Anticancer Activity Assay of Nano-Fractional Compounds that Purified from Soil Actinomycetes

Background and objectives: Cancer remains a global problem of health, and has been recorded as one of the causes of death after heart disease Natural products from plants, the environment and microorganisms are leveraged for the purpose of fighting cancer. Actinobacteria have been recognized as main sources of bioactive natural products as early as in the 1950s, for which about half of the secondary metabolites revealed, including enzymes, antibiotics, immunosuppressive, and anti-tumour agents Materials and methods:The methods of this study included isolated and identification of bacteria from soil samples and identified by morphology characters and biochemical test. Subjected extract of actinomycetes to HPLC purification then collected purified fractions then analyzed by GC-mass. After the fractions were mixed with liposome nanoparticals which tested activity on HT29 colon cancer cell line. Results: The results of identification of bacterial isolates showed the colonies growing on a SNA medium were morphologically identified where the colonies were well-growth and had a gray color, not producing dyes in the medium. The results of the biochemical tests indicated that isolates were amylase, catalase, and gelatinase producing isolates and non-lipase producing, non H2S production and consuming urea, while the carbon consumption test indicated the isolates' ability to consume starch, glucose and sucrose respectively. While The results of preparative HPLC revealed that 4 fractions analytical HPLC with (50 % HPLC-grade acetonitrile) at 254 nm and cycling up was employed to increase the separation efficiency. The chemical composition of the HPLC fractions using GC-MS showed the identification of many components example ( Hexadeconic acid , Octadecanoic acid,ethyl ester and Fumaric acid). The results of In vitro anti-tumor cytotoxicity showed that all four nano purified fractions were applied on HT 29 colon cancer cells and exhibited significantly differences compared with control.

Development and Validation of RP-HPLC Method for the Estimation of Clomiphene Citrate in Pharmaceutical Dosage Form

A simple, specific, precise, and accurate RP-HPLC method has been developed and validated for the estimation of Clomiphene citrate in bulk and pharmaceutical dosage form using C18 column Shimadzu (250mm × 4.5mm × 5μm) with a mobile phase consisting of 900mL of HPLC grade methanol and 100mL of HPLC grade acetonitrile. The mobile phase was sonicated for 10 min and filtered through a 0.45μm membrane filter at a flow rate of 1.0mL/min. The detection was carried out at 295nm and retention time of Clomiphene citrate was found to be 3.44 min. Linearity was observed in the concentration range of 10–50μg/mL (coefficient of determination R2=0.999) with regression equation y =20321x + 60021. The method was validated as per ICH guidlines.

RP-HPLC method development and validation for the quantification of Efonidipine hydrochloride in HME processed solid dispersions

Background Efonidipine hydrochloride (EFO) is a poorly water-soluble drug and, hence, has poor bioavailability. Solid dispersions (SDs) of EFO using Eudragit EPO were prepared using hot-melt extrusion (HME) for the first time. The current study aims at developing a simple RP-HPLC method to quantify EFO in the developed SDs. Results The chromatographic separation was carried out on an Agilent Eclipsed XDB-C18 column (4.6 × 250 mm), packed with 5 μm particles. The optimized mobile phase consisted of HPLC grade acetonitrile and 0.020 mol/L KH2PO4 (pH 2.5) buffer in the ratio of 85:15 v/v with a flow rate optimized at 1.2 ml/min. The developed method was validated for system suitability, linearity, accuracy, precision, and robustness. The linearity results showed an excellent linear relationship between the drug concentration and peak area, indicating the peak area is directly proportional to the analyte concentration within a specific range and an excellent correlation coefficient of 0.9998. Intermediate precision and repeatability confirmed that the method provides precise results with %RSD value less than 2% for EFO. The assay results of the developed formulations were in the acceptable range with RSD less than 2%. The enhanced drug dissolution from the Eudragit EPO carrier with 10% Citric Acid (CA) is attributed to the conversion of the drug from crystalline to amorphous form, and microenvironmental acidic pH provided by CA. Conclusion In a nutshell, the developed RP-HPLC method showed excellent ability to differentiate the formulations and highlights the role of the polymer and the plasticizer. Graphical abstract

DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR SIMULTANEOUS QUANTIFICATION OF VASICINE, GLYCYRRHIZIN AND PIPERINE IN POLY HERBAL COUGH SYRUP

Objective: The present study was undertaken to develop a rapid, simple, specific and economic high performance liquid chromatographic (HPLC) method has been developed, validated and used for simultaneous quantification of Vasicine, Glycyrrhizin and Piperine in poly herbal cough syrup Methods: An Agilent technologies 1200 Series quaternary pump combined with an Agilent 1200 series photo diode array detector (USA), an Agilent 1200 series vacuum degasser (USA) and an Agilent autosampler injector. Chromatographic separation was performed on a Hiber, prepacked column, C18, Size 250x 4.60 mm, 5µ maintained at 25 °C. PJ (solvent A) and HPLC grade Acetonitrile (solvent B). Results: The HPLC developed for quantization was simple, accurate and specific. The drug follows the beer’s lambert’s law in the concentration range of of Vasicine in concentration range 25–250 μg/ml, glycyrrhizin in concentration range 100–1000 μg/ml and Piperine in concentration range of 20–100μg/ml and exhibited good correlation coefficient and excellent mean recovery. Percentage RSD for precision and accuracy of the method was found to be less than 2%. Conclusion: The present standardization provides a specific and rapid tool in the herbal research, permitting to set quality specifications for identity, transparency and reproducibility of these Phytoconsituents in the herbal Cough syrup.

Pharmacokinetics of citicoline after intravenous and intramuscular administration in dogs

Background: New studies have confirmed the role of citicoline in reversing different pathological conditions in canine medicine, but dosing regimens of this drug has not yet been investigated in dogs. Using a high-performance liquid chromatography (HPLC) system, this study aims to quantify the levels of citicoline in dogs plasma after intravenous (IV) and intramuscular (IM) administration.Design and methods: The subjects of this study were 12 male, mixed breed healthy dogs that were approximately 2 to 3 years old and had a body weight between 15 and 25 kg. Plasma samples were extracted following protein precipitation. Samples were eluted from the column at flow rate of 0.7ml min-1. Solvents were degassed. The PH of the mobile phase of HPLC grade acetonitrile: water (20:80, v/v) was adjusted to 3.0 using 1% orthophosphoric acid. Both sample and standard solutions were filtered through 0.22 µm membrane filter. A sample volume of 20 microliter was injected to HPLC to obtain a standard curve.Results: No clinical signs or drug adverse effect was noticed in animals during and after the period of administration of citicoline. Measuring plasma concentration of citicoline sodium after IV and IM administration showed biphasic plasma peaks which occured at 15 minutes and 5 hours after the drug administration in dogs. Conclusions: Giving the drug once a day with 30-50 mg/kg dosage or twice daily with 15-30 mg/kg dosage would cause their levels to remain elevated for much of the day and doesn’t have any serious adverse effect.

Novel contribution to the simultaneous monitoring of pramipexole dihydrochloride monohydrate and levodopa as co-administered drugs in human plasma utilizing UPLC–MS/MS

An efficient, selective, sensitive, and rapid ultra-performance liquid chromatography tandem mass spectrometry method was established and validated for the quantification of pramipexole dihydrochloride monohydrate and levodopa simultaneously in human plasma with the aid of diphenhydramine as an internal standard. A simple protein precipitation technique with HPLC grade acetonitrile was efficiently utilized for the cleanup of plasma. The analysis was performed using a Hypersil gold 50 mm × 2.1 mm (1.9 µm) column and a mobile phase of 0.2% formic acid and methanol (90: 10 v/v). The triple-quadrupole mass spectrometer equipped with an electrospray source operated in the positive mode was set up in the selective reaction monitoring mode (SRM) to detect the ion transitions m/z 212.15 →153.01, m/z 198.10→ 135.16, and m/z 255.75 → 166.16 for pramipexole dihydrochloride monohydrate, levodopa, and diphenhydramine, respectively. The method was thoroughly validated according to FDA guidelines and proved to be linear, accurate, and precise over the range 100–4000 pg/mL for pramipexole dihydrochloride monohydrate and 60–4000 ng/mL for levodopa. The proposed method was effectively applied for monitoring both drugs in plasma samples of healthy volunteers.

Development and Validation of an HPLC Method for Determination of Thiamethoxam and Its Metabolites in Cotton Leaves and Soil

An easy and simple analytical method was standardized and validated for the estimation of residues of thiamethoxam and its metabolites in cotton. The samples were extracted with acetonitrile, water, and methanol; diluted with brine solution;partitioned into dichloromethane and ethyl acetate; dried over anhydrous sodium sulfate; and cleaned up by glass column chromatography. Final clear extracts were concentrated under vacuum and reconstituted into HPLC grade acetonitrile, and residues were estimated using an HPLC instrument equipped with a C18 column and photodiode array detector system. Acetonitrile–1% formic acid in HPLC grade water (30 + 70) was used as mobile phase at 0.2 mL/min. Consistent recoveries ranging from 82 to 97% for thiamethoxam and its metabolites were observed when samples were spiked at 0.05–1.0 mg/kg levels. The LOQ of the method was determined to be 0.05mg/kg. The analytical method was validated in terms of the selectivity, linearity, precision, and accuracy of the detection system.

Sensitive Methodology for Simultaneous Determination of Residues of Imidacloprid and its Metabolites in Sugarcane Leaves and Soil

An analytical method to quantify imidacloprid and its metabolites in sugarcane leaves and soil using HPLC has been developed. The samples were extracted with acetonitrile + water (80 + 20, v/v), soil samples partitioned with dichloromethane, and leaf samples with hexane + ethyl acetate (9 + 1, v/v) and dichloromethane. Further, the extracts were dried, filtered, and concentrated under vacuum into HPLC-grade acetonitrile. Residues were estimated using an HPLC equipped with a photodiode array detector system, C18 column with a mobile phase of acetonitrile + water (40 + 60, v/v) at 0.3 mL/min to separate imidacloprid and its six metabolites in single run of 20 min. The mean percent recoveries of imidacloprid and its metabolites ranged from 80.45 to 99.80 from sugarcane leaves and 80.20 to 99.70 from sugarcane soils. The analytical method was validated in terms of selectivity, linearity, reproducibility, repeatability, and accuracy. The repeatability values ranged from 0.24 to 3.15% and 1.69 to 4.94%, along with 2.73 to 3.82% and 1.12 to 4.96%, for imidacloprid and its metabolites in leaves and soil, respectively. The reproducibility of imidacloprid and its metabolites in leaves and soil ranged from 2.20 to 4.27% and 2.53 to 4.08%, respectively, and all measurements were within 15% at all concentration levels.

Development and Validation of an HPLC Method for Determination of Spirotetramat and Spirotetramat cis enol in Various Vegetables and Soil

An easy and simple analytical method was standardized and validated for the estimation of residues of spirotetramat and its metabolite spirotetramat cis enol in various substrates: okra fruits, brinjal leaves and fruits, green chili, red chili, and soil. The samples were extracted with acetonitrile, diluted with brine solution, partitioned into dichloromethane, dried over anhydrous sodium sulfate, and cleaned up by treatment with activated charcoal powder. Final clear extracts were concentrated under vacuum and reconstituted with HPLC grade acetonitrile. Residues were estimated using HPLC with a photodiode array detector and a C18 column, and confirmed by HPTLC. Acetonitrile was used as the mobile phase at 0.4 mL/min. Both spirotetramat and spirotetramat cis enol presented distinct peak at retention times of 8.518 and 7.598 min, respectively. Consistent recoveries ranging from 82 to 97% for spirotetramat and spirotetramat cis enol were observed when samples were spiked at 1.00 to 0.03 mg/kg levels. The LOQ of the method was found to be 0.03 mg/kg. The analytical method was validated in terms of parameters, including selectivity, linearity, precision, and accuracy.