Therapeutic drug monitoring of six contraindicated/co-administered drugs by simple and green RP-HPLC-PDA; application to spiked human plasma.

Therapeutic drug monitoring is an important clinical testing of the drugs to monitor their concentrations in plasma in order to guarantee their optimal impact, and to avoid any side effects resulting from drug-drug interactions. A green reversed-phase high-performance liquid chromatographic method using a photodiode array detector (RP-HPLC-PDA) was developed for the simultaneous determination of three carbapenem antibiotics (Imipenem, ertapenem, and meropenem) with the co-formulated drug (cilastatin) and contraindicated drugs (probenecid and warfarin) in spiked human plasma. The separation was achieved at 25 °C using a gradient elution of a mixture of mobile phase A: methanol and mobile phase B: phosphate buffer (pH 3.0). The photodiode array detector was adjusted at 220 nm. Bioanalytical method validation was carried out as per the FDA guidelines, and the method showed good linearity ranges for the six drugs that included their Cmax levels along with low limits of quantification. Based on the results, the method was found to be accurate and precise; with high % recovery and good % RSD, respectively. The method was successfully applied to spiked human plasma, signifying a good potential to be implemented in future TDM studies of these drugs when co-administered together.

Stability-indicating UPLC assay coupled with mass spectrometry for the analysis of vilanterol degradation products in human urine.

Vilanterol is a once-daily dose inhaler prescribed for asthma and chronic obstructive pulmonary disease. This study involved an investigation of vilanterol stability under acidic, basic, oxidative, thermal, and photolytic stress conditions. UPLC method was developed and validated for the analysis of vilanterol with its degradants. The drug was stable under photolytic and thermal stress conditions and degraded under acidic, basic, and oxidative stress conditions. Degradation kinetics was performed for acidic, basic and oxidative stress conditions. Kinetics parameters, K, half-life time (t1/2) and shelf-life time (t90) were assessed, and the degradation followed first order reaction. The method was linear from 0.10 to 100.00 µg mL-1 with accuracy, inter-day and intra-day precision from 99.45 to 100.02%, 0.391-0.694 and 0.041-0.345, respectively. Mass spectrometry was employed to elucidate the structure of the degradants, and the results revealed that certain degradation products were comparable to vilanterol metabolites. The World Anti-Doping Agency has prohibited the presence of vilanterol and its metabolites in athletes' urine except for exercise bronchoconstriction with limited dose. So, quantification of vilanterol in the presence of its degradants was performed in human urine. The results revealed that the method was linear in range of 1.00 to 100.00 µg mL-1. Samples collection and experimental protocol was performed according to the guidelines of the Research Ethics Committee of the Faculty of Pharmacy, the British University in Egypt with approval No. CH-2305.

Design of Experiment-Based Green UPLC-DAD Method for the Simultaneous Determination of Indacaterol, Glycopyrronium and Mometasone in their Combined Dosage Form and Spiked Human Plasma.

Indacaterol, is an ultra-long-acting ß2 agonist, glycopyrronium is a long-acting muscarinic-antagonist and mometasone is a synthetic corticosteroid. They were used recently in combination for the treatment of severe asthma symptoms and chronic obstructive pulmonary disease. In this work, it was the first time to develop a green and environment friendly ultra-performance liquid chromatographic method using design expert program for the analysis of the three drugs in their combined dosage form. Also, the method was bioanalytically validated for the analysis of the three drugs in spiked human plasma samples. The method was linear in range from 0.50 to 100.0 µg mL-1 for indacaterol and mometasone and from 1.0 to 150.0 µg mL-1 for glycopyrronium. It showed high accuracy where, the % recovery for indacaterol, glycopyrronium and mometasone in plasma were ranged from 94.27 to 97.86%, 96.43 to 98.75% and 96.86 to 98.43%, respectively. Also, it was precise where, the % relative standard deviation for the inter-day precision was ranged from 2.571 to 3.484%, 3.180 to 4.123% and 3.150 to 3.984% and the intra-day precision was ranged from 2.351 to 3.125%, 2.512 to 3.544% and 2.961 to 3.983% for indacaterol, glycopyrronium and mometasone, respectively. The limit of detection and the limit of quantification for indacaterol and mometasone were 0.03 and 0.10 µg mL-1 while for glycopyrronium, they were 0.16 and 0.50 µg mL-1.

Management of recurrent aphthous ulcers exploiting polymer-based Muco-adhesive sponges: in-vitro and in-vivo evaluation.

Recurrent aphthous ulcer (RAU) is a well-known painful, inflammatory disease with uncertain etiology for which local symptomatic therapy is only available. The aim of this study was to formulate and characterize muco-adhesive sponges containing a mixture of tenoxicam and miconazole nitrate to manage pain, inflammation and avoid candida infection that may accompany RAU due to poor oral hygiene. Two polymers at different concentrations were used to prepare sponges applying simple freeze-drying. Medicated chitosan (2%) sponges (mC2) showed acceptable physical appearance, surface pH (6.3 ± 0.042), porosity (25.7% ± 1.8), swelling index (5.7 ± 0.11), in-vivo and ex-vivo muco-adhesion time (115 min.±0.813 and 155 min.±1.537, respectively), ex-vivo muco-adhesion force (0.09 N ± 0.002) and scanning electron microscope (SEM) images. For concurrent clear-cut determination of tenoxicam and miconazole nitrate from mC2, a new UPLC method was developed and validated. mC2 sponges exhibited superior in-vitro drug release profiles where ∼100% of tenoxicam released within 5 min for fast pain relief with a more prolonged miconazole nitrate release. Furthermore, in-vivo animal study revealed that mC2 caused a significant decrease in the acetic acid-induced ulcer size in rats after 6 days of treatment (p < .0001) compared to negative and positive controls. Additionally, histopathological examination showed faster healing with complete restoration of the normal oral histology in rats. The present study concludes that chitosan sponge loaded with a combination of tenoxicam and miconazole nitrate could improve healing of RAU cases.

A Validated Ultra-Performance Liquid Chromatographic Method for the Simultaneous Determination of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate in Their Combined Dosage Form and Spiked Human Plasma Samples.

Nadifloxacin, mometasone furoate and miconazole nitrate are formulated together as a topical antifungal dosage form. In this work, a reversed-phase ultra-performance liquid chromatographic method coupled with a diode array detector (RP-UPLC-DAD) was developed and validated to determine nadifloxacin, mometasone furoate and miconazole nitrate simultaneously in their bulk powder, in pharmaceutical preparation and in spiked human plasma samples. Separation was achieved on an ACQUITY UPLC C18 column of 2.2 µm particle size (2.1 × 100 mm) via isocratic elution using a mobile phase consisting of methanol, acetonitrile and water with ratio (50:20:30; v/v/v) and 0.1 g ammonium acetate, then pH was adjusted to (7.00) using acetic acid, flow rate 0.6 mL/min, temperature 30°C and UV detection at 220 nm. The method is linear in a range from 5 to 400 µg/mL for both nadifloxacin and miconazole nitrate and from 20 to 500 µg/mL for mometasone furoate. The method was validated according to the ICH guidelines then applied successfully to determine the mentioned drugs in their pharmaceutical preparation and spiked human plasma samples. For plasma samples, the results showed that the method can determine nadifloxacin, mometasone furoate and miconazole nitrate in human plasma samples with high accuracy and precision.

Author Correction: Verbascoside: Identification, Quantification, and Potential Sensitization of Colorectal Cancer Cells to 5-FU by Targeting PI3K/AKT Pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Validated Reverse Phase-Ultra-Performance Liquid Chromatography Method for the Determination of Gemifloxacin Mesylate in Bulk and its Pharmaceutical Preparation.

OBJECTIVES: Gemifloxacin Mesylate is a fourth generation fluoroquinolone antibacterial agent. A simple, accurate, and precise reversed phase (RP)-ultra performance liquid chromatography (UPLC) method was developed and validated for short time analysis of Gemifloxacin Mesylate in its bulk and pharmaceutical preparation. MATERIALS AND METHODS: The optimum separation was achieved at 0.5±0.03 min using an AcclaimTM RSLC 120 C18 column 2.2 µm (2.1×100 mm) at 30°C by isocratic mobile phase at pH 3.0 composed of acetonitrile:phosphate buffer (25 mM) in a ratio of 75:25 (v/v). The column effluents were monitored at 276 nm using a photodiode array detector at a flow rate of 0.5 mL/min. The method was validated according to International Conference on Harmonization guidelines. RESULTS: The linearity of the calibration curve ranged from 0.5 µg/mL to 10 µg/mL and the square of the regression coefficient (r2) was 0.9991. The % relative standard deviation (RSD) of inter-day precision ranged from 0.081% to 1.233%, while for intra-day it ranged from 0.364% to 1.018%. The method was accurate with % recovery ranging from 93.71% to 100.29% and % RSD ranging from 1.054 to 2.722. The limit of detection and the limit of quantification were 0.066 and 0.2 µg/mL, respectively. CONCLUSION: The validated method proved its ability for the assay of Gemifloxacin Mesylate in its bulk and dosage form in a short time (less than 1 min). To the best of our knowledge, this is the first RP-UPLC method for the determination of Gemifloxacin Mesylate.

Verbascoside: Identification, Quantification, and Potential Sensitization of Colorectal Cancer Cells to 5-FU by Targeting PI3K/AKT Pathway.

Colorectal cancer (CRC) is the third most common cancer mortality worldwide. Although, 5-Fluorouracil (5-FU)-based chemotherapeutic regimens remain the mainstay for treatment of CRC, intrinsic and acquired resistance to 5-FU is the main reason for treatment failure and relapse. Adjunct or add-on therapy, therefore, should be thought of to enhance responsiveness to 5-FU. Verbascoside (VER) is a phenylethanoid glycoside ingredient present in many Plantago species and was widely used in traditional medicine. VER showed antiproliferative effects in many cancer types including CRC. In the present study, VER in Plantago seeds was identified using UPLC-MS/MS and quantified using newly developed and validated UPLC-DAD followed by investigating its potential sensitization of CRC cells to 5-FU in vitro. The potential impact on PI3K/AKT pathway was also investigated. A synergistic cytotoxic interaction between 5-FU and VER besides G1 cell cycle arrest were detected. Enhanced apoptosis mainly by affecting Bax and Bcl-2 and to a lesser extent Bcl-xL and p53 was also observed. Additionally, 5-FU combined to VER was capable of significantly reducing PI3K and p-AKT/total AKT ratio. Overall, these results suggest a potential role of VER as an adjuvant treatment to decrease the resistance of CRC cells to 5-FU possibly by targeting the PI3K/AKT pathway.

Enantiomeric separation of underivatized amino acids: predictability of chiral recognition on ristocetin a chiral stationary phase.

The present work aimed to investigate the predictability of the chromatographic behavior for the separation of underivatized amino acids on ristocetin A, known as Chirobiotic R, using a DryLab high-performance liquid chromatography (HPLC) method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. After implementing the basic runs, and judging the predictability via the computed resolution map, it can be deduced that the chiral recognition mechanisms tend towards a hydrophilic interaction chromatography rather than the reversed phase mode, which limits the ability of DryLab software to predict separations on Chirobiotic R.

Predictability of enantiomeric chromatographic behavior on various chiral stationary phases using typical reversed phase modeling software.

Pharmaceutical companies worldwide tend to apply chiral chromatographic separation techniques in their mass production strategy rather than asymmetric synthesis. The present work aims to investigate the predictability of chromatographic behavior of enantiomers using DryLab HPLC method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. Three different types of chiral stationary phases were tested for predictability: macrocyclic antibiotics-based columns (Chirobiotic V and T), polysaccharide-based chiral column (Chiralpak AD-RH), and protein-based chiral column (Ultron ES-OVM). Preliminary basic runs were implemented, then exported to DryLab after peak tracking was accomplished. Prediction of the effect of % organic mobile phase on separation was possible for separations on Chirobiotic V for several probes: racemic propranolol with 97.80% accuracy; mixture of racemates of propranolol and terbutaline sulphate, as well as, racemates of propranolol and salbutamol sulphate with average 90.46% accuracy for the effect of percent organic mobile phase and average 98.39% for the effect of pH; and racemic warfarin with 93.45% accuracy for the effect of percent organic mobile phase and average 99.64% for the effect of pH. It can be concluded that Chirobiotic V reversed phase retention mechanism follows the solvophobic theory.

Determination of the design space of the HPLC analysis of water-soluble vitamins.

Analysis of water-soluble vitamins has been tremendously approached through the last decades. A multitude of HPLC methods have been reported with a variety of advantages/shortcomings, yet, the design space of HPLC analysis of these vitamins was not defined in any of these reports. As per the food and drug administration (FDA), implementing the quality by design approach for the analysis of commercially available mixtures is hypothesized to enhance the pharmaceutical industry via facilitating the process of analytical method development and approval. This work illustrates a multifactorial optimization of three measured plus seven calculated influential HPLC parameters on the analysis of a mixture containing seven common water-soluble vitamins (B1, B2, B6, B12, C, PABA, and PP). These three measured parameters are gradient time, temperature, and ternary eluent composition (B1/B2) and the seven calculated parameters are flow rate, column length, column internal diameter, dwell volume, extracolumn volume, %B (start), and %B (end). The design is based on 12 experiments in which, examining of the multifactorial effects of these 3 + 7 parameters on the critical resolution and selectivity, was carried out by systematical variation of all these parameters simultaneously. The 12 basic runs were based on two different gradient time each at two different temperatures, repeated at three different ternary eluent compositions (methanol or acetonitrile or a mixture of both). Multidimensional robust regions of high critical R(s) were defined and graphically verified. The optimum method was selected based on the best resolution separation in the shortest run time for a synthetic mixture, followed by application on two pharmaceutical preparations available in the market. The predicted retention times of all peaks were found to be in good match with the virtual ones. In conclusion, the presented report offers an accurate determination of the design space for critical resolution in the analysis of water-soluble vitamins by HPLC, which would help the regulatory authorities to judge the validity of presented analytical methods for approval.