A Polymer Coating Transfer Enrichment Method for Direct Mass Spectrometry Analysis of Lipids in Biofluid Samples.

A porous polymer coating transfer enrichment method is developed for the direct mass spectrometry (MS) analysis of lipids. The enrichment is fast (ca. 1 min) and enables the profiling and quantitation of lipids in small-volume biofluid samples. Coupled with a photochemical Paternò-Büchi reaction, this method enables the fast determination of lipid structure at the C=C location level and point-of-care lipid biomarker analysis.

Electroosmotic pump-supported molecularly imprinted monolithic column for capillary chromatographic separation of nitrophenol isomers.

In this work, a novel molecularly imprinted polymer (MIP) monolithic column with integrated in-column electroosmotic pump (EOP) was designed and successfully prepared to facilitate the capillary chromatography with MIP column. A silica-based EOP was synthesized at the detection end of the MIP monolithic capillary column by so-gel to provide the hydrodynamic driven force for the capillary chromatography. Because of large surface area and low fluidic resistance of the silica monolith,a strong and steady EOF was generated by silica-based EOP, indicating that the EOP was quite compatible with MIP capillary column. With the sufficient EOF provided by EOP, the electro-driven based capillary chromatographic separation of nitrophenol isomers was achieved in 4-vinylpyridine-based MIP monolithic capillary, which was originally proved infeasible because of the EOF shortage. No significant influence upon the specific recognition of the MIP was found due to the setting of EOP after the detection window of the column. The influence of experimental parameters on the EOF such as voltage and pH value of running buffer was investigated. The column was also evaluated by capillary liquid chromatographic mode to compare with EOP-driven capillary chromatography. Higher column efficiency was obtained by EOP-driven separation with improved peak shape. The results suggested that EOP-supported technique would be a good way to solve the problem of weak EOF generation in electro-driven capillary chromatography.

Preparation of a novel molecularly imprinted polymer for the highly selective extraction of baicalin.

The selective extraction of baicalin is important to its quality control especially when the matrices are complicated. In this work, a novel molecularly imprinted polymer was prepared for the selective extraction of baicalin in herbs. The molecularly imprinted polymer was synthesized by the copolymerization of 4-vinyl pyridine and ethylene glycol dimethacrylate in the presence of baicalin by a precipitation polymerization method. After the optimization of parameters for molecularly imprinted polymer preparation, including the functional monomer, porogen, sampling solvent, and washing solvent, good selectivity was obtained, with an imprinting factor of about 4, which is much better than that achieved by the bulk-polymerization method. The performances of the prepared molecularly imprinted polymers were systematically investigated, including adsorption kinetics, isotherm experiment, and Scatchard analysis. On the basis of the good adsorptive capability of the prepared molecularly imprinted polymer, it was also applied for the pretreatment of baicalin in Scutellaria baicalensis Georgi. The result showed that most of the matrices were removed and baicalin was selectively enriched.

Novel molecularly imprinted magnetic nanoparticles for the selective extraction of protoberberine alkaloids in herbs and rat plasma.

In this work, a novel magnetic nanomaterial functionalized with a molecularly imprinted polymer was prepared for the extraction of protoberberine alkaloids. Molecularly imprinted polymers were made on the surface of Fe3 O4 nanoparticles by using berberine as template, acetonitrile/water as porogen, acrylamide as functional monomer and ethylene glycol dimethacrylate as cross-linker. The optimized molar ratio of template/functional monomer was 1:7. The polymeric magnetic nanoparticles were characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. The stability and adsorption capacity of the molecularly imprinted polymers were investigated. The molecularly imprinted polymers were used as a selective sorbent for the magnetic molecularly imprinted solid-phase extraction and determination of jatrorrhizine, palmatine, and berberine. Extraction parameters were studied including loading pH, sample volume, stirring speed, and extraction time. Finally, a magnetic molecularly imprinted solid-phase extraction coupled to high-performance liquid chromatography method was developed. Under the optimized conditions, the method showed good linear range of 0.1-150 ng/mL for berberine and 0.1-100 ng/mL for jatrorrhizine and palmatine. The limit of detection was 0.01 ng/mL for berberine and 0.02 ng/mL for jatrorrhizine and palmatine. The proposed method has been applied to determine protoberberine alkaloids in Cortex phellodendri and rat plasma samples. The recoveries ranged from 87.33-102.43%, with relative standard deviation less than 4.54% in Cortex phellodendri and from 102.22-111.15% with relative standard deviation less than 4.59% in plasma.

Mussel-inspired polydopamine-assisted hydroxyapatite as the stationary phase for capillary electrochromatography.

A novel capillary with hydroxyapatite (HAP) as the stationary phase was prepared for open-tubular capillary electrochromatography (OT-CEC). To immobilize HAP, a mussel-inspired polydopamine method was utilized to modify the capillary firstly, generating a polydopamine layer; and then a layer of HAP would be formed on the polydopamine layer by a biomineralization process, to produce a HAP-modified capillary (HAP@capillary). Scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDS) and X-ray photoelectron spectroscopy (XPS) provided evidence of nanostructured HAP grown on the surface of the capillary wall. The electroosmotic flow (EOF) characteristic of the HAP@capillary was investigated by varying the percentage of acetonitrile and pH value of the buffer with thiourea as a marker, and a pH-dependent EOF from anode to cathode was observed. The HAP@capillary exhibits high column efficiency for methylbenzene, up to 151,138 plates per meter. Different kinds of compounds including alkylbenzenes, phenols and amines have been successfully separated by the HAP@capillary in CEC mode. The HAP@capillary also possessed good separation ability in capillary liquid chromatography (CLC) mode because of the relatively large ratio of HAP in the capillary; however, the separation efficiency was not as good as that in CEC mode. The reproducibilities of the HAP@capillary were evaluated, and the relative standard deviations were found to be lower than 5%.

Graphene/polydopamine-modified polytetrafluoroethylene microtube for the sensitive determination of three active components in Fructus Psoraleae by online solid-phase microextraction with high-performance liquid chromatography.

Determination of bioactive compounds in traditional Chinese medicines and biological samples is usually interfered with by coexisting components in matrices. In this work, we prepared novel multilayer functional graphene/polydopamine-modified polytetrafluoroethylene microtube for selective solid-phase microextraction of three bioactive compounds in Fructus Psoraleae. Functional graphene/polydopamine-modified polytetrafluoroethylene microtube showed good extraction efficiency toward bavachin, isobavachalcone, and bavachinin; enrichment from 357- to 737-fold was obtained for these compounds. For qualitative analysis, an online solid-phase microextraction with high-performance liquid chromatography method was developed, which showed low limits of detection of 0.02 ng/mL by using UV detection, which is significantly more sensitive than previously reported methods. The proposed method has been used to determine bavachin, isobavachalcone, and bavachinin in Fructus Psoraleae, the contents of three compounds were quantified to be 64.0, 324.0, and 384.5 µg/g; recoveries were 93.4-101.1%. The proposed method has also been applied to determine bavachin, isobavachalcone, and bavachinin in rat plasma samples after oral administration of Fructus Psoraleae.

Development and in vivo evaluation of intranasal formulations of parathyroid hormone (1-34).

For efficient intranasal transport of parathyroid hormone (1-34) [PTH(1-34)], there is a great medical need to investigate permeation enhancers for intranasal formulations. In this study, the development of PTH(1-34) intranasal formulations was conducted. Based on conformation and chemical stability studies, the most preferable aqueous environment was determined to be 0.008 M acetate buffer solution (ABS). Subsequently, citric acid and Kolliphor® HS·15 were compared as permeation enhancers. The mechanisms of action of citric acid and Kolliphor® HS·15 were investigated using an in vitro model of nasal mucosa, and Kolliphor® HS·15 led to higher permeability of fluorescein isothiocyanate-labeled PTH(1-34) (FITC-PTH) by enhancing both the transcellular and paracellular routes. Moreover, citric acid showed severe mucosal toxicity resulting in cilia shedding, while Kolliphor® HS·15 did not cause obvious mucosa damage. Finally, Kolliphor® HS·15 was studied as a permeation enhancer using a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The results showed that 5% and 10% Kolliphor® HS·15 increased the bioavailability of PTH(1-34) to 14.76% and 30.87%, respectively. In conclusion, an effective and biosafe PTH(1-34) intranasal formulation was developed by using 10% Kolliphor® HS·15 as a permeation enhancer. Intranasal formulations with higher concentrations of Kolliphor® HS·15 for higher bioavailability of PTH(1-34) could be further researched.

Polydopamine-Modified Substrates for High-Sensitivity Laser Desorption Ionization Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) serves as a powerful tool for biological research, and laser desorption ionization (LDI) is used as a major sampling ionization method. Study of materials for LDI represents a major field in the MSI research, either for matrices in matrix-assisted LDI (MALDI) or sample substrates allowing matrix-free LDI. In this study, we developed a composite substrate using polydopamine (PDA) film to coat an antireflection (AR) surface for LDI-MSI. The AR material has been previously shown to confine UV energy within the micro-/nanostructures, leading to a highly localized temperature rise to facilitate analyte thermal desorption. PDA coating on the AR material further enhances the light-to-heat conversion and improves the contact between the substrate surface and the biological sample materials. With this substrate, desorption and ionization of lipids from raw human plasma samples and biological tissue sections have been achieved. Matrix-free LDI-MSI of around 30 lipid species in mouse brain sections was achieved with a significantly simplified MSI procedure at a spatial resolution of 50 µm. This method was applied to determine mouse fatty liver disease through monitoring the abundances and distributions of triacylglycerols and glycerophospholipids. Dramatic differences in the lipid profiles were subsequently identified between the liver tissues from the wild-type and obese mice.

Polydopamine-based immobilization of zeolitic imidazolate framework-8 for in-tube solid-phase microextraction.

Zeolitic imidazolate frameworks (ZIFs), a subfamily of metal-organic frameworks (MOFs), have received considerable attention as a novel porous material for sample preparation due to their fascinating structures and unique properties. Here, we developed a novel bio-inspired polydopamine (PDA) method to immobilize ZIFs inside the chemical resistance poly(ether ether ketone) (PEEK) tube for online solid-phase microextraction (SPME). Firstly, PDA layer was assembled inside the PEEK tube. Secondly, attributing to its noncovalent adsorption and covalent reaction ability, PDA could attract and anchor ZIF-8 nutrients onto the inner surface of PEEK tube to promote the nucleation and growth of ZIF-8. The morphology and surface properties of the prepared ZIF-8-PDA-PEEK tube were characterized by scanning electron microscopy and energy dispersive X-ray spectrometry. The ZIF-8-PDA-PEEK tube exhibits excellent extraction efficiency toward six polycyclic aromatic hydrocarbons (PAHs), with enrichment factor from 550 to 734. The developed online SPME-HPLC method shows good linearity (10-5000pg/mL) and low detection limits (0.5-5 pg/mL) for six PAHs. It also has been used to determine PAHs in environmental samples, with recoveries in the range of 82.5-98.6%.

Jacket-free stir bar sorptive extraction with bio-inspired polydopamine-functionalized immobilization of cross-linked polymer on stainless steel wire.

Stainless steel wire (SSW) is a good substrate for stir bar sorptive extraction (SBSE). However, it is still a challenge to immobilize commonly used cross-linked polymers onto SSW. In this work, we present a new approach for immobilization of the cross-linked organic polymer onto SSW for jacket-free SBSE. A dopamine derivative was firstly synthesized; by introducing a mussel-inspired polydopamine process, a stable coating layer was finally generated on the surface of SSW. Secondly, the cross-linked polymer was synthesized on the polydopamine-modified SSW by using acetonitrile as the porogen, acrylamide (AA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker and 2,2'-azobis (2-methylpropionitrile) as the initiator. A diluted pre-polymerization solution was carefully prepared to generate a thin layer of the polymer. The prepared poly(EGDMA-AA)-modified stir bar showed high stability and good tolerance toward stirring, ultrasonication, organic solvents, and strong acidic and basic conditions. Morphology and structure characterization of coatings were performed by scanning electron microscopy and Fourier transform infrared spectra, respectively. The prepared poly(EGDMA-AA)-modified stir bar showed great extraction efficiency toward protoberberines, with enrichment factors of 19-42. An SBSE-HPLC method was also developed for quantitative analysis of protoberberines. The method showed low limits of detection (0.06-0.15 ng mL(-1)), wide linear range (0.5-400 ng mL(-1)), good linearity (R≥0.9980) and good reproducibility (RSD≤3.60% for intra-day, RSD≤4.73% for inter-day). The developed method has been successfully applied to determine protoberberines in herb and rat plasma samples, with recoveries of 88.53-114.61%.

Enhancement of capillary electrochromatographic separation performance by conductive polymer in a layer-by-layer fabricated graphene stationary phase.

In this work, we fabricated a novel graphene-based capillary column for open-tubular capillary electrochromatography (OT-CEC) by a layer-by-layer strategy. To immobilize graphene onto the inner surface of silica capillary, a bio-inspired method was first used to functionalize the capillary surface with a layer of polydopamine (PDA). Graphene oxide (GO) was then introduced and can covalently react with polydopamine, realizing immobilization of graphene as a result. To enhance the modification efficiency of polydopamine, a conductive polymer, polyaniline (PANI) was introduced to be a sub-layer; polydopamine was then introduced following with GO, to generate a multilayer GO-PDA-PANI@capillary. Interestingly, separation efficiency of the graphene-based capillary was enhanced significantly by using conductive PANI as a sub-layer. The morphology of different layers modified on the capillary column was characterized by scanning electron microscopy (SEM). The electroosmotic flow (EOF) characteristics of capillaries modified with different layers were also investigated by varying the pH value of mobile phase. GO-PDA-PANI@capillary showed good separation efficiency towards alkylbenzenes by OT-CEC mode, with theoretic plate numbers up to 133,918 for benzene. The separation was found to follow a reversed-phase chromatographic retention mechanism. Repeatability of the GO-PDA-PANI@capillary was studied, with relative standard deviations for intra-day and inter-day runs less than 2.89%, and column-to-column runs less than 6.17%. The separation performance of GO-PDA-PANI@capillary was also compared with that of the reported graphene modified capillary.

Adsorptive behavior and solid-phase microextraction of bare stainless steel sample loop in high performance liquid chromatography.

In this work, we interestingly happened to observe the adsorption of stainless steel sample loop of HPLC. The adsorptive behaviors of the stainless steel loop toward different kinds of compounds were studied, including polycyclic aromatic hydrocarbons (PAHs), halogeno benzenes, aniline derivatives, benzoic acid derivatives, phenols, benzoic acid ethyl ester, benzaldehyde, 1-phenyl-ethanone and phenethyl alcohol. The adsorptive mechanism was probably related to hydrophobic interaction, electron-rich element-metal interaction and hydrogen bond. Universal adsorption of stainless steels was also testified. Inspired by its strong adsorptive capability, bare stainless steel loop was developed as a modification-free in-tube device for solid-phase microextraction (SPME), which served as both the substrate and sorbent and possessed ultra-high strength and stability. Great extraction efficiency toward PAHs was obtained by stainless steel loop without any modification, with enrichment factors of 651-834. By connecting the stainless steel loop onto a six-port valve, an online SPME-HPLC system was set up and an SPME-HPLC method has been validated for determination of PAHs. The method has exceptionally low limits of detection of 0.2-2pg/mL, which is significantly lower than that of reported methods with different kinds of sorbents. Wide linear range (0.5-500 and 2-1000pg/mL), good linearity (R(2)≥0.9987) and good reproducibility (RSD≤2.9%) were also obtained. The proposed method has been applied to determine PAHs in environmental samples. Good recoveries were obtained, ranging from 88.5% to 93.8%.

Preparation of micropipette tip-based molecularly imprinted monolith for selective micro-solid phase extraction of berberine in plasma and urine samples.

A novel berberine-imprinted polymer (MIP) monolith was prepared for extraction of berberine in aqueous medium. The MIP monolith was prepared inside a polypropylene micropipette tip by using dimethylsulfoxide as porogen, acrylamide (AA) as functional monomer and ethyleneglyol dimethacrylate (EGDMA) as cross-linker. Polymerization conditions were optimized and good permeability and selectivity was obtained when the ratio of berberine/AA/EGDMA was 1:5:30. Cross-reaction was also studied by three compounds (palmatine, coptisine, and jatrorrhizine) with similar structure. A molecularly imprinted micro solid-phase extraction (MI-µ-SPE) method was developed for selective extraction of berberine in aqueous solutions. Extraction parameters were investigated, such as sample pH value, sample flow rate, sample volume and elution solvent. By combining with HPLC/UV, MI-µ-SPE method showed a good linear range of 3-800 ng/mL with a low limit of detection limit of 1.0 ng/mL. The method was also applied for the pretreatment of berberine in human plasma and urine samples. The result showed that proteins and other biological matrix were successfully eliminated and berberine was selectively enriched. Recoveries were tested in plasma and urine samples, and calculated to be 90.6-103.2% with relative standard deviations less than 4.7%.

Mussel inspired polydopamine functionalized poly(ether ether ketone) tube for online solid-phase microextraction-high performance liquid chromatography and its application in analysis of protoberberine alkaloids in rat plasma.

A novel and simple poly(ether ether ketone) (PEEK) tube-based solid-phase microextraction (SPME)-HPLC method was developed in this work. A mussel inspired polydopamine method was used to functionalize the chemically resistant surface of PEEK tube and a poly(acrylamide-ethylene glycol dimethacrylate) monolith was synthesized inside the PEEK tube and chemically bonded with its surface. The stability of the polydopamine layer was investigated and found to be well enduring to most of commonly used organic solvents and strong acidic conditions. The PEEK tube-based monolith was coupled with HPLC system through a six-port valve, and the performance of the online SPME-HPLC system was demonstrated by analyzing three protoberberine alkaloids in aqueous samples, namely jatrorrhizine (Jat), palmatine (Pal) and berberine (Ber). These analytes were well extracted and separated within 10 min, and enrichment factors of about 400 were obtained. The limit of detection of the proposed method was decreased to 0.01 ng/mL by using UV detection. Finally, the online SPME-HPLC method was applied for determining Jat, Pal and Ber in rat plasma samples. Reproducibility was obtained by evaluating the intra- and inter-day precisions with RSDs less than 6.1% and 8.5%, respectively, and recoveries of the analytes in spiked plasma samples ranged from 89.8% to 96.7%. The proposed method was also applied to determine the concentration of Jat, Pal and Ber in rat plasma after oral administration of Cortex Phellodendri Chinensis.

Delivery of calf thymus DNA to tumor by folate receptor targeted cationic liposomes.

Calf thymus DNA (ctDNA) has been shown to stimulate macrophages to produce cytokines both in vitro and in vivo when complexed with cationic liposomes. In addition, direct cytotoxicity of ctDNA has been found in tissue culture and in mice. In this study, ctDNA and folate receptor targeted cationic liposome complexes (ctDNA-F-CLs) were prepared and evaluated in FR (+) tumors. In addition, the underlying mechanism for the anti-cancer activity of ctDNA-F-CLs was investigated. Selective uptake of ctDNA-F-CLs was observed in FR (+) KB and L1210JF cells using flow cytometry. In RAW264.7 cells and DBA/2 mice, ctDNA-F-CLs and ctDNA-N-CLs significantly induced TNF-α and IL-6 production compared to free ctDNA. However, no significant difference in cytokine production was observed between ctDNA-N-CLs and ctDNA-F-CLs. In tumor bearing DBA/2 mice, ctDNA-F-CLs significantly increased INF-γ and IL-6 production compared to ctDNA-N-CLs. Furthermore in L1210JF cells, ctDNA-F-CLs had significantly increased cytotoxicity compared to ctDNA-N-CLs. Tumor cell apoptosis was also found in co-culture of RAW264.7 cells and ctDNA-F-CLs treated L1210JF cells. In L1210JF tumor bearing mice, ctDNA-F-CLs were found to significantly inhibit tumor growth and prolong the median survival time (MeST). In contrast, ctDNA-N-CLs and free ctDNA showed similar activities for tumor inhibition and animal survival. Moreover, the anti-cancer effect of ctDNA-F-CL was further enhanced by combination with anti-cancer drug doxorubicin. These results suggest that ctDNA-F-CLs are a promising agent for treatment of FR-positive tumors.