Monolithic Column Research Articles

Preparation of chiral metal organic framework modified silica monolithic capillary column and its application in enantioseparations of chiral drugs

Metal organic frameworks (MOFs) are crystalline compounds composed of metal ions (or metal clusters) and organic ligands. Chiral MOFs have been successfully utilized as novel materials for the separation of chiral enantiomers by chromatography, demonstrating excellent chiral separation performance. In this study, a chiral MOF-modified silica monolithic capillary column was used for pressurized capillary electrochromatography. First, a chiral MOF (Co-glycyl-L-glutamic acid, Co-L-GG) was synthesized. This MOF was then used to prepare a chiral capillary monolithic column via a one-step in situ polymerization method. The optimal conditions for preparing the chiral capillary monolithic column were determined as follows: Co-L-GG amount, 5 mg; polyethylene glycol amount, 0.96 mg; tetramethoxysilane dosage, 3.6 mL; trimethoxymethylsilane dosage, 0.4 mL. Next, the effects of the separation conditions on the separation of chiral drugs were investigated. Under the conditions of an applied voltage of -20 kV and a mobile phase consisting of acetonitrile and 20 mmol/L disodium hydrogen phosphate (80∶20, v/v), six chiral drugs were separated within 3 min, with baseline separation achieved for amlodipine, fluvastatin, and tryptophan. Moreover, the prepared chiral capillary monolithic column exhibited good reproducibility and stability. Finally, molecular docking studies were conducted using AutoDock to explore the chiral recognition mechanism, and the results were analyzed using Discovery Studio. The results indicated that larger differences in binding free energy between Co-L-GG and the enantiomers of the chiral drugs were correlated with higher enantioselectivity factors. However, this correlation did not necessarily lead to an increase in resolution. Co-L-GG, which is enriched with primary amines, secondary amines, and carbonyl groups, demonstrated enantiomeric recognition capability. In conclusion, this study demonstrates that chiral MOFs can be effectively used as chiral functional monomers to prepare chiral monolithic capillary columns, highlighting their significant potential for the separation and analysis of chiral compounds. The comprehensive exploration of the synthesis, characterization, and applications of these MOFs will help provide valuable insights into the development of advanced separation technologies.

Preparation of monolithic silica HPLC columns with truss-structured skeletons and embedded surfactant-templated mesopores

AbstractMonolithic macro/mesoporous silica gels have been prepared via a sol-gel process using triblock copolymer Pluronic P123 (EO20PO70EO20) as a structure-directing agent. In this synthesis, P123 not only induces phase separation to form macroporous structure but also acts as a supramolecular template to form mesopores with precisely controlled shape and size. Obtained was a monolithic silica composed of continuous truss-like columnar skeletons in which cylindrical mesopores are arranged in a 2D-hexagonal symmetry. These monolithic silica gels have extremely high porosity approaching 90% and exhibited high specific surface area and sharp pore size distribution as revealed by N2 sorption measurements. Combinations of the initial composition and the post-gelation treatment on wet gels allowed the control of physical properties of meso- and macropore structures. The monolithic HPLC columns prepared using these silica gels surface-modified by ODS (octadecylsilyl) ligands gave as many as 140,000 theoretical plates/m for the separation of alkylbenzenes in a reversed-phase mode. Very weak dependence of height equivalent to theoretical plate, H, on the mobile phase velocity was also recognized in comparison with conventional particle-packed columns. Graphical Abstract

Multiscale simulation of liquid chromatography: Effective diffusion in macro–mesoporous beds and the B-term of the plate height equation

We performed multiscale simulations of analyte sorption and diffusion in hierarchical porosity models of monolithic silica columns for reversed-phase liquid chromatography to investigate how the mean mesopore size of the chromatographic bed and the analyte-specific interaction with the chromatographic interface influence the analyte diffusivity at various length scales. The reproduced experimental conditions comprised the retention of six analyte compounds of low to moderate solute polarity on a silica-based, endcapped, C18 stationary phase with water‒acetonitrile and water–methanol mobile phases whose elution strength was varied via the volumetric solvent ratio. Detailed information about the analyte-specific interfacial dynamics received from molecular dynamics simulations was incorporated through appropriate linker schemes into Brownian dynamics diffusion simulations in three hierarchical porosity models received from physical reconstructions of silica monoliths with a mean macropore size of 1.23 µm and mean mesopore sizes of 12.3, 21.3, or 25.7 nm. The mean mesopore size was found to have a similar influence on the effective mesopore diffusivity as the analyte polarity and the mobile-phase elution strength, which together determine the analyte residence time on a column. A smaller mesopore size attenuated the increase of the effective mesopore diffusivity with increasing mobile-phase elution strength significantly. The effective bed diffusivity was limited by the analyte residence time rather than by morphological details of the mesopore space. The stronger an analyte was retained by the chromatographic interface inside the mesopores, the slower became the mass transfer between the pore space hierarchies and the lower was the effective bed diffusivity. The B-terms of the plate height equation were finally generated with the bed diffusivities and phase-based retention factors derived from the hierarchical porosity models using additional information about the stationary-phase limit obtained from the analysis of analyte–bonded phase contacts. The B-terms highlight analyte- and mobile phase-specific behavior relevant to isocratic and gradient elution conditions in chromatographic practice. In particular, U-shaped B-term curves are observed due to the dominating contribution of the retention factor and the bed diffusivity to the B-term at low and high elution strength of the mobile phase, respectively.

Carboxyl hybrid monolithic column in-tube solid-phase microextraction coupled with UPLC-QTRAP MS/MS for the determination of amphetamine-type stimulants

Carboxyl hybrid monolithic column in-tube solid-phase microextraction coupled with UPLC-QTRAP MS/MS for the determination of amphetamine-type stimulants

Quantitative assessment of the 3D pore space and microglobule clustering network to understand chromatographic transport phenomena in polymeric monolithic columns

The 3D pore space and microglobule clustering network of polymer monolithic columns, which exhibited similar external porosity but significantly different chromatographic dispersion and permeability characteristics, were subjected to tomographic imaging followed by stereological analyses. The morphologies of the monolithic support structures were examined using serial-block-face scanning electron microscopy. The statistically computed characteristic chord-length, hydraulic radius and tortuosity of the pore are strongly associated to chromatographic transport processes, in particular, eddy dispersion and mass-transfer resistance contributing to chromatographic dispersion, and permeability of monolithic columns. Moreover, Giddings’ trans-column velocity bias has been quantified in monoliths for the first time. With demonstrated method robustness, the proposed morphological descriptors and the streamlined analysis workflow provide novel insights bridging the structure-performance relationship for future chromatography column design.

Design and Implementation of a Proof-of-Concept Robotic-Microfluidic Interface to Bridge Spatial Comprehensive Three-Dimensional Liquid Chromatography with Mass Spectrometry.

A proof-of-concept system is presented for the hyphenation of spatial comprehensive three-dimensional liquid chromatography (3D-LC) to mass spectrometry (MS) detection via a robotic-microfluidic interface. A three-dimensional fractal microflow distributor, incorporating 16 parallel RP monolithic capillary columns arranged in a 4 × 4 configuration, was connected to an X-Y-Z robotic system. This setup facilitated the deposition of successive arrays of microdroplets onto an MS target plate. To minimize carryover during droplet deposition, a strategy was implemented in which the distance between the target plate and the capillary was gradually increased during the deposition process. System-level variation in travel time and subsequent flow rates across parallel columns was assessed and translated in retention alignment based on injection of a protein standard. The successful separation of intact proteins was demonstrated through a parallel 4 × 4 column configuration, applying MALDI-MS detection after microdroplet spotting on an MS target plate. Furthermore, the discussion encompasses high-throughput MS imaging detection within the framework of spatial 3D-LC.

Fan assisted extraction and low pressure chromatographic system with a phenyl monolithic column for amperometric determination of volatile α-dicarbonyl compounds in coffee brews

In this work, a low pressure chromatographic system featuring a single 0.5 cm length phenyl monolithic column and an amperometric detector was developed to determine volatile ⍺-dicarbonyl compounds in coffee brews. The analytical strategy relied on the extraction of the volatile ⍺-dicarbonyl compounds and their derivatization with o-phenylenediamine, prior to the determination of the resulting quinoxaline derivative compounds in the designed chromatographic system. The studies of the mobile phase composition and electrochemical conditions are presented. The extraction of the volatile ⍺-dicarbonyl compounds was performed resourcing to a recent approach based on the fan assisted extraction system. This system performs the full evaporation technique and allowed the quantitative recovery (>85 %) of diacetyl and 2,3-pentanedione from the coffee brew based on the following experimental conditions: sample volume 30 µL, extraction period 15 min, acceptor solution (water, acetonitrile, 90:10, v/v) volume 400 µL, and sample temperature 50 °C. The developed method showed no matrix effect for different coffee brews and the quantification of the studied analytes can be performed through the external calibration method. Detection limits of 3.6 × 10−6 mol/L for diacetyl and 12.9 × 10−6 mol/L for 2,3-pentanedione were obtained. Sample analysis rate was of 5 h−1 and the acetonitrile consumption was of ca. 450 µL per chromatographic run.

De novo synthesis of α-Alk-β-CD: An organic–inorganic hybrid chiral stationary phase for enhanced enantioseparation efficiency and its recognition mechanism research

Herein, two highly stable β-CD and α-Alkenyl-β-CD (α-Alk-β-CD) monolithic columns were fabricated by copolymerizing β-CD and α-Alk-β-CD with GMA onto the pretreated capillary by a simple one-pot strategy. 1H NMR, SEM, FT-IR, TGA and BET surface area analyses were applied to characterize the prepared monolithic columns. The monoliths were evaluated on the enantioseparations of antibiotic drugs, antihistamine drugs, anticholinergic agents, antifungal drugs and pesticides. In contrast to the native β-CD monolith, the derived α-Alk-β-CD reveals significantly improved enantiodiscrimination performance for the analytes especially for pesticides. Through run-to-run (n = 6), day-to-day (n = 9), and column-to-column (n = 9) investigations, the RSDs values were all lower than 3.72 %. Molecular simulation reveled that the chiral recognition between α-Alk-β-CD and chiral drugs was an exothermic process mainly governed by interaction hydrogen bindings, resulting in different binding strength between a pair of isomers compounds and α-Alkenyl-β-CD, and then leading to different migration and finally achieving effective chiral separation.

Quantitation of Host Cell Proteins by Capillary LC/IMS/MS/MS in Combination with Rapid Digestion on Immobilized Trypsin Column Under Native Conditions.

Host cell protein (HCP) impurities are considered a critical quality attribute of biopharmaceuticals because of their potential to compromise safety and efficacy, and LC/MS-based analytical methods have been developed to identify and quantify individual proteins instead of employing enzyme-linked immunosorbent assay to assess total HCP levels. Native digestion enables highly sensitive detection of HCPs but requires overnight incubation to generate peptides, limiting the throughput of sample preparation. In this study, we developed an approach employing native digestion on a trypsin-immobilized column to improve the sensitivity and throughput. We examined suitable databases for the identification of HCPs derived from Chinese hamster ovary (CHO) cells and selected RefSeq's Chinese Hamster as the optimal database. Then, we investigated methods to identify HCPs with greater efficiency than that of denatured in-solution digestion. Native in-column digestion not only reduced the digestion time from overnight to 10 min but also increased the number of quantified HCPs from 154 to 226. In addition to this rapid digestion methodology, we developed high-throughput LC/MS/MS with a monolithic silica column and parallel reaction monitoring-parallel accumulation-serial fragmentation. The optimized system was validated with synthetic peptides derived from high-risk HCPs, confirming excellent linearity, precision, accuracy, and low limit of detection (LOD) and limit of quantification (LOQ) (1-3 ppm). The optimized digestion and analysis method enabled high-throughput quantification of HCPs, and is expected to be useful for quality control and characterization of HCPs in antibody drugs.

Fabrication and application of gold nanoparticles functionalized polymer monolith in spin column for the determination of S-nitrosoglutathione in meat

S-nitrosoglutathione (GSNO) is the most important S-nitrosothiol in vivo, which could affect the quality of meat by participating in calcium release, glucose metabolism, proteolysis and apoptosis, therefore may potentially serve as a marker for meat freshness. In this work, a solid-phase extraction (SPE) monolithic spin column modified with gold nanoparticles was prepared for GSNO extraction. The optimized SPE-LC-MS/MS method for GSNO quantification displays low limit of detection (0.01 nM), good precision (RSD < 15 %) and acceptable recovery (> 77.7 %). Furthermore, this approach has been applied to monitor GSNO levels in beef and pork stored at −20 °C for different days, showing that endogenous GSNO level increases during prolonged storage and could be employed as a marker to evaluate the freshness of ice stored meat. Additionally, the monolithic spin column remains in good quality after a half-year storage, which is promising to develop into commercial enrichment kit for endogenous GSNO analysis.

Thiol-functionalized MOF modified 3D printed monolithic microextraction array for analysis of trace Cd and Pb in human urine

Controlling the position, size, and shape of pores is a limitation of traditional monolithic preparation methods. The application of 3D printing technology offers high customizability, allowing the precise printing of pore positions, sizes, and shapes according to the designer's 3D model. Herein, by using Projection Microstereolithography (PμSL), we prepared a 3D-printed monolithic array with post-modification of thiol-functionalized metal-organic framework (MOF), and combined it with inductively coupled plasma mass spectrometry (ICP-MS) for the online analysis of trace Cd and Pb in human urine. To achieve array monolithic microextraction, six 3D-printed monolithic columns were modified with thiol-functionalized MOF-808 (MOF-808-SH), and were then assembled in the 3D printed extraction device incorporating gas valve and scaffold. The MOF-808-SH modified 3D-printed monolithic column exhibits excellent extraction performance to Cd2+ and Pb2+ due to rich active adsorption sites and hierarchical porous structure, and has long life span (>100 reused times). Under the optimized conditions, the limits of detection (LODs) are 3.5 and 17.6 ng L−1 for Cd2+ and Pb2+, respectively, with the relative standard deviations of 4.9 % and 8.2 % (0.1 μg L−1, n = 7), and the sample throughput is 11 h−1. To validate the accuracy of the method, the method was used to determine Cd and Pb in Certified Reference Materials of freeze-dried human urine, the determined results agree well with the certified values. This method was also successfully applied to the determination of trace Cd and Pb in real human urine samples. The developed method offers low LODs, robust anti-interference capability, high sample throughput, long reuse cycles, and automation analysis, showing great potential for the analysis of trace heavy metals in biological samples.

Solid-Phase microextraction on Self-Driven microfluidic chip using capillary micropump and microvalves for saliva electrochemical analysis

The real-time detection of biomarkers in saliva is of great significance for early non-invasive diagnostic of related diseases. In this work, molecular imprinting technology was used to prepare a lactic acid monolithic column, which could realize specific adsorption and elution of lactic acid, achieving solid phase microextraction (SPME) of lactic acid in samples, and purify the analytes. Lactic acid monolithic column was then embedded into the microfluidic channel, and passive microfluidic structures such as stop valve, retention valve, trigger valve and capillary pump were utilized to construct the SPME self-driven microfluidic chip. A series of complex operations such as lactic acid adsorption, rinse and elution on the monolithic column could be realized automatically without external power. Since the purification of the analytes was achieved by the monolithic column, the low-cost nano copper oxide modified electrode was used to achieve the specific electrochemical analysis of lactic acid. The screen-printed electrode was designed and fabricated, and the integrated electrochemical detection circuit was used to realize the portable analysis of Na+, K+ and lactic acid simultaneously. The microfluidic chip, screen-printed electrode and miniaturized detection circuit are integrated into a Point-of-care testing (POCT) device, which realizes the automatically real-time detection of saliva. By adjusting the template molecule in monolithic column polymerization, the application of this physiological fluid POCT device can be extended, and may provide some implications for the development of portable non-invasive health monitoring devices.

Effect of anionic groups in zwitterionic hydrophilic stationary phases on their chromatographic characteristics

The structure of zwitterion has great impact on the separation properties of zwitterionic hydrophilic stationary phases. To better understand the role of anionic groups of zwitterions, a novel carboxybetaine-based zwitterionic monolithic column was first prepared through thermo-initiated copolymerization of functional monomer (3-acrylamidopropyl)-dimethyl-(2-carboxymethyl) ammonium (CBAA) and crosslinker ethylene dimethacrylate (EDMA) within 100 μm ID capillary. The optimal poly(CBAA-co-EDMA) monolithic column exhibited satisfactory mechanical and chemical stability, good repeatability, high column efficiency (96,000 plates/m), and excellent separation performance for different classes of polar compounds (i.e., phenols, monophosphate nucleotides, urea and allantoin). A comparative study was then performed among three zwitterionic hydrophilic stationary phases containing different anionic groups, i.e. poly(CBAA-co-EDMA) (carboxybetaine), poly(2-{2-(methacryloyloxy) ethyldimethylammonium}ethyl n-butyl phosphate-co-EDMA) (phosphocholine), and poly(N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl) ammonium betaine-co-EDMA) (sulfobetaine) using benzoic acid derivatives, amine compounds, nucleobases and nucleosides as model analytes. The carboxybetaine-based monolithic column exhibited much higher positive zeta-potential and hydrophilicity, which endows it with a stronger retention capacity for acidic and neutral compounds, but sulfobetaine-based monolithic column exhibited much higher selectivity and retention capacity for the amines. Moreover, their enrichment efficiencies for N-glycopeptides were also evaluated based on their different hydrophilicity, and it was observed that the poly(CBAA-co-EDMA) monolithic material captured 4-8 times more N-glycopeptides compared to the other two materials.

Experimental and numerical study on cyclic behavior of precast segmental railway bridge columns with lap-spliced rebar connections using UHPC wet joints

A novel lap-spliced connection with ultra-high performance concrete (UHPC) was proposed to connect precast railway bridge column segments with footings or cap beams. Two 1:3 scaled specimens were tested under cyclic loads. Specimen PC-LS was a precast column with lap-spliced rebar connection and UHPC wet joints. Specimen CIP was a monolithic column as a reference specimen. The test results revealed that the plastic hinge region of the precast specimen shafted to the upper surface of the UHPC wet joint. The failure mode of the precast specimen was exhibited as the crushing of the core concrete and the buckling/fracture of the longitudinal rebars. Specimen PC-LS experienced larger lateral force, initial stiffness, comparable ductility, and energy dissipation compared to specimen CIP. Additionally, the validated finite-element (FE) models considering the bond-slip effect between concrete and reinforcements were developed. The parametric studies were carried out to further investigate the cyclic behavior of specimen PC-LS. Analytical results demonstrated that the variation of the compressive and tensile strength of UHPC showed little influence on the cyclic behavior of the precast segmental column, which indicated that UHPC with natural curing and lower volume content steel fibers could be used as the wet joint. The precast segmental column had similar lateral force and stiffness with the cast-in-place column when the lap splice length was 10 times of the rebar diameter.

Hybrid organic monolithic column containing MIL-68(Al) for the separation of small molecules by capillary HPLC

A hybrid organic monolithic column made of poly(lauryl methacrylate-co-1,6-hexanediol dimethacrylate) and the metal-organic framework MIL-68(Al) was prepared for the first time. The column was used in capillary liquid chromatography, both in isocratic and gradient elution modes. Separation performance towards small molecules of different chemical nature (polycyclic aromatic hydrocarbons, alkylbenzenes, phenols, etc.) was studied. Monte Carlo simulations were made to both select the proper precursors to obtain empty metal-organic framework micropores in the monolithic polymer and also, to analyze the potential free access of the studied analytes into the micropores (necessary to improve mass transfer and column efficiency). The hereby synthesized metal-organic framework microcrystals allowed obtaining homogeneous hybrid monolithic columns. Adding of MIL-68(Al) (1030 m2 g-1 BET specific surface area) increased the surface area from 3.9 m2 g-1 for the parent monolith to 18.2 m2 g-1 for the hybrid column containing 8 mg mL-1 of the microcrystals. Chromatographic performance of this new column was evaluated by studying retention factors, resolution, and plate counts at room temperature. Different compounds, not completely resolved in the parent monolith, were partially or completely separated after metal-organic framework addition. Using the monolithic column with only 2 mg mL-1 of MIL-68(Al), five alkylbenzenes were completely separated with very symmetrical peak shapes, resolution factors up to 3.60 and plate counts of 4300 plates m-1 for n-hexylbenzene. This value is higher than those obtained by other authors who used organic monolithic columns with embedded metal-organic frameworks to perform separations at room temperature. Additionally, nine polycyclic aromatic hydrocarbons were partially or completely resolved in gradient elution mode. The hybrid monolithic columns exhibited very good intra-day (%RSD=1.9), inter-day (%RSD=2.6), and column-to-column (%RSD=4.3) reproducibility values. Easy and fast column preparation, and versatility to efficiently separate several compounds of different chemical nature in isocratic and gradient mode, makes this new hybrid column a very good option for the analysis of small molecules in capillary (or nano) HPLC.

Design and fabrication of fluorous monoliths with tunable surface property for capillary liquid chromatography

Hierarchically porous monoliths with satisfactory properties have been employed in diverse fields, especially separation. In this study, pentafluorophenyl acrylate (PFPA), pentaerythritol tetraacrylate (PETA) and trimethylolpropane tris(3-mercaptopropionate) (TTMP) were selected as precursors to fabricate a novel monolithic column by thermally initiated polymerization in the presence of a binary porogenic system containing tetrahydrofuran and 1-propanol. The fabricated poly(PFPA-co-PETA-co-TTMP) monolithic column revealed excellent permeability and mechanical stability. Additionally, baseline separation of the mixture of small molecules can be achieved, involving alkylbenzene and fluorobenzene in chromatographic assessment, and the theoretical plate number is up to 60,500 plates/m for butylbenzene with a linear velocity of 0.14 mm/s. Tryptic digest of HeLa as an analyte was used to investigate the possibility of the poly(PFPA-co-PETA-co-TTMP) monolith in biological separation by cLC-MS/MS. Moreover, benefiting from the existence of pentafluorophenyl groups, the cucurbit[8]uril (CB[8]) could be modified on the prepared poly(PFPA-co-PETA-co-TTMP) monolith through host-guest interaction to obtain poly(PFPA-co-PETA-co-TTMP)-CB[8] monolith. It could be observed that significant changes in retention behavior of analytes appeared after immobilizing CB[8] on the monolith. It offered an innovative approach by utilizing host-guest interaction to fabricate monolithic columns with different chromatographic behaviors.

Study of the Enantioselectivity and Recognition Mechanism of Allyl-β-CD Modified Organic Polymer Monolithic Capillary Column.

Allyl-β-CD was synthesized and used as the chiral functional monomer to prepare chiral organic polymer monolithic columns in capillary HPLC. First, the enantioselectivity of the prepared allyl-β-CD modified organic polymer monolithic capillary columns was investigated. Then, the influences of enantioseparation conditions of chiral drugs were further explored. Finally, the recognition mechanism was studied by molecular docking with AutoDock. Complete enantioseparations of four chiral drugs as well as partial enantioseparations of eight chiral drugs have been achieved. Results showed that the RSD values for run-to-run, day-to-day, and column-to-column variations ranged from 1.2% to 4.6%, 1.4% to 4.7%, and 2.0% to 6.1%, respectively. The enantioselectivity factor rather than resolution is correlated with the binding free energy difference between enantiomers with allyl-β-CD. Furthermore, the abundant ether bonds, hydroxyl groups, and hydrophobic cavities in cyclodextrin are responsible for the enantioseparation ability of the chiral monolithic capillary columns.

One-Step Synthesis and Oriented Immobilization of Strep-Tag II Fused PDGFRβ for Screening Intracellular Domain-Targeted Ligands.

Accurate orientations and stable conformations of membrane receptor immobilization are particularly imperative for accurate drug screening and ligand-protein affinity analysis. However, there remain challenges associated with (1) traditional recombination, purification, and immobilization of membrane receptors, which are time-consuming and labor-intensive; (2) the orientations on the stationary phase are not easily controlled. Herein, a novel one-step synthesis and oriented-immobilization membrane-receptor affinity chromatography (oSOMAC) method was developed to realize high-throughput and accurate drug screening targeting specific domains of membrane receptors. We employed Strep-tag II as a noncovalent immobilization tag fused into platelet-derived growth factor receptor β (PDGFRβ) through CFPS, and meanwhile, the Strep-Tactin-modified monolithic columns are prepared in batches. The advantages of oSOMAC are as follows: (1) targeted membrane receptors can be expressed independent of living cell within 1-2 h; (2) orientation of membrane receptors can be flexibly controlled and active sites can expose accurately; and (3) targeted membrane receptors can be synthesized, purified, and orientation-immobilized on monolithic columns in one step. Accordingly, three potential PDGFRβ intracellular domain targeted ligands: tanshinone IIA (Tan IIA), hydroxytanshinone IIA, and dehydrotanshinone IIA were successfully screened out from Salvia miltiorrhiza extract through oSOMAC. Pharmacological experiments and molecular docking further demonstrated that Tan IIA could attenuate hepatic stellate cells activation by targeting the protein kinase domain of PDGFRβ with a KD value of 9.7 μM. Ultimately, the novel oSOMAC method provides an original insight for accurate drug screening and interaction analysis which can be applied in other membrane receptors.

A Green Liquid Chromatography Method for Simultaneous Quantification of Caffeine and its Three Major Metabolites in Urine, Drinks, and Herbal Products

ABSTRACTA green reversed‐phase liquid chromatography method was developed to determine caffeine (CF), theobromine (TB), theophylline (TP), and paraxanthine (PX). The stationary phase was a silica‐based C18 monolithic column (100 × 3.0 mm2 i.d.), eluted with a gradient from 6% to 10% (v/v) of ethanol in water, at a flow rate of 0.75 mL/min (35°C) and UV detection at 272 nm. The compounds were separated in less than 6 min, with a total time of 10 min for column reconditioning. The effect of gradient composition, flow rate, injection volume, and column temperature was evaluated in the separation efficiency, especially regarding the critical pair PX/TP, for which the best resolution was 1.37. The linear range was between 0.5 and 100 µg/mL. The LODs were 0.065, 0.057, 0.040, and 0.044 mg/mL for TP, TB, PX, and CF, respectively. The method was applied to green tea and yerba mate infusions, soft drinks, energy drinks, and soluble coffee. Urine samples were analyzed before and after solid‐phase extraction. The accuracy was evaluated by the spiking/recovery strategy standing in the 79.6% to 116.4% range. The separation performance and the mobile phase consumption were compared to those of another method using a gradient of methanol in water.

Development of an organic polymer monolith column for the nano liquid chromatography fast analysis of monoclonal antibody in infusion bags prepared in a hospital pharmacy.

Poly(butyl methacrylate-co-ethylene dimethacrylate) monolith was in situ prepared in a liquid chromatography capillary column with a 75 μm internal diameter. This monolith offered high permeability (5.3 ± 10-14m2) and good peak capacity (140 for a 15 cm column length at 300 nl/min with a 20 min gradient time). This is exemplified by its separation ability in reversed mode for subunit analysis of monoclonal antibodies after IdeS digestion (middle-up analysis). The potential of this column was also illustrated for the fast analytical control of therapeutic monoclonal antibodies in standardized infusion bags prepared in advance in a pharmacy department. Linearity analysis revealed the column's capability for accurate quantification analysis of the different dose bandings (in mg) of monoclonal antibodies in <2min. In addition, lifetime analysis data indicated that the column can be highly reproducible and has a long lifetime with stable and low back pressure. The variations observed on the peak shape and area between unstressed (intact) and stressed monoclonal antibodies indicated that our nano liquid chromatographic method was stability indicating. In addition, using a gradient elution mode, the presence of minor components in the infusion bags was visualized.