Journal of Pharmaceutical Analysis

2018, 8(2)

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Advancements in the preparation of high-performance liquid chromatographic organic polymer monoliths for the separation of small-molecule drugs

Xiali Ding, Jing Yang, Yuming Dong

2018, 8(2): 75-85.

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The various advantages of organic polymer monoliths, including relatively simple preparation processes, abundant monomer availability, and a wide application range of pH, have attracted the attention of chromatographers. Organic polymer monoliths prepared by traditional methods only have macropores and mesopores, and micropores of less than 50 nm are not commonly available. These typical monoliths are suitable for the separation of biological macromolecules such as proteins and nucleic acids, but their ability to separate small molecular compounds is poor. In recent years, researchers have successfully modified polymer monoliths to achieve uniform compact pore structures. In particular, microporous materials with pores of 50 nm or less that can provide a large enough surface area are the key to the separation of small molecules. In this review, preparation methods of polymer monoliths for high-per-formance liquid chromatography, including ultra-high cross-linking technology, post-surface modifica-tion, and the addition of nanomaterials, are discussed. Modified monolithic columns have been used successfully to separate small molecules with obvious improvements in column efficiency.

Novel degradation products of argatroban:Isolation, synthesis and extensive characterization using NMR and LC-PDA-MS/Q-TOF

Vinodh Guvvala, Venkatesan Chidambaram Subramanian, Jaya Shree Anireddy, Mahesh Konda

2018, 8(2): 86-95.

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Forced degradation study of argatroban under conditions of hydrolysis (neutral, acidic and alkaline), oxidation, photolysis and thermal stress, as suggested in the ICH Q1A (R2), was accomplished. The drug showed sig-nificant degradation under hydrolysis (acidic, alkaline) and oxidation (peroxide stress) conditions. The drug remained stable under thermal and photolytic stress conditions. In total, seven novel degradation products (DP-1 to DP-7) were found under diverse conditions, which were not reported earlier. The chemical structures of these degradation products were characterized by 1H NMR, 13C NMR, 2D NMR, Q-TOF-MSn and IR spectral analysis and the proposed degradation products structures were further confirmed by the individual synthesis.

Structural confirmation of sulconazole sulfoxide as the primary degradation product of sulconazole nitrate

Qun Xu, Ashraf Khan, Di Gao, Kristie M. Adams, Fatkhulla Tadjimukhamedov, Shane Tan, John T. Simpson

2018, 8(2): 96-102.

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Sulconazole has been reported to degrade into sulconazole sulfoxide via sulfur oxidation; however, structural characterization data was lacking and the potential formation of an N-oxide or sulfone could not be excluded. To clarify the degradation pathways and incorporate the impurity profile of sulconazole into the United States Pharmacopeia–National Formulary (USP–NF) monographs, a multifaceted approach was utilized to confirm the identity of the degradant. The approach combines stress testing of sulco-nazole nitrate, chemical synthesis of the degradant via a hydrogen peroxide-mediated oxidation reaction, semi-preparative HPLC purification, and structural elucidation by LC―MS/MS and NMR spectroscopy. Structural determination was primarily based on the comparison of spectroscopic data of sulconazole and the oxidative degradant. The mass spectrometric data have revealed a McLafferty-type rearrange-ment as the characteristic fragmentation pathway for alkyl sulfoxides with aβ-hydrogen atom, and was used to distinguish the sulfoxide from N-oxide or sulfone derivatives. Moreover, the generated sulco-nazole sulfoxide was utilized as reference material for compendial procedure development and valida-tion, which provides support for USP monograph modernization.

Physicochemical characterization, the Hirshfeld surface, and biological evaluation of two meloxicam compounding pharmacy samples

Luciana F.A. Romani, Maria I. Yoshida, Elionai C.L. Gomes, Renes R. Machado, Felipe F. Rodrigues, Márcio M. Coelho, Marcelo A. Oliveira, Maria B. Freitas-Marques, Rosane A.S. San Gil, Wagner N. Mussel

2018, 8(2): 103-108.

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Meloxicam (MLX) is an anti-inflammatory drug susceptible to variations and crystalline transitions. In compounding pharmacies, the complete crystallographic evaluation of the raw material is not a routine procedure. We performed a complete crystallographic characterization of aleatory raw MLX samples from compounding pharmacies. X-ray diffraction indicated the presence of two crystalline forms in one sample. DSC experiments suggested that crystallization, or a crystal transition, occurred differently be-tween samples. The FTIR and 1H NMR spectra showed characteristic assignments. 13C solid-state NMR spectroscopy indicated the presence of more than one phase in a sample from pharmacy B. The Hirshfeld surface analysis, with electrostatic potential projection, allowed complete assignment of the UV spectra in ethanol solution. The polymorph I of meloxicam was more active than polymorph Ⅲ in an experi-mental model of acute inflammation in mice. Our results highlighted the need for complete crystal-lographic characterization and the separation of freely used raw materials in compounding pharmacies, as a routine procedure, to ensure the desired dose/effect.

Anti-diabetic activity of quercetin extracted from Phyllanthus emblica L. fruit:In silico and in vivo approaches

Prabhu Srinivasan, S. Vijayakumar, Swaminathan Kothandaraman, Manogar Palani

2018, 8(2): 109-118.

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In this study, molecular interactions of the ligands, quercetin, gallic acid, and metformin with various diabetes mellitus-related protein targets, such as glycogen phosphorylase and peroxisome proliferator-activated receptor gamma, were assessed. It was revealed that quercetin possesses good binding affinity to both targets. Quercetin is a major constituent of methanolic extracts of Phyllanthus emblica fruit. The antihyperglycemic effect of quercetin in streptozotocin (STZ)-induced diabetic rats was examined. The isolated quercetin administered at a dose of 75 mg/kg body weight produced a maximum decrease of 14.78%in blood glucose levels in the diabetic rats after 7 days of treatment. Furthermore, quercetin doses of 50 and 75 mg/kg were shown to significantly improve the profiles of triglycerides, high-density li-poprotein, very-low-density lipoprotein, low-density lipoprotein, and total cholesterol at the end of the study in STZ-induced diabetic rats. The administration of quercetin (25, 50, and 75 mg/kg body weight) daily for 28 days in STZ-induced diabetic rats resulted in a significant decrease in blood glucose and urine sugar levels, with a considerable rise in plasma insulin and hemoglobin levels. Therefore, quercetin is a potential drug with antidiabetic and antihyperglycemic action mediated by changes in the levels of glucose, cholesterol, and triglycerides as indicated by in silico and in vivo studies.

Quantitative determination of erlotinib in human serum using competitive enzyme-linked immunosorbent assay

Yuta Yamamoto, Tetsuya Saita, Yutaro Yamamoto, Masashi Shin

2018, 8(2): 119-123.

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A selective and sensitive competitive enzyme-linked immunosorbent assay (ELISA) method was developed and validated for the quantification of erlotinib in 50μL of samples of human serum. Anti-erlotinib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and 3,4-bis(2-methoxyethoxy)benzoic acid using the N-succinimidyl ester method. Enzyme labeling of erlotinib with horseradish peroxidase was similarly performed using 3,4-bis(2-methoxyethoxy)benzoic acid. A simple competitive ELISA for erlotinib was developed using the principle of direct competition between erlotinib and the enzyme marker for anti-erlotinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Serum erlotinib concentrations lower than 40 ng/mL were reproducibly measurable using the ELISA. This ELISA was specific to erlotinib and showed very slight cross-reactivity (6.7%) with a major metabolite, O-desmethyl erlotinib. Using this assay, drug levels were easily measured in the blood of mice after oral administration of erlotinib at a single dose of 30 mg/kg. ELISA should be used as a valuable tool for therapeutic drug monitoring and in pharmacokinetic studies of erlotinib.

Ultrasensitive electrochemical determination of metronidazole based on polydopamine/carboxylic multi-walled carbon nanotubes nanocomposites modified GCE

Satar Tursynbolat, Yrysgul Bakytkarim, Jianzhi Huang, Lishi Wang

2018, 8(2): 124-130.

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An ultrasensitive electrochemical sensor based on polydopamine/carboxylic multi-walled carbon nano-tubes (MWCNTs-COOH) nanocomposites modified glassy carbon electrode (GCE) was presented in this work, which has been developed for highly selective and highly sensitive determination of an anti-microbial drug, metronidazole. The preparation of polydopamine/MWCNTs―COOH nanocomposites/GCE sensor is simple and possesses high reproducible, where polydopamine can be coated on the surface of MWCNTs―COOH via a simple electropolymerization process. Under optimized conditions, the proposed sensor showed ultrasensitive determination for metronidazole with a wide linear detection range from 5 to 5000μmol/dm3 and a low detection limit of 0.25μmol/dm3 (S/N = 3). Moreover, the proposed sensor has been successfully applied for the quantitative determination of metronidazole in real drug samples. This work may provide a novel and effective analytical platform for determination of me-tronidazole in application of real pharmaceutical and biological samples analysis.

Eco-friendly reduced graphene oxide for the determination of mycophenolate mofetil in pharmaceutical formulations

Prashanth S. Narayan, Nagappa L. Teradal, Seetharamappa Jaldappagari, Ashis K. Satpati

2018, 8(2): 131-137.

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Graphene oxide (GO) was synthesized and characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), atomic force microscopy (AFM), X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). GO was then electro-chemically reduced and used for electrochemical study of mycophenolate mofetil (MMF). The electro-chemically reduced graphene oxide (ERGO) film on glassy carbon electrode (GCE) showed enhanced peak current for electrooxidation of MMF. MMF exhibited two irreversible oxidation peaks at 0.84 V (peak a1) and 1.1 V (peak a2). Effects of accumulation time, pH and scan rate were studied and various electro-chemical parameters were calculated. A differential pulse voltammetric method was developed for the determination of MMF in bulk samples and pharmaceutical formulations. Linear relationship was ob-served between the peak current and concentration of MMF in the range of 40 nM―15μM with a limit of detection of 11.3 nM. The proposed method is simple, sensitive and inexpensive and, hence, could be readily adopted in clinical and quality control laboratories.

Surrogate potency assays:Comparison of binding profiles complements dose response curves for unambiguous assessment of relative potencies

Robert Karlsson, Veronica Fridh, ?sa Frostell

2018, 8(2): 138-146.

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Surface plasmon resonance (SPR) systems are widely used for detailed characterization of antibody ac-tivities including antigen and Fc-receptor binding. During the later stages of development, where the focus is to ensure that established critical quality attributes (CQAs) are maintained during cell culture, purification and formulation processes, analysis is simplified, and relative potencies are often de-termined. Here, simulation of binding data revealed that relative potency values, determined via parallel line analysis (PLA) and half maximal effective concentration (EC50) analysis accurately reflect changes in active concentration only if binding kinetics remain unchanged. Changes in the association rate constant shifted dose response curves, and therefore relative potencies, in the same way as changes in analyte concentration do. However, for interactions characterized by stable binding, changes in the dissociation rate constant did not result in any shift, suggesting that this type of change may go unnoticed in the dose response curve. Thus, EC50 and PLA analyses of dose response curves obtained with an anti-TNF-αan-tibody were complemented with the Biacore functionality for sensorgram comparison analysis, whereby changes in antigen and Fc-receptor binding profiles could be detected. Next, analysis of temperature stressed TNF-α antibody revealed that calibration free concentration analysis (CFCA) data correlated perfectly with relative potency values. Together, these results demonstrate that combinations of SPR based dose response curves, sensorgram comparison and CFCA can be used to strengthen the confidence in relative potency assessments, and suggest that SPR can potentially be used as a surrogate potency assay in the quality control of biotherapeutic medicines.

2018 Vol. 8, No. 2