (10 mM, pH=7).

Fig. 6. Extracted chromatograms of PFOA and PFOS obtained from analysis of (A) urine sample no. 4 and (B) serum sample no. A.