Chromatographic Method Research Articles

A comparative metabolomic investigation of different sections of Sicilian Citrus x limon (L.) Osbeck, characterization of bioactive metabolites, and evaluation of in vivo toxicity on zebrafish embryo.

Citrus fruits are a diverse and economically important group of fruit crops known for their distinctive flavors and high nutritional value. Their cultivation and consumption contribute significantly to the global agricultural economy and offer a wide range of health benefits. Among the genetic diversity of citrus species, Citrus x limon (L.) Osbeck is particularly relevant due to its chemical composition and potential health benefits. Two cultivars from the Sicily region (southern Italy) were compared for their phenolic content and preliminary antioxidant activity to select the distinctive extract with potential biological activity. A detailed characterization revealed the occurrence of phenolics, coumarins, and flavonoids. The quantification of metabolites contained in the selected extract was performed by an ultrahigh-performance liquid chromatographic method coupled with an ultraviolet detector. Different concentrations were tested in vivo through the fish embryo acute toxicity test, and the 50% lethal dose of 107,833µgmL-1 was calculated. Finally, the effect of the extract on hatching was evaluated, and a dose-dependent relationship with the accelerated hatching rate was reported, suggesting a Femminello Zagara Bianca green peel upregulating effect on the hatching enzymes. PRACTICAL APPLICATION: Citrus fruits and their products continue to be one of the natural food sources with the highest waste output. In this study, we demonstrate how food industry waste, particularly lemon peel, is rich in bioactive compounds with anti-inflammatory and antioxidant properties that may be used in the nutraceuticals industry.

Two chromatographic methods for determination of cytarabine and dexamethazone; two co-administered drugs for the treatment of leukemia: green analytical perspective

Two sensitive, straightforward and repeatable chromatographic techniques were developed for the determination of Cytarabine HCl and Dexamethazone in their pure form and spiked human plasma without prior separation. The drugs are used co-administered for the treatment of Leukemia, a certain type of blood cancer. Method (A) is an isocratic chromatographic HPLC method; separation was accomplished on C18 column using the eluting mixture of 6.9 g/L Monobasic Sodium Phosphate pH 3: methanol (70:30, v/v) and detection was at 275 nm. Concentrations were in the range of 0.2–15 μg/mL for both CYT and DEX. Method (B) is a HPTLC method in which separation was attained on HPTLC F254 plates using methanol: ethyl acetate: ammonia, (7.8:2:0.2, by volume) as eluting solvents and detection was at 275 nm. Concentrations were in the range of 0.1–4 μg/band for both CYT and DEX. The parameters for system suitability testing were evaluated to determine the effectiveness of the developed chromatographic procedures in terms of performance. The recently developed techniques were applied for the determination of the drugs under investigation in spiked human plasma. Validation parameters were examined in accordance with US-FDA criteria. All results were found to be within the acceptable ranges. To evaluate the greenness characters of the proposed methods to the environment; three greenness assessment tools including eco-scale assessments (ESA), green analytical procedure index (GAPI), and Analytical Greenness calculator (AGREE) were used. Acceptable and satisfying results that demonstrated the greenness characteristics of the suggested methods were attained.

In-depth Exploration of the Pharmacological, Analytical, and Pharmaceutical Attributes of Irbesartan

Irbesartan, an angiotensin II receptor blocker (ARB), has gained prominence in the management of hypertension and diabetic nephropathy due to its potent antihypertensive and Reno protective effects. This review provides a comprehensive overview of the pharmacological, analytical, and pharmaceutical characteristics of Irbesartan. Pharmacologically, Irbesartan selectively antagonizes the angiotensin II type 1 (AT1) receptors, leading to vasodilation, reduced aldosterone secretion, and consequently blood pressure lowering. The drug also exhibits favourable effects on renal function, making it a cornerstone therapy for diabetic nephropathy. Analytically, various chromatographic methods including high-performance liquid chromatography (HPLC) and Ultra-performance liquid chromatography (UPLC) have been developed and validated for the quantification of Irbesartan in biological samples and pharmaceutical formulations, owing to its importance in pharmacokinetic studies and quality control processes. Moreover, spectroscopic techniques such as UV-visible spectrophotometry have been utilized for Irbesartan determination due to their simplicity and cost-effectiveness. Pharmaceutical considerations encompass formulation strategies, stability studies, and bioavailability enhancement techniques aimed at ensuring the efficacy and safety of Irbesartan formulations. The regulatory approval of Irbesartan-containing products by major health authorities underscores its clinical significance and quality assurance.

Validated chromatographic methods for determination of teriflunomide and investigation of its intrinsic stability

Two rapid, precise, and sensitive stability-indicating high performance chromatographic methods for the measurement of Teriflunomide in its degradation products’ existence were developed. These were RP-HPLC and HPTLC using UV detector. HPLC separation was accomplished utilizing Thermo BDS hypercil C18 column (250 × 4.6 mm, 5 μm) and acetonitrile: 0.03 M potassium dihydrogen phosphate: triethylamine (50:50:0.1%, by volume) as mobile phase at flow rate of 1mL/min. The separated peaks were detected at 250.0 nm. The densitometric approach was conducted utilizing HPTLC 60 F254 silica gel plates, and a developing system of benzene: ethanol: acetic acid (7.5:1:0.25, by volume) and detection was done at 250.0 nm. The developed approaches were evaluated regarding the International Conference on Harmonization (ICH) instructions. The calibration curves of both techniques were constructed with linearity ranges of (5-100) µg/mL and (2–10) µg /band, for HPLC and densitometric determination, consecutively. Teriflunomide was exposed to base and acid hydrolysis, oxidation using H2O2 and finally, thermal degradation as stated in ICH guidelines. The degradation product structures’ elucidation was achieved through LC-MS.

Chromatographic Methods for the Determination of Glyphosate in Cereals together with a Discussion of Its Occurrence, Accumulation, Fate, Degradation, and Regulatory Status

The European Union’s recent decision to renew the authorization for the use of glyphosate until 15 December 2033 has stimulated scientific discussion all around the world regarding its toxicity or otherwise for humans. Glyphosate is a chemical of which millions of tons have been used in the last 50 years worldwide to dry out weeds in cultivated fields and greenhouses and on roadsides. Concern has been raised in many areas about its possible presence in the food chain and its consequent adverse effects on health. Both aspects that argue in favor of toxicity and those that instead may indicate limited toxicity of glyphosate are discussed here. The widespread debate that has been generated requires further investigations and field measurements to understand glyphosate’s fate once dispersed in the environment and its concentration in the food chain. Hence, there is a need for validated analytical methods that are available to analysts in the field. In the present review, methods for the analytical determination of glyphosate and its main metabolite, AMPA, are discussed, with a specific focus on chromatographic techniques applied to cereal products. The experimental procedures are explained in detail, including the cleanup, derivatization, and instrumental conditions, to give the laboratories involved enough information to proceed with the implementation of this line of analysis. The prevalent chromatographic methods used are LC-MS/MS, GC-MS/SIM, and GC-MS/MS, but sufficient indications are also given to those laboratories that wish to use the better performing high-resolution MS or the simpler HPLC-FLD, HPLC-UV, GC-NPD, and GC-FPD techniques for screening purposes. The concentrations of glyphosate from the literature measured in wheat, corn, barley, rye, oats, soybean, and cereal-based foods are reported, together with its regulatory status in various parts of the world and its accumulation mechanism. As for its accumulation in cereals, the available data show that glyphosate tends to accumulate more in wholemeal flours than in refined ones, that its concentration in the product strictly depends on the treatment period (the closer it is to the time of harvesting, the higher the concentration), and that in cold climates, the herbicide tends to persist in the soil for a long time.

Accurate and rapid quantification of tea saponins using a thin-layer chromatography (TLC) method based on hemolysis and machine vision

Tea saponins are increasingly used in the food industries due to their biological activities. Current methods for the quantification of tea saponins are either inaccurate or time consuming and costly. In this study, a quantitative thin-layer chromatographic (TLC) method using machine vision and based on the hemolytic activity (HA) of tea saponins was developed. The optimized method consisted of ethanol extraction, sample loading, separation, color development, and smartphone-machine vision quantification. The new HA-TLC method was compared with UV spectrophotometric and liquid chromatographic methods, and achieved higher precision (RSD% = 0.75%) and accuracy (96.4%-106.2% recovery), with a limit of detection of 0.285 μg and limit of quantification of 0.95 μg. The method enables simultaneous qualitative and quantitative determination of saponins from different parts of tea plant extracts with high specificity, yet rapid and inexpensive, with high throughput and low solvent consumption, providing new options for saponin analysis in the food industry.

A novel derivatization method for the determination of ethyl carbamate in spirits by liquid chromatography with spectrophotometric detection

A novel derivatization method for the ethyl carbamate (EC) determination is introduced, representing a significantly more promising approach to the only previously described and time-demanding EC derivatization with the expensive 9–xanthydrol agent. The presented derivatization procedure utilizes a 4–(dimethylamino)benzaldehyde (p-DMAB) reagent to form a derivative with a recovery of 99.4 %. The quantitative EC reaction with p-DMAB proceeds within five minutes without any considerable by-products and the resulting derivative was confirmed by mass spectrometry. The derivatization procedure led to the development of a new chromatographic method with spectrophotometric detection at 420 nm for EC determination in spirits. The achieved quantification limit of 582.5 µg L–1 complies with the recommendation of the European Commission from 2010, demonstrating its practical use in food safety control. Within the optimization of detection, the electrochemical behaviour was included to reveal the possibility of amperometric detection. Compared to the method used so far, the proposed approach significantly reduces the cost and time required to screen this potential carcinogen and could lead to the manufacture of commercial colorimetric assay kits detecting hazardous EC concentration levels.

Linear solvent strength model on porous graphitic carbon stationary phase using high temperature liquid chromatographic method for Allopurinol related substances analysis

Linear solvent strength model on porous graphitic carbon stationary phase using high temperature liquid chromatographic method for Allopurinol related substances analysis

Research of saccharides and related biocomplexes: A review with recent techniques and applications.

Saccharides and biocompounds as saccharide (sugar) complexes have various roles and biological functions in living organisms due to modifications via nucleophilic substitution, polymerization, and complex formation reactions. Mostly, mono-, di-, oligo-, and polysaccharides are stabilized to inactive glycosides, which are formed in metabolic pathways. Natural saccharides are important in food and environmental monitoring. Glycosides with various functionalities are significant in clinical and medical research. Saccharides are often studied with the chromatographic methods of hydrophilic interaction liquid chromatography and anion exchange chromatograpy, but also with capillary electrophoresis and mass spectrometry with their on-line coupling systems. Sample preparation is important in the identification of saccharide compounds. The cases discussed here focus on bioscience, clinical, and food applications.

Chromatographic purification of C-Phycocyanin from Spirulina platensis: assessing antioxidant activity and stability.

Efficient separation and purification of proteins like C-phycocyanin (C-PC) from Spirulina platensis are essential for their commercialization, yet remain challenging. This study investigates three chromatographic methods: weak anion exchange chromatography (DEAE), strong anion exchange chromatography (Q Sepharose), and hydrophobic interaction chromatography (HIC) for C-PC purification. DEAE chromatography achieved a recovery of 36.80 mg unit (57.08%) with a purity of 3.23, outperforming Q Sepharose (yield: 23.21 mg unit means that 46.33%, purity: 2.76) and HIC (yield: 22.95 mg unit means that 17.57%, purity: 3.02). The purified C-PC consisted of α and β subunits with molecular masses of 16 kDa and 17 kDa, respectively. Further assessment revealed its antioxidant capacity through an ABTS assay. C-PC stability was tested at different pH levels and temperatures. Maximum stability was observed at pH 7, while pH 4 showed the lowest stability. Glutaraldehyde-treated C-PC (GC-PC) demonstrated gradual degradation up to 50°C, retaining 73.25% after 30 min. Notably, GC-PC exhibited stability even at higher temperatures, with degradation rates of 57.32% at 70°C and 50.96% at 80°C. DEAE chromatography proved superior for C-PC purification, offering higher yields and purity compared to Q Sepharose and HIC. The purified C-PC showed promising antioxidant capacity and stability, particularly GC-PC, which exhibited resistance to degradation even at elevated temperatures. These findings underscore the potential of C-PC as a valuable compound for various applications, with DEAE chromatography being an efficient method for its production and commercialization. This article is protected by copyright. All rights reserved.

Quartz Crystal Microbalance as a Holistic Detector for Quantifying Complex Organic Matrices during Liquid Chromatography: 1. Coupling, Characterization, and Validation.

A matrix in highly complex samples can cause adverse effects on the trace analysis of targeted organic compounds. A suitable separation of the target analyte(s) and matrix before the instrumental analysis is often a vital step for which chromatographic cleanup methods remain one of the most frequently used strategies, particularly high-performance liquid chromatography (HPLC). The lack of a simple real-time detection technique that can quantify the entirety of the matrix during this step, especially with gradient solvents, renders optimization of the cleanup challenging. This paper, along with a companion one, explores the possibilities and limitations of quartz crystal microbalance (QCM) dry-mass sensing for quantifying complex organic matrices during gradient HPLC. To this end, this work coupled a QCM and a microfluidic spray dryer with a commercial HPLC system using a flow splitter and developed a calibration and data processing strategy. The system was characterized in terms of detection and quantification limits, with LOD = 4.3-15 mg/L and LOQ = 16-52 mg/L, respectively, for different eluent compositions. Validation of natural organic matter in an environmental sample against offline total organic carbon analysis confirmed the approach's feasibility, with an absolute recovery of 103 ± 10%. Our findings suggest that QCM dry-mass sensing could serve as a valuable tool for analysts routinely employing HPLC cleanup methods, offering potential benefits across various analytical fields.

Quality by Design-driven Analytical Method: A Quality Risk Management-Based Liquid Chromatography Method for Daclatasvir and Characterization of its Putative Degradants by LC-MS/MS

Background:: Antiviral drugs can cure more than 95 percent of people with hepatitis C, but the inaccessibility of quality affordable medicines and the lack of their uninterrupted supply pose a major challenge. Impurities in drugs have a significant impact on their quality and are one of the substantial causes of drug recalls, ultimately leading to the unavailability of the drug in the market. Hence, there is a need for a robust, quality risk management and quality by design-driven analytical method that can detect the antiviral drug, Daclatasvir dihydrochloride, in the presence of its probable impurities. Objective:: This study aimed to develop a Quality by Design-driven stability- indicating liquid chromatography method for Daclatasvir dihydrochloride and the characterization of its putative degradants by LC-MS. Method:: The fishbone diagram and quality risk assessment investigated twenty-four process parameters and concluded that three risk parameters, i.e., flow rate, buffer pH, and stationary phase type, were the critical process parameters. The critical quality attributes viz. resolution between impurity 6 and DCV and impurity 2 & 3 (Rs˃1.5), the shape of the peak of DCV which is decided by the Number of Theoretical Plates (NTP˃5000), and the retention time of Daclatasvir (tR14-23 mins) were optimized using a two-level three-factor full factorial design with five center points. Results:: The optimized method is stability-indicating in its true sense as it can separate the sample with its degradants generated in basic (three), acidic (two), oxidative (H2O2: three, Azobisisobutyronitrile: one), photo (three), and dry heat (one) conditions. Degradants structures were elucidated, and degradation routes were established, using LC-MS and LC-MS/MS analyses. Conclusion:: The drug is highly susceptible to acid, base hydrolysis, and oxidation degradation conditions and poses a significant risk to the analytical method to fail in system suitability criteria. Hence, a robust and flexible chromatographic method with the capacity for continuous improvement was developed and successfully validated within the criteria of design space.

Saponins from seeds of Zizyphus jujuba Mill. var. spinosa exhibit vasorelaxant effects on rat isolated aorta

AbstractAimThe vasorelaxant saponins jujuboside A(1), A1(2), B(3), and acetyljujuboside B(4) were isolated from 50% MeOH extracts of seeds of Zizyphus jujuba Mill. var. spinosa Hu ex H.F. Chou (Rhamnaceae), and their effects were studied using isolated rat aortic rings.MethodDried jujube seeds were crushed and extracted with 50% MeOH, and the extracts were concentrated under reduced pressure. Vasorelaxant effects of this extracts were assessed on rat isolated aorta rings. The aorta rings were placed in a well‐oxygenated bath of modified Krebs–Henseleit solution and the mechanical tension was measured isometrically.ResultsThe crude extract was fractionated by chromatographic methods with the guidance of vasorelaxant activity, and four saponins (1~4) were isolated. These compounds were identified using nuclear magnetic resonance (NMR) studies. Compounds 1~4 showed slow relaxation activity against norepinephrine (NE)‐induced contractions of rat aorta with/without endothelium. Treatment with 1~4 did not significantly inhibit NE‐induced vasoconstrictions. Compounds 1~4 inhibited vasoconstriction induced by high‐concentration potassium (60 mM) and NE in the presence of nicardipine but did not affect phorbol 12‐myristate 13‐acetate‐induced vasoconstriction.ConclusionThese results suggest that inhibition of NE‐induced vasoconstriction by 1~4 is due to the inhibition of receptor‐operated Ca2+ channels and voltage‐dependent Ca2+ channels or inhibition of intracellular calcium modulations.

A novel isoquinoline alkaloid HJ-69 isolated from Zanthoxylum bungeanum attenuates inflammatory pain by inhibiting voltage-gated sodium and potassium channels

Ethnopharmacology relevanceZanthoxylum bungeanum Maxim. (Z. bungeanum), a member of the Rutaceae family, has a rich history of traditional use in Asia for treating arthritis and toothache conditions. As characteristic chemical components, numerous kinds of alkaloids have been extracted from plants and their diverse biological activities have been reported. However, research on the isoquinoline alkaloid, a specific type of alkaloids, in Z. bungeanum was scarce. Aim of the studyThe study aimed to isolate a novel isoquinoline alkaloid from Z. bungeanum and explore its pharmacological activity in vitro and analgesic activity in vivo. Materials and methodsIsoquinoline alkaloid isolation and identification from Z. bungeanum were conducted using chromatographic and spectroscopic methods. The whole-cell patch-clamp technique was applied to assess its impact on neuronal excitability, and endogenous voltage-gated potassium (Kv) and sodium (Nav) currents in acutely isolated mouse small-diameter dorsal root ganglion (DRG) neurons. Its inhibitory impacts on channels were further validated with HEK293 cells stably expressing Nav1.7 and Nav1.8, and Chinese hamster ovary (CHO) cells transiently expressing Kv2.1. The formalin inflammatory pain model was utilized to evaluate the potential analgesic activity in vivo. ResultsA novel isoquinoline alkaloid named HJ-69 (N-13-(3-methoxyprop-1-yl)rutaecarpine) was isolated and identified from Z. bungeanum for the first time. HJ-69 significantly suppressed the firing frequency and amplitudes of action potentials in DRG neurons. Consistently, it state-dependently inhibited endogenous Nav currents of DRG neurons, with half maximal inhibitory concentration (IC50) values of 13.06 ± 2.06 μM and 30.19 ± 2.07 μM for the inactivated and resting states, respectively. HJ-69 significantly suppressed potassium currents in DRG neurons, which notably inhibited the delayed rectifier potassium (IK) currents (IC50 = 6.95 ± 1.29 μM) and slightly affected the transient outward potassium (IA) currents (IC50 = 523.50 ± 39.16 μM). Furtherly, HJ-69 exhibited similar potencies on heterologously expressed Nav1.7, Nav1.8, and Kv2.1 channels, which correspondingly represent the main components in neurons. Notably, intraperitoneal administration of 30 mg/kg and 100 mg/kg HJ-69 significantly alleviated pain behaviors in the mouse inflammatory pain model induced by formalin. ConclusionThe study concluded that HJ-69 is a novel and active isoquinoline alkaloid, and the inhibition of Nav and Kv channels contributes to its analgesic activity. HJ-69 may be a promising prototype for future analgesic drug discovery based on the isoquinoline alkaloid.

Stability-Indicating UPLC-PDA-QDa Methodology for Carvedilol and Felodipine in Fixed-Dose Combinations Using AQbD Principles

The development of analytical procedures, in line with the recent regulatory requirements ICH Q2 (R2) and ICH Q14, is progressing, and it must be able to manage the entire life cycle of the methodology. This is also applicable to and especially challenging for combinations of drug substances and dosage form. A reliable and efficient, stability-indicating, MS-compatible, reverse-phase ultra-performance liquid chromatographic (UPLC®) method was developed for the determination of carvedilol and felodipine in a combination oral dosage form. The development of the method, performed using analytical quality by design (AQbD) principles, was in line with the future regulatory requirements. Furthermore, the fixed-dose combination dosage forms are a clear solution to the polypharmacy phenomenon in the elderly population. The main factors evaluated were the mobile phase buffer, organic modifier, column, flow, and column temperature. The optimum conditions were achieved with a Waters Acquity HSS T3 (100 × 2.1 mm i.d., 1.8 µm) column at 38 °C, using ammonium acetate buffer (5 mM, pH 4.5) (Solution A) and MeOH (Solution B) as mobile phases in gradient elution (t = 0 min, 10% B; t = 1.5 min, 10% B; t = 12.0 min, 90% B; t = 13.0 min, 10% B; t = 15.5 min, 10% B) at a flow rate of 0.2 mL/min and UV Detection of 240 and 362 nm for carvedilol (CAV) and felodipine (FLP), respectively. The linearity was demonstrated over concentration ranges of 30–650 µg/mL (R2 = 0.9984) (CAV) and 32–260 µg/mL (R2 = 0.9996) (FLP). Forced degradation studies were performed by subjecting the samples to hydrolytic (acid and base), oxidative, and thermal stress conditions. Standard solution stability was also performed. The proposed validated method was successfully used for the quantitative analysis of bulk, stability, and fixed-dose combination dosage form samples of the desired drug product. Using the AQbD principles, it is possible to generate methodologies with improved knowledge, leading to high-quality data, lower operation costs, and minimum regulatory risk. Furthermore, this work paves the way for providing a platform of robust analytical methods for the simultaneous quantification of innovative on-demand new dose combinations.

Antiproliferative, antioxidant and phytochemical analysis of Loranthus europaeus fruits

AbstractLoranthus europaeus with numerous therapeutic usages was chosen for further bioactivity assays and phytochemical analysis. The air‐dried grounded L. europaeus fruits were solvent extracted via Soxhlet apparatus. Afterwards, methanol extract was fractionated via different chromatographic methods and structures of the isolated pure compounds were elucidated with relative nuclear magnetic resonance spectra. Additionally, L. europaeus fruits essential oil was achieved through hydrodistillation and analyzed via gas chromatography‐mass spectrometry. Besides, the in vitro DPPH and nitric oxide antioxidant property of L. europaeus methanol extract alongside its phenolic and flavonoids content were evaluated. MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay was carried out in A549 cancer cells and normal HUVECs cells for evaluating cytotoxicity of the plant extract and essential oil. Structure of the isolated major compounds of the extract were determined as two flavonoids. Analysis of the L. europaeus essential oil showed that it principally contained germacrene D (32.8%), β‐caryophyllene (19.2%) and alloaromadendrene (11.0%). In the MTT assay, 300 µg/mL of methanol extract caused ≈60% A549 cells death after 48 h with IC50 of 226 µg/mL. Likewise, the IC50 of the essential oil against A‐549 cell line for the 100 µg/mL L. europaeus fruits in 48 h was equal to 69 µg/mL. Overall, the present study is the first report on the phytochemical analysis of L. europaeus revealing occurrence of rhamnocitrin‐3‐O‐rhamnoside and rhamnetin‐3‐O‐rhamnoside in the plant extract alongside its high content of sesquiterpenoids in its essential oil. The observed antiproliferative activity of L. europaeus methanol extract against A549 cancer cells rather than healthy cells indicated its anti‐tumor properties which might be related to the high values of phenolic and flavonoids content in the methanol extract of the plant associated with its superior in vitro antioxidant activities.

Development of natural dye photosensitizers for dye-sensitized solar cells: a review.

Dye-sensitized solar cell (DSSC) is a photovoltaic device that can be produced from natural source pigments or natural dyes. The selection of natural dyes for DSSC application is currently under research. The utilization of natural dye materials that are easy to obtain, cost-effective, and non-toxic can reduce waste during DSSC fabrication. Natural dyes can be extracted from plants through extraction and chromatography methods. The suitability and viability of utilizing natural dyes as photosensitizers in DSSCs can be predicted using appropriate software simulation by varying related parameters to produce high power conversion efficiency. In this context, the purpose of the review is to highlight the evolution of performance improvement in the development of DSSCs with consideration of natural dye extraction and software simulation. This review also focuses on the results of extracting natural dyes from herbal ingredients, which are still very limited in information, and several parts of herbal plants that can be used as natural dye sources in the future of solid-state DSSCs have been identified. Based on the results of this review, the highest efficiency was obtained for the DSSC that used chlorophyll pigments as natural dyes using Peltophorum pterocarpum leaves with 6.07%, followed by anthocyanin pigments as natural dyes using raspberries (black) fruits with 1.5%, flavonoid pigments as natural dyes using Curcuma longa herbs with 0.64%, and flavonoid pigments as natural dyes using Indigofera tinctoria flowers with 0.46%.

Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

A new gas chromatographic method for quantification of Metformin hydrochloride and Vildagliptin in bulk and pharmaceutical dosage form: development and validation

BackgroundMetformin, an antidiabetic drug, assists in reducing the creation of glucose inside the liver. Vildagliptin, a DPP-4 inhibitor, enhances insulin release from the pancreas and reduces the hormones that elevate blood sugar levels. The combined medications work synergistically to lower blood sugar levels. This study was designed to develop and validate a reliable method of simultaneous assessment of Metformin and Vildagliptin in bulk and pharmaceutical dosage forms. For the chromatographic separation, a Gs-Tek INNOWAX column was utilized. This column has a length of 30 m, an internal diameter of 0.25 mm, and a 1.8 µm film thickness. For the detection, a Flame Ionization Detector was utilized. The ideal conditions included an injection volume of 1 µL with a split mode of 10 to 1 ratio, a flow rate of 1 mL/minute for the nitrogen carrier gas, an injector temperature of 300 °C, a detector temperature of 250 °C, an initial oven temperature of 100 °C that was maintained for seven minutes and then programmed to climb at a rate of 10 °C per minute up to a temperature of 300 °C.ResultsA gas chromatographic method that is simple, precise, accurate, robust, and reliable has been developed and implemented for the simultaneous estimation of Metformin and Vildagliptin in the tablet dosage form. The retention time for Metformin and Vildagliptin was 10.203 and 22.021 min. respectively. Validation studies were performed on the method’s Linearity, detection limit (LOD), and quantitation limit (LOQ), as well as its accuracy, precision, system suitability, and robustness, using the norms established by the International Conference on Harmonization (ICH). The mean recovery value for Metformin and Vildagliptin was 100.31% (% R.S.D. = 0.6743%) and 100.33% (% R.S.D. = 0.6900%). All the results are within the acceptable range.ConclusionValidation of the developed method revealed that all the results were within an acceptable range, and techniques can be employed to analyze these two medications in combined dosage forms. It is the first method used for simultaneous estimation of these two drugs.Graphical abstract

A comparative greenness assessment of chromatographic methods for cilnidipine analysis and its combination

The greenness assessment of analytical methods is an important aspect of green analytical chemistry, which focuses on making analytical procedures eco-friendly and safer. This study conducts a thorough comparative analysis of chromatographic methods utilized in the analysis of Cilnidipine (CLN), a commonly employed antihypertensive medication. The research aims to not only evaluate the analytical performance of different chromatographic techniques but also assess their environmental impact, with a focus on promoting sustainable and eco-friendly practices in pharmaceutical analysis.The greenness profiles of twelve chromatographic methods for CLN and its derivatives were evaluated utilizing multiple assessment metrics including the Green Analytical Procedure Index (GAPI), Analytical GREEness (AGREE), Analytical eco-scale (ESA), Chloroform-oriented Toxicity Estimation (ChlorTox scale), Blue Applicability Grade Index (BAGI), and Red-Green-Blue 12 (RBG 12). By quantifying the environmental implications, the study aims to highlight the importance of adopting greener practices in pharmaceutical analysis. This assessment encompasses considerations such as solvent used, energy consumed, and waste generated by the method. The outcomes of this research contribute valuable insights into the selection of environmentally friendly chromatographic methods for CLN, promoting a balance between analytical efficiency and ecological responsibility in the pharmaceutical field.