ASSESSMENT OF PHENOLIC COMPOUNDS, ANTIMICROBIAL ACTIVITY AND FREE RADICAL SCAVENGING POTENCY OF THREE SELECTED VEGETABLES

Phytochemicals are compounds derived from plants that are assumed to have defensive role against certain disease. They have antioxidants, anticancer, antimicrobial, antifungal, antiviral, antithrombic and anti-inflammatory properties. They have a high specificity to boost the immune system and play important role in the metabolism of hormones. The current study is based on qualitative and quantitative evaluation of total phenolics contents, phenolic compounds, antioxidant potential, free and bound phenolic acids in selected vegetables available at the local market of Hyderabad, Sindh, Pakistan. Two different extraction procedures ultrasonic-assisted base hydrolysis extraction and sonication extraction were used. Total 13 phenolic compounds were found and quantified by high-performance liquid chromatography (HPLC), in which Ferulic acid was quantified in a higher amount of 16.71 mg/g in bitter gourd. Total phenolic contents were determined by using Perklin-Elmer lambda UV/Visible spectrophotometer and higher concentration was found in Bitter gourd 92.56 mg 100/g as compared to Luffa and Brinjal with 79.03 and 66.56 mg 100/g, respectively. The antioxidant activity (DPPH assay) was measured at ?max of 517 nm, results revealed that Bitter gourd possessed the higher antioxidant activity with 182.61 µMol/g followed by Luffa and Brinjal with 112.94 and 82.96 µMol/g. The total Flavonoid contents were higher in Brinjal with 44.32 mg g-1 whereas Luffa and Bitter gourd possess the Flavonoid concentration in the range 38.02 and 34.64mg g-1 respectively, the total tannin contents also higher in Brinjal 31.40 mg/g follwed by in Luffa and Bitter gourd with 25.17 and 21.19 mg/g respectively. Antimicrobial activity showed that, all the extracts are the highly effective against S. aureus as compared to E. coli. Finally, it is concluded that all the selected vegetables are very good sources of Phenolic compounds as well as phytochemicals and should be included in the daily human diet for good health. On the basis of obtained results, it is also suggested that these samples will be further investigated for the determination of fatty acids by GC-MS and liquid chromatography-mass spectrum (LC-MS).

Optimisation of conditions for deacetylation of chitin-containing raw materials

The paper describes the differences between chitosan and chitin, and reviews works by foreign scientists on obtaining chitosan from various raw materials. Methods of modifying chitosan and obtaining combined sorbents have been analysed. It has been studied whether chitosan is applicable in the technology of wines and alcoholic beverages as a sorbent. The purpose of the study was determining the optimal conditions of the deacetylation stage to obtain chitosan with the best sorption properties from Aspergillus niger biomass. A three-factor experiment has been carried out. It involved obtaining 27 samples of chitosan using sequential four-step acid-base hydrolysis under various conditions of the deacetylation stage. The deacetylation process was optimised under alkaline conditions depending on the alkali concentration, processing temperature, and exposure. For each of the samples obtained, the adsorption activity, specific surface area, and distribution coefficient in the sorbent–sorbate system have been determined. The degrees of deacetylation of all chitosan samples have been determined by potentiometric titration. The study has resulted in determining the optimal conditions for the deacetylation stage: processing temperature 110–130°C, sodium hydroxide concentration 27–36 g/dm3, exposure 45 to 65 minutes. The sample deacetylated at the temperature 120 °C, alkali concentration 30 g/dm3, and exposure 45 minutes has shown the best adsorption activity values: the adsorption activity for methyl orange 347.96 mg/g, the specific surface area of the sorbent samples 0.52·105 m2/g, the distribution coefficient in the sorbent–sorbate system 3.29·10-3 ml/g. This sample had the highest degree of deacetylation, 43.6%. The sample has been analysed using IR spectroscopy, and its main characteristic frequencies have been studied. It has been concluded that the sample obtained was equivalent to the reference chitosan

Analytical Method Development and Validation for the Analysis of Nepafenac and its related substances using Ultra Performance Liquid Chromatography

A novel, economic and time-efficient reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method has been developed for the analysis of Nepafenac in the presence of both impurities and degradation products generated by forced degradation. When Nepafenac was subjected to acid hydrolysis, oxidative, base hydrolysis, photolytic, and thermal stress, observed degradation only in oxidative and base hydrolysis. The drug was found to be stable to other stress conditions. Various method development trails were performed for the separation of drug from impurities. However, best chromatographic separation was achieved on a Waters Acquity CSHC18, 100mm x 2.1mm, 1.7µ particle size column, UV detection at 245nm, a gradient elution of Ammonium formate (pH 4.0), mixture of organic solvents (Acetonitrile, Methanol) as mobile phase for drug, its impurities and it was captured. The method was validated for specificity, precision, linearity, accuracy, robustness and can be used in quality control during manufacture and for assessment of the stability samples of Nepafenac. Total elution time was about 6.5 min and equilibration time of about 1.5 min which allowed analysis of more than 100 samples per day. The analytical method discussed in United states Pharmacopoeia was pH sensitive and compatible to LC-MS analysis.

FORCED DEGRADATION STUDIES OF TIZANIDINE HYDROCHLORIDE AND DEVELOPMENT OF VALIDATED STABILITY-INDICATING RP-HPLC METHOD

Simple and precise stability indicating RP-HPLC method for the quantitative determination of tizanidinehydrochloride (TZN) in pharmaceutical dosage form was developed and validated. The separation was achieved using Atlantis C18 column (250 x 4.6 mm, 5µm) at 25 °C using a mobile phase containing 20mM KH2 PO4 (pH 3.5):methanol in the ratio of 30:70 V/V at 0.8 mL/min flow rate and 315nm detection wavelength. The retention time for TZN was 3.7 min and showed linearity in the 5-40µg/mL range with R2 >0.99. The drug was subjected to acid/ base hydrolysis, oxidative and thermal degradation to establish stability indicating method. The method was validated as per ICHQ2 (R1) guidelines. The method was accurate, precise, and specific for TZN estimation. In stress studies, the drug was found to be stable to acid/alkaline hydrolysis, oxidation, and thermal degradation conditions. Thus, the reported method can be used as a stability-indicating method for quality control and routine analysis of TZN.

INVESTIGATION OF THE COMPOSITION OF THE ORGANIC AND MINERAL PARTS OF SOLID WASTE FROM THE PRODUCTION OF HUMIC PREPARATIONS

The composition of the organic and mineral parts of solid residues from the production of humic preparations Hydrohumate, Oxyhumate, peat oxidate and Consil was studied. Significant differences in the component composition of these wastes, depending on the technologies for obtaining drugs, have been established. Redox-hydrolytic processing of peat leads to almost complete (acid-base hydrolysis) or partial (oxidation) destruction of hemicelluloses and the relative accumulation of cellulose and "lignin" in solid waste. The organic parts of the residues contain up to 30 % humic substances, due to the fact that the technologies for the production of humic preparations do not provide for the stage of washing the residues after separation in a centrifuge and some of the humic preparations remain in the solid phase. It is shown that the mineral part of solid residues includes a wide range of biogenic macro-and microelements, since humates of monovalent metals pass into solution, and salts of humic substances with metals of higher valence are insoluble and remain in the solid residue from hydrolysis or oxidation of peat. The study of the chemical composition of solid residues from the production of humic preparations showed possible directions for their effective disposal. Waste products are humate-containing products with a wide range of biogenic macro-and microelements, so they can be effectively used as biologically active additives to soils, compost, fertilizers, as well as in pond fish farming to stimulate the development of components of the natural food base of fish and increase the fish productivity of reservoirs.

Cannabinoids in Oral Fluid: Limiting Potential Sources of Cannabidiol Conversion to Δ9- and Δ8-Tetrahydrocannabinol

In late 2019 the National Laboratory Certification Program (NLCP) published an article reporting on the potential analytical conversion of 7-carboxy-cannabidiol (CBD-COOH) to 11-nor-9-carboxy-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. (1) The same conversion is possible in oral fluid with the parent analyte cannabidiol (CBD) converting to Δ9-tetrahydrocannabinol (Δ9-THC) and Δ8-tetrahydrocannabinol (Δ8-THC) under strong acidic conditions. With the recent rise in states legalizing the use of THC and the availability of products containing only CBD, unless the analytical in vitro conversions are controlled, the detection of Δ9-THC or Δ8-THC in oral fluid may not clarify whether the donor was using a CBD product, licit or illicit THC product. Authentic oral fluid samples submitted for cannabinoid analysis were subjected to multiple sample preparation procedures and extraction methods to determine the conditions that allow CBD to convert to THC. CBD single analyte controls prepared from a certified THC-free source were added to the batch to monitor the rate of conversion. Samples were prepared using a base hydrolysis, solid phase extraction, derivatization, and analysis by liquid chromatography with tandem mass spectrometry (LC–MS-MS). The base hydrolysis and derivatization were tested independently and did not contribute to the conversion rate. Adjusting the pH of the sample preparation and extraction from pH 2.0 to pH 5.0 changed the conversion rate from 5% to 1%. A pH of 6.0 was not strong enough to extract the cannabinoids efficiently. Removing the acid component of the preparation and extraction procedure eliminated the conversion to THC; however, this did reduce the analyte recovery depending on which extraction column was used. Processing time also contributed to the conversion rate. With smaller trial runs, conversion was not always seen but with larger validation batches low level conversion of 1–2% was observed. A fully validated LC–MS-MS method utilizing solid-phase extraction was developed for CBD, Δ9-THC, Δ8-THC, and cannabinol (CBN). The method specifically targets those analytes found in oral fluid after CBD administration and those that are seen during in vitro CBD conversion. CBD administration was performed using a certified THC-free CBD control.

A Newly Developed Reverse Phase-High Performance Liquid Chromatography Method for the Assay of Dexmethylphenidate and Serdexmethylphenidate with PDA

Aims: The present application is a Newly Validated Reverse Phase-High Performance Liquid Chromatography Method for the Assay of Dexmethylphenidate and Serdexmethylphenidate with PDA. Study design: Mentioned study is a quick, rapid, economical, precise, and accurate reverse phase- high performance liquid chromatographic method for estimating Dexmethylphenidate and Serdexmethylphenidate. Place and duration of study: The present assay was carried out at the Shree icon Pharma laboratories PVT.ltd, Vijayawada, AP, and India, from December 2020 to February 2021. Methodology: The stationary phase Agilent C18 column with dimensions of 150x4.6mm, 3.5 was used for chromatography and pH-2.5 ammonium acetate buffer with orthophosphoric acid: acetonitrile in a 50:50 ratio used as a buffer. The detection wavelength was 265nm, and the flow rate was 1mL/min. The strategy was justified according to ICH guidelines Results: Dexmethylphenidate and Serdexmethylphenidate had retention periods of 4.258 and 5.629 minutes, respectively. For the estimation of Dexmethylphenidate and Serdexmethylphenidate, the method has been validated for linearity, accuracy, precision, stability tests, and forced degradation studies including acid, base, hydrolysis, peroxide, and thermal degradation. By multiplying the quality six times, the system's suitability parameter was investigated, and they were well within reasonable limits. The regression coefficient of the two drugs was found to be 0.999 during the linearity study, which was performed at 10% to 150 percentage points. Precision results for Dexmethylphenidate and Serdexmethylphenidate were 0.54 and 1.24, respectively. The drugs were recovered at a rate of 98-102 percent, which is within the acceptable range. Conclusion: The validation results were found to be satisfactory. It was clear that the proposed method was suitable for routine quality control and analysis of pharmaceutical preparations.

Phenolic Compounds Profile and Antioxidant Capacity of Pitahaya Fruit Peel from Two Red-Skinned Species (Hylocereus polyrhizus and Hylocereus undatus)

Pitahaya peel is a good source of bioactive polyphenols. However, the bound phenolics and their antioxidant activity remain unclear. The bound phenolics of pitahaya peel from two red-skinned species with red pulp (RP) and white pulp (WP) were released with different methods (acid, base, and composite enzymes hydrolysis). The results revealed that base hydrolysis was the most efficient method for releasing the bound phenolics from RP (11.6 mg GAE/g DW) and WP (10.5 mg GAE/g DW), which was 13.04-fold and 8.18-fold for RP and 75.07-fold and 10.94-fold for WP compared with acid hydrolysis and enzymatic hydrolysis, respectively. A total of 37 phenolic compounds were identified by UPLC-TOF/MS with most chlorogenic acid, caffeic acid, ferulic acid and p-coumaric acid in RP, whereas chlorogenic acid, caffeic acid, ferulic acid, rutin and isoquercitrin were the main compounds in WP. Regardless of the hydrolysis method, the extracts having the highest phenolic content showed the strongest antioxidant activities. The work shows that hydrolysis methods have a significant effect on the release of phenolics, and the contents of major characteristic bound phenolic compounds are related to the ecological type of pitahaya.

Reduksi Bahan Organik Kulit Kopi dan Eceng Gondok Terhidrolisis Menggunakan Proses Anaerbik

Coffee pulp and water hyacinth are a biomass source that can be used to feeding material for biogas production as energy an anaerobic digester. But coffee pulp and water hyacinth contain lignin. The Alkaline or base hydrolysis is a method of the solving chemical structure of lignin compounds using a strong acid and base. The focus of research investigated the base hydrolysis with NaOH in coffee pulp and water hyacinth an anaerobic process for organic material reduction. The research design in laboratory conduct of organic materials reduction on coffee pulp and water hyacinth used Completely Random Design (CRD). Anaerobic treatments were without hydrolysis (H1), only the coffee pulp with hydrolysis (H2), only water hyacinth with hydrolysis (H3) and all with hydrolysis (H4). The highest NaOH concentration for lignin reduction on the base hydrolysis was 60 ppm. The analysis of variants with significantly (p<0.05) showed all treated differently. Anerobic treatment of the coffee pulp and water hyacinth (H4) had the highest value organic material reduction. The efficiency of organic material reduction i.e. C/N, BOD and COD was in sequence namely 64.22 ± 0.02; 75.23 ± 0.02 dan 52.55 ± 0.04.

DOMINATED RP-HPLC METHOD FOR QUALITY CONTROL OF TERNARY PHARMACEUTICAL FORMULATION: GUAIFENESIN/LEVOCETIRIZINE/ AMBROXOL HCL AND STABILITY STUDIES

The present investigation was aimed to establish a liquid chromatographic method for simultaneous quantification of guaifenesin, levocetirizine and ambroxol HCl in ternary fixed dosage form. The three drugs were swiftly resolved using ODS C18 reverse phase column with mobile phase consisting of ammonium phosphate buffer, pH 4.5: acetonitrile (60:40, v/v) at a flow rate of 1.0 mL/min and UV detection at 236 nm. The retention time values were 2.231, 2.772 and 6.309 min, respectively, for guaifenesin, levocetirizine and ambroxol HCl. The response was a linear function of analyte over the concentration range of 12.5-75 µg/mL, 0.625-3.750 µg/mL and 3.75-22.5 µg/mL with correlation co-efficient value of 0.999. Maximum recovery (99.12-101.2 %) was obtained in ternary liquid oral formulation (Leoriv plus syrup). Three drugs were well resolved from their degradation products and net degradation was calculated in acid hydrolysis, base hydrolysis, oxidation, thermal and UV conditions. The proposed method enables the simultaneous quantification of guaifenesin, levocetirizine and ambroxol HCl in routine QC laboratories.