Journal of Pharmaceutical Analysis

2018, 8(5)

Abstract(76) PDF(0)

Abstract:

3D biofabrication of vascular networks for tissue regeneration: A report on recent advances

M.D. Sarker, Saman Naghieh, N.K. Sharma, Xiongbiao Chen

2018, 8(5): 277-296.

Abstract(212) PDF(2)

Abstract:
Rapid progress in tissue engineering research in past decades has opened up vast possibilities to tackle the challenges of generating tissues or organs that mimic native structures. The success of tissue en-gineered constructs largely depends on the incorporation of a stable vascular network that eventually anastomoses with the host vasculature to support the various biological functions of embedded cells. In recent years, significant progress has been achieved with respect to extrusion, laser, micro-molding, and electrospinning-based techniques that allow the fabrication of any geometry in a layer-by-layer fashion. Moreover, decellularized matrix, self-assembled structures, and cell sheets have been explored to replace the biopolymers needed for scaffold fabrication. While the techniques have evolved to create specific tissues or organs with outstanding geometric precision, formation of interconnected, functional, and perfused vascular networks remains a challenge. This article briefly reviews recent progress in 3D fab-rication approaches used to fabricate vascular networks with incorporated cells, angiogenic factors, proteins, and/or peptides. The influence of the fabricated network on blood vessel formation, and the various features, merits, and shortcomings of the various fabrication techniques are discussed and summarized.

A rapid reporter assay for recombinant human brain natriuretic peptide (rhBNP) by GloSensor technology

Lei Yu, Xinchang Shi, Chunmei Han, Chunming Rao, Junzhi Wang

2018, 8(5): 297-301.

Abstract(113) PDF(3)

Abstract:
Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide (rhBNP). In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGlo-G1 was constructed by transfecting pGloSensor?40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGlo-G1 showed high precision with intra-assay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R2of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay (paired t test, p=0.630). All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP.

Solution pH jump during antibody and Fc-fusion protein thaw leads to increased aggregation

Kevin P. Kent, Chad E. Schroeder, Chandana Sharma

2018, 8(5): 302-306.

Abstract(133) PDF(2)

Abstract:
Freeze-thaw cycles impact the amount of aggregation observed in antibodies and Fc-fusion proteins. Various formulation strategies are used to mitigate the amount of aggregation that occurs upon putting a protein solution through a freeze-thaw cycle. Additionally, low pH solutions cause native antibodies to unfold, which are prone to aggregate upon pH neutralization. There is great interest in the mechanism that causes therapeutic proteins to aggregate since aggregate species can cause unwanted im-munogenicity in patients. Herein, an increase in aggregation is reported when the pH is adjusted from pH 3 up to a pH ranging from pH 4 to pH 7 during the thaw process of a frozen antibody solution. Raising the pH during the thaw process caused a significant increase in the percent aggregation observed. Two antibodies and one Fc-fusion protein were evaluated during the pH jump thaw process and similar ef-fects were observed. The results provide a new tool to study the kinetics of therapeutic protein ag-gregation upon pH increase.

Primula vulgaris extract induces cell cycle arrest and apoptosis in human cervix cancer cells

Selim Demir, Ibrahim Turan, Rezzan Aliyazicioglu, Serap Ozer Yaman, Yuksel Aliyazicioglu

2018, 8(5): 307-311.

Abstract(197) PDF(1)

Abstract:
Primula vulgaris belongs to the genus Primula, members of which are frequently used in folk medicine. Various studies have investigated the cytotoxic effect of different Primula species, but there have been limited studies on the cytotoxic effect of P. vulgaris. The aim of this study was to investigate the cytotoxic effects, and possible mechanisms involved, of P. vulgaris flower extract on human cervical cancer (HeLa) cells. The cytotoxic effect of the extract on HeLa cells was revealed using the MTT assay. Mechanisms involved in the extract's cytotoxic effect were then investigated in terms of apoptosis, mitochondrial membrane potential, and the cell cycle, using fluorometric methods. P. vulgaris flower extract exhibited selective cytotoxic effects against HeLa cells by arresting their cell cycle at the S phase, and inducing the number of apoptotic cells compared to normal fibroblast cells by reducing mitochondrial membrane potential in a concentration-dependent manner. This is the first study to reveal the antiproliferative effect of P. vulgaris flower extract. Further studies are now needed to identify the cytotoxic molecules in the extract and their mechanisms.

Discoursing on Soxhlet extraction of ginseng using association analysis and scanning electron microscopy

Yuan Yue, Zi-Dong Qiu, Xian-You Qu, Ai-Ping Deng, Yuan Yuan, Lu-Qi Huang, Chang-Jiang-Sheng Lai

2018, 8(5): 312-317.

Abstract(73) PDF(0)

Abstract:
Soxhlet extraction is a common method of sample preparation. However, there has been no discussion about the efficiency of Soxhlet extraction from different batches and the factors that cause content fluctuation. In this study, Panax ginseng was selected as a model sample. Soxhlet extraction by means of a water bath, which has always been neglected, was identified as a novel key factor in the poor repeat-ability in different batches of Soxhlet extraction, as it can affect the siphon times and reflux time, which have been positively correlated with the ginsenoside contents. By substituting round bottom flasks in the same column, the relative standard deviation of the most fluctuated compound, ginsenoside Rb1, was decreased from 24.6% to 5.02%. Scanning electron microscopy analysis confirmed that the breakdown of the surface of the ginseng powder in the Soxhlet extraction led to a better dissolution of ginsenosides, indicating that chloroform may promote the extraction of ginsenosides by disrupting the cell structure. Moreover, 70% methanol was regarded as the better solvent for extracting the ginsenosides. Overall, this work offers a practical and effective protocol for improving the accuracy and repeatability of Soxhlet extraction methodology for ginsenosides and other analytes.

Molecular docking studies of human MCT8 protein with soy isoflavones in Allan-Herndon-Dudley syndrome (AHDS)

Divya Shaji

2018, 8(5): 318-323.

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Abstract:
Monocarboxylate transporter-8 (MCT8) is a specific thyroid hormone transporter, essential for the uptake of thyroid hormone into target tissues. Mutations in the MCT8 gene have been identified as the cause of Allan-Herndon-Dudley syndrome (AHDS). It has been reported that soy isoflavones influence thyroid hormone system and can interact with thyroid hormone transporter proteins. Therefore, the present study aimed to find out whether soy isoflavones (genistein, daidzein and glycitein) can be used as a natural inhibitor to target MCT8 in AHDS. Docking studies were performed for soy isoflavones in order to evaluate their binding affinity to MCT8 protein using AutoDock4 (version 4.2.6) and AutoDock Vina. After docking, the ligands were ranked according to their binding energy and the best lead compound was selected based on the least binding energy. The docking results indicated that daidzein possesses the lowest binding energy against MCT8. Moreover, it was found that the residues PRO-338, HIS-341, and GLU-348 were involved in hydrogen bond interactions with genistein and daidzein. This study suggests that daidzein is a promising natural inhibitor to target MCT8 in AHDS.

Mass spectrometry detection of basic drugs in fast chiral analyses with vancomycin stationary phases

Hongyue Guo, M. Farooq Wahab, Alain Berthod, Daniel W. Armstrong

2018, 8(5): 324-332.

Abstract(105) PDF(3)

Abstract:
Current trends in chiral analysis of pharmaceutical drugs are focused on faster separations and higher separation efficiencies. Core-shell or superficially porous particles (SPP) based chiral stationary phases (CSPs) provide reduced analysis times while maintaining high column efficiencies and sensitivity. In this study, mobile phase conditions suitable for chiral analyses with electrospray ionization LC-MS were systematically investigated using vancomycin as a representative CSP. The performance of a 2.7 μm SPP based vancomycin CSP (SPP-V) 10 cm × 0.21 cm column was compared to that of a corresponding 5 μm fully porous particles based analogue column. The results demonstrated that the SPP-V column provides higher efficiencies, 2–5 time greater sensitivity and shorter analysis time for a set of 22 basic pharma-ceutical drugs. The SPP-V was successfully applied for the analysis of the degradation products of racemic citalopram whose enantiomers could be selectively identified by MS.

Simultaneous determination of indapamide, perindopril and perindoprilat in human plasma or whole blood by UPLC-MS/MS and its pharmacokinetic application

Yi Tao, Sheng Wang, Lei Wang, Min Song, Taijun Hang

2018, 8(5): 333-340.

Abstract(132) PDF(0)

Abstract:
Simple and sensitive methods were developed for the determination of indapamide, perindopril and its active metabolite perindoprilat in human plasma or whole blood by hyphenated ultra-performance li-quid chromatography-mass spectrometry (UPLC-MS/MS). Indapamide-d3, perindopril-d4 and perindo-prilat-d4 were used as the internal standards. The separation was performed on a Thermo BDS Hypersil C18column (4.6 mm × 100 mm, 2.4 μm) for indapamide and perindopril simultaneously following a protein precipitation pretreatment of the biosamples. The separation of perindoprilat was achieved in-dependently on a phenomenex PFP column (4.6 mm × 150 mm, 5 μm). All the analytes were quantitated with positive electrospray ionization and multiple reactions monitoring mode. The assay exhibited a linear range of 1–250 ng/mL for indapamide, 0.4–100 ng/mL for perindopril and 0.2–20 ng/mL for peri-ndoprilat. The methods were fully validated to meet the requirements for bioassay in accuracy, precision, recovery, reproducibility, stabilities and matrix effects, and successfully applied to the pharmacokinetic study of perindopril tert-butylamine/indapamide compound tablets in Chinese healthy volunteers and the comparative pharmacokinetic study between plasma and whole blood.

Determination of asenapine in presence of its inactive metabolites in human plasma by LC-MS/MS

Nirav P. Patel, Mallika Sanyal, Naveen Sharma, Dinesh S. Patel, Pranav S. Shrivastav, Bhavin N. Patel

2018, 8(5): 341-347.

Abstract(91) PDF(2)

Abstract:
A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been described for the determination of asenapine (ASE) in presence of its inactive metabolites N-desmethyl asenapine (DMA) and asenapine-N-glucuronide (ASG). ASE, and ASE 13C-d3, used as in-ternal standard (IS), were extracted from 300 μL human plasma by a simple and precise liquid-liquid extraction procedure using methyl tert-butyl ether. Baseline separation of ASE from its inactive meta-bolites was achieved on Chromolith Performance RP8e(100 mm × 4.6 mm) column using acetonitrile-5.0 mM ammonium acetate-10% formic acid (90:10:0.1, v/v/v) within 4.5 min. Quantitation of ASE was done on a triple quadrupole mass spectrometer equipped with electrospray ionization in the positive mode. The protonated precursor to product ion transitions monitored for ASE and ASE 13C-d3 were m/z 286.1 → 166.0 and m/z 290.0 → 166.1, respectively. The limit of detection (LOD) and limit of quantitation (LOQ) of the method were 0.0025 ng/mL and 0.050 ng/mL respectively in a linear con-centration range of 0.050–20.0 ng/mL for ASE. The intra-batch and inter-batch precision (% CV) and mean relative recovery across quality control levels were ≤5.8% and 87.3%, respectively. Matrix effect, eval-uated as IS-normalized matrix factor, ranged from 1.03 to 1.05. The stability of ASE under different storage conditions was ascertained in presence of the metabolites. The developed method is much simpler, matrix free, rapid and economical compared to the existing methods. The method was suc-cessfully used for a bioequivalence study of asenapine in healthy Indian subjects for the first time.

2018 Vol. 8, No. 5