Single-Particle Microplastics Analysis via Laser-Assisted MicroPyrolysis Flowing Atmospheric-Pressure Afterglow Mass Spectrometry Imaging

Abstract

Microplastics (MPs) have emerged as ubiquitous contaminants in the environment. Thus, novel MP characterization techniques are in urgent demand to achieve the necessary environmental monitoring, as well as studying the impact of MPs on human health. Herein, laser-assisted micropyrolysis flowing atmospheric pressure afterglow high-resolution mass spectrometry (LAMP FAPA MS) is developed toward direct imaging of single-particle MPs collected on filters. The use of a CO₂ laser (10.6 μm) is shown to yield up to >100× higher signal intensity for polyethylene terephthalate fragment ions compared to a diode laser (405 nm) at the same power. Also, a mass spectral analysis algorithm was developed and implemented for fast polymer identification in MPs based on the corresponding fragment ion series separated by the repeating base unit. Furthermore, additive identification is shown for single-particle MPs. Finally, LAMP FAPA MS is demonstrated for quantitative analysis of polymer mass with a detection limit of 0.083 ng for polyethylene microspheres, which corresponds to a spherical particle with a 5.4 μm diameter.

Introduction

Microplastics (MPs) have become an emerging environmental issue and are causing concerns worldwide because MPs are found in environments such as oceans, rivers, soils, as well as drinking water, food, and human tissues. MPs are small polymer debris less than 5 mm² and down to 1 μm. , While the risk of MPs to human health is currently not well understood due to the lack of data on exposure and hazard, some studies show that MPs may pose human cytotoxicity. Also, the polymer additives in MPs, as well as other pollutants adsorbed in the environment due to their large surface area, pose a potential risk for human health. Therefore, the thorough characterization of MPs in both environmental and biological samples is important. However, the detection, identification, and quantification of MPs are challenging due to their wide size range over several orders of magnitude, diverse chemical compositions (including additives and adsorbed species), and complex sample matrices. Therefore, analytical methods that can provide rich information on MP samples are in demand.

Vibrational spectroscopy methods, such as Fourier transformed infrared (FTIR) spectroscopy and Raman spectroscopy, are widely used for MP characterization. These techniques coupled with microscopy system can provide significant information on MPs, including chemical composition, particle number, and size. Also, these techniques are nondestructive and allow analysis of MPs directly on collection filters, which is an advantage because MPs in the environment are typically collected and separated through processes including digestion, sieving, density separation, and eventually collected by filtration. Thus, analysis of MPs directly on filters can minimize sample loss and contamination during sample transportation. However, FTIR and Raman microscopy have several limitations. For example, MP color can affect identification accuracy in FTIR, and samples must be dry to avoid interference from the broad absorption band of water. This limitation is particularly significant for tire–road wear particles (TRWP), which are complex mixtures of synthetic rubber, carbon black, and mineral debris; the high carbon black content strongly absorbs infrared radiation, leading to weak or distorted FTIR signals and reduced spectral quality. In addition, ATR-FTIR is inherently surface-sensitive, with a penetration depth typically on the order of 0.5–5 μm depending on wavelength and crystal properties, which may not be representative of the bulk composition of heterogeneous TRWP particles. Both traditional FTIR and Raman techniques have limited sensitivity for detecting and characterizing additives within MPs. Consequently, neither technique readily enables accurate quantification of MP mass concentration.

Mass spectrometry (MS) is a powerful tool for MP characterization. Compared with vibrational spectroscopy methods, MS is much more sensitive to less abundant components, such as additives. Also, the identification capability of MS methods would not be affected by the color or fluorescence of MPs. For example, pyrolysis gas chromatography–MS (py-GC/MS) is a well-established method for bulk MP characterization with good quantitative capability for MP mass concentration. However, it suffers from a long chromatographic separation time. Also, it loses the size and particle-number information, since many MPs are pyrolyzed simultaneously. Matrix-assisted laser desorption/ionization-MS (MALDI-MS) has also been used for MP characterization, and it has the capability to provide size and particle-number information when MS imaging (MSI) is performed. However, MALDI-MS has limitations when it is used to characterize polyolefins. Nonpolar polymers with a simple hydrocarbon structure do not ionize well by using traditional metal cationization methods. This makes it difficult to obtain useful MALDI spectra. To enable detection, charged groups such as ammonium or phosphonium salts must first be chemically attached to the polymer chain ends. These additional modification steps make sample preparation more time-consuming and less straightforward. As a result, MALDI-MS is not always a convenient method for the routine characterization of polyolefins; secondary ion mass spectrometry (SIMS) has also been investigated for qualitative microplastic imaging owing to its exceptional surface sensitivity and submicrometer spatial resolution, which enables detailed investigation of microplastic formation, weathering, degradation, and interactions with biological systems. − Nevertheless, SIMS is susceptible to matrix effects that complicate quantitative analysis, and polymer identification may have to rely on ion ratios rather than characteristic ions. , In addition, the requirement for the sample to be in an ultrahigh vacuum necessitates involved sample preparation, particularly for complex environmental samples, while substantial instrument capital and maintenance costs, as well as the need for highly trained personnel, may limit the use of SIMS for routine microplastic analysis. Single-particle ICP-MS (SP-ICP-MS) enables the rapid quantification of microplastics in suspension by striving to detect the elemental signal from individual particle events to relate to mass and particle number concentration, while assuming spherical dimensions to yield a size distribution. , While the technique is originally limited to suspension-based analysis and does not preserve the spatial distribution of particles, it can be coupled to laser ablation sampling (LA-SP-ICP-MS) to also enable the direct analysis of particles deposited on surfaces and identify their spatial distribution. In addition, traditional LA-ICP-MS foregoes the advantages of the SP detection mode to maintain the capability to obtain particle shape information from elemental maps of microplastic solid samples. , Nevertheless, ICP-MS-based techniques detect the ions of atomized analytes to provide elemental analysis, which prevents the direct acquisition of molecular information, therefore limiting the molecular characterization of polymers, as well as additives and potentially adsorbed contaminants.

Ambient MS (AMS) allows direct sample desorption and ionization in real time with minimal-to-no sample preparation. Various AMS techniques have been developed since 2004 showing advantages of easy-to-use, as well as fast and cost-efficient analysis. The capability of AMS to analyze the solid sample directly in its native state makes it a promising tool for MP analysis. For example, direct analysis in real time (DART) MS has been reported for bulk MP characterization coupled with thermal degradation techniques, but similar to py-GC/MS, it pyrolyzes many MPs simultaneously, so it could not provide single-particle information, such as MP size or particle number. Recently, atmospheric solid analysis probe (ASAP) MS multimodal analysis was reported for MP characterization with single-particle and imaging capabilities, coupled with fluorescence microscopy. However, it takes a long time to analyze several particles, since each particle must be placed by itself on the needle tip to perform a single analysis. Alternatively, MSI with laser-assisted sampling can provide significantly improved spatial confinement of the sampling area. Flowing atmospheric pressure afterglow (FAPA), − which is also a plasma-based AMS source, has shown the capability for fast bulk polymer and additive characterization with spatially resolved information in three dimensions when coupled with the laser-assisted micropyrolysis (LAMP) technique for pyrolysis. , Therefore, LAMP FAPA MS is a promising tool for MP characterization with rich information such as particle number, size, chemical composition, and mass concentration.

Herein, the LAMP FAPA MS imaging technique is demonstrated for MP characterization directly on filters. A CO₂ laser was implemented for significantly better pyrolysis efficiency for transparent and translucent MPs compared with the previously reported diode laser. Various MPs, including polar and nonpolar ones, were analyzed. In addition, immobilization sample preparation methods were studied to prevent the sample loss during sampling. Also, a mass spectral analysis algorithm was developed for fast MP identification by searching for characteristic mass differences. Furthermore, single-particle characterization capabilities are demonstrated by MSI using polyethylene microspheres.

Experimental Section

Chemicals and Materials

MPs including high-density polyethylene (HDPE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), and Polyamide/Nylon-6 (PA6) were part of Polymer Kit 1.0 obtained from the Center for Marine Debris Research at Hawaii Pacific University. An HDPE sheet was purchased from Small Parts, Inc. (ZAM2966-B0070Z87US). Blue PE microspheres with different sizes 45–53 μm, 106–125 μm, and 180–212 μm were purchased from Cospheric LLC. Glass microfiber filters were purchased from Whatman (934-AH, binder-free).

Sample Preparation

All MPs from Polymer Kit 1.0 were cut down to ∼1 mm pieces before analysis by LAMP FAPA MS. A glass microfiber filter piece (4 mm × 6 mm) was adhered to a glass slide with a 40 μL DI water droplet on the glass surface. These larger MP samples were deposited and gently pressed on the filter using a stainless-steel rod with a flat surface to minimize particle movement during analysis. Another set of MP samples were also covered with another glass microfiber filter on top. An aliquot of 20 μL of DI water was then deposited on the filter to facilitate adherence between the top and bottom filters.

The PE microspheres (∼32 mg) were suspended in 10 mL DI water. The suspensions were shaken by hand to avoid particle settling right before an aliquot of 20 μL of the suspension was deposited on the filter for LAMP FAPA analysis.

LAMP FAPA Source

The FAPA ionization source has a pin-to-capillary configuration where a pin cathode and capillary anode are separated by 6 mm. A glow discharge is generated between the electrodes by applying a negative DC voltage to the pin cathode through a 4.5 kΩ ballast resistor by a power supply (model 210-01R, Bertan Associates, Inc.), while the capillary is grounded. The operating voltage ∼450 V, resulting in a current of 30 mA. Helium is used as the discharge gas with a flow rate of 0.7 standard liters per minute (SLPM). The geometry of the FAPA/sample/MS inlet has been described previously. ,

A 405 nm diode laser (J Tech Photonics, Inc.) or a 10.6 μm CO₂ laser (model DIAMOND C-20, Coherent Corp.) was used for LAMP. The laser power of the CO₂ laser is controlled in remote mode by modulating the laser duty cycle and gating via an Arduino board (Adafruit Industries, LLC) with a digital-to-analog converter (model MCP 4725, Adafruit Industries, LLC). The CO₂ laser beam is steered into by a mirror (25 mm DIA BB IR Laser Mirror, Edmund Optics) and focused onto the sample surface by a lens (PCX ZnSe 12.7*50.8 Unctd, Edmund Optics), resulting in a spot diameter of ∼300 μm (Figure A). The diode laser was operated at 100% duty cycle with the laser power controlled via the applied current as previously described.

1.

A) LAMP FAPA MS setup featuring a CO₂ laser. Mass spectra from a PET microplastic particle with (B) a diode laser at 405 nm compared to (C) the CO₂ laser at 10.6 μm, while keeping the laser power at 0.6 W. The PET fragment ions are easily distinguished in the CO₂ mass spectra by the mass difference corresponding to the polymer base unit.

Mass Spectrometry

Most experiments were performed on an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA) in the positive ion mode. The standard ESI housing was replaced with the LAMP FAPA housing. The capillary temperature (275 °C) and voltage (+4 V), as well as the tube lens voltage (+50 V), were set for the positive ion mode. High-resolution spectra were acquired with a mass range of typically 100–800 Da in the Orbitrap analyzer with the resolution set at 30,000. One microscan was applied for all experiments with 500 ms maximum ion injection time. Experiments with the PE microspheres were also performed on an Exploris 240 orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) in the positive ion mode. For these experiments, the automatic gain control (AGC) target was set to 100%, with a maximum injection time of 100 ms per single scan. The RF lens was operated at 100%, and the multi-RF threshold ratio was set to 8. The resolving power was reduced to 15,000 to improve imaging throughput while maintaining sufficient mass-resolving capability for identification. Ion identities were confirmed by HCD MS/MS for selected ions included for MS imaging. Spectra were acquired over a mass range of m/z 100–800, consistent with the conditions used in the LTQ Orbitrap experiments. For all experiments, samples were placed on a motorized stage (M30XY, Thorlabs) and scanned at 0.04 or 0.2 mm/s along the X direction and then moved 200 μm toward the MS inlet along the Y direction for the next scan, and so on. Mass spectra were processed with Thermo Excalibur software (Thermo Scientific). MSI was reconstructed in MATLAB R2019a (The MathWorks, Inc.) and ImageJ. KMD plots were plotted in Excel (Microsoft). The MP identification program was developed using Python 3.10.

Results and Discussion

Effect of Laser Wavelength on Ion Signals of Interest

LAMP FAPA MS has been used for synthetic polymer characterization using a 405 nm diode laser previously. In that study, a graphite layer had to be applied to the surface of transparent or translucent samples to enhance the laser absorption and obtain better pyrolysis efficiency. Such a simple approach is particularly useful for bulk polymer samples, such as polymer sheets and films; however, MPs can be so small that graphite deposition is not practical. It has been reported that most polymers have high optical absorption at wavelengths typical of the CO₂ laser, including 10.6 μm, which is often used in polymer laser processing. Therefore, a 10.6 μm CO₂ laser is implemented here to do away with the graphite-layer sample preparation requirements, and its LAMP performance toward FAPA MS is compared with the diode laser.

As shown in Figure B–C, the mass spectra with the CO₂ laser clearly show the PET pyrolysis fragment series with a mass difference of 193.0501 Da, which corresponds to the repeating unit (C₁₀H₈O₄) of PET, including the protonated PET monomer [(C₁₀H₈O₄)­H]⁺, dimer [(C₁₀H₈O₄)₂H]⁺, and trimer [(C₁₀H₈O₄)₃H]⁺. It is difficult to observe these ions in the mass spectra obtained with the diode laser on the same intensity scale because the corresponding intensity is 2 to 3 orders of magnitude lower. Thus, the CO₂ laser was selected for use in the rest of the development of LAMP FAPA MS toward MP samples.

MP Characterization Directly on Filters

Another challenge for MP analysis is avoiding sample alteration, including loss or contamination, during both MP collection and sample processing. Typically, binder-free nonpolymer filters, such as Al₂O₃ and/or glass microfiber filters, are used for collecting MPs after sample processing. Thus, developing LAMP FAPA MS for the direct analysis of MPs on filters can significantly improve the sample throughput and prevent the further possibility of sample alteration before analysis. On the other hand, many ambient mass spectrometry techniques that implement gas flow, including many plasma-based or spray-based techniques, are limited in the analysis of loose particulate material, such as powders, because the sample is liable to be blown away, such that the sample must be immobilized onto a surface. However, typical immobilizing surfaces, such as those from double-sided pressure-sensitive adhesive tapes, are not compatible with the analysis of MPs because they contain many polymers, such as polyethylene and polyisoprene, which can interfere with the analysis. Here, a filter sandwich sample preparation method was developed to immobilize the larger MPs (0.5–5 mm). It consists of covering the MPs collected on a glass microfiber filter with another filter to prevent their loss during transport or analysis. The glass microfiber filters were also adhered to the glass slide by surface tension with DI water. Two layers of the glass microfiber filter sandwich were also adhered together in the same manner.

In the filter sandwich configuration, the laser power must be high enough to burn through the upper filter layer to reach the MPs underneath. An HDPE sheet sample, ∼2 mm in the longest dimension, was used for easier optimization of the laser power, with a single laser line-scan measurement across the particle. At 0.6 W, the laser can burn the top surface of the filter, but it is not high enough to burn through, such that no signal from the HDPE can be detected (data not shown). Further increasing the laser power to 1.2 W enables the laser to burn through the full filter thickness, such that reproducible mass spectra of HDPE pyrolysis products are obtained. Furthermore, measurements of the HDPE were compared with/without the filter cover at 1.2 W to evaluate the effect of the filter cover on the mass spectra. As shown in Figure A and B, the intensity of the major HDPE fragment ions is comparable, but more fragment ion series are observed without the filter cover. This is confirmed in the Kendrick mass defect (KMD) plots (Figure C and D, base unit: CH₂, 14.01565 Da) that show the bubbles from the PE fragment ion series are aligned along horizontal straight lines. It is evident that the KMD plot of the mass spectra without the filter cover has many more parallel horizontal straight lines with different y-intercepts, which indicates PE fragment ion series containing different end groups. It is possible that the higher amount of laser energy interacting with the polymer sample without the filter cover leads to more fragmentation and other reactions that yield different end groups. Nevertheless, the simple filter sandwich approach proved to be very successful in enabling the analysis of larger MPs while preventing them from being blown away by the FAPA gas flow, and without significantly affecting the detection sensitivity.

2.

Mass spectra of HDPE without (A) and with (B) filter cover. KMD plots (base unit: CH₂) from mass spectra of HDPE without (C) and with (D) filter cover.

Various single MP particles with the longest dimension between 1 and 2 mm including both polar (PA6) and nonpolar (HDPE, PP, PS) were analyzed by LAMP FAPA HRMS to evaluate its performance for different polymers. As shown in Figure A–D, it is evident that the MPs can be successfully identified by the mass spectra with specific mass difference that matches the repeating unit (CH₂ for HDPE, C₃H₆ for PP, C₈H₈ for PS, and C₆H₁₁NO for PA6). In addition, the intensity of PA6 fragments is about 2 orders of magnitude higher than that of HDPE, PP, and PS, which is likely due to the higher ionization efficiency of the amide moiety compared with the nonpolar MPs. However, the identification of MPs by inspecting each individual mass spectrum manually even with the aid of a KMD plot is very time-consuming, which is not practical for a larger number of diverse MPs and could become the sample-throughput-limiting factor when coupled with methods that feature fast sampling, such as LAMP FAPA. Also, it is important to extract the intensity information of MPs’ fragment ions for quantitative study. However, unlike typical targeted analysis that has one or several ions with specific known m/z to monitor, the specific m/z values of MPs’ pyrolysis fragment ions are not known prior to analysis even for MPs with the same repeating unit because different end groups and side-chain modifications can shift the specific m/z to different values. Therefore, a mass spectral analysis algorithm that can automatically identify and extract the intensity of MPs’ fragment ions is necessary. Unlike Raman and FTIR techniques that have many spectral databases and spectral analysis algorithms developed for MP identification, , polymer automatic analysis tools for mass spectra are rare. A mass spectral analysis tool has been developed for per- and polyfluoroalkyl substances (PFASs) screening. However, most data analysis tools for polymer identification are commercial and require proprietary licenses, such as the repeat finder implemented in JEOL software. Although Poly-Calc is an open-access tool, it cannot visualize homologous repeating units without knowledge of end-group identity or KMD plots. It is also limited in integrating the cumulative intensities of repeat-related ions relevant for quantitative analysis. To address these limitations, an open-access data analysis algorithm based on searching for a specific mass difference (Δm), rather than a specific m/z value, within the mass spectra was developed. This approach enables the identification of microplastic-derived polymer signatures and allows the extraction and integration of the total intensities of all corresponding pyrolysis fragment ions for relative quantitative analysis (Figure ). Users only need to import the spectral information (containing the m/z peak list and corresponding intensities) and input the chemical formula of the MP polymer repeating unit to perform the m/z difference search. The algorithm outputs the series of m/z values of the pyrolysis fragment ions for fast MP polymer identification. It also counts the total number of fragments and extracts the total intensity of fragments, which can be used for MP quantitation studies. It is worth mentioning that this program is not limited to LAMP FAPA HRMS; it can be used for MP characterization with other MS-based techniques, such as MALDI MS and pyrolysis-based MS, if the mass spectra contain the repeating unit information. The program is available on https://github.com/ejokeakatugba/polymer_ms_analysis. More features, such as differentiation of MPs that contain CH₂ repeating unit (LDPE, MDPE, HDPE, and PP), data analysis coupled with MSI, and a graphical user interface (GUI), will be developed in the future to facilitate MS-based studies of MPs and polymers.

3.

Mass spectra and extracted fragment list with corresponding sum-count total intensity of HDPE (A), PP (B), PS (C), and PA6 (D).

Mass Spectrometry Imaging of MPs

MSI can be a powerful tool to obtain spatial information on single-particle samples, such as size and particle number; thus, LAMP FAPA MSI is developed here for MPs. For this purpose, a few PE microspheres from a sample with a nominal diameter range of 180–212 μm were dispersed on a filter by depositing 20 μL of the corresponding aqueous particle suspension. It should be mentioned that even without a filter cover these smaller size MP particles are not blown away by the FAPA effluent gas, probably due in part to their size being closer to the scale of the filter’s surface roughness that provides a stronger interaction. Thus, the filter cover is not necessary, and the laser power was lowered to 0.6 W. The sample was scanned at 0.04 mm/s along the direction of Y and then moved 0.2 mm along the X direction toward the MS for the next scan (top-down, flyback). The images were reconstructed in MATLAB using intensities of selected peaks with a median filter (radius = 3) applied along the scan dimension. As shown in Figure , the spatial distribution of the PE microspheres observed in the white-light image of the sample obtained with an inspection microscope (Figure A) compares very favorably with the MS image of the peak at m/z 576.0872 (Figure B), which shows the analyte is confined to the position of the microspheres. This peak is tentatively identified as protonated copper phthalocyanine, commonly known as phthalo blue, which would account for the blue color of the PE microspheres, and was confirmed by HCD MS/MS (Figure S1). A second measurement was performed on a few polyethylene (PE) microspheres from a sample with a nominal diameter range of 106–125 μm and, as per its white-light image (Figure C), the positions of the PE microspheres on the filter correlate well with the spatial distribution of the PE fragment-ion intensities in the corresponding MS image (Figure D). All three particles are clearly detected based on the cumulative intensity of fragment ions (sum-count image) within the m/z range of 123.1169–241.2165. These ions were tentatively assigned to homologous series of alkyl-substituted vinyl cyclohexenes (Table S1) and vinyl-terminated carboxylic acids (Table S2), and were confirmed using HCD MS/MS experiments of a representative ion from each respective series (Figure S2 and Figure S3). In addition, a fragment-ion series of alkyl-substituted cyclic diketones (Table S3) was also identified from the HCD MS/MS of a representative ion (Figure S4), but it was not included in the sum-count image due to the lower intensities. These assignments are consistent with products formed during the thermal oxidation of polyethylene. − One should bear in mind that the samples implemented in the proof-of-concept work here present relatively simple matrices, such that the potential for spectral overlaps is significantly reduced. Nevertheless, analysis of samples with more complex matrices, as is the case with environmental or biological samples, may require higher mass resolving power to prevent potential isobaric interferences, which would correspondingly affect sample throughput, or selection of a different set of analyte ions. It should be noted that the MS images display some areas in the vicinity of certain particles that resemble a “shadow”, which corresponds to the sample scanning direction. It is possible that this may be due to redeposition of the pyrolysis products onto the filter substrate. For example, pyrolysis-product vapor is blown by the FAPA effluent source toward the MS (top of image) but may be redeposited onto the filter substrate toward the direction of the sample scan, such that it would be resampled on the next scan. Additionally, it merits consideration that the spatial resolution of the current LAMP FAPA MS imaging configuration is governed by the laser spot size, as well as the scanning step length. Thus, small particles that simultaneously come to be under the laser-beam spot could not be resolved. Nevertheless, the proof-of-concept work presented here is implemented on filter samples with low number density of microplastic particles that are separated by distances significantly longer than the ∼300 μm laser spot size and scanning step length, which allows each particle to be resolved. Accordingly, implementation of the current LAMP FAPA MS imaging configuration for environmental MP samples would be limited to low number-density conditions, such that it may require diluting the suspension prior to collecting it onto the filter for analysis or filtering a lower suspension volume. Even so, this is not a fundamental limitation of the LAMP FAPA MS imaging technique, and current and future work in our laboratory involves implementing better commercially available laser focusing optics, which will eventually approach a diffraction-limited spot size of ∼5.3 μm (∼λ/2, in air) with the current laser wavelength (10.6 μm), thereby improving significantly the spatial resolution and enabling the analysis of filter samples with a high number density of microplastic particles. In addition, further systematic optimization of instrumental parameters, including the relative geometry of the FAPA source to the mass spectrometer, FAPA discharge current, helium flow rate, laser power, and scan speed, is expected to further enhance SNR and the overall quality of the mass spectrometry images.

4.

Optical microscope image of PE microspheres (180–212 μm) on glass microfiber filter (A), MSI of the additive peak at m/z 576.0872 (B), optical microscope image of PE microspheres (106–125 μm) on a glass microfiber filter (C), and sum-count MSI of PE microspheres fragment ions at m/z 123.1169, 137.1325, 151.1481, 157.1221, 165.1636, 171.1337, 179.1794, 185.1532, 213.1849, 227.2006, and 241.2165 (D). A ten-step interpolation was applied to the height dimension (steps between scans), while a 3:1 binning was applied to the width dimension (scan direction) to match the pixel dimensions in the MS images displayed.

Single-Particle Polymer Mass Quantitative Analysis

LAMP FAPA MS imaging of MPs shows the capability for single-particle detection, which carries the potential for single-particle quantitation. For example, single-particle quantitation in terms of particle mass may be possible from its relationship to the intensity of the polymer fragment ions when the particle is fully consumed. Thus, a calibration curve was constructed to study the possibility of single-particle mass quantitation using the following strategy. The two visibly intense fragment ion series (m/z 185.1534–423.4191 and m/z 183.1378–421.403) from PE mass spectra (Figure A) were selected, and their intensities were summed to generate the mass spectrometry images (MSI), which would then be used for quantification. The pixels corresponding to each microsphere were identified in the X and Y dimensions, and their intensities integrated across the area of each particle to obtain the total signal per particle. PE microspheres from samples with different diameter size ranges (45–53 μm, 106–125 μm, and 180–212 μm; 6 microspheres from each size range/18 microspheres total) were analyzed. The average intensities per six particles were plotted against particle mass, which was obtained from the diameter of the microspheres by calculating the corresponding volume and using the density provided by the PE microsphere manufacturer (1.00 g/cm³). It is noteworthy that in terms of diameter the largest size here is ∼4× the lowest, but in terms of mass it is approximately 2 orders of magnitude. The corresponding calibration curve (Figure B) displays a linear response (R ² = 0.9989). Furthermore, the LOD in terms of particle mass is 0.083 ng, which would be equivalent to a spherical single particle of 5.4 μm diameter. As mentioned earlier, the samples here contain relatively simple matrices, but samples collected from environmental or biological sources will have more complex matrices that may affect the analytical figures of merit, for example, such as from ionization suppression. Future work involves evaluating the technique’s performance under such conditions.

5.

Mass spectra of PE microspheres (180–212 μm) (A) and the calibration curve of intensity vs particle mass (B).

It is important to keep in mind that the MS imaging mode also provides information on the particle shape. For example, an MP with a greater length than width, such as fiber, would be easily distinguished from a pellet. Compared to single-particle event detection techniques like SP-ICP-MS, on the one hand, LAMP FAPA MS imaging overcomes the limitation of having to assume all particles are spherical to relate mass to shape. ,, On the other hand, the novel technique presented here is more restricted with respect to microplastic particle sample throughput and detection limits, which are reported to be an order of magnitude better (∼0.1 μm) in terms of diameter for SP-ICP-MS. , In this sense, LAMP FAPA MS is more comparable to conventional LA-ICP-MS for analysis of MPs because they both preserve spatial distribution and particle morphology while extending the upper limit in terms of particle size. , Nevertheless, ICP-MS-based techniques provide data on elemental composition and cannot directly provide molecular information, whereas LAMP FAPA MS is demonstrated here to yield molecular and structural information on microplastic polymers and organic additives. Thus, LAMP FAPA MS imaging should be regarded as a complementary analytical technique to ICP-MS-based techniques to expand the analytical toolbox for comprehensive microplastic characterization.

Conclusions

In this study, LAMP FAPA MS imaging is demonstrated for the characterization of microplastics (MPs) at the single-particle level. The results show that a CO₂ laser enables significantly more efficient pyrolysis-based sampling of transparent and translucent MPs compared to a 405 nm diode laser. The method developed allows MP characterization directly on filter substrates, which is compatible with widely implemented MP collection protocols, thus avoiding further sample preparation to minimize potential sample loss and contamination. In addition, a mass spectral analysis algorithm was built to automatically identify series of polymer fragment ions in MPs based on their mass differences corresponding to the repeating/base unit. Both polar and nonpolar polymers in MPs were successfully characterized. Furthermore, LAMP FAPA MS is also shown for the analysis of additives and, for the first time, was developed to enable quantitation of polymer mass by imaging single-particle polyethylene microspheres, with a limit of detection equivalent to 5.4 μm diameter spherical particle. Ultimately, the newly developed capabilities of LAMP FAPA MS toward single-particle MP characterization have the potential for significant impact in the field, given that currently different techniques are needed to obtain information on the type of polymer, identification of additives, particle mass, or particle size. In fact, some of the most popular techniques for MP analysis, such as IR microscopy, have limitations that could be overcome via LAMP FAPA MS, for example, in the analysis of tire–road ware particles (TRWP), where IR signals are severely limited.

Ongoing and future work will focus on developing the technique’s single-particle quantitative capabilities toward a broader range of environmentally relevant microplastic polymers. Also, the figures-of-merit will be characterized for samples collected from environmental and biological sources to evaluate the performance with complex matrices. Moreover, the potential for the technique to be implemented in analyzing adsorbed contaminants on MPs, as well as quantifying copolymer fractions in TRWP, will be explored. In addition, the imaging capabilities will be enhanced, including the spatial resolution by reducing the laser spot size and optimizing the LAMP FAPA source geometry.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.6c02632.

• Additional tables of tentatively identified LAMP FAPA MS polymer fragment-ion series from single microparticles and HCD-MS/MS spectra of selected ions from each series (PDF)

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D.Z. and E.A. contributed equally to this work. D.Z.: Methodology, investigation, data curation, validation, formal analysis, writingoriginal draft. E.A.: Methodology, investigation, data curation, validation, formal analysis, writingoriginal draft. G.G.: Conceptualization, methodology, supervision, writingoriginal draft, writingreview and editing, resources, project administration, funding acquisition.

The authors declare no competing financial interest.