Quaternary Pump Research Articles

Antioxidant Activities and Phenolic Composition of Sarcococca saligna Leaves

Abstract: Sarcococca saligna (D. DON) Muel is a vital plant with several steroidal alkaloids and is effective against syphilis, liver diseases, gastrointestinal tract disease, infections, and inflammation. The present study aimed to investigate the in vitro antioxidant activities and phenolic profile of S. saligna. High-performance liquid chromatography (HPLC) analysis has shown the presence of twelve phenolics namely kaempferol-3-O-glucoside, proanthocyanidin B1, quercetin-3-malonylglucoside- 7-glucoside, kaempferol-3-O-sophorotioside, Isorhamnetin-3-O-glucoside-7-Orhamnoside, kaempferolhexoside, 4-O-Caffeoyl-5-O-p-coumaroylquinic acid, 3,4-Di-O-caffeoyl-5- O-feruloylquinic acid, 3,4-Di-O-feruloyl-5-O-caffeoylquinic acid, flavogalloyl-HHDP-gluconic acid (lagerstannin B), 3,4-di-O-caffeoylquinic acid and kaempferol-3-(caffeoyldiglucoside)-7-glucoside. The hot water extract showed the highest anti-lipid peroxidative activities. The IC50 value for 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was 71.5 ± 2.1 μg/mL. The extract could chelate the iron and possess reducing activities on phosphomolybenum assay. It is concluded that extracts of S. saligna are rich in antioxidants and contain essential phytochemicals. objective: The present study was aimed to investigate thein vitro antioxidant activities and phenolic profile of S. saligna. HPLC analysis has shown the presence of twelve phenolics namely kaempferol-3-O-glucoside, proanthocyanidin B1, quercetin-3-malonyl-glucoside-7-glucoside, kaempferol-3-O-sophorotioside,Isorhamnetin-3-O glucoside-7-O-rhamnoside,kaempferolhexoside, 4-O-Caffeoyl-5-O-p-coumaroylquinic acid, 3,4-Di-O-caffeoyl-5-O-feruloylquinic acid, 3,4-Di-O-feruloyl-5-O-caffeoylquinic acid, flavogalloyl-HHDP-gluconic acid (lagerstannin B), 3,4-di-O-caffeoylquinic acid and kaempferol-3-(caffeoyldiglucoside)-7-glucoside. method: The chemical used were 1,10-phenathroline, Thibarbituric acid, TRIS-HCl, amominium molybdate, Iron, sodium nitroprusside. HPLC standards were purchased from Sigma-Aldrich. All the reagents were of analytical grade. Double beam spectrophotometer was used for UV-VIS spectrophotometery. The HPLC system used was Agilent 1260 Infinity HPLC system which consists of quaternary pump, degasser, auto-sampler and diode array detector (DAD). conclusion: The results have shown that S. saligna antioxidant rich extract can be effectively utilized in food and pharmaceutical industries with additional income. However, more detailed understanding of utilization of extract against degenerative diseases is required. There is also need to find out the safety and bioavailability of the plant extract for its use as nutraceutical and as a plant products.

A UPLC method development and validation study of Upadacitinib and its impurities in extended – release oral tablet dosage forms

The primary objective was to develop a concomitant isocratic ultra-performance liquid chromatographic photo-diode array detection method to estimate Upadacitinib and its process-related impurities: impurity-1 and impurity-2. Further validation was conducted and studied for possible degradants under stress environments. All the chemicals and reagents used were of HPLC (acetonitrile, methanol) and analytical grade (trifluoro acetic acid). The ultra-performance liquid chromatography (Agilent 1290 Infinity II LC system) consists of a quaternary pump, a BEH C18 (50×2.1mm, 1.7μ) column, and photo-diode array detector. The method was developed with acetonitrile: methanol: 0.1% v/v trifluoro acetic acid (50:20:30 v/v/v) mobile phase at 0.2mL/min flow rate within a run time of 5.5min The detection was carried at 231.2nm. The respective retention times achieved were 2.289min (Upadacitinib), 0.972min (Upadacitinib impurity-1), and 3.508min (Upadacitinib impurity-2). The optimized method was validated further, and the linearity range was best fit at 15.0-180.0μg/mL for Upadacitinib and 1.0-12.0μg/mL for both Upadacitinib impurity-1 and 2 respectively. The detection and quantification limits were 4.50μg/mL, 15.00μg/mL (Upadacitinib) and 0.30μg/mL, 1.0μg/mL (Upadacitinib impurity-1 and 2). A fast, isocratic, specific, and reproducible ultra-performance liquid chromatographic method was developed and validated for various parameters according to the ICH Q2 (R1) guidelines studies. Stress studies were conducted exposing the sample dilution to various treatments (acid, alkali, peroxide, HPLC water, heat, and UV light). The degradants were well-separated apart from the peaks of the active substance. The stability indicating nature was observed during the degradation. The optimized method can be applied for the separation and estimation of Upadacitinib and its process-related impurities in pharma sector in tablet dosage forms.

Analysis of Secondary Metabolites of Elaeagnus Angustifolia Leaves Based on UPLC-Q-TOF-MS.

The leaves of Elaeagnus angustifolia, belonging to the Elaeagnaceae Juss. family, are known for their medicinal properties for relieving cough and asthma, as well as treating dysentery and diarrhea. To establish a rapid qualitative method for the detection of secondary metabolites in leaves of Elaeagnus angustifolia, including the identification and analysis of various secondary metabolites in leaves of Elaeagnus angustifolia. Samples were separated using a Waters ACQUITY H-Class ultra-performance liquid chromatography (UPLC) system (FTN autosampler, quaternary LC pump) and ACQUITY UPLC® BEH C18 column (1.7 μm, 2.1 mm× 100 mm). The flow rate was set to 0.4 mL/min, the injection volume was 1.0 μL, and the column temperature was set to 45 °C. The mobile phase was methanol (A) with) -0.1% formic acid in water (B). Samples were analyzed by quadrupole time-of-flight mass spectrometry (Q-TOF-MS). A total of 182 different secondary metabolites were detected from 10 varieties of leaves of Elaeagnus angustifolia, including 77 flavonoids, 20 steroids, 7 alkaloids, 15 amino acids, 18 organic acids, and 45 other compound types. A method for the rapid analysis of leaves of Elaeagnus angustifolia by UPLC-Q-TOF-MS was established, and the secondary metabolites in leaves of Elaeagnus angustifolia were identified. The enrichment of secondary metabolites in leaves of different varieties of Elaeagnus angustifolia was clarified. The UPLC-Q-TOF-MS method is very fast and possesses a high degree of selectivity, precision, and sensitivity. These findings provide a reliable foundation for the development of medicinal resources derived from Elaeagnus angustifolia leaves.

Method Development and Validation of Indigenously Isolated Caspofungin Acetate and its related Substances using RP-HPLC Method

Caspofungin is prepared from a precursor PneumocandinB0 derived from a fungus Glarea lozoyensis. Prepared crude caspofungin acetate has many impurities, namely, A, B, D, E and PneumocandinB0, and these impurities vary slightly in chemical or structural composition and need to be eliminated by purification. The present study involves the preparation of caspofungin acetate from PneumocandinB0, followed by purification and method development for detecting caspofungin and its related substances.”We used a high-tech liquid chromatograph system, which included components like the Agilent 1260 infinity quaternary pump module, a sophisticated MWD detector, and the Empower 3 data handling system. To separate and analyze substances, we employed an analytical column made of stainless steel. This column was 150 mm long and had an internal diameter of 3.0 mm. It was filled with octadecyl silane chemically bonded to porous silica particles, each with a tiny diameter of 3 micrometers. The test method underwent validation to ensure it could accurately identify specific elements, detect minimal quantities, establish the lowest measurable amounts, maintain linearity, demonstrate precision, achieve accuracy, and suitably handle sample solutions and the system.” and the developed test method is suitable and can be introduced into the routine use for the detection of caspofungin acetate and its related substances.

Analytical method development and validation for the determination of Rifampicin, Isoniazid and Pyrazinamide in “Rifampicin, Isoniazid, Pyrazinamide and Ethambutol hydrochloride” tablets

A simple and efficient method for the quantification of rifampicin, isoniazid and pyrazinamide in a fixed dose combination of anti-tuberculosis drug product was developed and validated by using HPLC system. The separation of rifampicin, isoniazid and pyrazinamide was carried out on a Waters 2690, composed of a quaternary pump, autosampler and PDA detector by chromatographic technique. Empower 3 software was used for data acquisition and analysis. Optimization of chromatographic separations was achieved using Phenomenex curosil-pfp column (250 × 4.6 mm, 5 μm particle size), with gradient program using disodium hydrogen phosphate and sodium 1- octane sulfonate buffer solution (pH adjusted to 7.0) as mobile phase A and acetonitrile (100%) as mobile phase B at a flow rate of 0.8 mL/min and UV detection at 238 nm. Approximate relative retention times of isoniazid, pyrazinamide and rifampicin were found to be 0.89, 1.00 and 2.63 respectively.

A Universal Eluent System for Method Scouting and Separation of Biotherapeutic Proteins by Ion-Exchange, Size-Exclusion, and Hydrophobic Interaction Chromatography.

Characterization and quality control of biotherapeutic proteins commonly require the application of several orthogonal separation techniques in order to establish product identity and purity. Many of the techniques used rely on a buffered aqueous mobile phase system to maintain the native conformation of the protein and its variants. Optimal pH, buffer substance(s), and chromatography methods vary with each protein of interest and result in tedious method development for each new drug product. Linear controlled pH gradient systems from pH 5.6 to pH 10.2 has been shown to provide a global method for the separation of charge variants of monoclonal antibodies. This can be realized using two balanced zwitterionic buffer blends. The pH linearity of the resulting system, with a cation ion exchange column in place, can generate any pH value in this accessible pH range. This study expands the scope of this buffer system and demonstrates its application in conjunction with a quaternary HPLC pump for several analytical techniques: the pH optimization of salt gradient-based anion and cation exchange during method development, as well as performing pH gradient elution. In addition, the same universal buffers are used for hydrophobic interaction and size exclusion chromatography. This eluent system omits the need to prepare different buffers for each method and flushing of the HPLC system between method changes. The implementation of this concept is further demonstrated to allow an automated method scouting approach and selection of different methods that requires minimal manual intervention.

Development and Validation of Stability Indicating Assay for Simultaneous Determination of Bupivacaine and Meloxicam in Bulk and Pharmaceutical Formulations by Using Rp-Hplc Method

A simple, rapid, precise, sensitive, and reproducible reverse phase high-performance liquid chromatography (RPHPLC) method has been developed for the quantitative estimation of Bupivacaine and Meloxicam in pharmaceutical dosage form. Aim and objective of our study is to determine the amount of bupivacaine and meloxicam by using RP-HPLC using Waters alliance HPLC system, Quaternary gradient pump of e2695 series equipped with an auto sampler injector with 10μl is injected, and eluted with the mobile phase containing Acetonitrile and Water in the ratio of 60:40 v/v which is pumped at a flow rate of 1 ml/min and detected by UV detector at 225 nm. The peak of Bupivacaine and Meloxicam was eluted at retention times of 2.646 min and 3.136 min, respectively. Chromatographic separation of Bupivacaine and Meloxicam was achieved on Waters Alliancee2695by using Chiralcel ODH 150x4.6mm, 5μcolumn and the mobile phase containing Acetonitrile and water in the ratio of 60:40% v/v. The flow rate was 1.0 ml/min; detection was carried out by absorption at 225 nm using a photodiode array detector at ambient temperature. The number of theoretical plates and tailing factor for Bupivacaine and Meloxicamwere NLT 2000 and kept not more than two respectively. Peak areas, Percentage relative standard deviation of all measurements always less than 2.0. This method was validated according to ICH guidelines. The method was a simple, economical, suitable, precise, accurate & robust method for quantitative analysis of Bupivacaine and Meloxicam study of its stability.

P076 The promising antimycotic activities of a novel cyclic antifungal lipopeptide against Human Dermatophyte Isolates

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesTo determine the minimum inhibitory concentrations (MICs) of a novel antifungal lipopeptide against clinical isolates of dermatophytes of human origin.MethodsTo perform antifungal susceptibility testing (AFST) by CLSI microbroth dilution method, a reversed-phase high-performance liquid chromatography (RP-HPLC) purified lipopeptide of 1071.4 Da from a wild-type soil isolate Bacillus subtilis was used and compared with the standard allylamine terbinafine MICs. Briefly, 1200 ml of cell-free culture supernatant was extracted using a solvent mixture and silica gel (230-400 mesh size)-based adsorption chromatography. The semi-preparative RP-HPLC system consisted of an Agilent quaternary pump and a variable wavelength detector equipped with a Phenomenex Luna C18 column (10 mm × 250 mm, 5 μm). The solvent system for RP- HPLC was (A) water with 0.1% trifluoroacetic acid (TFA) and (B) acetonitrile containing 0.1% TFA. The gradient of solvent B used for purification was as follows: 0%-54% for 0-20 min at the flow rate of 1 ml/min, 54%-60% from 20-48 min at 0.5 ml/min, 60%-100% from 48-58 min at 0.5 ml/min, 100%-0% from 58-65 min at 1 ml/min and monitored at 210 nm.ResultsSuperficial skin infections are caused by dermatophytes including Trichophyton spp. Nowadays, resistance to terbinafine in Trichophyton spp. isolates with higher MICs have been documented in India. We report here the antifungal prowess of a novel small antifungal lipopeptide against 20 clinical isolates of Trichophyton spp. of human origins (from human skin scrapings and nails) with the clinical diagnosis of tinea corporis/cruris and tinea unguium. The representative photographs of T. tonsurans, T. rubrum, and T. mentagrophytes complex are provided below. A total of 6 isolates of T. mentagrophytes and T. rubrum carry point mutations at F397L of squalene epoxidase (SQLE) protein. The in vitro antifungal efficacies determined by AFST revealed that the lipopeptide showed less or equivalent MICs (100% inhibition) in the case of five dermatophytes. The lipopeptide drug has exhibited improved MICs against two T. mentagrophytes complex and two T. rubrum isolates with amino acid substitution F397L in SQLE protein. Trichophyton mentagrophytes complex and T. rubrum with F397L mutations were inhibited at MICs 4-32 μg/mL of terbinafine. In comparison, the lipopeptide showed 4-16 μg/ml (100% inhibition). In the case of all four Trichophyton tonsurans, the MICs ranged between 2-4 μg/ml for the lipopeptide.ConclusionsThe broad-spectrum lipopeptide showed promising antifungal activity against dermatophytes and may be considered for nano-emulsion formulation and tested for topical application in a mice model.

Validated stability-indicating RP-HPLC method for simultaneous estimation of candesartan and pioglitazone in human plasma

Objective: The focus of this study was to establish a simple, robust, and validated RP-column approach to analyze Candesartan and Pioglitazone in a bilayer tablet dosage form. To broaden patient compliance in conditions such as diabetes and hypertension, a combination of drugs is often prescribed. As a result, a bilayer tablet containing a fixed-dose combination of 8 mg of Candesartan and 15 mg of Pioglitazone was developed. Both drugs were analyzed and chromatographically separated using the liquid chromatography technique. Methodology: waters 2695 with a quaternary pump and Inertsil C18 column mobile phase as buffer: acetonitrile (60:40) and flow rate of 1ml per minute and detection at 220nm was used for performing Chromatography. Results: Internal standard, Candesartan, and Pioglitazone eluted with retention times of 2.208, 2.569, and 3.553 minutes, respectively. There was no interference found between the peaks. With a correlation value of 0.999, the approach is verified across a linear range of 0.5 µg/ml – 20.0µg/ml for Candesartan and 0.08 µg/ml – 3.2 µg/ml for Pioglitazone. 6 samples at LLOQ level were analyzed for accuracy and precision and found to be in limit and all the analytes were stable at 28 °C and -80 °C in human plasma.

Development and Validation of a Selective Reversed-phase High Performance Liquid Chromatography Method for the Quantification of 1 ALPHA -Hydroxy Vitamin D2 and its Impurities

A selective reversed-phase high-performance liquid chromatography (RP-HPLC) technique was developed for 1α-hydroxy vitamin D2 (Doxercalciferol) related substances analysis. The technique is proficient of eluting various impurities related to process and degradation. To separate both the vitamin D2 Analogs i.e., doxercalciferol and ergocalciferol. HPLC of Shimadzu, provided with autosampler, column oven, quaternary pump and UV detector, were used for development studies. The mobile phase used water and acetonitrile. The diluent used was methanol and detection was examined at wavelength 265 nm. The method is sensitive for doxercalciferol, ergocalciferol, impurity-A and impurity-B; the limit of detection (LoD), value is 0.01% and limit of quantitation (LoQ) value is 0.03%. Linearity of doxercalciferol and related impurities at various concentrations was performed and the obtained correlation coefficient for doxercalciferol is 0.995, ergocalciferol is 0.997, impurity-A is 0.990 and impurity-B is 0.993. The optimized method was validated and demonstrated that the technique is selective, precise, linear, sensitive and accurate. The method is simple, inexpensive and useful for routine testing of doxercalciferol API. In addition, the technique was effectively applied for the elution of both the vitamin D2 Analogs i.e., doxercalciferol and ergocalciferol. Further, the same method is applicable for the determination of assay for these two API’s and the same assay method was validated.

Stability of a standardized preparation of methotrexate, cytarabine, and methylprednisolone hemisuccinate for intrathecal use.

Data about the feasibility or stability of drugs prepared for intrathecal administration are scarce, especially concerning the possibility of mixing two or more compounds in the same syringe. We evaluated the stability of an extemporaneously prepared triple intrathecal therapy containing methotrexate, cytarabine, and methylprednisolone hemisuccinate. Six mixtures containing 12.5 mg methotrexate, 50 mg cytarabine, and 40 mg methylprednisolone hemisuccinate, diluted to a final volume of 5 ml with water for injection, were prepared in polypropylene syringes on six different days. Syringes were stored protected from light either at room temperature (20°C) (n = 3) or refrigerated temperature (4°C) (n = 3). Samples were analyzed immediately after preparation and again at 0.5, 2, 4, 6, 8, and 24 h. The analysis was conducted with a high-performance liquid chromatography instrument equipped with a quaternary pump and diode array detector. pH was also assessed before every sample analysis. When mixed in a polypropylene syringe, the three drugs were stable at both temperatures tested. No degradation >10% was observed in any sample and pH remained between 7.0 and 7.5 over time. No precipitation or color change occurred. Among the three compounds, methylprednisolone hemisuccinate was the most labile as a slight temperature- and time-dependent degradation was observed. Triple intrathecal solution of methotrexate, cytarabine, and methylprednisolone hemisuccinate is stable for up to 24 h when stored in polypropylene syringes protected from light at 4°C and 20°C.

DEVELOPMENT AND VALIDATION OF A STABILITY INDICATING RELATED SUBSTANCES OF ATENOLOL AND NITRENDIPINE BY RP-HPLC

Objective: A validated stability-indicating RP-HPLC method for Atenolol and Nitrendipine was developed by separating its related impurities. Methods: By using Waters HPLC e-2695 quaternary pump with a PDA detector of 2998 instrument, the chromatographic separation of Atenolol, Nitrendipine and its related impurities was achieved on the column of Agilent eclipse C18 (150x4.6 mm, 3.5 µ) using gradient elution with a buffer containing 0.1percent formic acid and acetonitrile as a mobile phase with a flow rate of 1 ml/min at ambient temperature. A detector wavelength of 218 nm utilizing the PDA detector was given in the instrumental settings. The linearity was studied between the concentration range of 6.25-37.5 µg/ml of Atenolol, 0.75-4.5 µg/ml each of Atenolol imp-A, imp-B and 5-30 µg/ml of Nitrendipine, 0.5-3 µg/ml each of Nitrendipine imp-1, imp-2 were injected with a run time of 40 min. Validation of the proposed method was carried out according to an International Conference on Harmonization (ICH) guidelines. Results: LOD and LOQ for the Atenolol and its impurities were established with respect to test concentration. The plotted calibration curves were linear with a regression coefficient of R2>0.999, indicating that the linearity was with in the limit. As a part of method validation the parameters like specificity, linearity, accuracy, ruggedness, robustness were determined and the results were found to be within the allowable limit. Conclusion: The method developed was found to be applicable to routine analysis and to be used for the measurement of active pharmaceutical ingredients (i. e, Atenolol, Nitrendipine and their related impurities). Since there is no HPLC method reported in the literature for the estimation of Atenolol, Nitrendipine and their related impurities, there is a need to develop quantitative methods under different conditions to achieve improvement in specificity selectivity etc.

Development and validation of a HPLC-UV method for methadone hydrochloride quantification in a new oral solution with preservatives to be implemented in physicochemical stability studies

PurposeThe Pharmacy Service of the Infanta Leonor University Hospital acquires, compounds, distributes and dispenses more than 3000 L of methadone oral solution to Drug Addiction Patients Centers per year. Our purpose is to develop and validate an improved high performance liquid chromatography (HPLC) method to quantify methadone hydrochloride in a new oral solution with methylhydroxybenzoate (methylparaben) and propylhydroxybenzoate (propylparaben) to be implemented in physicochemical stability studies that allow to provide more information and even to increase the beyond-use date.MethodsA HPLC-Agilent® 1100 equipment, comprising a quaternary pump and an ultraviolet diode-array-detector (DAD) was used. An analytical method development and validation was completed. The curve was constructed from methadone working concentrations of 75–125% (7.5, 9.0, 10.0, 11.0 and 12.5 mg/mL) to assess the linear relationship between the concentration of the analyte and the obtained areas. Precision and accuracy were calculated. Detection and quantification limit (LD, LQ) were estimated using the EURACHEM method. Forced-degradation studies were also performed.ResultsChromatographic conditions were: flow rate 1.6 mL/min; mobile phase 55% acetonitrile and 45% sodium phosphate 25 mM (pH = 10); injection volume was 5 µL. The column was a Waters-XTerra™ RP18, maintained at 40 °C. DAD was λ = 254 nm. Retention times for methadone, methylparaben and propylparaben were 4.34, 0.70 and 0.88 min respectively.The method was linear (y = 284.3x − 97.8, r = 0.996). Instrumental precision was 0.33% for standards (n = 10); intra-assay precision 0.53% (n = 6) and inter-assay precision 1.95% (n = 12). The relative standard deviation percentage for accuracy was 1.28%. The recovery percentage was 101.5 ± 1.5%. LQ and LD were 2.18 µg/mL and 2.0 µg/mL respectively. The most destabilizing conditions were oxidizing and alkaline. The chromatograms confirmed no interference with the methadone signal.ConclusionsThe HPLC method has proved to be valid and reproducible for methadone quantification in a new oral solution with methylparaben and propylparaben. This assay is a rapid, simple and reliable technique that can be used in daily analysis and physicochemical stability studies.

Quantitative Determination of Related Substances in Formoterol Fumarate and Tiotropium in Tiomate Transcaps® Dry Powder Inhaler

Tiotropium (TIO) and formoterol fumarate (FF) combination in dry powder inhaler (DPI) dosage form used for treating asthma, bronchospasm, chronic bronchitis, emphysema and chronic obstructive pulmonary diseases. Aim to develop an analytical method for estimating emerging and advancing dry powder inhaler combination toward enhanced therapeutics for the estimation of related substances but for this it is foremost to have a sensitive, simple, robust and validated method therefore, a new reverse phase-high performance liquid chromatography (HPLC) method has been developed for the determination of related substances in FF and TIO DPI. The analytical method was performed on schimadzu HPLC with a quaternary pump, the separation achieved using BDS Hypersil C18 (250×4.6 mm, 5 μm) column with mobile phase consisting of sodium phosphate buffer pH 3.2 and acetonitrile 1.0 mL min-1 flow rate in the gradient elution. Diluent consists of a mixture of buffer pH 3.2 and acetonitrile in the ratio of (70:30) %v/v. 30°C column temperature and photodiode-array detection detector at a wavelength 240 nm. The run time was 50 min. The Retention time of FF and TIO was found to be at 7.8 and 10.3 min respectively. Both the analyte peaks were found to be free from interference. The method was validated as per the International Council on Harmonisation guidelines, the linearity was performed on 0.015 to 1.089 ppm for TIO and 0.01 to 0.728 ppm for FF concentration with correlation coefficient of 1,000. The precision and accuracy were also performed at the limit of quantification level were within the limits. Forced degradation study was also conducted. The recommended method for the related substance determination of FF and TIO is simple, selective, specific and precise. It also demonstrates the study of the degradation pattern. Moreover, the above developed related substance analytical method was applied to the bulk analysis and pharmaceutical dosage form for routine analysis and stability study.

FORCED INDICATING UPLC-DAD METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF APALUTAMIDE IN BULK AND IN PHARMACEUTICAL DOSAGE FORM

A new analytical method was developed for the estimation of apalutamide in bulk and its pharmaceutical formulation. The sensitive, précise and accurate method was developed by using Waters Acquity UPLC system equipped with quaternary gradient pump. The column used was Waters C18 150 X 2.1 mm X 1.7 µm and mobile phase was 0.2 % OPA buffer in water : acetonitrile in the ratio of 25:75 V/V. The buffer pH was maintained at 4.5. The fl ow rate of mobile phase was 0.5 mL min-1 and detection was at 272 nm by using PDA detector. The method was performed at ambient temperature. The retention time of the apalutamide was 1.27 min. The % RSD value in precision was >2 %. The accuracy of the method was found to be between 99.54 % - 100.01 %. The limit of detection and limit of quantifi cation values were found to be 0.14 µg mL-1 and 0.48 µg mL-1, respectively. The linearity concentration range was found to be 11.25 - 67.5 µg mL-1, it show wide linearity concentration range. The method was proved to have good robustness after changing parameters of fl ow rate, temperature and mobile phase composition. The method showed good ability towards different stress conditions of acidity, alkalinity, peroxide and UV-light. The method can be used for the routine analysis of apalutamide in bulk and its pharmaceutical dosage form by using UPLC.

New Validated Method for the Estimation of Pioglitazone and Rosiglitazone Using RP-HPLC

New, simple and economical high pressure liquid chromatography method has been developed for the simultaneous quantification of Pioglitazone and Rosiglitazone.By using Waters HPLC e-2695 quaternary pump with a PDA detector of 2998 instrument the chromatographic separation of Pioglitazone and Rosiglitazone was achieved on the column of Inertsil ODS (150x4.6mm, 3.5 µ) using an isocratic elution with a buffer containing 0.1percentformic acid and acetonitrile at a rate of 30:70 as a mobile phase with a flow rate of 1 ml/min at ambient temperature. A detector wavelength of 261 nm utilizing the PDA detector were given in the instrumental settings. The linearity was studied between the concentration range of 3-45 µg/ml of Pioglitazone and 1-15 µg/ml of Rosiglitazone were injected.The plotted calibration curves were linear with a regression coefficient of R2> 0.999, indicates that the linearity was with in the limit. As a part of method validation the parameters like specificity, linearity, accuracy, ruggedness, robustness were determined and the results were found to be within the allowable limit. The method developed was found to be applicable to routine analysis and to be used for the measurement of both active pharmaceutical ingredients (i.e, Pioglitazone and Rosiglitazone).Validation of the proposed method was carried out according to an International Conference on Harmonization (ICH) guidelines. Since, there is no HPLC method reported in the literature for the estimation of Pioglitazone and Rosiglitazone, there is a need to develop quantitative methods under different conditions to achieve improvement in specificity, selecivity etc.

DEVELOPMENT AND VALIDATION OF A STABILITY INDICATING RELATED SUBSTANCES OF TRANDOLAPRIL BY RP-HPLC AND ITS DEGRADATION

Objective: A validated stability-indicating RP-HPLC method for Trandolapril was developed by separating its related impurities. Methods: By using Waters HPLC e-2695 quaternary pump with a PDA detector of 2998 instrument the chromatographic separation of Trandolapril and its related impurities was achieved on the column of Agilent eclipse C18 (150x4.6 mm, 3.5 µ) using gradient elution with a buffer containing 0.1percent formic acid and acetonitrile as a mobile phase with a flow rate of 1 ml/min at ambient temperature. A detector wavelength of 213 nm utilizing the PDA detector were given in the instrumental settings. The linearity was studied between the concentration range of 4-60 µg/ml of Trandolapril and 0.5-7.5 µg/ml of imp-E, imp-A, imp-B and 0.7-10.5 µg/ml of imp-D were injected with a run time of 17 min. Validation of the proposed method was carried out according to an International Conference on Harmonization (ICH) guidelines. Results: LOD and LOQ for the Trandolapril and its impurities were established with respect to test concentration. The plotted calibration curves were linear with a regression coefficient of R2>0.999, indicates that the linearity was with in the limit. As a part of method validation the parameters like specificity, linearity, accuracy, ruggedness, robustness were determined and the results were found to be within the allowable limit. Conclusion: The method developed was found to be applicable to routine analysis and to be used for the measurement of active pharmaceutical ingredients (i. e, Trandolapril and its related impurities). Since, there is no HPLC method reported in the literature for the estimation of Trandolapril and its related impurities, there is a need to develop quantitative methods under different conditions to achieve improvement in specificity, selecivity etc.

NEW VALIDATED METHOD FOR THE ESTIMATION OF ALLANTOIN AND PERMETHRIN USIGN RP-HPLC IN BULK AND PHARMACEUTICAL DOSAGE FORM

Objective: Special, effective high pressure liquid chromatography method has been developed for the simultaneous quantification of Allantoin and Permethrin. Methods: By using Waters HPLC e-2695 quaternary pump with a PDA detector of 2998 instrument the chromatographic separation of Allantoin and Permethrin was achieved on the column of Symmetry C18 (150x4.6 mm, 3.5 µm) using an isocratic elution with a buffer containing 0.1percent ortho phosphoric acid and acetonitrile at a rate of 40:60 as a mobile phase with a flow rate of 1 ml/min at ambient temperature. A detector wavelength of 226 nm utilizing the PDA detector were given in the instrumental settings. The linearity was studied between the concentration range of 1-15 µg/ml of Allantoin and 25-375 µg/ml of Permethrin were injected with a run time of 6 min. As a part of method validation the parameters like specificity, linearity, accuracy, ruggedness, robustness were determined and the results were found to be within the allowable limit. Validation of the proposed method was carried out according to an International Conference on Harmonization (ICH) guidelines. Results: LOD and LOQ for the two active ingredients were established with respect to test concentration. The plotted calibration curves were linear with a regression coefficient of R2>0.999, indicates that the linearity was with in the limit. As a part of method validation the parameters like specificity, linearity, accuracy, ruggedness, robustness were determined and the results were found to be within the allowable limit. Conclusion: The method developed was found to be applicable to routine analysis and to be used for the measurement of both active pharmaceutical ingredients (i. e, Allantoin and Permethrin). Since, there is no HPLC method reported in the literature for the estimation of Allantoin and Permethrin, there is a need to develop quantitative methods under different conditions to achieve improvement in specificity, selecivity etc.

HPLC-DAD METHOD FOR DETECTION OF SOME PESTICIDE RESIDUES IN SOIL AND CROPS CULTIVATED IN BANAT COUNTY

High productivities and economic benefits in modern agriculture have been accomplished only by the use of pesticides. The introduction of these compounds in the food chain can be considered a risk for human health due to their toxicity. So, pesticides residues and their degradation products resulted by hydrolytic, photochemical and microbial processes in soil and cereals must be monitored and controlled. The aim of this research was to develop rapid and sensitive HPLC-DAD methods for residues detection of several pesticides, imidacloprid, amidosulfuron, bromoxynil and deltamethrin, from soil and some crops, maize, wheat and rape seeds, cultivated in Banat County-Moravita Zone. In order to perform the pesticides extraction, the samples were subjected to an ultrasonic technique, at 59 kHz and 30°C, during 30-45 min., using acetonitrile and acetonitrile-water mixture as solvents. Acetonitrile alone proved to be more appropriate for this purpose. The HPLC analyses were conducted on HPLC-DAD apparatus, Dionex Ultimate 3000 (Dionex Corp., USA), equipped with quaternary pump LPG 3400A, thermostat of columns TCC-3000 and a C-18 Acclaim® 120 Silica-reversed-phase (4.6x150 mm, 5 µm) column, using different compositions of acetonitrile-water as mobile phase. The presented HPLC - Dionex system proved to be very suitable for determination of pesticides residues in soil and crops.

Simultaneous separation of insulin and six therapeutic analogues on a mixed mode column: HPLC-UV method development and application

Human insulin and six most used therapeutic analogues are very similar in terms of retention on a reversed-phase column. Thus, the LC methods prescribed in the European Pharmacopoeia monographs for insulin and insulin analogues include many similar separation methods, which tend to be time consuming when separating individual products of insulins or are inadequate when handling a mixture. In this study, we present a simple, robust, versatile and accessible HPLC-UV separation method for identification and quantification of human insulin and its analogues in one run. The simultaneous separation and detection is possible by fine-tuning the mobile phase properties that affect the separation mechanism on a mixed mode column combining anion exchange and reversed-phase characteristics. Also developed was a simple and effective SPE sample cleaning procedure with insulin recoveries ranging from 80 to 100% for all analogues. On the other hand, the concentration of major excipients such as phenol and m-cresol fall below 1%. The two developed and validated separation methods differ in their compatibility with the use of a quaternary or binary pump, thus enabling sample characterisation independent of the HPLC solvent delivery system. The methods are compatible with the use of a mass spectrometric detector for an indisputable identification.