Reversed-phase Chromatographic Method Research Articles

Diversity and Activity of Bacteria Cultured from a Cup-The Sponge Calyx nicaeensis.

Marine sponges are well-known for hosting rich microbial communities. Sponges are the most prolific source of marine bioactive compounds, which are frequently synthesized by their associated microbiota. Calyx nicaeensis is an endemic Mediterranean sponge with scarce information regarding its (bioactive) secondary metabolites. East Mediterranean specimens of mesophotic C. nicaeensis have never been studied. Moreover, no research has inspected its associated bacteria. Thus, we studied the sponge's bacterial diversity and examined bacterial interspecific interactions in search of a promising antibacterial candidate. Such novel antimicrobial agents are needed since extensive antibiotic use leads to bacterial drug resistance. Bacteria cultivation yielded 90 operational taxonomic units (OTUs). A competition assay enabled the testing of interspecific interactions between the cultured OTUs. The highest-ranked antagonistic bacterium, identified as Paenisporosarcina indica (previously never found in marine or cold habitats), was mass cultured, extracted, and separated using size exclusion and reversed-phase chromatographic methods, guided by antibacterial activity. A pure compound was isolated and identified as 3-oxy-anteiso-C15-fatty acid-lichenysin. Five additional active compounds await final cleaning; however, they are lichenysins and surfactins. These are the first antibacterial compounds identified from either the C. nicaeensis sponge or P. indica bacterium. It also revealed that the genus Bacillus is not an exclusive producer of lichenysin and surfactin.

Validated chromatographic approach for determination of two ternary mixtures in newly approved formulations for helicobacter pylori eradication: assessment of greenness profile and content uniformity

A new, sensitive, and rapid isocratic reversed phase chromatographic method (RP-HPLC–UV) was developed for simultaneous separation of two newly co-formulated antiulcer mixtures; Amoxicillin, Vonoprazan and Clarithromycin [Mixture (I)], and Amoxicillin, Lansoprazole and Clarithromycin [Mixture (II)]. Analytical separation was performed using a Promosil C18 column and ultraviolet detection at 210 nm. The separation was achieved within only 8 min. For both mixtures, an aqueous solution, composed of (Acetonitrile: Methanol: 0. 2 M phosphoric acid) within ratio of (30: 30: 40) adjusted to final pH 3.0, was the mobile phase. This method was validated as per the International Conference on Harmonization guidelines. The linearity ranges of these proposed method of the (Mixture (I)) were 25.0–400.0 µg/mL Amoxicillin, 0.5–8.0 µg/mL Vonoprazan, and 12.5–200.0 µg/mL Clarithromycin. And the linearity ranges of the (Mixture (II)) were 10.0–300.0 µg/mL Amoxicillin, 0.3–9.0 µg/mL Lansoprazole and 5.0–150.0 µg/mL Clarithromycin. This method was firstly applied for effective separation of Amoxicillin, Vonoprazan and Clarithromycin [Mixture (I)]. It fulfilled good repeatability, sensitivity, and accuracy (R.S.D. < 2.0%). The mean recoveries of the analytes in their Tri-Pak formulations were acceptable. The greenness of the developed chromatographic methods was assessed using an Eco-scale method and it was applied for content uniformity testing as per the United States Pharmacopoeia (USP) and the acceptance value of Amoxicillin, in Mixture (I) was 2.88, the acceptance values for Amoxicillin, Lansoprazole in Mixture (II) were 2.592, 2.424, respectively.

Whiter and Greener RP-HPLC Method for Simultaneous Determination of Dorzolamide, Brinzolamide, and Timolol Using Isopropanol as a Sustainable Organic Solvent in the Mobile Phase

A sustainable reversed-phase chromatographic method has been developed and validated for the simultaneous determination of three active pharmaceutical ingredients, dorzolamide, brinzolamide, and timolol, used to treat glaucoma. The eco-friendly solvent isopropanol has been used as an organic mobile phase constituent. According to the Hansen space green solvent selection tool, isopropanol has a G score of 6.5, comparable to ethanol, which has a G score of 6.6. The mobile phase consists of isopropanol: aqueous sodium acetate buffer (0.1 M, pH 4.25) in the ratio of 10:90 (v/v). The flow rate was maintained at 1 mL/min. Dorzolamide and brinzolamide were detected at 254 nm, and timolol was detected at 295 nm. A high-purity silica with a polymeric C18 modification column (150 × 4.6 mm, 5 µm particle size) was used for this separation. The three compounds were eluted within 8 min. The method was validated according to ICH guidelines. The calibration curves were linear in the range of 20–70 µg/mL, 40–140 µg/mL, and 20–70 µg/mL for dorzolamide, brinzolamide, and timolol, respectively. The LODs were found to be 1.61 µg/mL, 1.60 µg/mL, and 3.16 µg/mL for dorzolamide, brinzolamide, and timolol, respectively. Good accuracy and precision were obtained for the three compounds. The greenness and whiteness of the method were indicated using the AGREE, ChlorTox, and RGB12 tools.

High-throughput method for Peptide mapping and Amino acid sequencing for Calcitonin Salmon in Calcitonin Salmon injection using Ultra High Performance Liquid Chromatography – High Resolution Mass Spectrometry (UHPLC-HRMS) with the application of Bioinformatic tools

BackgroundTandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic peptide drugs, and peptide drug products including side chain modifications in the Peptide drugs. This article explains a step-by-step guide to developing a high-throughput method using high resolution mass spectrometry for characterization of Calcitonin Salmon injection containing high proportion of UV-active excipients. MethodsThe major challenge in the method development of Amino acid sequencing and Peptide mapping was presence of phenol in drug product. Phenol is a UV-active excipient and reacts with both Dithiothreitol (DTT) and Trypsin. Hence Calcitonin Salmon was extracted from the Calcitonin Salmon injection using solid phase extraction after the extraction, Amino acid sequencing and peptide mapping study was performed. Upon incubation of Calcitonin Salmon with Trypsin and DTT, digested fragments were generated which were separated by mass compatible reverse phase chromatography and the molecular mass of each fragment was determined using HRMS. ResultsA reverse phase chromatographic method was developed using UHPLC-HRMS for the determination of direct mass, peptide mapping and to determine the amino acid sequencing in the Calcitonin Salmon injection. The method was found Specific and fragments after trypsin digest are well resolved from each other and the molecular mass of each fragment was determined using HRMS. Sequencing was performed using automated identification of b and y ions annotation and identifications based on MS/MS spectra using Biopharma finder and Proteome discoverer software. ConclusionUsing this approach 100% protein coverage was obtained and protein was identified as Calcitonin Salmon and the observed masses of tryptic digest of peptide was found similar with theoretical masses. The method can be used for both UV and MS based Peptide mapping and whereas the UV based peptide mapping method can be used as identification test for Calcitonin Salmon drug substance and drug product in quality control.

A Strategy for assessing peak purity of pharmaceutical peptides in reversed-phase chromatography methods using two-dimensional liquid chromatography coupled to mass spectrometry. Part II: Development of second-dimension gradient conditions

The importance of therapeutic peptides continues to increase in the marketplace for treating a range of diseases including diabetes and obesity. Quality control analyses for these pharmaceutical ingredients usually depends on reversed-phase liquid chromatography, and it is critically important to ensure that no impurities coelute with the target peptide at levels that would compromise the safety or effectiveness of the drug products. This can be challenging due to the broad range of properties of impurities that can be present on one hand (e.g., amino acid substitutions, chain cleavages, etc.), and the similarity of other impurities on the other hand (e.g., d-/l-isomers). Two-dimensional liquid chromatography (2D-LC) is a powerful analytical tool that is well suited to address this particular problem; the first dimension can be used to detect impurities over a broad range in properties, while the second dimension can be used to focus specifically on those species that might coelute with the target peptide in the first dimension. While hundreds of papers have been published on the use of 2D-LC for proteomics applications, there are very few papers that have focused on its use for characterisation of therapeutic peptides. This paper is the second in a two-part series. In Part I of the series, we studied several different column / mobile phase combinations that could be useful in 2D-LC separations of therapeutic peptides, with a focus on selectivity, peak shape, and complementarity to other combinations, particularly for isomeric peptides under mass spectrometry-friendly conditions (i.e., volatile buffers). In this second part in the series, we describe a strategy to derive second-dimension (2D) gradient conditions that both, ensure elution from the 2D column, and increase the likelihood of resolving peptides with very similar properties. We find that a two-step process yields conditions that place the target peptide in the middle of the 2D chromatogram. This process begins with two scouting gradient elution conditions in the second dimension of a 2D-LC system, followed by building and refining a retention model for the target peptide using a third separation. The process is shown to be generically useful by developing methods for four model peptides, and application to a sample of degraded model peptide to demonstrate its utility for resolving impurities in a real sample.

A strategy for assessing peak purity of pharmaceutical peptides in reversed-phase chromatography methods using two-dimensional liquid chromatography coupled to mass spectrometry. Part I: Selection of columns and mobile phases

The current study describes the development of a 2D-LC-MS-based strategy for assessing main peak purity in the analysis of pharmaceutical peptides. The focus is on 2D-LC using reversed-phase (RP) separations in both dimensions, and particularly peptide isomer selectivity, since compounds with the same mass to charge ratio are not readily differentiated by mass spectrometry and therefore must be separated chromatographically. Initially, 30 column / mobile phase combinations were evaluated for both general separation performance (i.e., selectivity and peak shape) and isomer selectivity using forcibly degraded peptide samples and mixtures of synthetic diastereomers. A ranking of more than 300 UV and MS chromatograms suggests that when developing a new method, screening a set of four columns and four volatile mobile phases with differing characteristics should be adequate to both cover the selectivity space, and yield good separation performance. When 2D-LC-MS is to be used to evaluate peak purity for a new method, our results show that a second-dimension separation comprising a C8/C18 column possessing no ionic functionality, and an acetic acid / ammonium acetate mobile phase buffered at pH 5, provides good selectivity at 25 °C for peptide isomers with a MW <10 kDa.Retention data for 29 diverse peptides (1 < MW < 14 kDa, 3.7 < pI < 12.5) measured in this study using a variety of column and mobile phase conditions (i.e., 30 in total) are consistent with the classification of these various chromatographic conditions using the previously reported Peptide RPC Column Characterisation Protocol. For the investigated peptides trifluoroacetic acid was found to reduce selectivity differences between columns of diverse properties, probably due to its potential to form ion-pairs with peptides. Trifluoroacetic acid often improves peak shape for very large peptides (i.e. MW > 10 kDa). In the current dataset which also contain smaller peptides it received the highest ranking for 40% of the column and mobile phase combinations due to better selectivity and/or peak shape.The reported work here constitutes part one of a series of two papers. The second paper focuses on the use of retention modelling for rapid and accurate selection of the shallow gradients (i.e., << 1% ACN/min) required to obtain sufficient peptide isomer retention and separation in the second dimension. The overall results presented in this series of papers provides the guidance needed to develop a 2D-LC-MS method from start to finish for the analysis of main peak purity of therapeutic peptides.

Batroxin I: A Novel Bradykinin-Potentiating Peptide with Cytotoxic Activity Isolated from Bothrops atrox Snake Venom

Venom peptides are interesting molecular models for the development of biotechnological strategies applicable in generating therapeutic agents and/or experimental tools for basic and applied research. The present study aimed to search for peptides from Bothrops atrox snake venom with anticancer potential activity against HepG2 liver tumor cell line, determine their cytotoxic action, and analyze the structure–function relationship. The novel peptide Batroxin I (M.W. 1.38 kDa) was isolated by molecular exclusion and reversed phase chromatography methods. The Batroxin I presented a selective cytotoxicity towards tumor cells, reducing the viability of HepG2 cells by 94.6% with IC50 of 0.72 μg/mL, and showing a low toxicity against peripheral blood mononuclear cells. Analysis of the apoptotic and necrotic peptide effects revealed that it induced apoptosis by intrinsic pathway activation. The amino acid sequence of Batroxin I was determined by de novo sequencing as < EKWPRPDAPIPP (where < E = pyroglutamic acid); hence, it is an unpublished peptide that belongs to the class of bradykinin-enhancing peptides and cell penetration peptide. This is one of the first reports on the cytotoxic antitumor activity of a bradykinin-enhancing peptide. Our results indicate that this peptide could serve not only as a template for the development of new drugs, but also as an adjuvant to less effective marketed drugs to treat cancer and other diseases.

The use of reversed-phase chromatography methods for the determination of di-tert-butylphenols in biological fluids

The use of reversed-phase chromatography methods for the determination of di-tert-butylphenols in biological fluids

Method development and validation of spray content uniformity for ipratropium bromide and budesonide in nasal spray

Ipratropium bromide and Budesonide is combined Pharmaceutical Nasal dosage form. The label claim of this combined dosage form is 21 mcg of Ipratropium Bromide and64 mcg of Budesonide per Spray. It is for treatment of allergic rhinitis. Spray content uniformity is in-vitro investigational test which provides idea of uniformity of product throughout product life cycle. The present study aimed to validate High Performance Liquid Chromatography method for determination of spray content uniformity of these analytes. This validation study performed as per guidelines of International Conference on Harmonization and covers Precision, Linearity, Accuracy, Robustness, Ruggedness, stability of analytical solution and Specificity. The reversed phase chromatographic method uses a mobile phase prepared by mixing Buffer containing Sodium dihydrogen orthophosphate with Acetonitrile, Zorb ax CN150 mm column, 1.5 ml per min flow rate, 220 nm wavelength, 25°C column oven and 50 μL injection volume. The method found linear over the range of 50% to 150% of target concentration with correlation coefficient of 0.9997 for Ipratropium bromide and 0.9999 for Budesonide. The recoveries obtained were 103.1% - 103.8% for Ipratropium bromide and 97.0% - 100.1% for Budesonide. %Related Standard Deviation found well within the acceptance criteria for precision. No interference found at the retention time of analyte peaks in blank, placebo and degradation conditions describe specificity of method. The method showed a successful application for determination of Ipratropium bromide and Budesonide in Nasal spray pharmaceutical formulation.

Number of Detected Proteins as the Function of the Sensitivity of Proteomic Technology in Human Liver Cells.

The main goal of the Russian part of C-HPP is to detect and functionally annotate missing proteins (PE2-PE4) encoded by human chromosome 18. To achieve this goal, it is necessary to use the most sensitive methods of analysis. However, identifying such proteins in a complex biological mixture using mass spectrometry (MS)-based methods is difficult due to the insufficient sensitivity of proteomic analysis methods. A possible solution to the problem is the pre-fractionation of a complex biological sample at the sample preparation stage. This study aims to measure the detection limit of SRM SIS analysis using a standard set of UPS1 proteins and find a way to enhance the sensitivity of the analysis and to, detect proteins encoded by the human chromosome 18 in liver tissue samples, and compare the data with transcriptomic analysis of the same samples. Mass spectrometry, data-dependent acquisition, selected reaction monitoring, highperformance liquid chromatography, data-dependent acquisition in combination with pre-fractionation by alkaline reversed-phase chromatography, selected reaction monitoring in combination with prefractionation by alkaline reversed-phase chromatography methods were used in this study. The results revealed that 100% of UPS1 proteins in a mixture could only be identified at a concentration of at least 10-9 М. The decrease in concentration leads to protein losses associated with technology sensitivity, and no UPS1 protein is detected at a concentration of 10-13 М. Therefore, the two-dimensional fractionation of samples was applied to improve sensitivity. The human liver tissue was examined by selected reaction monitoring and shotgun methods of MS analysis using onedimensional and two-dimensional fractionation to identify the proteins encoded by human chromosome 18. A total of 134 proteins were identified. The overlap between proteomic and transcriptomic data in human liver tissue was ~50%. The sample concentration technique is well suited for a standard UPS1 system that is not contaminated with a complex biological sample. However, it is not suitable for use with a complex biological protein mixture. Thus, it is necessary to develop more sophisticated fractionation systems for the detection of all low-copy proteins. This weak convergence is due to the low sensitivity of proteomic technology compared to transcriptomic approaches. Also, total mRNA was used to perform RNA-seq analysis, but not all detected mRNA molecules could be translated into proteins. This introduces additional uncertainty in the data; in the future, we plan to study only translated mRNA molecules-the translatome. Data is available via ProteomeXchange with identifier PXD026997.

Determination of lipid content and stability in lipid nanoparticles using ultra high-performance liquid chromatography in combination with a Corona Charged Aerosol Detector.

For many years, lipid nanoparticles (LNPs) have been used as delivery vehicles for various payloads (especially various oligonucleotides and mRNA), finding numerous applications in drug and vaccine development. LNP stability and bilayer fluidity are determined by the identities and the amounts of the various lipids employed in the formulation and LNP efficacy is determined in large part by the lipid composition which usually contains a cationic lipid, a PEG‐lipid conjugate, cholesterol, and a zwitterionic helper phospholipid. Analytical methods developed for LNP characterization must be able to determine not only the identity and content of each individual lipid component (i.e., the parent lipids), but also the associated impurities and degradants. In this work, we describe an efficient and sensitive reversed‐phase chromatographic method with charged aerosol detection (CAD) suitable for this purpose. Sample preparation diluent and mobile phase pH conditions are critical and have been optimized for the lipids of interest. This method was validated for its linearity, accuracy, precision, and specificity for lipid analysis to support process and formulation development for new drugs and vaccines.

UHPLC analysis and in vitro biological activities of Areca triandra Roxb. ex Buch.-Ham.: a substitute of areca nut

Areca triandra Roxb. ex Buch.-Ham. (Arecaceae) distributed in Assam, North Bengal plains, Meghalaya andaman and Nicobar Islands is used as a substitute for A. catechu L. (Areca Nut). A. catechu is widely used as masticator in South Asia. The present study was conducted to find out the phenolic acids present in A. triandra and potential of it in inhibiting the reactive oxygen species (ROS). Plant extracts obtained from leaf and nut were analyzed qualitatively, quantitatively and phenolic acids were identified and quantified using reverse phase chromatography method developed on through Hypersil Gold aQ C18 column in Thermo Scientific Dionex Ultimate 3000 UHPLC. Three antioxidant assays viz. DPPH, ABTS and reducing power assay were performed to assess the potential of A. triandra as an antioxidant. Antifungal susceptibility test was performed by Clinical Laboratory Standard Institute (CLSI) recommended broth microdilution assay and disk diffusion susceptibility assay using Tinidazole as standard. Total catechin content was 19.51% of total phenolic content in NME. The antioxidant property was observed in NME only for DPPH, ABTS and RPA assays. In antifungal activity, minimum effective concentration (MEC) was observed in NME at the concentration of 1.0mg/ml and above whereas in LME, no MEC was observed up to tested concentrations. A. triandra possesses high catechin content and has promising potential as an antioxidant agent.

Determination of Chromatographic Assay of Deferasirox in Bulk and Pharmaceutical Dosage Form

Deferasirox belongs to the class antidote, which is not official in any pharmacopoeia. But only a few reported studies were availableemploying RP-HPLC technique. To validate the analytical process, we chose bulk powder and tablets of different intensity deferasirox tablets.The objective of the research or analysis is to develop a sensitive reverse phase chromatography method and validate quantitative perseverance of deferasirox in bulk and marketed formulation. A Simple, precise, accurate, economical and reliable UVspectrometric method has been developed toestimate deferasirox bulk and its tablet dosage form. The method was validated as per the international conference on harmonization (ICHQ2AR1) the validated dosage type was tested for device suitability, precision, linearity, specificity, robustness, and ruggedness.Statistical analysis proved that the method is repeatable and specific for the determination of the said drug. The established procedure or technique was done using Inertsil ODS 3V (C18 250mm X 4.6mm, 5µm) column oven temperature 40°C and sample temperature is 8°C with a flow rate of 1.5mL/min. By using HPLC waters PU-2487 pump and photodiode array detector at 245nm. The separation was carried out using a mobile phase consisting of amixture of sodium di hydrogen phosphate monohydrate buffer (pH3.0) and acetonitrile in the ratio of 45:55. The retention time of Deferasirox peak was considered to be about 7.5 min and the total run time was found to be 20 minutes.The calibration plot gave linear relationship over the concentration range of 6.28- 811.480 µg/ml The average percent recovery was proved to be 96.1,97.6&103.The correlation coefficient was found to be 0.999. The relative standard deviation (R.S.D) was found <2.0% for the UV-spectroscopic RP-HPLC method. The percent assessment of the drug was around 100% indicating the accuracy of the procedure. The suggested techniques were further confirmed and employed for the testing of the drug in tablet preparation.

Detection and Isolation of Emetic Bacillus cereus Toxin Cereulide by Reversed Phase Chromatography.

The emetic toxin cereulide is a 1.2 kDa dodecadepsipeptide produced by the food pathogen Bacillus cereus. As cereulide poses a serious health risk to humans, sensitive and specific detection, as well as toxin purification and quantification, methods are of utmost importance. Recently, a stable isotope dilution assay tandem mass spectrometry (SIDA–MS/MS)-based method has been described, and an method for the quantitation of cereulide in foods was established by the International Organization for Standardization (ISO). However, although this SIDA–MS/MS method is highly accurate, the sophisticated high-end MS equipment required for such measurements limits the method’s suitability for microbiological and molecular research. Thus, we aimed to develop a method for cereulide toxin detection and isolation using equipment commonly available in microbiological and biochemical research laboratories. Reproducible detection and relative quantification of cereulide was achieved, employing reversed phase chromatography (RPC). Chromatographic signals were cross validated by ultraperformance liquid chromatography–mass spectrometry (UPLC–MS/MS). The specificity of the RPC method was tested using a test panel of strains that included non-emetic representatives of the B. cereus group, emetic B. cereus strains, and cereulide-deficient isogenic mutants. In summary, the new method represents a robust, economical, and easily accessible research tool that complements existing diagnostics for the detection and quantification of cereulide.

Assessment of solid phase microextraction as a sample preparation tool for untargeted analysis of brain tissue using liquid chromatography-mass spectrometry

This work presents an evaluation of solid-phase microextraction (SPME) SPME in combination with liquid chromatography-high resolution mass spectrometry (LC-HRMS) as an analytical approach for untargeted brain analysis. The study included a characterization of the metabolite coverage provided by C18, mixed-mode (MM, with benzene sulfonic acid and C18 functionalities), and hydrophilic lipophilic balanced (HLB) particles as sorbents in SPME coatings after extraction from cow brain homogenate at static conditions. The effects of desorption solvent, extraction time, and chromatographic modes on the metabolite features detected were investigated. Method precision and absolute matrix effects were also assessed. Among the main findings of this work, it was observed that all three tested coating chemistries were able to provide comparable brain tissue information. HLB provided higher responses for polar metabolites; however, as these fibers were prepared in-house, higher inter-fiber relative standard deviations were also observed. C18 and HLB coatings offered similar responses with respect to lipid-related features, whereas MM and C18 provided the best results in terms of method precision. Our results also showed that the use of methanol is essential for effective desorption of non-polar metabolites. Using a reversed-phase chromatographic method, an average of 800 and 1200 brain metabolite features detected in positive and negative modes, respectively, met inter-fibre RSD values below 30% (n=4) after removal of fibre and solvent artefacts from the associated datasets. For features detected using a lipidomics method, a total of 900 and 1800 features detected using C18 fibers in positive and negative mode, respectively, met the same criteria. In terms of absolute matrix effects, the majority of the model metabolites tested showed values between 80 and 120%, which are within the acceptable range. Overall, the findings of this work lay the foundation for further optimization of parameters for SPME-LC-HRMS methods suitable for in vivo and ex vivo brain (and other tissue) untargeted studies, and support the applicability of this approach for non-destructive tissue metabolomics.

A methodology employing retention modeling for achieving control space in liquid chromatography method development using quality by design approach

This study reports the application of retention modeling and quality by design practices for reverse-phase liquid chromatographic method development of a new chemical entity. Prior to the retention modeling, preliminary screening experiments were performed for the selection of stationary phase, organic modifiers, and method parameters. Based on the results of preliminary method conditions, tG-T (gradient time - temperature) 2-D modeling with 4 input runs, and tG-T-tc (gradient time-temperature-ternary composition) 3-D modeling with 12 input runs were designed to build a model for achieving the optimized separation. Modeling of reverse phase separations was based on the measurement of both retention times and peak areas. A design space with appropriate input variables and control strategy was established prior to optimization and robustness evaluation following the quality by design framework. DryLabⓇ was used to predict the optimized gradient profile and separation temperature. The robustness evaluation was carried out using the multiple factors at a time approach and the control space was established. The interdependence of control space and the control strategy was demonstrated by evaluating method robustness using two levels of system suitability criteria. The predictive accuracy of the retention modeling was established through experimental verification of the in-silico predictions. The quality by design based method development approach demonstrated the in-silico optimization as an integral component of reverse-phase chromatographic method development to evaluate the interplay of factors such as organic modifiers, separation temperature and gradient time, which greatly integrated and enhanced method robustness during method development.

Development and validation of simultaneous estimation of drugs in combination from pharmaceutical formulation by RP-HPLC method

Metformin is recommended as a first-line agent for monotherapy and combination therapy for patients with type 2 diabetes mellitus (T2DM). Patients whose glycaemic control deteriorates over time with metformin monotherapy will require additional anti-diabetic medication. The development of HPLC method for simultaneous estimation of anti-diabetic drugs in combination from solid dosage form by HPLC method. To validate the developed HPLC method as per ICH guidelines. The System suitability test ,Capacity factor, Tailing factor, Resolution, Selectivity, Separation factor, Theoretical plates, Regression coefficient, STD for intercept, LOD (limit of detection), LOQ (limit of quantification, Repeatability, Precision studies (Intra-day and Interday/Intermediate), Linearity/Calibration studies, Robustness, Force degradation/Stability indicating studies, Specificity, Drug recovery/accuracy studies are performed. The system suitability test performed for saxagliptin and metformin hydrochloride has achieved all guideline criteria; including tailing factor (T), separation factors (α), theoretical plates (N), capacity factor (k’), resolution (R) and RSD (%), force degradation studies were also performed for both these drugs. So combinedly we concluded that the proposed reverse phase chromatographic (RP-HPLC) analytical method for the simultaneous estimation in both bulk and tablet formulation have complied the ICH and US-FDA guidelines.

Analytical QbD Approach for Development and Validation of RP-High Performance Liquid Chromatography Method for Determination of Tofisopam in Pure Form and Tablets

A novel, simple, optimized reversed-phase chromatography method for assay of Tofisopam in pure and tablet form is developed. The experimental trial was by Box Behnken design using the Design Expert® software 10 version. The attributes selected were peak symmetry, number of theoretical, and peak purity. The predicted data satisfied with actual experimental data. The optimized chromatographic conditions required a quaternary pump with a mobile phase of Water: Acetonitrile 25:75 v/v at 1 mL/min, oven temperature at 25oC at 310 nm using C18(250 × 4.6 mm Id, 5μm) column and PDA detector with a run time of 5 min. The method was validated for linearity, precision, accuracy, and specificity. The method produced a linear response over a concentration range of 4–24 ppm with an overall average accuracy of 99.98%. The method was robust, reproducible, and specific with respect to the retention time of tofisopam.

Development and validation of HPLC assay method for marbofloxacin determination in veterinary chewable tablets

The present study aimed to develop and validate an analytical method for determination of marbofloxacin (MAR) in veterinary chewable tablets. The isocratic reversed-phase chromatographic method was developed and validated using a Vertisep®, RP C18 column (150 mm × 4.6 mm, 5.0 μm). The mobile phase was composed of water–acetonitrile (55:45, v/v) with pH adjusted to 3.0 with ortho-phosphoric acid and a flow rate set at 0.4 mL/min. The proposed method was validated for linearity in a concentration range of 2.5 to 17.5 μg/mL with a correlation coefficient of 0.99991. The mean content of MAR found in chewable tablets was 104.40% with RSD below 2%. The accuracy expressed as average recovery of the proposed method was 98.74%, and the precision expressed as relative standard deviation among repeated analysis was 0.55%. The method has adequate sensitivity with detection and quantitation limits of 0.25 and 0.81 μg/mL, respectively. Based on the presented results and according to the ICH and AOAC guidelines on validation of analytical methods, the proposed method was considered precise, accurate with adequate sensitivity, and robust in the MAR quantitative analysis. Therefore, the method can be used in the quality control of chewable veterinary tablets containing MAR.

Molybdenum and lanthanum as alternate burn-up monitors – development of chromatographic and mass spectrometric methods for determination of atom percent fission

Abstract A rapid high performance liquid chromatography (HPLC) and thermal ionisation mass spectrometric (TIMS) methods have been developed for the separation and estimation of fission product elements molybdenum and lanthanum for the burn-up measurements on the dissolver solution of Indian pressurised heavy water reactor (PHWR) spent fuel. Reverse phase chromatography method was developed to separate molybdenum from dissolver solution using mandelic acid as mobile phase and a dynamic ion exchange chromatography technique was used for the separation of lanthanum as well as neodymium from a dissolver solution. Sample loading methods which resulted in enhanced ionisation efficiency have been developed for the TIMS analysis of HPLC separated molybdenum and lanthanum fractions. Ascorbic acid mixed with silicic acid in HCl medium was used for loading the molybdenum on to a rhenium filament to obtain stable and intense ion beam. A novel sample loading method for lanthanum in which a mixture of graphite + boric acid (H3BO3) + silica gel was employed to achieve enhanced and steady ion beam formation of LaO+. Concentrations of species of interest were determined employing suitable spikes by isotope dilution mass spectrometry (IDMS) method. The developed methods were adopted for PHWR dissolver solution to establish molybdenum and lanthanum as alternate burn-up monitors. The burn-up data obtained were compared with the well established method of neodymium as fission product monitor. This is a first study of its kind where the data obtained by using molybdenum and lanthanum as fission product monitors were compared with that obtained by Nd-148 method.