Current Chromatography - Volume 7, Issue 2, 2020

Background: Quercetin (3’,3’,4,5,7-pentahydroxyflavonol), a natural flavonoid found in fruits, vegetables, beverages, and other phytoproducts, exerts multiple health benefits including a reduction in hypoxia-induced oxidative stress, inflammation, lipid peroxidation, allergic disorders, neurodegenerative disorders, and cardiovascular diseases. Objective: Despite knowledge of such therapeutic efficacy of quercetin to human health, there is limited literature available that sheds light on an organ-wise distribution of quercetin. Therefore, the current study was performed to accurately estimate the distribution of quercetin in its supplemented form in different tissues of a mammalian model, i.e., male Sprague Dawley (SD) rats. Materials and Methods: The rats were exposed to different durations (1 h, 3 h, 6 h, and 12 h) of hypoxia in a simulated hypobaric hypoxia chamber, with parameters maintained at 8 % O2 and 282 mm Hg, following which they were sacrificed. Plasma and different tissue samples were duly collected. A high-performance thin layer chromatography (HPTLC) approach was employed for the first time, using our own reported method, along with an optimized sample preparation procedure for quercetin determination. Briefly, the samples were developed in a mobile phase constituted of ethyl acetate, dichloromethane, methanol, formic acid, and glacial acetic acid. Results and Dicussion: Distinct bands of quercetin in resultant HPTLC profiles verified that the amount of quercetin varied among different tissues, with varying durations to hypoxia exposure. Quercetin was substantially retained in vital organs namely, lungs, liver, and heart for relatively longer durations. Conclusion: The present study established HPTLC as an efficient and high throughput tool, leading to a satisfactory evaluation of the amount of quercetin present in various tissue samples under hypoxia.

Background: Motion of the two mutually immiscible liquids in hydrodynamic countercurrent chromatographic systems is speculated based on the observation of their behavior in a closed coiled tube rotating in unit gravity. Materials and Methods: The experiment revealed an up and down pattern of four stages of twophase volume ratio occupied at the head end of the coil according to the rotation speed. These two-phase behaviors are comprehensively explained on the bases of interplay between the unit gravity and centrifugal force generated by rotation of the coil. This theory is successfully extended to explain the two-phase behavior in a coil undergoing the type-I and type-J planetary motions. Results and Discussion: The type-I planetary motion produces the centrifugal force distribution similar to that of slowly rotating coil in unit gravity (Stage I), where both phases competitively move toward the head of the coil. In contrast, the type-J planetary motion displays complex distribution patterns of centrifugal force according to the location of the coil on the holder hence the two-phase motion varies with the ß values. When ß is 0.5 – 0.75, the force pattern simulates that of the rotating coil in unit gravity at 120 rpm (Stage III) where the lighter phase moves toward the head leaving the heavier phase behind. Conclusion: This clearly demonstrates the importance of the proper choice of ß values in highspeed countercurrent chromatography utilizing the type-J planetary motion.

Aims: To develop and validate a new ecological HPLC method for the determination of vancomycin dosage form. Background: Vancomycin is an important antimicrobial. According to the literature, there are many methods that use HPLC, but none of these methods follow the green analytical chemistry principles. Objective: Therefore, a green analytical method to quantify vancomycin in lyophilized powder for injectable solution by HPLC was developed. Materials and Methods: It uses less quantity of toxic solvents, minimizing the costs and optimizing the time of analysis. Water + 0.1% acetic acid and ethanol (85:15, v/v), 0.5 mL min-1, and C18 column (15 cm) at 280 nm were used. Results and Discussion: The method was linear in the range of 40 to 140 μg mL-1, with a correlation coefficient of 0.9998. It was selective when subjected to acid 0.1M, basic 0.01M, oxidative 0.3%, UV light and neutral degradation in a bath of 60 ºC for 8 hours. The precision of the method was proved at intraday (RSD 1.08%), interday (RSD 0.47%) and intermediate levels (RSD 2.35%). It was accurate with a mean recovery of 100.19% and robust when changes were performed in seven parameters of the method and analyzed by the Youden and Steiner test. Conclusion: The method can be applied to routine quality control of vancomycin lyophilized powder for injectable solution as an ecological and sustainable alternative that contemplates the green analytical chemistry and the current pharmaceutical analyses.

Background: Polysaccharide based chiral stationary phases (CSPs) were used to perform enantiomeric separation of Orphenadrine Citrate by Ultra-Fast Liquid Chromatography (UFLC) technique. Trials were conducted using the polar mode, reverse phase mode and normal phase mode. Amylose and Cellulose-based CSPs were used for the same. Materials and Methods: Eight Amylose-based CSPs and four Cellulose-based CSPs were used in the reverse phase mode. Five Amylose-based CSPs and two Cellulose-based CSPs were used in polar mode. The only Cellulose-based CSP used in the normal phase mode could effectively separate Orphenadrine Citrate enantiomers with a good resolution. Results and Discussion: Successful enantioseparation was obtained using Chiralcel OD-H containing Cellulose tris (3, 5- dimethylphenylcarbamate) as a chiral selector and n-hexane: Ethanol: Diethylamine (95: 05: 0.1, v/v/v) as the mobile phase. The developed method was validated in accordance with ICH guidelines (Q2R1). Conclusion: The proposed objectives were successfully accomplished as the developed method could effectively resolve Orphenadrine Citrate enantiomers.

Background: Cinnarizine is used to treat nausea and vomiting accompanied by motion sickness. Dimenhydrinate is used in the treatment of nausea and vomiting and dizziness. The coformulation of the two drugs showed the lowest rate of adverse effects compared to single dimenhydrinate. Objective: A fully validated ultra-performance liquid chromatographic method has been conducted for the simultaneous estimation of cinnarizine (CIN) and dimenhydrinate (DIM). Materials and Methods: The UPLC method used Acquity Column as stationary phase and mobile phase methanol: buffer (pH = 3.5 ± 0.05) and acetonitrile in the ratio of 50: 25: 25 at a flow rate of 0.2 mL/min. Detection was performed by DAD at 260 nm. Results and Discussion: Retention time was 0.71 and 1.12 min for DIM and CIN, respectively. The linearity was found to be 1-40 μg/mL and 2-80 μg/mL for CIN and DIM; respectively. Conclusion: The method was appropriately used for the quantitation of both drugs in pure form, synthetic mixtures and tablet preparation.

Background: Withanolide glycosides in Ashwagandha (Withania somnifera), are important metabolites attributed with widely acclaimed therapeutic potential for which validated methods for quantitative determination are limited. Objective: The primary objective was to develop and validate a Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method for simultaneous quantification of total withanolide glycosides (WG), withanoside IV and withaferin A present in ashwagandha extract.The study also aimed to identify various other constituents present in the extract. Materials and Methods: Aqueous methanol extract (AME) of Ashwagandha was prepared and fractionated into two viz. flavonoid rich fraction (FF) and withanolide rich fraction (WF). RP-HPLC method was developed and validated for the estimation of total WG in ashwagandha extract according to ICH guidelines. Preparative HPLC based purification of major compounds from WF fraction was carried out and constituents were identified using spectroscopic techniques. HPLC chemical profiling of WF before and after acid hydrolysis under controlled conditions was carried out to further confirm the glycosidic compounds. Results and Discussion: The RP-HPLC method gave a precise differentiation of flavonoids, withanolides and WG present in ashwagandha extract. The method demonstrated good reliability and sensitivity, and can be conveniently used for the quantification of total WG, withanoside IV and withaferin A present in ashwagandha extracts. According to this method, a purified fraction (WF) prepared from roots and leaves of Ashwagandha comprise 35% of total WG, 3.27% of withanoside IV and 2.40% of Withaferin A. The method was also applied to different products prepared from Ashwagandha with total withanolide glycosides ranged from 1.5% to 60%, and the results were found to be reproducible. Identification of the individual chemical constituents as well as the acid hydrolytic pattern of the extract further supported the reliability of the developed method for the quantitative determination of total WG. This study also reported a new withanolide glycoside named, cilistol V-6'-O-glucoside (Aswanoside) along with some other known withanolide glycosides. Conclusion: A Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the quantitative determination of total WG, withanoside IV and withaferin A present in ashwagandha extract according to ICH guidelines. This study also reported a new withanolide glycoside named, cilistol V-6'-O-glucoside (Aswanoside) along with some other known WG.

Background: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.