Validation of high-performance liquid chromatography methods for pharmaceutical analysis. Understanding the differences and similarities between validation requirements of the US Food and Drug Administration,the US Pharmacopeia and the International Conference on Harmonization

One of the most critical factors in developing pharmaceutical drug substances and drug products today is ensuring that the HPLC analytical test methods that are used to analyze the products generate meaningful data. The US Food and Drug Administration (FDA) and United States Pharmacopeia (USP) have each recognized the importance of this to the drug development process and have separately increased validation requirements in recent years. A third source, the International Conference on Harmonization (ICH), has added requirements that, when combined with the previous two sources, have led to three different sets of validation requirements leaving the industry in a state of confusion. This paper is written to clear up the confusion over the validation requirements that are presented by each of these three sources.     

Development and validation of a liquid chromatography-mass spectrometry method for the determination of 4,5-diazafluoren-9-one

A new liquid chromatography-mass spectrometry (LC-MS) method is developed and validated for the identification and determination of novel 4,5-diazafluoren-9-one compound. The method employs a Waters XTerra RP-18 column (150 mmx4.6 mm, i.d. 5 microm) with a mobile phase comprised of a (50:50, v/v) mixture of deionized water containing 0.2% acetic acid (solvent A) and methanol (solvent B) at a flow rate of 1 mL/min, at 35 degrees C. The detection is performed with photodiode-array (PDA) set at 210-400 nm and single quadropole mass spectrometer with electrospray ionization (ESI) positive ion mode. The chromatographic separation is achieved in less than 3 min. The linearity is established over the concentration range of 0.1-0.5 mg/mL (r2=1.000). The mean RSD values for intra- and inter-day precision studies are <2%. The recovery of 4,5-diazafluoren-9-one ranged between 99.84 and 99.97%. The limits of detection and quantitation are determined to be 0.58 and 0.1 mg/mL, respectively.     

A UPLC-DAD-Based Bio-Screening Assay for the Evaluation of the Angiotensin Converting Enzyme Inhibitory Potential of Plant Extracts and Compounds: Pyrroquinazoline Alkaloids from Adhatoda vasica as a Case Study

Angiotensin converting enzyme (ACE) plays a crucial role in regulating blood pressure in the human body. Identification of potential ACE inhibitors from medicinal plants supported the idea of repurposing these medicinal plants against hypertension. A method based on ultra-performance liquid chromatography (UPLC) coupled with a diode array detector (DAD) was used for the rapid screening of plant extracts and purified compounds to determine their ACE inhibitory activity. Hippuryl-histidiyl-leucine (HHL) was used as a substrate, which is converted into hippuric acid (HA) by the action of ACE. A calibration curve of the substrate HHL was developed with the linear regression 0.999. The limits of detection and quantification of this method were found to be 0.134 and 0.4061 mM, respectively. Different parameters of ACE inhibitory assay were optimized, including concentration, incubation time and temperature. The ACE inhibition potential of Adhatoda vasica (methanolic-aqueous extract) and its isolated pyrroquinazoline alkaloids, vasicinol (1), vasicine (2) and vasicinone (3) was evaluated. Compounds 1–3 were characterized by various spectroscopic techniques. The IC₅₀ values of vasicinol (1), vasicine (2) and vasicinone (3) were found to be 6.45, 2.60 and 13.49 mM, respectively. Molecular docking studies of compounds 1–3 were also performed. Among these compounds, vasicinol (1) binds as effectively as captopril, a standard drug of ACE inhibition.     

Exploring the Phytochemicals and Anti-Cancer Potential of the Members of Fabaceae Family: A Comprehensive Review

Cancer is the second-ranked disease and a cause of death for millions of people around the world despite many kinds of available treatments. Phytochemicals are considered a vital source of cancer-inhibiting drugs and utilize specific mechanisms including carcinogen inactivation, the induction of cell cycle arrest, anti-oxidant stress, apoptosis, and regulation of the immune system. Family Fabaceae is the second most diverse family in the plant kingdom, and species of the family are widely distributed across the world. The species of the Fabaceae family are rich in phytochemicals (flavonoids, lectins, saponins, alkaloids, carotenoids, and phenolic acids), which exhibit a variety of health benefits, especially anti-cancer properties; therefore, exploration of the phytochemicals present in various members of this family is crucial. These phytochemicals of the Fabaceae family have not been explored in a better way yet; therefore, this review is an effort to summarize all the possible information related to the phytochemical status of the Fabaceae family and their anti-cancer properties. Moreover, various research gaps have been identified with directions for future research.     

Flow-injection Spectrophotometric Determination of Uric Acid in Urine via Prussian Blue Reaction

A simple and sensitive flow injection(FI) spectrophotometric method was reported for the determination of uric acid based on the reduction of Fe(III)/ferricyanide in the presence of uric acid. The in situ reduced ions reacted with unreduced portion of ferricyanide/Fe(III) to form soluble Prussian blue, which was monitored at an absorption wavelength of 735 nm. The optimized conditions allow a linear calibration graph in a concentration range of 1―100 μmol/L. The relative standard deviation was in a range of 0.5%―2.5%, with a detection limit(3σ blank) of 0.3 μmol/L and a sampling frequency of 60 injection/h was obtained. The effect of common substances present in human physiological fluids on the determination of uric acid was examined. The method was applied to determining uric acid in human urine samples with the recoveries in a range of 96%―105%. The results agree well with those by spectrophotometric reference method at a confidence level of 95%. Spectrophotometric procedures for uric acid determination in clinical samples were reviewed briefly.     

A Note on Classification of Spatially Homogeneous Rotating Space-Times According to Their Teleparallel Killing Vector Fields in Teleparallel Theory of Gravitation

In this paper we classify spatially homogeneous rotating space-timesaccording to their teleparallel Killing vector fields using direct integration technique. It turns out that the dimension of the teleparallel Killing vector fields is 5 or 10. In the case of 10 teleparallel Killing vector fields the space-time becomes Minkowski and all the torsion components are zero. Teleparallel Killing vector fields in this case are exactly the same as in general relativity. In the cases of 5 teleparallel Killing vector fields we get two more conservation laws in the teleparallel theory of gravitation. Here we also discuss some well-known examples of spatially homogeneous rotating space-times according to their teleparallel Killing vector fields.     

Applications of Innovative Non-Thermal Pulsed Electric Field Technology in Developing Safer and Healthier Fruit Juices

The deactivation of degrading and pectinolytic enzymes is crucial in the fruit juice industry. In commercial fruit juice production, a variety of approaches are applied to inactivate degradative enzymes. One of the most extensively utilized traditional procedures for improving the general acceptability of juice is thermal heat treatment. The utilization of a non-thermal pulsed electric field (PEF) as a promising technology for retaining the fresh-like qualities of juice by efficiently inactivating enzymes and bacteria will be discussed in this review. Induced structural alteration provides for energy savings, reduced raw material waste, and the development of new products. PEF alters the α-helix conformation and changes the active site of enzymes. Furthermore, PEF-treated juices restore enzymatic activity during storage due to either partial enzyme inactivation or the presence of PEF-resistant isozymes. The increase in activity sites caused by structural changes causes the enzymes to be hyperactivated. PEF pretreatments or their combination with other nonthermal techniques improve enzyme activation. For endogenous enzyme inactivation, a clean-label hurdle technology based on PEF and mild temperature could be utilized instead of harsh heat treatments. Furthermore, by substituting or combining conventional pasteurization with PEF technology for improved preservation of both fruit and vegetable juices, PEF technology has enormous economic potential. PEF treatment has advantages not only in terms of product quality but also in terms of manufacturing. Extending the shelf life simplifies production planning and broadens the product range significantly. Supermarkets can be served from the warehouse by increasing storage stability. As storage stability improves, set-up and cleaning durations decrease, and flexibility increases, with only minor product adjustments required throughout the manufacturing process.     

Microbial transformation of (+)-adrenosterone

The microbial transformation of (+)-adrenosterone (1) by Cephalosporium aphidicola afforded three metabolites identified as androsta-1,4-diene-3,11,17-trione (2), 17beta-hydroxyandrost-4-ene-3,11-dione (3) and 17beta-hydroxyandrosta-1,4-diene-3,11-dione (4). The fermentation of 1 with Fusarium lini also produced metabolites 2 and 4, while the fermentation with Trichothecium roseum afforded metabolite 3. The structures of transformed products were determined by spectroscopic methods.     

Synthesis of 1‐(p‐Sulphophenyl)‐3‐methyl‐5‐pyrazolone Based Acid Dyes and Their Applications on Leather

A new series of pyrazolone based azo acid dyes (3a–g) has been synthesized starting from 1‐(p‐sul‐phophenyl)‐3‐methyl‐5‐pyrazolone (1). The synthetic methodology included the nitrosation of p‐sulphophenyl methyl pyrazolone followed by reduction, diazotization and coupling with Naphthol AS derivatives (2a–f), in alkaline medium to yield different acid dyes. Multichromic metal complexes of these dyes (5a–f, 6a–f and 7a–f) with 3d transition metals Chromium, Iron and Copper were also synthesized. The structures of all of newly synthesized compounds were confirmed by analytical data and spectroscopic techniques. The synthesized dyes were applied on leather to assess their light fastness, wash fastness and rubbing fastness and were shown to exhibit high values of 4–5 for majority of dyes.