Key structural requirements of benzamide derivatives for histone deacetylase inhibition: design, synthesis and biological evaluation.

Background: Histone deacetylase inhibitors (HDACIs) are important as anticancer agents. Objective: This study aimed to investigate some key structural features of HDACIs via the design, synthesis and biological evaluation of novel benzamide-based derivatives. Methods: Novel structures, designed using a molecular modification approach, were synthesized and biologically evaluated. Results: The results indicated that a subset of molecules with CH3/NH2 at R2 position possess selective antiproliferative activity. However, only those with an NH2 group showed HDACI activity. Importantly, the shorter the molecule length, the stronger HDACI. Among all, 7j was the most potent HDAC1-3 inhibitor and antiproliferative compound. Conclusion: The results of the present investigation could provide valuable structural knowledge applicable for the development of the HDACIs and benzamide-based antiproliferative agents in the future.

Design, synthesis, in vitro and in silico evaluations of new isatin-triazine- aniline hybrids as potent anti- Alzheimer multi-target directed lead compounds.

Multi target directed ligands (MTDLs) are one of the promising tools for treatment of complex disease like Alzheimer's disease (AD). In this study, using rational design, we synthesized new 15 hybrids of the s-triazine, isatin and aniline derivatives as anti- AD compounds. The design was as way as that new compounds could had anti cholinesterase (ChE), antioxidant and biometal chelation ability. In vitro biological evaluation against ChE enzymes showed that these molecules were excellent inhibitors with IC50 values ranging from 0.2 nM to 734.5 nM for acetylcholinesterase (AChE), and 0.02 µM to 1.92 µM for butyrylcholinesterase (BChE). Among these compounds, 8 l with IC50 AChE = 0.7 nM, IC50 BChE = 0.09 µM and 8n with IC50 AChE = 0.2 nM, IC50 BChE = 0.03 µM were the most potent compounds. In silico studies showed that these molecules had key and effective interactions with the corresponding enzymes residues. The molecules with hydroxyl group on aniline moiety had also good antioxidant activity with EC50 values ranging from 64.2 µM to 103.6 µM. The UV-Vis spectroscopy study revealed that molecule 8n was also able to chelate biometals such as Zn2+, Cu2+and Fe2+ properly. It was concluded that these molecules could be excellent lead compounds for future studies.

Determination of indoxyl sulfate by spectrofluorimetric method in human plasma through extraction with deep eutectic solvent.

A rapid and efficient analytical method was established to quantify indoxyl sulfate (IS) in plasma through extraction technique with a deep eutectic solvent (DES) and spectrofluorimetric method. DES (choline chloride: urea) was mixed with plasma samples for the extraction of IS, followed by the addition of dipotassium hydrogen phosphate (K2HPO4) solution to form an aqueous two-phase system. The fluorescence intensity of IS which was first extracted to the DES-rich-phase and then back-extracted into the salt-rich-phase, was measured by spectrofluorimetric method. Some key factors such as pH, centrifugation speed and time, the volume ratio of DES/salt, and salt concentration were optimized. Under the optimized conditions, the suggested method had a dynamic range between 20 and 160 µg/mL with a coefficient of determination (R2) of 0.99. Precision (relative standard deviation) was less than 15% and accuracy (% relative recovery) was ± 15% at the nominal concentration level. In addition, results showed that IS levels in real samples were higher than 40 µg/mL which was compatible with reported IS levels in end-stage renal disease (ESRD) patients. Overall, all the results reflect the fact that the presented analytical method can potentially be used for the determination of IS in real plasma samples.

Benchmarking different docking protocols for predicting the binding poses of ligands complexed with cyclooxygenase enzymes and screening chemical libraries.

Introduction: Non-steroidal anti-inflammatory drugs (NSAIDs) constitute an important class of pharmaceuticals acting on cyclooxygenase COX-1 and COX-2 enzymes. Due to their numerous severe side effects, it is necessary to search for new selective, safe, and effective anti-inflammatory drugs. In silico design of novel therapeutics plays an important role in nowadays drug discovery pipelines. In most cases, the design strategies require the use of molecular docking calculations. The docking procedure may require case-specific condition for a successful result. Additionally, many different docking programs are available, which highlights the importance of identifying the most proper docking method and condition for a given problem. Methods: In the current work, the performances of five popular molecular docking programs, namely, GOLD, AutoDock, FlexX, Molegro Virtual Docker (MVD) and Glide to predict the binding mode of co- crystallized inhibitors in the structures of known complexes available for cyclooxygenases were evaluated. Furthermore, the best performers, Glide, AutoDock, GOLD and FlexX, were further evaluated in docking-based virtual screening of libraries consisted of active ligands and decoy molecules for cyclooxygenase enzymes and the obtained docking scores were assessed by receiver operating characteristics (ROC) analysis. Results: The results of docking experiments indicated that Glide program outperformed other docking programs by correctly predicting the binding poses (RMSD less than 2 Å) of all studied co-crystallized ligands of COX-1 and COX-2 enzymes (i.e., the performance was 100%). However, the performances of the other studied docking methods for correctly predicting the binding poses of the ligands were between 59% to 82%. Virtual screening results treated by ROC analysis revealed that all tested methods are useful tools for classification and enrichment of molecules targeting COX enzymes. The obtained AUCs range between 0.61-0.92 with enrichment factors of 8 - 40 folds. Conclusion: The obtained results support the importance of choosing appropriate docking method for predicting ligand-receptor binding modes, and provide specific information about docking calculations on COXs ligands.

Application of Carbon-based Nano Adsorbents in the Extraction of Amphetamines from Urine Samples.

Amphetamines, as psychoactive drugs, are extensively abused in society and cause serious mental and physical disorders among young people. Furthermore, the extremely euphoric and excited sense of stimulant consumption leads to dramatic social problems. Determination of various analytes and related metabolites in the complex biological matrices at trace levels has made sample preparation an indispensable part of forensic sciences. According to the problems above, providing high sensitivity, solving some analytical problems like matrix effects in LCMS-MS, and needing a cleaner extract are remarkable aspects of novel sample preparation methods in drug analysis. Application of nanotechnology and carbon-based nanocomposites seems to bring the above properties in developed and novel sample preparation methods. This review will try to provide an overview of different carbonic nano adsorbents used in sample preparation methods of amphetamines and discuss their superiority over the other nanomaterials.

Construction of a bacteriophage-derived vector with potential applications in targeted drug delivery and cell imaging.

There is a strong relationship between the dysregulation of epidermal growth factor receptor (EGFR) and the development of epithelial-derived cancers. Therefore, EGFR has usually been considered the desired target for gene therapy. Here, we propose an approach for targeting EGFR-expressing cells by phage particles capable of displaying EGF and GFP as tumor-targeting and reporting elements, respectively. For this purpose, the superfolder GFP-EGF (sfGFP-EGF) coding sequence was inserted at the N-terminus of the pIII gene in the pIT2 phagemid. The capability of the constructed phage to recognize EGFR-overexpressing cells was monitored by fluorescence microscopy, fluorescence-activated cell sorting (FACS), and cell-based ELISA experiments. FACS analysis showed a significant shift in the mean fluorescence intensity (MFI) of the cells treated with phage displaying sfGFP-EGF compared to phage displaying only sfGFP. The binding of phage displaying sfGFP-EGF to A-431 cells, monitored by fluorescence microscopy, indicated the formation of the sfGFP-EGF-EGFR complex on the surface of the treated cells. Cell-based ELISA experiments showed that phages displaying either EGF or sfGFP-EGF can specifically bind EGFR-expressing cells. The vector constructed in the current study has the potential to be engineered for gene delivery purposes as well as cell-based imaging for tumor detection.

17ß-Estradiol-Loaded Exosomes for Targeted Drug Delivery in Osteoporosis: A Comparative Study of Two Loading Methods.

Purpose: Exosomes are natural nanoparticles that participate in intercellular communication through molecular transport. Recently, due to their membrane vesicular structure and surface proteins, exosomes have been used extensively in the research field of drug delivery. Osteoporosis is an inflammation in which the cellular balance of bone tissue is disturbed that reduces bone density and making bone prone to abnormal fractures with small amount of force. Utilizing estrogen is one of the main therapeutic strategies for osteoporosis. Despite the positive effects of estrogen on bone tissue, changes in the natural estrogen levels of the body can cause a number of diseases such as different types of cancer. Therefore, designing a therapeutic system which controls more accurate tissue targeting of estrogen seems to be a rational and promising practical approach. Methods: In this study, bone marrow mesenchymal stem cells (BMMSCs)-derived exosomes were loaded by estradiol using two different methods of drug loading, namely incubation and sonication methods and then the survival effects of the drug loaded exosomes on BMMSCs was investigated. Results: Examination of size, shape, and surface factors of exosomes in different states (pure exosomes and drug-loaded exosomes) showed that the round morphology of exosomes was preserved in all conditions. However, the particles size increased significantly when loaded by sonication method. The increased survival of BMMSCs was noted with estradiol-loaded exosomes when compared to the control group. Conclusion: The results suggest that estradiol-loaded exosomes have potential to be used as nano-drug carriers in the treatment of osteoporosis.

Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli.

Purpose: Medical usage of L-asparaginase (ASNase), the first-line of acute lymphoblastic leukemia treatment, is linked to allergic responses and toxicities, which necessitates the development of new bio-better ASNases. The aim of the current study was in silico design of a novel ASNase with predicted improved enzymatic properties using strategies encompassing sequence-function analysis of known ASNase mutants. Additionally, current study aimed to show that the new enzyme is active. Methods: Based on 21 experimentally reported mutations for ASNase, a virtual library of mutated enzymes with all 7546 possible combinations of up to 4 mutations was generated. Three-dimensional models of proposed mutant enzymes were built and their in silico stabilities were calculated. The most promising mutant was selected for preparing a genetic construct suitable for expression of the designed ASNase in bacterial cells. Results: Computational study predicted that Y176F/S241C double mutation of Escherichia coli ASNase may increase its folding stability. The designed ASNase was expressed in two different E. coli strains (Origami B(DE3) and BL21(DE3)pLysS) and then the soluble fractions prepared from the cell lysates of the host cells were used in enzyme activity assay. Results showed that enzyme activity of soluble fraction from Origami (95.4 ± 7.5 IU/0.1 mL) was four times higher than that of soluble fraction from pLysS (25.8 ± 2.5 IU/0.1 mL). Conclusion: A novel functional double mutant ASNase with predicted improved enzymatic properties was designed and produced in E. coli. The results of the current study suggest a great commercial potential for the identified enzyme in pharmaceutical and industrial applications.

Expression, Purification and Characterization of Functional Teduglutide Using GST Fusion System in Prokaryotic Cells.

Purpose: Teduglutide is the first and only FDA-approved drug for long-term treatment of short bowel syndrome (SBS). The current study aimed to present an approach for production of teduglutide using recombinant DNA technology. Methods: The coding gene for teduglutide was cloned into pGEX-2T vector, where coding sequence for factor Xa cleavage site was added between GST and teduglutide coding genes. The GST-teduglutide protein was overexpressed in E. coli BL21 (DE3) strain and affinity purified using glutathione sepharose affinity column. Results: On-column proteolytic activity of factor Xa followed by size exclusion chromatography resulted in the pure teduglutide. Circular dichroism (CD) spectropolarimetry showed that the produced teduglutide folds into mainly α-helical structure (>50%), as expected. In mass spectroscopy analysis, the fragments of teduglutide resulted by cyanogen bromide cleavage as well as those expected theoretically due to mass fragmentation were identified. The functionality of the produced peptide was evaluated by measuring its proliferative effect on Caco2 intestinal epithelial cells, and the results indicated that produced teduglutide induces cell proliferation by 19±0.30 and 33±7.82 % at 1.21 and 3.64 µM concentrations, respectively, compared to untreated cells. Conclusion: Teduglutide was successfully expressed and purified and its functionality and structural integrity were confirmed by in vitro experiments. We believe that the experimental-scale method presented in the current study can be useful for pilot-scale and also industrial-scale production of teduglutide.

Design, Synthesis, and Biological Evaluation of Novel Indanone Derivatives as Cholinesterase Inhibitors for Potential Use in Alzheimer's Disease.

Indanone derivatives containing meta/para-substituted aminopropoxy benzyl/benzylidene moieties were designed based on the structures of donepezil and ebselen analogs as the cholinesterase inhibitors. The designed compounds were synthesized and their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were measured. Inhibitory potencies (IC50 values) for the synthesized compounds ranged from 0.12 to 11.92 µM and 0.04 to 24.36 µM against AChE and BChE, respectively. Compound 5 c showed the highest AChE inhibitory potency with IC50 value of 0.12 µM, whereas the highest BChE inhibition was achieved by structure 7 b (IC50 =0.04 µM). Structure-activity relationship (SAR) analysis revealed that there is no significant difference between meta and para-substituted derivatives in AChE and BChE inhibition. However, the most potent AChE inhibitor 5 c belongs to meta-substituted compounds, while the most active BChE inhibitor is para-substituted derivative 7 b. The order of enzyme inhibition potency based on the substituted amine group is dimethyl amine>piperidine>morpholine. Compounds containing C=C linkage are more potent AChE inhibitors than the corresponding saturated structures. Molecular docking studies indicated that 5 c interacts with AChE in a very similar way to that observed experimentally for donepezil. The introduced indanone-aminopropoxy benzylidenes could be used in drug-discovery against Alzheimer's disease.

Dispersive solid-phase extraction of risperidone from plasma samples using graphene oxide aerogels and determination with liquid chromatography.

A graphene oxide-based aerogel was synthesized and applied to the extraction and the determinations with the high-performance liquid chromatography-ultraviolet detector. After the characterization of the produced graphene-aerogel, it was utilized as a dispersive solid-phase extraction sorbent for risperidone extraction from plasma samples. Aerogels are materials with a large surface area-to-mass ratio and plenty of core with functional groups which can easily attach to the analytes to extract them to the second phase. The suggested method determined risperidone in plasma samples in the wide dynamic range from 20 ng/ml to 3 µg/ml. The limits of detection and quantification of the developed method were calculated as 2.4 and 8.2 ng/ml, respectively. As a novel feature, the developed method has no need to precipitate plasma proteins, improving the analytical performance of the analysis. Also, for the first time, the produced materials were utilized for the extraction of risperidone from the plasma samples. The obtained results revealed that the developed approach could be employed as an accurate method for the quantification of risperidone in real plasma samples.

Efficient dispersive solid-phase extraction of methylprednisolone from exhaled breath of COVID-19 patients.

In the current study, bismuth ferrite nano-sorbent was synthesized and utilized as a sorbent for the dispersive solid-phase extraction of methylprednisolone from exhaled breath samples. The size and morphology of the nano-sorbent were characterized by X-ray diffraction analysis and scanning electron microscopy. Following its desorption with acetonitrile, methylprednisolone was quantified by a high-performance liquid chromatography-ultraviolet detector. Factors affecting the extraction of methylprednisolone were optimized. Under optimized experimental conditions, a linear relationship between the analytical signals and methylprednisolone concentration was obtained in the range of 0.001-0.2 µg mL-1 for exhaled breath condensate samples and 0.002-0.4 µg per filter for filter samples. A pre-concentration factor of 6.4-fold, corresponding to an extraction recovery of 96.0%, was achieved. The validated method was applied for the determination of methylprednisolone in real samples taken from the exhaled breath of COVID-19 patients under mechanical ventilation.

S1PR1 modulators in multiple sclerosis: Efficacy, safety, comparison, and chemical structure insights.

Multiple sclerosis (MS) is a neurological disease that leads to severe physical and cognitive disabilities. Drugs used in the treatment of MS vary from small synthetic molecules to large macromolecules such as antibodies. Sphingosine 1-phosphate receptor modulators are frequently used for the treatment of MS. These medicines prevent the egress of lymphocytes from secondary lymphoid organs leading to immune system suppression. Currently, four S1PR modulators are on the market and several potential drug candidates are in clinical trials for the treatment of MS. These compounds differ in chemical structure, adverse effects, and efficacy points of view. The current article reviews the latest studies on S1PR1 modulators and compares them with other MS drugs in terms of efficacy, tolerability, and safety. A special focus was dedicated to discussing the structure-activity relationships of these compounds and performing a three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis to gain better insight into the ligand-receptor interaction mode.

Drug Repurposing for Identification of S1P1 Agonists with Potential Application in Multiple Sclerosis Using In Silico Drug Design Approaches.

Purpose: Drug repurposing is an approach successfully used for discovery of new therapeutic applications for the existing drugs. The current study was aimed to use the combination of in silico methods to identify FDA-approved drugs with possible S1P1 agonistic activity useful in multiple sclerosis (MS). Methods: For this, a 3D-QSAR model for the known 21 S1P1 agonists were generated based on 3D-QSAR approach and used to predict the possible S1P1 agonistic activity of FDA-approved drugs. Then, the selected compounds were screened by docking into S1P1 and S1P3 receptors to select the S1P1 potent and selective compounds. Further evaluation was carried out by molecular dynamics (MD) simulation studies where the S1P1 binding energies of selected compounds were calculated. Results: The analyses resulted in identification of cobicistat, benzonatate and brigatinib as the selective and potent S1P1 agonists with the binding energies of -85.93, -69.77 and -67.44 kcal. mol-1, calculated using MM-GBSA algorithm based on 50 ns MD simulation trajectories. These values are better than that of siponimod (-59.35 kcal mol-1), an FDA approved S1P1 agonist indicated for MS treatment. Furthermore, similarity network analysis revealed that cobicistat and brigatinib are the most structurally favorable compounds to interact with S1P1. Conclusion: The findings in this study revealed that cobicistat and brigatinib can be evaluated in experimental studies as potential S1P1 agonist candidates useful in the treatment of MS.

Recent Advances in Structural Modification Strategies for Lead Optimization of Tyrosine Kinase Inhibitors to Explore Novel Anticancer Agents.

Lead optimization as a bottleneck in the process of drug discovery is conducted to tackle problems associated with poor pharmacokinetics, continuous emergence of drugresistance, adverse side effects and drug-drug interactions of known pharmaceuticals. Due to the intensive application of multi-targeted tyrosine kinase inhibitors (MTKI) in various pathological conditions, optimization of their structures has always been the focus of intensive medicinal chemistry research efforts. The current review portrays the application of scaffold hopping, bioisosterism, structure-based, and hybrid-based drug design methods in the optimization of lead compounds aiming to enhance their usefulness as novel drugs. Then, the review proceeds with examples of structural modifications carried out, particularly on multi-targeted drugs already available on the market. The demonstrated examples cover structural modifications on 7 well-known drugs during the last twenty years. The application of the above-mentioned strategies has led to the generation of 52 new multitargeted tyrosine kinase inhibitors. Most of the optimized compounds showed improved properties compared to their parent lead compound. The rationales behind the applied modifications and the achieved outcomes were discussed to present practical examples to the researchers engaged in the area.

The impact of simulation time in predicting binding free energies using end-point approaches.

The profound impact of in silico studies for a fast-paced drug discovery pipeline is undeniable for pharmaceutical community. The rational design of novel drug candidates necessitates considering optimization of their different aspects prior to synthesis and biological evaluations. The affinity prediction of small ligands to target of interest for rank-ordering the potential ligands is one of the most routinely used steps in the context of virtual screening. So, the end-point methods were employed for binding free energy estimation focusing on evaluating simulation time effect. Then, a set of human aldose reductase inhibitors were selected for molecular dynamics (MD)-based binding free energy calculations. A total of 100[Formula: see text]ns MD simulation time was conducted for the ligand-receptor complexes followed by prediction of binding free energies using MM/PB(GB)SA and LIE approaches under different simulation time. The results revealed that a maximum of 30[Formula: see text]ns simulation time is sufficient for determination of binding affinities inferred from steady trend of squared correlation values (R2) between experimental and predicted [Formula: see text]G as a function of MD simulation time. In conclusion, the MM/PB(GB)SA algorithms performed well in terms of binding affinity prediction compared to LIE approach. The results provide new insights for large-scale applications of such predictions in an affordable computational cost.

Preparation and Antiproliferative Activity Evaluation of Juglone-Loaded BSA Nanoparticles.

Purpose: Today, the discovery of novel and effective chemotherapeutic compounds is the main challenge in cancer therapy. In recent years, the anti-tumoral activity of natural naphthoquinone juglone (JUG), present in different parts of walnut trees, has received considerable interest. The purpose of the current study was to prepare and evaluate the in vitro antiproliferative activity of JUG-loaded bovine serum albumin nanoparticles (JUG-BSA NPs). Methods: BSA NPs and JUG-BSA NPs were prepared using the desolvation technique. The NPs were characterized for their particle size (PS), zeta potential (ZP), drug loading (DL) capacity and encapsulation efficiency (EE). The anti-proliferative activity of JUG-BSA NPs was evaluated on A431 and HT29 cancer cell lines using cellular uptake and MTT assays. Results: The PS and ZP values of JUG-BSA NPs were 85 ± 6.55 nm and -29.6 mV, respectively. The DL capacity and EE were 3.7% to 5% and 50.4% to 94.6%, respectively. The cytotoxicity of JUG-BSA NPs was significantly less on both cultured A431 and HT29 cells at the studied concentrations when compared to free JUG. However, the effect was not very substantial, particularly at high levels. Conclusion: In conclusion, BSA NPs can be used as a suitable and safe carrier for the delivery of JUG, a cytotoxic hydrophobic natural compound.

Current trends in development of HDAC-based chemotherapeutics.

BACKGROUND: Histone deacetylases (HDACs) are one of the essential epigenetic targets in cancer treatment. These enzymes play key roles in post-translation modification (PTM) and gene expression, and consequently, their inhibitors are about to find their place in pharmacotherapy. Most of the currently approved HDAC inhibitors (HDACIs) are wide-spectrum with poor clinical outcomes and numerous side effects. Therefore, new generations of HDAC-based chemotherapeutics with better clinical outcomes are emerging, e.g., isoform-selective inhibitors, multitargeted HDACIs, as well as HDAC degraders. AIM: The review intended to introduce drug design approaches which were used for designing novel agents which can be beneficial in the process of finding new and more effective HDACI-based therapeutics. METHODS: PubMed and other databases were searched for literature regarding the structure-function of HDAC isoforms, and strategies used to design HDAC inhibitors. Also, all clinical trials available from the ClinicalTrials site for years 2021-2022 were investigated. KEY FINDINGS: It is expected that the future of drug discovery projects in HDAC field will concentrate mostly on issues such as isoform-selectivity, multitargeted HDAC inhibitors and HDAC degraders. Deeper knowledge of the 3D structure of HDACs complexed with inhibitors and extensive delineation of biological roles of HDACs are needed for efficient investigations leading to the discovery of novel potent inhibitors. SIGNIFICANCE: Histone deacetylases (HDACs) are one of the important epigenetic targets in cancer treatment drug discovery. Comprehending the structure of HDAC isoforms along with applied drug design strategies can inspire new ideas.

Microextraction Techniques for Sample Preparation of Amphetamines in Urine: A Comprehensive Review.

Psychological disorders and dramatic social problems are serious concerns regarding the abuse of amphetamine and its stimulant derivatives worldwide. Consumers of such drugs experience great euphoria along with serious health problems. Determination and quantification of amphetamine-type stimulants are indispensable skills for clinical and forensic laboratories. Analysis of low drug doses in bio-matrices necessitates applications of simple and also effective preparation steps. The preparation procedures not only eliminate adverse matrix effects, but also provide reasonable clean-up and pre-concentration benefits. The current review presents different methods used for sample preparation of amphetamines from urine as the most frequently used biological matrix. The advantages and limitations of various sample preparation methods were discussed focusing on the miniaturized methods.

Clinical Application Study of Polymeric Nanospheres Network in Methylphenidate Extraction from Urine Samples by Dispersive Solid Phase Extraction Adsorbent.

Purpose: This research introduces a polymeric nanosphere as a new dispersive solid phase extraction (DSPE) adsorbent for the extraction of methylphenidate (MPH) from urine and its high performance liquid chromatography (HPLC) analysis. Methods: Polymeric nanosphere is a kind of copolymeric network obtained by copolymerization of an ionic liquid monomer and styrene in the presence of vinyltriethoxysilane and 2-hydroxyethylmethacrylate. HPLC coupled with ultra violet detector was applied for the determination and quantification of MPH. Dominant parameters in extraction were modified by the one-parameter-at-a-time method. The results are as follow: 10 mg of polymeric nanospheres (PNS), 400 µL of acetonitrile (ACT), 5 mL of urine with the pH value of 9, and the extraction and desorption times of 2 and 5 minutes, respectively, which can be selected as the optimum extraction conditions. Results: Calibration curve was plotted through optimized conditions, and the proposed method was validated. The results demonstrated that the method presented linearity in the concentration range of 30-1200 ng/mL. Selectivity, matrix effect and metabolites interference effect were investigated and the method presented no obvious interference effect during the analysis run time. Repeatability, limit of detection (LOD) and limit of quantification (LOQ) values of the method can be reported in this section as well. The method showed satisfactory results with 98.8% relative recovery in the analysis of positive urine samples. Conclusion: The findings convinced the applicability of the introduced method for DSPE and HPLC analysis of the positive urine samples in different laboratories.