Ligand recognition and G-protein coupling of trace amine receptor TAAR1.

Trace-amine-associated receptors (TAARs), a group of biogenic amine receptors, have essential roles in neurological and metabolic homeostasis1. They recognize diverse endogenous trace amines and subsequently activate a range of G-protein-subtype signalling pathways2,3. Notably, TAAR1 has emerged as a promising therapeutic target for treating psychiatric disorders4,5. However, the molecular mechanisms underlying its ability to recognize different ligands remain largely unclear. Here we present nine cryo-electron microscopy structures, with eight showing human and mouse TAAR1 in a complex with an array of ligands, including the endogenous 3-iodothyronamine, two antipsychotic agents, the psychoactive drug amphetamine and two identified catecholamine agonists, and one showing 5-HT1AR in a complex with an antipsychotic agent. These structures reveal a rigid consensus binding motif in TAAR1 that binds to endogenous trace amine stimuli and two extended binding pockets that accommodate diverse chemotypes. Combined with mutational analysis, functional assays and molecular dynamic simulations, we elucidate the structural basis of drug polypharmacology and identify the species-specific differences between human and mouse TAAR1. Our study provides insights into the mechanism of ligand recognition and G-protein selectivity by TAAR1, which may help in the discovery of ligands or therapeutic strategies for neurological and metabolic disorders.

Catecholamine Derivatives: Natural Occurrence, Structural Diversity, and Biological Activity.

Catecholamines (CAs) are aromatic amines containing a 3,4-dihydroxyphenyl nucleus and an amine side chain. Representative CAs included the endogenous neurotransmitters epinephrine, norepinephrine, and dopamine. CAs and their derivatives are good resources for the development of sympathomimetic or central nervous system drugs, while they also provide ligands important for G-protein coupled receptor (GPCR) research. CAs are of broad interest in the fields of chemical, biological, medical, and material sciences due to their high adhesive capacities, chemical reactivities, metal-chelating abilities, redox activities, excellent biocompatibilities, and ease of degradability. Herein, we summarize CAs derivatives isolated and identified from microorganisms, plants, insects, and marine invertebrates in recent decades, alongside their wide range of reported biological activities. The aim of this review is to provide an overview of the structural and biological diversities of CAs, the regularity of their natural occurrences, and insights toward future research and development pertinent to this important class of naturally occurring compounds.

A LysLysLys-tag as trigger in polynorepinephrine epitope imprinting: The case study of soluble PD-L1 detection in serum by optical-based sensing.

Polycatecholamines (pCAs)-based molecularly imprinted polymers (MIPs) represent the new performing generation of biocompatible ligand/receptor mimetics. In this context, dealing with MIPs synthesis for bio-macromolecules detection/extraction, one of the critical steps in ensuring effective binding affinity for the parent molecule is the selection of suitable epitopes for pCAs imprinting. To address this challenge, here we investigated the ability of lysine (K) residues to trigger the epitope imprinting process into a polynorepinephrine (PNE) matrix. To this aim, we first designed a set of model epitopes composed of three K and six alanine (A) residues to investigate the influence of each 'KA' combination on the imprinting process and the resulting binding performance by Surface Plasmon Resonance (SPR). Only the case of three flanking K residues in N-terminus arose as an excellent trigger for epitope imprinting. The efficacy of the 3K-tag strategy was then evaluated on two peptide templates belonging to soluble programmed cell death protein 1 ligand (PD-L1), which is of great interest as a cancer biomarker in liquid biopsies. These templates were selected due to their negligible natural ability to be imprinted into the PNE matrix and were modified with 3K-tags, in N-, C-, and N/C- positions, respectively. The SPR sensor developed by exploiting the N-3K tag strategy allowed us to achieve excellent sensitivity (0.31 ± 0.04 ng mL-1) and repeatability (avCV% = 4.5) in human serum samples. This strategy opens new insights both for epitopes' design for pCAs-based mimetics and as triggering tags when native epitopes display negligible imprinting capabilities.

Boronate affinity paper spray mass spectrometry for determination of elevated levels of catecholamines in urine.

The analysis of catecholamines, such as dopamine, epinephrine and norepinephrine in urine can be used in the diagnosis of certain pathologies, such as hormone-producing tumors. Here, a fast and simple quantitative boronate affinity paper spray tandem mass spectrometric (PS-MS/MS) method is established, which can improve selectivity and reduce ion suppression without needing any instrumental chromatography. We use here the property of boronic acids, which can selectively bind ortho-diol-containing compounds under alkaline conditions. Paper tip modification and catechol enrichment protocols were developed to selectively bind, clean up and subsequently desorb such catecholamines. Standard catecholamine solutions, as well as human urine samples were analyzed with the PS-MS(/MS) method, which is fast, cheap and easy-to-operate compared to HPLC-MS/MS. Despite its high simplicity, boronate affinity PS-MS/MS exhibits good performance compared to HPLC-MS/MS in human urine analysis in terms of precision (2.1%-7.2% vs. 1.1%-2.9%) and accuracy (-10.2%-9.3% vs. -4.8%-5.1%), and a physiologically relevant limit of detection (0.027-0.12 µg mL-1). The boronate affinity PS-MS/MS clearly achieved the detection limits that would allow the fast analysis of urine samples for clinical purposes, such as screening for pheochromocytoma (exceeding 0.5 µg mL-1).

Many Drugs of Abuse May Be Acutely Transformed to Dopamine, Norepinephrine and Epinephrine In Vivo.

It is well established that a wide range of drugs of abuse acutely boost the signaling of the sympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis, where norepinephrine and epinephrine are major output molecules. This stimulatory effect is accompanied by such symptoms as elevated heart rate and blood pressure, more rapid breathing, increased body temperature and sweating, and pupillary dilation, as well as the intoxicating or euphoric subjective properties of the drug. While many drugs of abuse are thought to achieve their intoxicating effects by modulating the monoaminergic neurotransmitter systems (i.e., serotonin, norepinephrine, dopamine) by binding to these receptors or otherwise affecting their synaptic signaling, this paper puts forth the hypothesis that many of these drugs are actually acutely converted to catecholamines (dopamine, norepinephrine, epinephrine) in vivo, in addition to transformation to their known metabolites. In this manner, a range of stimulants, opioids, and psychedelics (as well as alcohol) may partially achieve their intoxicating properties, as well as side effects, due to this putative transformation to catecholamines. If this hypothesis is correct, it would alter our understanding of the basic biosynthetic pathways for generating these important signaling molecules, while also modifying our view of the neural substrates underlying substance abuse and dependence, including psychological stress-induced relapse. Importantly, there is a direct way to test the overarching hypothesis: administer (either centrally or peripherally) stable isotope versions of these drugs to model organisms such as rodents (or even to humans) and then use liquid chromatography-mass spectrometry to determine if the labeled drug is converted to labeled catecholamines in brain, blood plasma, or urine samples.

Catecholamine-Copper Redox as a Basis for Site-Specific Single-Step Functionalization of Material Surfaces.

Realization of robust and facile surface functionalization processes is critical to biomaterials and biotechnology yet remains a challenge. Here, we report a new chemical approach that enables operationally simple and site-specific surface functionalization. The mechanism involves a catechol-copper redox chemistry, where the oxidative polymerization of an alkynyl catecholamine reduces Cu(II) to Cu(I), which in situ catalyzes a click reaction with azide-containing molecules of interest (MOIs). This process enables drop-coating and grafting of two- and three-dimensional solid surfaces in a single operation using as small as sub-microliter volumes. Generalizability of the method is shown for immobilizing MOIs of diverse structure and chemical or biological activity. Biological applications in anti-biofouling, cellular adhesion, scaffold seeding, and tissue regeneration are demonstrated, in which the activities or fates of cells are site-specifically manipulated. This work advances surface chemistry by integrating simplicity and precision with multipurpose surface functionalization.

Electroless deposition of gold nanoparticles on carbon nanopipette electrode for electrochemical detection of catecholamines released from PC12 cells.

An electroless deposition method is reported for the fabrication of gold nanoparticles (Au NPs) modified carbon nanopipette electrode (CNPE) for sensitive electrochemical detection of dopamine (DA) in aqueous solution and catecholamines released from PC12 cells. A CNPE is fabricated by chemical vapor deposition with a carbon layer onto nanocapillary and then contacted with copper (Cu) wire. Cu wire of CNPE is able to serve as reducing agent for electroless deposition of Au NPs on the CNPE because the potential of Cu2+/Cu is more negative than that of AuCl4-/Au. The method is simple, time-saving, and environmentally friendly. Field emission scanning electron microscopy, energy-dispersive X-ray analysis, and electrochemical techniques confirm the successful fabrication of the Au NPs/CNPE. Furthermore, Au NPs/CNPE exhibits a good sensing activity for DA oxidation with a wide linear determination range of 0.1-8 µmol/L and a low detection limit of 6 nmol/L. The Au NPs/CNPE can be potentially applied for measurement of catecholamines released from PC12 cells. This present work is believed to be beneficial to the design and development of active metal catalysts onto nanoelectrodes for the detection of electroactive biological molecules in living cells.Graphical abstract An electroless deposition method was developed for the fabrication of gold nanoparticles onto the carbon nanopipette electrode, which was served as an enhanced electrochemical sensing platform for highly sensitive detection of dopamine with a linear range of 0.1-8 µmol/L and a detection limit of 6 nmol/L, and was also applied in the detection of catecholamines released from PC12 cells.

Correlating Molecule Count and Release Kinetics with Vesicular Size Using Open Carbon Nanopipettes.

In this work, open carbon nanopipettes (CNPs) with radius between 50 and 600 nm were used to control translocation of different-sized vesicles through the pipette orifice followed by nanoelectrochemical analysis. Vesicle impact electrochemical cytometry (VIEC) was used to determine the number of catecholamine molecules expelled from single vesicles onto an inner-wall carbon surface, where the duration of transmitter release was quantified and correlated to the vesicle size all in the same nanotip. This in turn allowed us to both size and count molecules for vesicles in a living cell. Here, small and sharp open CNPs were employed to carry out intracellular VIEC with minimal invasion and high sensitivity. Our findings with VIEC reveal that the vesicular content increases with vesicle size. The release kinetics of vesicular transmitters and dense core size have the same relation with the vesicle size, implying that the vesicular dense core size determines the speed of each release event. This direct correlation unravels one of the complexities of exocytosis.

Kinetic characterization of the oxidation of catecolamines and related compounds by laccase.

The pathways of melanization and sclerotization of the cuticle in insects are carried out by the action of laccases on dopamine and related compounds. In this work, the laccase action of Trametes versicolor (TvL) on catecholamines and related compounds has been kinetically characterized. Among them, dopamine, l-dopa, l-epinephrine, l-norepinephrine, dl-isoprenaline, l-isoprenaline, dl-α-methyldopa, l-α-methyldopa and l-dopa methylester. A chronometric method has been used, which is based on measuring the lag period necessary to consume a small amount of ascorbic acid, added to the reaction medium. The use of TvL has allowed docking studies of these molecules to be carried out at the active site of this enzyme. The hydrogen bridge interaction between the hydroxyl oxygen at C-4 with His-458, and with the acid group of Asp-206, would make it possible to transfer the electron to the T1 Cu-(II) copper centre of the enzyme. Furthermore, Phe-265 would facilitate the adaptation of the substrate to the enzyme through Π-Π interactions. To kinetically characterize these compounds, we need to take into consideration that, excluding l-dopa, l-α-methyldopa and dl-α-methyldopa, all compounds are in hydrochloride form. Because of this, first we need to kinetically characterize the inhibition by chloride and, after that, calculate the kinetic parameters KM and VmaxS. From the kinetic data obtained, it appears that the best substrate is dopamine. The presence of an isopropyl group bound to nitrogen (isoprenaline) makes it especially difficult to catalyse. The formation of the ester (l-dopa methyl ester) practically does not affect catalysis. The addition of a methyl group (α-methyl dopa) increases the rate but decreases the affinity for catalysis. l-Epinephrine and l-norepinephrine have an affinity similar to isoprenaline, but faster catalysis, probably due to the greater nucleophilic power of their phenolic hydroxyl.

Two-Photon Antenna Sensitization of Curium: Evidencing Metal-Driven Effects on Absorption Cross Section in f-Element Complexes.

Two-photon-excited fluorescence spectroscopy is a powerful tool to study the structural and electronic properties of optically active complexes and molecules. Although numerous lanthanide complexes have been characterized by two-photon-excited fluorescence in solution, this report is the first to apply such a technique to actinide compounds. Contrasting with previous observations in lanthanides, we demonstrate that the two-photon absorption properties of the complexes significantly depend on the metal (4f vs 5f), with Cm(III) complexes showing significantly higher two-photon absorption cross sections than lanthanide analogues and up to 200-fold stronger emission intensities. These results are consistent with electronic and structural differences between the lanthanide and actinide systems studied. Hence, the described methodology can provide valuable insights into the interactions between f-elements and ligands, along with promising prospects on the characterization of scarce compounds.

Enhancement of Signal-to-Noise Ratio for Serotonin Detection with Well-Designed Nanofilter-Coated Potentiometric Electrochemical Biosensor.

In this paper, we proposed to enhance a signal-to-noise (S/N) ratio for detecting a primary stress marker, serotonin, using a potentiometric biosensor modified by a well-designed nanofilter film. An extended-Au-gate field-effect transistor (EG-Au-gate FET) biosensor exhibits highly sensitive electrochemical detection toward various small biomolecules, including serotonin. Therefore, to enhance the S/N ratio for the serotonin detection, we designed an appropriate nanofilter film on the Au electrode by combining the aryldiazonium salt reduction strategy and boronate affinity. That is, only serotonin can approach the Au sensing surface to generate an electrical signal; interfering biomolecules are prevented from penetrating through the nanofilter, either because large interfering biomolecules cannot permeate through the highly dense, nanoporous multilayer film, or because phenylboronic acids included in the nanofilter captures small interfering biomolecules (e.g., catecholamines). The potentiometric biosensor modified by such a nanofilter film detected serotonin in a model sample solution containing catecholamines, cortisol, and human serum albumin with a high S/N ratio for the serotonin levels in the blood. Furthermore, we found that the effect of the nanofilter directly reflects the binding affinity of the receptors such as phenylboronic acids included in the nanofilter; thus, the selectivity and dynamic range of small target biomolecules can be tuned freely by designing the appropriate receptors for the nanofilter. The results show that a well-designed nanofilter biointerface can be a versatile biosensing platform for point-of-care testing, particularly for a simple stress check.

Nanosized cation exchanger for the electrophoretic separation and preconcentration of catecholamines and amino acids.

The first example of application of nanosized polystyrene-based cation exchanger (NSCE) with sulfo groups as a dynamic coating of capillary walls was demonstrated. The conditions of dynamic coating formation were optimized and ensured the long-term stability of the coating. Capillary-to-capillary and day-to-day repeatabilities were 4% and 3%, correspondingly. The NSCE coating stability at various pH and influence of pH on the EOF mobility were investigated. The developed NSCE-modified coated capillaries provided improved resolution (Rs = 0.9-3.2 for catecholamines and Rs = 1.7-2.8 for amino acids) and efficiencies (330-520 ×103 t.p./m) of basic analytes, which are 1.5 times higher compared to untreated capillary. The optimized conditions were as follows: 50 mM phosphate buffer solution at pH 2.2 with 5 µM NSCE. The effect of the NSCE concentration in BGE on the electrophoretic mobilities of the analytes was investigated. The various online concentration techniques were tested in order to decrease the LODs. The simultaneous application of NSCE capillaries and field-amplified sample stacking provided the lowest LODs of catecholamines and amino acids and allowed to determine these analytes in human urine.

Automated on-line packed fiber solid phase extraction for determination of urinary catecholamines.

Despite the development of an off-line packed fiber solid phase extraction procedure (PFSPE) for urinary catecholamines, automation remains a challenge. Here, we propose an on-line PFSPE-HPLC procedure for automated sample processing and analysis of urinary catecholamines, with good recovery and precision, to avoid manual operation errors. The on-line PFSPE-HPLC procedure has been thoroughly optimized concerning the gradient, valve switch timing, the effects of complexing reagent and buffer solution, and the stability of the nanofibers. Validation of the developed on-line PFSPE-HPLC protocol in urine yielded satisfactory accuracies of 99.6-104.2%, precision below 7.0%, as well as a linear range from 1 ng/mL to 100 ng/mL with a correlation coefficient of 0.999. The developed protocol is herein presented as a potential technology for automated sample pretreatment for the determination of urinary catecholamines.

Engineering Shelf-Stable Coating for Microfluidic Organ-on-a-Chip Using Bioinspired Catecholamine Polymers.

The conceptualization of body-on-a-chip in 2004 resulted in a new approach for studying human physiology in three-dimensional culture. Despite pioneering works and the progress made in replicating human physiology on-a-chip, the stability, reliability, and preservation of cell-culture-treated microfluidic chips remain a challenge. The development of a reliable surface treatment technique to more efficiently and reproducibly modify microfluidic channels would significantly simplify the process of creating and implementing organ-on-a-chip (OOC) systems. In this work, a new flow-based coating technique using bioinspired polymers was implemented to create reliable, reproducible, ready-to-use microfluidic cell culture chips for OOC studies. Single-channel polydimethylsiloxane microfluidic chips were coated with the bioinspired catecholamine polymers, polydopamine (PDA) and polynorepinephrine (PNE), using a flow-based coating technique. The functionality of the resulting microfluidic chips was evaluated by extensive surface characterizations, at 130 °C, in the presence of various cleaning and culture media in static and flow conditions regularly used in OOCs and tested for shelf life by storing the coated microfluidic chips for 4 months at room temperature. Microfluidic chips coated with polycatecholamine were then seeded with the mouse cancer cell line Cath.a.differentiated (CAD) and with the normal human cerebral microvascular endothelial cell line human cerebral microvascular endothelial cells (hCMEC)/D3. Cell viability, cell phenotype, and cell functionality were assessed to evaluate the performance of both the coatings and the surface treatment technique. Both PDA- and PNE-coated microfluidic chips maintained high viability, phenotype, and functionality of CAD cells and hCMEC/D3 cells. In addition, CAD cells retained high viability when they were cultured in both the polymer-coated chips, which were stored at room temperature for up to 120 days. These results suggest that flow-based techniques to coat surfaces with polycatecholamines can be used to generate ready-to-use microfluidic OOC chips that offer long-term stability and reliability for the culture of cell types with application in pathophysiological studies and drug screening.

LC-MS determination of catecholamines and related metabolites in red deer urine and hair extracted using magnetic multi-walled carbon nanotube poly(styrene-co-divinylbenzene) composite.

A novel analytical methodology for the extraction and determination of catecholamines (dopamine, epinephrine and norepinephrine) and their metabolites DL-3,4-dihydroxyphenyl glycol and DL-3,4-dihydroxymandelic acid by LC-MS is developed and validated for its application to human and animal urine and hair samples. The method is based on the preliminary extraction of the analytes by a magnetic multi-walled carbon nanotube poly(styrene-co-divinylbenzene) composite. This is followed by a <9 min chromatographic separation of the target compounds in an Onyx Monolithic C18 column using a mixture of 0.01% (v/v) heptafluorobutyric acid in water and methanol at 500 µL min-1 flow rate. Detection limits within range from 0.055 to 0.093 µg mL-1, and precision values of the response and retention times of analytes were >90%. Accuracy values comprised the range 79.5-109.5% when the analytes were extracted from deer urine samples using the selected MMWCNT-poly(STY-DVB) sorbent. This methodology was applied to real red deer urine and hair samples, and concentrations within range from 0.05 to 0.5 µg mL-1 for norepinephrine and from 1.0 to 44.5 µg mL-1 for its metabolite 3,4-dihydroxyphenyl glycol were calculated. Analyses of red deer hair resulted in high amounts of 3,4-dihydroxyphenyl glycol (0.9-266.9 µg mL-1).

Adrenergic Innervation of the thyroid Gland, Blood and Lymph Vessels, and Lymph Nodes in Hypothyroidism.

Adrenergic innervation in the tissue of the thyroid gland, blood vessels of the thyroid gland, cervical lymphatic vessel, and lymph nodes in rats with hypothyroidism was studied by using a specific histochemical fluorescent-microscopic method of visualization of catecholamines. The presence of adrenergic innervation in the blood and lymph vessels and nodes was demonstrated. In hypothyroidism, diffusion of norepinephrine from nerve fibers and varicose thickenings was observed in the wall of the upper and lower thyroid arteries and adjacent cervical lymphatic vessels and nodes.

Hexadentate ß-Dicarbonyl(bis-catecholamine) Ligands for Efficient Uranyl Cation Decorporation: Thermodynamic and Antioxidant Activity Studies.

The special linear dioxo cation structure of the uranyl cation, which relegates ligand coordination to an equatorial plane perpendicular to the O═U═O vector, poses an unusual challenge for the rational design of efficient chelating agents. Therefore, the planar hexadentate ligand rational design employed in this work incorporates two bidentate catecholamine (CAM) chelating moieties and a flexible linker with a ß-dicarbonyl chelating moiety (ß-dicarbonyl(CAM)2 ligands). The solution thermodynamics of ß-dicarbonyl(CAM)2 with a uranyl cation was investigated by potentiometric and spectrophotometric titrations. The results demonstrated that the pUO22+ values are significantly higher than for the previously reported TMA(2Li-1,2-HOPO)2, and efficient chelation of the uranyl cation was realized by the planar hexadentate ß-dicarbonyl(CAM)2. The efficient chelating ability of ß-dicarbonyl(CAM)2 was attributed to the presence of the more flexible ß-dicarbonyl chelating linker and planar hexadentate structure, which favors the geometric arrangement between ligand and uranyl coordinative preference. Meanwhile, ß-dicarbonyl(CAM)2 also exhibits higher antiradical efficiency in comparison to butylated hydroxyanisole. These results indicated that ß-dicarbonyl(CAM)2 has potential application prospects as a chelating agent for efficient chelation of a uranyl cation.

Single-step functionalization of poly-catecholamine nanofilms for ultra-sensitive immunosensing of ubiquitin carboxyl terminal hydrolase-L1 (UCHL-1) in spinal cord injury.

Rapid, selective, and ultra-sensitive detection of brain and spinal cord injury markers in bodily fluids is an unmet clinical need. In this work, Polycatecholamine as a rich source of amine moieties was used for single-step fabrication of ultrasensitive immunosensors for the detection of Ubiquitin carboxyl-terminal hydrolase (UCHL-1) biomarker of brain and spinal cord injuries and address the clinical need. The surface of graphene electrodes was modified by electropolymerizing aqueous solution of dopamine (DA) and norepinephrine (NE) monomers for generating polycatecholamines nanofilms on the surface of graphene screen printed electrodes (GSPE) in a single functionalization step. Amine moieties of the polymer allowed immobilization of UCHL-1 antibody on the electrode. The single-step modification of GSPE offered a simple, ultrasensitive, and stable production of immunosensors for the detection of UCHL-1. The operational range of the UCHL-1 immunosensor developed with Polynorepinephrine pNE-modified is 0.1 pg mL-1 - 105 pg mL-1 (LOD: 1.91 pg mL-1), and 1 pg mL-1 - 105 pg mL-1 (LOD: 0.70 pg mL-1) with Polydopamine (pDA) modification, satisfying the clinical range. Both pNE and pDA modified immunosensors, detected UCHL-1 spiked in phosphate buffer saline, artificial cerebrospinal fluid, and serum. Along with the sensitive detections, selective performances were recorded in the above matrices in the presence of interfering neurotransmitters GABA and Glutamate as well as glial fibrillary acidic protein (GFAP). Upon testing clinical samples of spinal cord injury patients and healthy controls, both pNE and pDA immunosensors, delivered a comparable response for UCHL-1, thereby, making immunosensors useful for clinical settings.

Polymethacryloyl-L-Phenylalanine [PMAPA]-Based Monolithic Column for Capillary Electrochromatography.

The ability to detect catecholamines (CAs) and their metabolites is vital to understand the mechanism behind the neuronal diseases. Neurochemistry aims to provide an improved pharmacological, molecular and physiological understanding of complex brain chemistries by analytical techniques. Capillary electrophoresis (CE) is one such analytical technique that enables the study of various chemical species ranging from amino acids and peptides to natural products and drugs. CE can easily adapt the changes in research focus and in recent years remains an applicable technique for investigating neuroscience and single cell neurobiology. The prepared phenylalanine-based hydrophobic monolithic column, Polymethacryloyl-L-phenylalanine [PMAPA], was used as a stationary phase in capillary electrochromatography to separate CAs that are similar in size and shape to each other including dopamine (DA) and norepinephrine (NE) via hydrophobic interactions. Separation carried out in a short period of 17 min was performed with the electrophoretic mobility of 5.54 × 10-6 m2 V-1 s-1 and 7.60 × 10-6 m2 V-1 s-1 for DA and NE, respectively, at pH 7.0, 65% acetonitrile ratio with 100 mbar applied pressure by the developed hydrophobic monolithic column without needing any extra process such as imprinting or spacer arms to immobilize ligands used in separation.