Polypyrrole-based Extraction Materials for Microcystin Separation and Extraction

Inventors: Jon R. Kirchhoff, Amila Devasurendra, Dilruhshika S.W. Palagama, Dragan Isailovic, Joshua A. Young

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is national stage application filed under 35 USC §371 of international application PCT/US2019/xxxxxx filed February xx, 2019 which claims priority to US provisional application Ser. No. 62/629,874 filed February 13, 2018, the entire disclosure of which is expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The invention not was made with any U.S. Government support, and the United States Government has no rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Microcystins (MCs) are liver toxins produced by freshwater cyanobacteria often found in lakes or reservoirs during harmful algal blooms (HABs). They cause both acute and chronic health effects and can lead to death of humans and animals when exposed to MCs in high concentrations. MCs are cyclic heptapeptides with the common structure cyclo(Adda-D-Glu- Mdha-D-Ala-L-X-D-MeAsp-L-Z-), where Adda is 3-amino-9-methoxy-2,6,8,-trimethyl-10- phenyl-4,6-decadienoic acid, Mdha is N-methyl-dehydroalanine, D-MeAsp is 3-methylaspartic acid, and X and Z are variable L-amino acids (FIG 1).

[0004] MCs can also have structural differences such as the loss or addition of substituent groups. To date, more than 200 different MC variants have been identified. Among these variants, MC-LR is the most common MC found in freshwater sources during HABs.

[0005] During the summer of 2014, the City of Toledo (Ohio, USA), which relies on Lake Erie as its primary source of water, announced a“do not drink” water advisory for its residents after detecting elevated levels of MC-LR in their public drinking water system. Since then, HABs and their impact on water quality have become a serious topic of discussion during the summer months. The current United States EPA 10-day drinking water health advisories related to MC exposure for pre-school age children and for school-age children and adults are 0.3 pg/L and 1.6 pg/L, respectively, while the World Health Organization (WHO) 70-year lifetime guideline is 1 pg/L (free plus cell bound) for MC-LR. The effect of MCs on tourism, recreation, and appearance of aquatic ecosystems has become a significant concern around the world. Thus, growing attention to the health and societal impacts of MCs highlights the necessity to develop highly reliable, sensitive and selective analytical detection techniques for their identification and determination.

[0006] Several analytical detection methods have been previously evaluated to quantify MCs. Enzyme-linked immunosorbent assay (ELISA), protein phosphatase inhibition assay (PPIA), capillary electrophoresis (CE), and high performance liquid chromatography (HPLC) combined with ultraviolet (UV) or mass spectrometric (MS) detection are the most common techniques. However, detection of MCs at extremely low concentrations is still challenging due to matrix interferences in environmental samples. Therefore, sample preparation is considered a crucial step. In the aforementioned methods, preconcentration techniques such as dispersive liquid-liquid microextraction (DLLME), solid-phase microextraction (SPME), and solid-phase extraction (SPE) have been applied.

[0007] For SPE analyses, several different choices, such as C8 and Cl 8 _, ion-exchange resin, and silica gel cartridges, are commercially available. Although SPE is used for analyte preconcentration and clean-up in many trace level analyses, one of the major disadvantages of conventional sorbents materials (e.g., C8, C18, ion-exchange) is the lack of selectivity. Similar analytes often co-elute and thereby decrease the selectivity and sensitivity of the detection technique. As an alternative, researchers have developed target specific sorbent materials including immunosorbents and molecular imprinted polymers (MIPs). However, these sorbents still have limitations such as complex and expensive fabrication protocols, lack of stability, and selectivity. Moreover, they are highly specific and thus may not be applicable for extraction of a broad range of analytes.

[0008] As such, there is a great need to efficiently analyze and extract microcystins.

SUMMARY OF THE INVENTION

[0009] In one aspect, there is described herein a method for analysis and/or extraction of one or more microcystins from a sample, comprising: 1) exposing the sample to a gold-polypyrrole (Au-PPy) nanocomposite sorbent material; and, 2a) conducting a solid-phase extraction (SPE) protocol that includes one or more elution solutions, thereby analyzing and/or extracting the microcystins; or, 2b) conducting a liquid chromatography mass spectrometric (LC-MS) detection protocol, thereby analyzing and/or extracting the microcystins.

[0010] In certain embodiments, the SPE protocol allows for simultaneous microextraction of multiple types of microcystins from the sample.

[0011] In certain embodiments, the sample comprises liquid from one or more of: river, lake, pond, ditch, reservoir brown field site, treatment plant, waste water from a manufacturing process, and potable water.

[0012] In another aspect, there is described herein is the use of polypyrrole (PPy) and Au nanoparticles (NPs) together to make a novel SPE sorbent material for preconcentration, quantification, and selective determination of MCs in natural and public drinking water systems

[0013] A gold -polypyrrole (Au-PPy) nanocomposite sorbent is prepared and immobilized simultaneously on silica particles by chemical polymerization. The Au-PPy nanocomposite sorbent material shows excellent extraction efficiency for six of the most common MCs. By changing the elution solvent system and SPE bed size, full separation between hydrophobic and hydrophilic MCs is also achieved.

[0014] Such an off-column separation of MCs using a sample preparation and

preconcentration technique now provides a new methodology for determination of low levels of MCs.

[0015] In one particular aspect, there is provided a nanocomposite sorbent material, comprising silica particles at least partially coated with an Au-PPy matrix. It is also within the contemplated scope that different coatings beyond PPy coatings can be used. For example, in other embodiments, derivatives of PPy including, but not limited to ionic liquid (IL)-PPy coatings such as poly[pyrrole-C ₆ MIm] ⁺ , where MIm is methyl imidazole, and other heterocycle-based conducting polymers like thiophenes, useful.

[0016] In certain embodiments, the silica particles have a particle distribution range from about 40 to about 100 micrometers. In certain embodiments, the particle sizes range from 55-105 micro meters. In certain specific embodiments, the silica particles comprise silica gel-granular particles having a particle distribution of 40-100, and also 40-63 pm.

[0017] In another aspect, there is provided a method for analysis and/or extraction of one or more microcystins from a sample, comprising exposing the sample to an Au-PPy nanocomposite sorbent material, and conducting a solid-phase extraction (SPE) protocol.

[0018] In certain embodiments, the method allows for simultaneous microextraction of multiple microcystins from the sample.

[0019] In certain embodiments, the sample comprises liquid from one or more of: river, lake, pond, ditch, brown field site, treatment plant, waste water from a manufacturing process, etc.

[0020] In certain embodiments, the liquid sample comprises water, including, for example, potable water.

[0021] In certain embodiments, the microcystins (MC) comprise one or more of: cyclic heptapeptides with the common structure cyclo(Adda-D-Glu-Mdha-D-Ala-L-X-D-MeAsp-L-Z-); where Adda is 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyl-4,6-decadienoic acid; Mdha is N- methyl-dehydroalanine; D-MeAsp is 3-methylaspartic acid; X is Leu, Try or Arg; and, Z is Arg, Phe, Trp or Ala.

[0022] In certain embodiments, the microcystins comprise one or more of: MC-LR, MC-YR, MC-RR, MC-LA, MC-LF, and MC-LW, as shown in FIG. 1.

[0023] In certain embodiments, the MC’ s are detected at ng/L concentrations.

[0024] In another aspect, there is provided a method for detecting microcystins in a sample, comprising optimizing Au-PPy nanocomposite sorbent materials, and conducting a liquid chromatography mass spectrometric (LC-MS) detection protocol to detect the microcystins.

[0025] In another aspect, there is provided a method for separating hydrophilic (MC-LR, MC- YR, and MC-RR) and more hydrophobic (MC-LA, MC-LF, and MC-LW) MCs, comprising: selecting at least one elution solution during a solid-phase extraction (SPE) protocol with sequential elution.

[0026] In certain embodiments, the solvent combinations comprise one or more of solvent combinations found in FIG. 16.

[0027] In certain embodiments, the optimizing the elution solvent comprises combining one or more elution solvent combinations of ES-1, ES2, ES-3 and ES-4.

[0028] In certain embodiments, the solvent combination comprises ES-6.

[0029] In certain embodiments, the method further comprises equilibrating with 0.1% formic acid (FA) prior to sample loading to convert PPy to a fully protonated form to facilitate ionic interactions -with the -COO- groups of the MCs.

[0030] In another aspect, there is provided a method for efficient and selective recovery of diversified biological analytes, comprising using a nanocomposite sorbent material comprised of silica particles at least partially coated with an Au-PPy matrix.

[0031] In another aspect, there is provided a column packing material for LC separation of contaminated water samples, comprising Au-PPy nanocomposite sorbent materials.

[0032] Other systems, methods, features, and advantages of the present invention will be or will become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present invention, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The patent or application file may contain one or more drawings executed in color and/or one or more photographs. Copies of this patent or patent application publication with color drawing(s) and/or photograph(s) will be provided by the Patent Office upon request and payment of the necessary fee.

[0034] FIG. 1. The general structure of six common variants of MC. MC-LR (X = Leu, Z = Arg); MC-YR (X = Tyr, Z = Arg); MC-RR (X = Arg, Z = Arg); MC-LF (X = Leu, Z = Phe); MC- LW (X = Leu, Z = Trp); MC-LA (X = Leu, Z = Ala).

[0035] FIGS. 2A-2G. LM image of unmodified silica (FIG. 2A), and Au-PPy

nanocomposite-coated silica particles (FIG. 2B), SEM microphotographs of Au-PPy

nanocomposite-coated silica particles (FIG. 2C), an Au nanoparticle cluster within PPy (FIG. 2D), Au-PPy nanocomposite-coated silica particles before extraction (FIG. 2E), and after three successive extractions (FIG. 2F); and image of a SPE cartridge packed with Au-PPy

nanocomposite sorbent material (FIG. 2G).

[0036] FIG. 3. Optimization of the eluting solvent for MCs with the Au-PPy nanocomposite sorbent material (number of replicates, n=3). Solvent compositions ES-1 to ES-11 are shown in FIG. 16 - Table SI, and the dotted line in FIG. 3 depicts the optimum condition.

[0037] FIG. 4. Optimization of the equilibration solvent for MCs with the Au-PPy nanocomposite sorbent material (number of replicates, n=3).

[0038] FIG. 5. Optimization of the acidity during sample loading for MCs with the Au-PPy nanocomposite sorbent material (number of replicates, n=3).

[0039] FIG. 6. Graph of recovery vs. fractions collected during selective extraction of MCs (number of replicates, n=3). Total recoveries of individual MCs from selective separation (%): MC-LR (73.6); MC-YR (74.7); MC-RR (84.7); MC-LF (85.0); MC-FW (88.0); MC-FA (89.0).

[0040] FIG. 7 - Table 1. Average recoveries and relative standard deviations (RSDs) for MC determination using the Au-PPy nanocomposite sorbent coating for SPE purification and preconcentration for different spiked water matrices (number of cartridges, n=3).

[0041] FIG. 8 - Table 2. Quantification of MCs in spiked tap and lake water matrices.

[0042] FIGS. 9A-9F. SIM mass spectra of: MC-FR (FIG. 9A); MC-YR (FIG. 9B); MC-RR (FIG. 9C); MC-FA (FIG. 9D); MC-FW (FIG. 9E; and, MC-FF (FIG. 9F).

[0043] FIG. 10. FTIR spectra of: unmodified silica (a), and Au-PPy nanocomposite-coated silica particles (b).

[0044] FIG. 11. SEM-EDX spectrum of the Au-PPy nanocomposite sorbent material.

[0045] FIG. 12. PXRD spectrum of the Au-PPy nanocomposite sorbent material. The pattern was recorded with Cu K _a (1.54A) radiation in the range of 2Q from 35° to 85° with a standard goino experiment.

[0046] FIGS. 13A-13F. Calibration curves for quantitation of each MC in tap water samples (concentration range: 2.5-80.0 ng/F for MC-FR, MC-YR and MC-RR and 5.0-80.0 ng/F for MC- FA, MC-FF and MC-FW).

[0047] FIGS. 14A-14F. Calibration curves for quantitation of each MC in lake water samples (concentration range: 2.5-80.0 ng/F for MC-FR, MC-YR and MC-RR and 5.0-80.0 ng/F for MC-FA, MC-FF and MC-FW).

[0048] FIGS. 15A-15C. Graphs of percent recovery vs. SPE trials for reusability of the Au- PPy nanocomposite sorbent material with ten successive extractions with a single cartridge of: 10 ng/F (FIG. 15A), 50 ng/F (FIG. 15B) and, 500 ng/F (FIG. 15C) of MCs; black and red dotted line represents 100% and 95% recovery limits, respectively.

[0049] FIG. 16 - Table SI. Different solvent compositions used for optimization of the elution of MCs from the Au-PPy nanocomposite cartridge.

[0050] FIGS. 17A-17C. - Table S2 Recoveries and RSDs for MC determination using the Au-PPy nanocomposite sorbent coating for SPE purification and preconcentration: HPFC-grade water (FIG. 17A); tap water (FIG. 17B); and, (c) lake water (FIG. 17C). Number of replicates, n=3 and preconcentration factor, lOx. [0051] FIG. 18 - Table S3. Calibration data and calculated lower limit of detections (LODs) and limit of quantifications (LOQs) values for MC-spiked tap and lake water matrices.

[0052] FIG. 19A - Table S4. Comparison for the extraction of MC-LR in water samples using commercially available sorbent materials, and the Au-PPy nanoparticle SPE sorbent materials.

[0053] FIG. 19B. List of references compared in FIG. 19A.

[0054] FIG. 20 - Table S5. Sequential fraction collection procedure for selective separation of hydrophilic and hydrophobic MCs.

DETAILED DESCRIPTION

[0055] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

[0056] Solid-phase extraction (SPE) provides a simple, fast, easy to automate, and sensitive sample preparation method. SPE eliminates the disadvantages of conventional extraction methods such as using toxic organic solvents, large amounts of sample and solvent, and complex, time- consuming extraction and cleanup procedures. However, until the present invention, the application of SPE for the analysis of microcystins has been challenging.

[0057] An Au-PPy nanocomposite sorbent material with multi-mode interaction ability was synthesized on silica particles by chemical polymerization of pyrrole with HAuCl _4* 3H ₂ 0. SEM, EDX and PXRD investigations confirmed that the new sorbent material exhibited uniform coverage, while AuNPs were formed and aggregated to produce sphere shaped clusters embedded in the PPy coating. Using a minimal amount of sorbent material (20 mg) with careful optimization, an efficient, and sensitive LC-ESI-MS-based protocol was established to preconcentrate and quantify a mixture of MCs (MC-LR, MC-YR, MC-RR, MC-LA, MC-LF, and MC-LW) at ng/L concentrations. This method was then extended to analyze MCs spiked in water samples from Lake Erie and regular tap water. All MCs showed a high recovery and low LODs and LOQs with excellent precision. By changing cartridge bed size and solvent composition during the SPE protocol with sequential elution, complete separation between more hydrophilic (MC-LR, MC-YR, and MC-RR) and more hydrophobic (MC-LA, MC-LF, and MC-LW) MCs was accomplished.

[0058] The Au-PPy nanocomposite sorbent material is effective for purification and preconcentration of MCs in LC-ESI-MS analyses. It is important to further note that the LODs and LOQs for MC-LR and its variants are well below the recommended EPA short-term exposure guidelines that are critical for monitoring and assessing the health risks due to HABs, and are improved when compared to readily accepted procedures that utilize immunoassay -based methodology.

[0059] The SPE sorbent material is also useful for efficient and selective recovery of diversified biological analytes, and as a column packing material for LC separation of contaminated water samples.

[0060] EXAMPLES

[0061] The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. All publications, including patents and non-patent literature, referred to in this specification are expressly incorporated by reference. The following examples are intended to illustrate certain preferred embodiments of the invention and should not be interpreted to limit the scope of the invention as defined in the claims, unless so specified.

[0062] The value of the present invention can thus be seen by reference to the Examples herein.

[0063] Materials and Reagents

[0064] Standard stock solutions of MC-LR, MC-LA and MC-RR (concentrations of 500 mg/L, 500 mg/L and 100 mg/L, respectively) in ethanol were purchased from Cayman Chemical Company (Ann Arbor, MI). MC-LW, MC-LF, and MC-YR were purchased as solid substances from Enzo Life Sciences (Farmingdale, NY). HPLC-grade water and methanol were purchased from Fisher (Fair Lawn, NJ). HPLC-grade acetonitrile, formic acid (FA, 98-100%), pyrrole (98%), gold(III) chloride trihydrate (HAuCU*3H ₂ 0, >99.9%), non-fluorous polypropylene reversible SPE tubes with frits (0.5 mL), clear glass vials (2 mL) and glass inserts were purchased from Sigma (St. Louis, MO). Standard-grade silica gel-granular (particle distribution 40-63 pm) was obtained from Sorbent Technologies, Inc. (Norcross, GA). 10 mL and 3 mL syringes were purchased from Becton Dickinson and Company (Franklin Lakes, NJ). MC-free lake water from the shore of Lake Erie (Oregon, OH) and regular tap water at the University of Toledo (Toledo, OH) were collected and filtered through a nylon membrane filter from Sigma (pore size, 0.2 pm). Pyrrole was freshly distilled each time before use. All the other reagents and materials were used as received from the supplier. HPLC-grade water was used to prepare all solutions and mixtures when necessary.

[0065] Instrumentation

[0066] An Orbitrap Fusion (Thermo Scientific, San Jose, CA) mass spectrometer containing three mass analyzers (a quadmpole, a linear ion trap, and an orbitrap) and an electrospray ionization (ESI) source was used. Attached to the MS was an HPLC (Shimadzu Technologies, Addison, IL) equipped with two LC-20AD pumps, a DGU-20A3 degasser, a SIL-20A HT autosampler, and a CBM-20A system controller. HPLC separation of MCs was achieved on a XBridge C8 column (particle diameter (3.5 pm), i.d. (3.0 mm), and length (100 mm), Waters, Milford, MA) with a C8 guard column. MS data were acquired and analyzed using Xcalibur software (Thermo Scientific). Scanning electron microscopy (SEM) images were obtained with a JEOL JSM-7500F scanning electron microscope (JEOL USA, Inc., Peabody, MA) with a BRUKER XFlash 5010 series energy dispersive X-ray spectroscopy (EDX) detector (Billerica, MA). Light microscopy (LM) images were obtained with an Olympus SZX7 microscope coupled with an Olympus SC 100 camera (Olympus America, Inc., PA). Powder X-ray diffraction (PXRD) data were collected using a PANalytical X’Pert Pro diffractometer with a line focus incident beam and an X’celerator detector (Almelo, The Netherlands). Fourier transform infrared (FTIR) spectroscopic measurements were carried out in the attenuated total reflectance (ATR) mode with a PerkinElmer Frontier spectrophotometer (Shelton, CT).

[0067] SPE Procedure for MC Extraction

[0068] Au-PPy nanocomposite-coated silica particles were prepared and packed into a standard SPE cartridge. The detailed procedures for both the sorbent preparation and packing are described below.

[0069] Stock solutions of MCs were first diluted with HPLC-grade water, tap water or lake water to prepare mixtures of standard or control samples. SPE cartridges were then conditioned with 2 mL of an acidified aqueous-methanol solution (CH OfTlTO, 90:10 v/v with 0.1% FA) followed by equilibration with 2 mL of a 0.1% FA solution. Next, 2.0 mL of a MC mixture was loaded onto the SPE cartridge and further washed with 1.5 mL of 0.1 % FA to remove salts and other impurities. The extracted MCs were eluted with 1.75 mL of acidified aqueous-methanol (CtFOIFtbO, 90:10 v/v with 0.1% FA) solution and preconcentrated by an Eppendorf vacuum concentrator (Hauppauge, NY). MCs were then re-dissolved in 200 pL of acidified aqueous- methanol solution (CPFOI-htbO, 90:10 v/v with 0.1% FA) and used for LC-MS analyses.

[0070] LC-ESI-MS Protocol

[0071] LC separation was conducted with 0.05% FA (mobile phase A) and acetonitrile containing 0.05% FA (mobile phase B) as the mobile phases. The column was conditioned with a mobile phase ratio of 80:20% (A:B) for at least 30 min prior to separation. During each analysis, a solvent gradient consisting of 0.1-2 min 60% of B, 2-7 min 70% of B, 7-12 min 90% of B, 12-14 min 20% of B, and then an additional 6 min at 20% of B for equilibration was applied for a total of 20 min run time. The sample injection volume was 20 pL. The HPLC was coupled to the Orbitrap Fusion mass spectrometer for identification and quantification of MCs. The electron spectroscopic imaging (ESI)-selected ion monitoring (SIM)-MS positive ion mode was used to achieve higher sensitivity. Samples were analyzed in triplicate and extracted ion chromatogram (EIC) peak areas of monoisotopic MC ions were used for data extraction and calculation. Singly-charged protonated ions ([M+H] ⁺ ) of MC-LR (m/z 995.56), MC-LA (m/z 910.49), MC-LW (m/z 1025.53), MC-LF ( m/z 986.52), MC-YR ( m/z 1045.54), and doubly-charged protonated ([M+2H] ²⁺ ) ion of MC-RR ( m/z 519.79) were selected with a quadmpole isolation width of 5 m/z units (FIGS. 9A- 9F). A mass accuracy of less than 3 ppm was achieved. MS/MS was performed simultaneously and the Adda fragment ion [CeFE-CFECIhOCFE)] at m/z 135.08 was used for identification of MCs.

[0072] Preparation and Characterization of Au-PPy Nanocomposite Sorbent Material

[0073] Chemical polymerization was used to prepare the Au-PPy nanocomposite-coated silica particles by a one-pot synthesis procedure using HAuCU^FhO in an acetonitrile reaction medium. During the synthesis, HAuCEGFEO served as the oxidizing agent for pyrrole.

[0074] Other useful oxidizing agents can include, but are not limited to, AuCU, FcCF, potassium persulfate, hydrogen peroxide, and the like.

[0075] As an added benefit for analyte extraction, AuNPs were also formed simultaneously with PPy and trapped inside the polymer matrix to form the Au-PPy nanocomposite, which was deposited on the surface of the silica particles.

[0076] Once prepared, the Au-PPy coated silica was examined by in-situ FTIR analysis and compared to unmodified silica. As can be seen in FIG. 10, both unmodified and Au-PPy nanocomposite-coated silica showed strong bands at around 1085 cm ¹ and 824 cm ¹ , which correspond to the asymmetric and symmetric vibrations of Si-O-Si, respectively. In addition, unique absorption bands of PPy due to C=C stretching at 1570 cm ¹ and 1490 cm ¹ , =C-H out-of- plane vibrations at around 700 cm ¹ and 965 cm ¹ , and broad asymmetric stretching of -N-H at 3000-3700 cm ¹ can be seen in FIG. 10 graph line (b). Compared to the characteristic silica FTIR absorption bands, PPy polymer absorptions were less pronounced in the coated sample which is believed to be due to the high silica to nanocomposite ratio.

[0077] FIGS. 2A-2G illustrate LM and SEM images of the Au-PPy nanocomposite-coated silica particles and the packed cartridge for the extraction of MCs. FIG. 2A and FIG. 2B clearly contrast the difference between unmodified silica particles and the Au-PPy nanocomposite-coated black silica particles. Moreover in FIG. 2B, evenly distributed yellow colored bright spots were identified throughout the nanocomposite coating.

[0078] As shown in FIG. 2C and FIG. 2D with SEM imaging, these spots were identified as Au nanoparticle aggregates embedded in PPy to form sphere shaped nanocomposite clusters with diameters around 5 pm. EDX and PXRD analyses were further performed to confirm the presence of Au and the nanoparticle size distribution. The surface EDX spectrum obtained for the Au-PPy coating clearly shows peaks attributed to Au along with other constituents such as C, N, Si, and O (FIG. 11).

[0079] In addition, a peak for Cl can also be seen, which acts as the counter anion for PPy.

The PXRD pattern of the Au nanoparticles is shown in FIG. 12. The pattern was measured using Cu K _a (1.54A) radiation in the range of 20 from 35° to 85°, where 0 is the Bragg angle. Au nanoparticles show characteristic diffraction peaks of face-centered cubic metallic Au at 38.150° (0.003), 44.344° (0.007), 64.564° (0.008), 77.523° (0.009), and 81.696° (0.021) (Crystallography Open Database, COD: 96-900-8464). PPy did not produce any peaks in this range, which indicates its amorphous nature. Based on PXRD peak widths and the Scherrer equation (particle size = 0.9/./i/cos(7, where l is X-ray wavelength and d is line broadening at the half maximum intensity), the average crystal size of the Au nanoparticles was estimated to be 20-36 nm.

[0080] FIG. 2E and FIG. 2F compare the SEM images for the coating morphology before and after three successive extractions with vigorous organic solvent exposure (methanol, acetonitrile, formic acid, etc.). As can be seen, there was no significant change in morphology, which confirms the excellent stability of the sorbent coating.

[0081] FIG. 2G represents an image of a SPE cartridge containing the Au-PPy nanocomposite-coated silica particles. The particles were manually packed in between two polyethylene frits to avoid any disturbance to the SPE bed during analyte extraction. During this process, extra care was taken to maintain consistent packing density between cartridges.

[0082] Optimization of the SPE Protocol

[0083] SPE protocols involve conditioning and equilibration steps for the SPE cartridge, followed by sample loading, washing, and elution steps. Analytes are then concentrated by evaporation and redissolved in a suitable solvent for subsequent analysis. Therefore, these steps were optimized for the newly designed Au-PPy nanocomposite sorbent material for MCs.

Acidified aqueous-methanol was used for preliminary conditioning of the sorbent particles. For evaluation and optimization of the retention and recovery capabilities of the Au-PPy nanocomposite, a standard mixture of six MCs with an individual MC concentration of 50 ng/L was used. Recoveries were calculated by comparing LC-MS EIC peak areas of the monoisotopic peak of each MC ion in the extracted sample and MC standard solution. For highly efficient performance, in certain embodiments, the MCs can be quantitatively retained and then desorbed from the sorbent material in a minimal amount of elution solvent. In this step, different solvent combinations were investigated by changing the acidity and eluent polarity while maintaining a constant elution volume of 1.75 mL (FIGS. 17A-17C).

[0084] Based on these results, an acidified aqueous-methanol solution, which resulted in a recovery for all MCs > 95%, was selected as the best elution solvent (FIG. 3).

[0085] Prior to sample loading and elution, the Au-PPy nanocomposite sorbent was equilibrated with pure water or 0.1 % FA while keeping the other conditions constant. Compared to pure water, equilibration with 0.1% FA showed slightly better MC recoveries (FIG. 4).

[0086] Next, the effect of acidification of the sample was studied using 0.1% FA in the loading stage. In contrast to the equilibration step, acidity during sample loading dramatically reduced MC recovery (FIG. 5). [0087] By considering the properties of the Au-PPy nanocomposite materials described herein, the results for the optimization experiments are shown. Upon acidification, nitrogen atoms in PPy are protonated resulting in the sorbent material becoming positively charged. To maintain the overall neutrality of PPy, ion-pairing with anions present in the solution occurs. Existence of these positive charges on the sorbent material significantly enhances sorption of anionic analytes by coulombic interactions. Equilibration with 0.1% FA prior to sample loading converts PPy to a fully protonated form and facilitates ion-exchange of anions with the -COO groups of the MCs. On the other hand, acidic conditions during sample loading may enable the protonation of -COO groups and weaken the interaction of MCs with the sorbent material, resulting in reduced recovery. The best recoveries for elution with 0.1% FA can be attributed to the same phenomena. Addition of 0.1% FA facilitates the anion exchange and releases the MCs from the sorbent to yield higher recoveries. This is clearly seen during the optimization of the elution solvent (see elution solvent combinations ES-1 to ES-4). Different solvent compositions also displayed the optimum solvent polarity for each MC with ES-6 having the optimum conditions and highest recoveries for analysis.

[0088] Conjugated p-bonding of pyrrole rings in PPy also increases the binding affinity to aromatic groups via p-p interaction. All MCs have an aromatic Adda moiety in the structure such that they can strongly bind to PPy via p-p bonding. Particularly, the more hydrophobic MCs (MC- FF and MC-FW) have additional aromatic substitutes in their variable amino acid groups providing further interaction points. Independently, AuNP clusters embedded in PPy can have a strong affinity towards MCs due to hydrophobic and p-system interactions between the AuNP surface and different analyte moieties. The extent of all these multi-modal interactions happening together at the same time provide the basis for the selective separation of the individual MC structures with variable substituents at the X and Z positions.

[0089] SPE-LC-MS Quantification and Method Validation for MC Determination

[0090] To evaluate the accuracy and reproducibility of the Au-PPy nanocomposite as a sorbent material, water samples were analyzed by FC-MS after spiking the samples with MCs at different concentrations (10, 50, and 500 ng/F). Initial experiments were conducted using HPFC- grade water and followed by tap and lake water samples as real-world matrices. The sample loading was kept constant at 2 mL with a lOx preconcentration factor in all cases. This volume can be increased depending on the analysis requirement to achieve lower limit of detections (FODs) and limit of quantifications (FOQs). However, a 2 mL volume was maintained for easy handling and to minimize the amount of sample needed during the loading step. Recoveries of MCs were calculated from three replicates of each concentration using three independent cartridges in HPFC-grade water, tap and lake water (see FIGS. 17A-17C).

[0091] FIGS. 17A-17C summarize the average recoveries (of three cartridges) and relative standard deviations (RSDs) for all MCs in different matrices, which ranged from 94.1-103.2% and-1.6-5.4 %, respectively. RSDs related to recoveries were calculated based on the propagation of errors to determine the precision of the method. Comparatively, HPLC-grade water and lake water showed the highest and lowest recoveries, respectively. For all extractions, relatively low RSDs for the average recovery for three cartridges confirmed the good reproducibility of the extraction method. The excellent performance of the Au-PPy nanocomposite as a sorbent coating demonstrates its applicability in actual environmental and drinking water samples.

[0092] Accuracy of the method for quantification of lower concentrations was tested by constructing the calibration curve for each MC in tap and lake water samples between a concentration range of 2.5-80.0 ng/L for MC-LR, MC-YR and MC-RR and 5.0-80.0 ng/L for MC- LA, MC-LF and MC-LW (FIGS. 13A-13F) and 2.5-80.0 ng/L for MC-LR, MC-YR and MC-RR and 5.0-80.0 ng/L for MC-LA, MC-LF and MC-LW (FIGS. 14A-14F).

[0093] Calibration curves showed excellent linearity with R ² values equal to or greater than to 0.9955 for both sample matrices. LOD and LOQ data for MCs in tap and lake water were estimated from signal-to-noise (S/N) ratios of 3 and 10 times, respectively.

[0094] A summary of this data is given in FIG. 18 - Table S3. LODs and LOQs for six MCs in tap water ranged from 0.6-1.5 ng/L and 2.5-5.0 ng/L, while those for lake water were 0.7- 1.5 ng/L and 2.5-5.0 ng/L, respectively. MCs with one (MC-LR and MC-YR) or two (MC-RR) Arg residues at the X and Z positions tend to have lower LOD and LOQ values compared to the other MCs as Arg groups improve ionization of peptides by ESI. Validation of the calibration curves was further tested using control samples at three concentrations (15.0, 25.0, and 35.0 ng/L). All MCs in both tap and lake water samples revealed errors equal to or less than 7.5 % compared to control samples, indicating good reliability of the method (FIG. 8 - Table 2). These values for detection of MC-LR and its variants are well below the recommended EPA short-term exposure guidelines that are critical for monitoring and assessing the health risks due to HABs. Thus, SPE with the Au-PPy nanocomposite sorbent followed by LC-MS analysis exhibits excellent sensitivity and the ability to detect MCs at levels well below the levels considered to be hazardous.

[0095] For comparison of the Au-PPy nanocomposite sorbent coating with other materials described in literature, MC-LR was selected as the common MC analyte. FIGS. 19A-19B - Table S3 reviews the performance of MC-LR extraction in different water samples using a wide range of SPE sorbent materials. In most cases, the performance of the Au-PPy nanocomposite was greater than the other known SPE sorbent materials. Moreover, the SPE-LC-MS procedure described herein exhibits outstanding LODs and LOQs in comparison to many commercial immunoassay systems.

[0096] Reusability of the SPE Cartridges

[0097] Multiple extractions with a single commercial SPE cartridge are not often recommended due to the concern that repeated use of high concentrations of organic solvents and exposure of the sorbent material to sample contaminants, which can lead to irreversible changes in binding sites and decreased performance. Meanwhile, chemical robustness is considered one of the significant characteristics of the Au-PPy nanocomposite sorbent material. This allows repetitive cleaning and reactivation of the SPE cartridge with vigorous solvent conditions after multiple uses. Thus, the Au-PPy nanocomposite sorbent material was investigated for reusability.

[0098] To accomplish this, a single cartridge was tested with ten successive extractions of MCs at three different loading concentrations in lake water samples (10, 50, and 500 ng/L). Each cartridge was washed three times with 5 mL of 0.1% FA and 5 mL of an acidified aqueous- methanol solution in between individual sample loading to eliminate any carry-over effects of MCs. FIGS. 15A-15C summarize the data obtained for the ten repetitive extractions with the three concentrations of MCs in the more complex lake water matrix. To determine whether the performance is significantly reduced or not during use, a decrease in recovery equal to or less than 5.0% for any MC was set as the limit. For lower concentrations such as 10 and 50 ng/L, there is no significant decrease in recovery until the fifth and fourth trial, respectively. For the higher concentration, i.e., 500 ng/L, multiple attempts are possible for at least three repetitive uses. The decrease in recovery at later SPE trials could be due to the irreversible binding of interferences from the lake water. However, it is now shown herein that reusability of the cartridge is possible for a minimum of three trials without sacrificing the efficiency of analyte recovery.

[0099] Selective Recovery of MCs

[00100] Now described herein is a protocol to selectively separate more hydrophilic (MC-LR, MC-YR and MC-RR) vs. more hydrophobic (MC-LA, MC-LA and MC-LW) MCs in a mixture. To accomplish this, the bed size of the cartridge was increased to 40 mg and the elution solvent composition was changed while collecting sequential fractions (F-l to F-8). The increased bed size increased the retention time on the sorbent material and thus facilitated complete separation between the two groups of MCs. The detailed procedure and the optimized solvent conditions are given in FIG. 20 - Table S5.

[00101] Each fraction of eluent was independently concentrated and subjected to LC-ESI-MS analysis for detection and quantification. The results are presented in FIG. 6. More hydrophilic MCs eluted in early fractions with 100% methanol while the hydrophobic MCs stayed in the SPE cartridge until acidified aqueous-methanol was used as the elution solvent. Separation was achieved by manipulating the multi-mode interactions of the MCs with the sorbent material by changing the solvent polarity and acidity. The absence of 0.1% FA in the first elution solvent (100% methanol) allowed more hydrophobic MCs to be retained longer in the cartridge due to p-p and hydrophobic interactions. Meanwhile hydrophilic MCs eluted gradually throughout first few fractions (F-l to F-6). Washing steps with 0.1% FA in between fractions replenished the sorbent material and improved separation in subsequent fractions. In F-7, no significant amount of MCs was detected indicating complete separation was achieved. In F-8, the retained hydrophobic MCs were removed by application of acidified aqueous methanol. Total recovery was then calculated for each MC by adding up the individual recoveries in each fraction (FIG. 6).

[00102] For all MCs, the total recoveries ranged between 73.6 to 88.0%, which is less than when elution of all MCs occurs as a single fraction (FIG. 7 - Table 1). This is now believed to be due to the analyte losses during the continuous washing and transferring steps. However, to achieve complete separation between hydrophilic and hydrophobic MCs at the sample preparation and preconcentration stage prior to LC-ESI-MS analysis with a slight sacrifice in recovery can be, in certain embodiments, a reasonable compromise for many analysis situations. Attempts were also made to separate MCs with above elution strategy using conventional C18 cartridges and no such separation was achieved.

[00103] Preparation of Au-PPy Nanocomposite-Coated Silica Particles

[00104] 4.0 g of unmodified silica particles were dried overnight at 100°C and suspended in 25 mL of 0.125 M HAuCU*3H ₂ 0 in acetonitrile while stirring. Pyrrole was introduced using a KDS 220 syringe pump (Holliston, MA) at a flow rate of 5 mL/hour until a final concentration of 0.925 M was achieved. The reaction mixture was then stirred overnight at room temperature to complete the polymerization. Next, excess reagents and solvents were removed by vacuum filtration followed by exhaustive washing with acetonitrile and HPLC-grade water. Dark black Au-PPy coated silica particles were obtained upon air drying and stored in a glass container at room temperature.

[00105] Preparation of SPE Cartridge with Au-PPy Nanocomposite-Coated Silica Particles

[00106] 20 mg of Au-PPy nanocomposite-coated silica particles were suspended in 200 pL of

HPLC-grade water. The suspension was transferred carefully into an empty SPE cartridge and left for approximately 30 minutes to settle to the bottom. Finally, the cartridge was capped and washed sequentially with 5 mL each of HPLC grade water, methanol and acetonitrile. FIG. 17A- 17C show recoveries and RSDs for MC determination using the Au-PPy nanocomposite sorbent coating for SPE purification and preconcentration: HPLC-grade water (FIG. 17A); tap water (FIG. 17B); and, lake water (FIG. 17C). Number of replicates, n=3 and preconcentration factor, lOx).

[00107] MC recovery experiments were conducted in HPLC-grade water, tap water and lake water using three SPE cartridges. For the calculation of average recovery, individual cartridges (from 1 to 3) were considered at 10 ng/L, 50 ng/L, and 500 ng/L of MC concentrations. Three LC- MS runs were conducted for each preconcentrated and control sample. Recoveries were determined by comparing LC-MS EIC average peak areas of the monoisotopic MC ions in preconcentrated samples and control samples using equation (1). RSD for a cartridge at each concentration was calculated by considering the propagation of error associated with peak areas of preconcentrated and control samples using equation (2). Final SDs and RSDs for three cartridges at a particular concentration were calculated with equation (3) and (4), respectively. [00108] 10D ft ₍ l ₎

[00109] While the invention has been described with reference to various and preferred embodiments, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof.

[00110] Therefore, it is intended that the invention not be limited to the particular embodiment disclosed herein contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the claims.

[00111] The publication and other material used herein to illuminate the invention or provide additional details respecting the practice of the invention, are incorporated be reference herein, and for convenience are provided in the following bibliography.

Citation of the any of the documents recited herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.