This document describes the development and validation of an RP-HPLC method for the simultaneous estimation of anti-tuberculosis drugs isoniazid, rifampicin, pyrazinamide, and ethambutol in human plasma. It provides background on tuberculosis and the common drugs used to treat it. The document reviews several literature methods for analyzing these drugs and discusses the drug profiles. It states that the objective is to develop a sensitive analytical method to quantitatively determine the drugs and metabolites in biological fluids to evaluate pharmacokinetics and pharmacodynamics.
This document discusses Vierordt's method, a technique for simultaneous multicomponent analysis using UV-VIS spectrometry. It involves measuring absorbances at multiple wavelengths to generate equations that can be solved to determine the concentrations of each component in a mixture. The key steps include measuring calibration curves for each component at different wavelengths, measuring the sample mixture absorbances, and simultaneously solving the equations derived from Beer's law. Advantages include avoiding separation techniques and allowing simultaneous analysis, while requirements include components having distinct absorption profiles and obeying Beer's law. Applications described include pharmaceutical drug analysis using this spectroscopic method.
This document discusses nano liquid chromatography. It begins by describing different types of liquid chromatography techniques including rapid resolution liquid chromatography, ultra performance liquid chromatography, ultra fast liquid chromatography, and nano liquid chromatography. It then focuses on nano liquid chromatography, explaining that it uses capillary columns with diameters of 75 micrometers or less and flow rates between 10-700 nanoliters per minute. The document outlines the advantages of nano LC including reduced solvent usage and improved sensitivity. It also reviews the instrumentation involved including pumps, injection systems, columns, and detectors such as diode array detection. Finally, it discusses applications of nano LC in fields like food analysis and proteomics and concludes by noting its increasing use.
DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATIO...rahul ampati
This document describes the development and validation of an RP-HPLC method for the simultaneous determination of ramipril and amlodipine in tablets. The method utilizes a gradient elution with a C18 column, mobile phase of acetonitrile and sodium perchlorate buffer, and UV detection. The method was validated per ICH guidelines and showed good linearity, accuracy, precision, specificity and robustness. Forced degradation studies demonstrated the method can separate ramipril, amlodipine and their degradation products. The method was successfully applied to determine the content of ramipril and amlodipine in three tablet batches.
CE-MS is an analytical technique that combines capillary electrophoresis (CE) and mass spectrometry (MS). CE separates ions based on their charge and size, while MS identifies molecules based on their mass-to-charge ratio. CE-MS provides high separation efficiency, molecular mass information, and can analyze small sample volumes at high speeds. It has applications in proteomics, biomolecule analysis, and clinical medicine. Challenges include developing interfaces between CE and MS that maintain separation efficiency and sensitivity.
This document discusses analytical method development for pharmaceutical drug substances and products. It describes the importance of method development and outlines the key steps involved, including collecting sample information, selecting the chromatographic technique, stationary and mobile phases. It also discusses sources of impurities in drugs, control and qualification of impurities according to ICH guidelines, and pharmacopeial and ICH quality guidelines for impurity testing and thresholds.
Bioanalytical method development and validation .Shubham Bora
1) A bioanalytical method was developed and validated for the quantification of levodopa and carbidopa in rat plasma using LC-MS/MS. Derivatization and ion-pairing chromatography were used to improve the chromatographic retention of the polar analytes.
2) The method was fully validated as per FDA guidelines and demonstrated selectivity, linearity, accuracy, precision, recovery, matrix effects and stability in accordance with acceptance criteria.
3) The validated method was successfully applied to support toxicokinetic studies of levodopa and carbidopa in rats.
This document discusses Vierordt's method, a technique for simultaneous multicomponent analysis using UV-VIS spectrometry. It involves measuring absorbances at multiple wavelengths to generate equations that can be solved to determine the concentrations of each component in a mixture. The key steps include measuring calibration curves for each component at different wavelengths, measuring the sample mixture absorbances, and simultaneously solving the equations derived from Beer's law. Advantages include avoiding separation techniques and allowing simultaneous analysis, while requirements include components having distinct absorption profiles and obeying Beer's law. Applications described include pharmaceutical drug analysis using this spectroscopic method.
This document discusses nano liquid chromatography. It begins by describing different types of liquid chromatography techniques including rapid resolution liquid chromatography, ultra performance liquid chromatography, ultra fast liquid chromatography, and nano liquid chromatography. It then focuses on nano liquid chromatography, explaining that it uses capillary columns with diameters of 75 micrometers or less and flow rates between 10-700 nanoliters per minute. The document outlines the advantages of nano LC including reduced solvent usage and improved sensitivity. It also reviews the instrumentation involved including pumps, injection systems, columns, and detectors such as diode array detection. Finally, it discusses applications of nano LC in fields like food analysis and proteomics and concludes by noting its increasing use.
DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATIO...rahul ampati
This document describes the development and validation of an RP-HPLC method for the simultaneous determination of ramipril and amlodipine in tablets. The method utilizes a gradient elution with a C18 column, mobile phase of acetonitrile and sodium perchlorate buffer, and UV detection. The method was validated per ICH guidelines and showed good linearity, accuracy, precision, specificity and robustness. Forced degradation studies demonstrated the method can separate ramipril, amlodipine and their degradation products. The method was successfully applied to determine the content of ramipril and amlodipine in three tablet batches.
CE-MS is an analytical technique that combines capillary electrophoresis (CE) and mass spectrometry (MS). CE separates ions based on their charge and size, while MS identifies molecules based on their mass-to-charge ratio. CE-MS provides high separation efficiency, molecular mass information, and can analyze small sample volumes at high speeds. It has applications in proteomics, biomolecule analysis, and clinical medicine. Challenges include developing interfaces between CE and MS that maintain separation efficiency and sensitivity.
This document discusses analytical method development for pharmaceutical drug substances and products. It describes the importance of method development and outlines the key steps involved, including collecting sample information, selecting the chromatographic technique, stationary and mobile phases. It also discusses sources of impurities in drugs, control and qualification of impurities according to ICH guidelines, and pharmacopeial and ICH quality guidelines for impurity testing and thresholds.
Bioanalytical method development and validation .Shubham Bora
1) A bioanalytical method was developed and validated for the quantification of levodopa and carbidopa in rat plasma using LC-MS/MS. Derivatization and ion-pairing chromatography were used to improve the chromatographic retention of the polar analytes.
2) The method was fully validated as per FDA guidelines and demonstrated selectivity, linearity, accuracy, precision, recovery, matrix effects and stability in accordance with acceptance criteria.
3) The validated method was successfully applied to support toxicokinetic studies of levodopa and carbidopa in rats.
Related Substances-Method Validation-PPT_slideBhanu Prakash N
This document provides an overview of analytical method validation. It defines validation as demonstrating a method is suitable for its intended purpose. Key validation characteristics discussed include precision, accuracy, specificity, linearity, range, detection limit, quantitation limit, ruggedness and robustness. The document describes the methodology for evaluating each characteristic, such as spiking known concentrations of analytes and establishing acceptance criteria. It emphasizes that validation confirms a method consistently produces results meeting pre-defined standards of quality.
Stability indicating rp hplc method development and validation for simultaneo...Rajasekhar
The document describes the development and validation of a stability-indicating RP-HPLC method for the simultaneous estimation of levodropropizine and chlorpheniramine maleate in bulk and pharmaceutical dosage forms. The method utilizes a C18 column with a mobile phase of 45% 0.1% orthophosphoric acid and 55% acetonitrile at a flow rate of 1 mL/min. The method was validated for parameters such as linearity, accuracy, precision, specificity, limit of detection, limit of quantification and robustness according to ICH guidelines. Forced degradation studies were also performed to demonstrate the stability-indicating ability of the developed method.
This document discusses various reagents used in pharmaceutical analysis including PDAB, Folin Ciocalteau, and MBTH reagents. It provides details on the principles, mechanisms, procedures, examples, and applications of each reagent. PDAB is used to detect primary amine groups via a colorimetric reaction. Folin Ciocalteau is used to detect phenols and reduces tungstate-molybdate to form a blue complex. MBTH forms colored complexes with aldehydes, phenols, and amines through oxidative coupling and can be used to analyze samples containing these functional groups. The document concludes that optimizing reagent volume and concentration allows these reagents to be successfully used to quantify drugs in pharmaceutical formulations.
USFDA guidelines for bioanalytical method validationbhatiaji123
The document discusses guidelines for bioanalytical method validation from the USFDA. It describes key parameters that must be validated for a bioanalytical method, including selectivity, accuracy, precision, recovery, calibration curves, sensitivity, reproducibility and stability. Accuracy and precision are determined by analyzing quality control samples in replicates across multiple runs. Recovery experiments compare extracted samples to unextracted standards. A calibration curve consisting of multiple concentrations over the expected range must be precise and reproducible.
The document discusses hyphenated techniques, specifically liquid chromatography-nuclear magnetic resonance (LC-NMR). It begins by introducing hyphenated techniques as the combination of two analytical methods, usually a separation technique coupled with a spectroscopic technique. It then describes LC-NMR in more detail, explaining how it works to separate components with liquid chromatography and then uses NMR for identification. Key aspects covered include instrumentation, modes of data acquisition (continuous flow, stopped flow, time sliced), and advantages such as automation, reproducibility, and simultaneous separation and quantification.
This document discusses HPLC columns used for separation of compounds. It begins with an introduction describing typical HPLC column dimensions and materials. It then covers different types of columns based on scale of preparation such as analytical and preparative columns. The main types of columns based on mode of operation are reversed phase and normal phase. The document explains the chemistry and mechanisms involved in reversed phase and normal phase HPLC columns. It also discusses column efficiency, the Van Deemter equation, and the Lx table in USP which provides standards for HPLC columns based on particle size and chemical compounds.
This document presents information on preparative high pressure liquid chromatography. It begins with an introduction to chromatography and classification of column chromatographic methods. It then discusses the differences between analytical and preparative HPLC and the objectives, instrumentation, method development, applications, and commercially available instruments for preparative HPLC. The instrumentation section describes the major components of a preparative HPLC system including the solvent reservoir, pump, injector, columns, detectors, fraction collector and more. Method development and optimization factors like mobile phase selection, temperature, retention, selectivity, and column overloading techniques are also covered.
This document provides an overview of high performance liquid chromatography (HPLC). It begins by defining HPLC and explaining that it uses high pressure to pump the mobile phase, yielding faster separation than traditional column chromatography. The document then discusses the basic principles of chromatography and liquid chromatography. It provides details on the types of HPLC based on mode of separation, principle of separation, elution technique, scale of operation, and type of analysis. The key components of an HPLC instrument are described including the solvent reservoir, pump, injector, column, detectors, and data recording system. Various columns, stationary phases, and pumps used in HPLC are also outlined.
1) Ion pair chromatography is a type of column chromatography that uses ion pairing agents to neutralize charged analytes and allow their separation on a reversed-phase column.
2) By adding counter ions with the opposite charge to the mobile phase, ion pairs form between the counter ions and analytes, neutralizing their charge and increasing their hydrophobicity.
3) The use of ion-pairing reagents as mobile phase additives allows the separation of ionic and highly polar substances that cannot otherwise be separated by reversed-phase chromatography.
The document discusses bioanalytical sample preparation. It begins with an introduction to sample preparation as an essential step in the bioanalytical process. Sample preparation techniques discussed include protein precipitation, liquid-liquid extraction, and solid phase extraction. Protein precipitation involves denaturing proteins to isolate analytes. Liquid-liquid extraction uses differential solubility to separate analytes between immiscible liquid phases. Solid phase extraction selectively retains analytes on a solid sorbent under specific conditions. The document provides details on each technique's principles, steps involved, advantages, and disadvantages.
The document discusses the formulation and evaluation of gastro-retentive mucoadhesive microballoons of nizatidine for the management of peptic ulcer. It provides background information on peptic ulcers, drug delivery systems, gastroretentive drug delivery systems, and mechanisms of bioadhesion. The objective is to develop mucoadhesive microballoons of nizatidine to increase its retention time in the stomach and improve the treatment of peptic ulcers.
Spectrophotometric titration by Mr. Pradeep SwarnkarPradeep Swarnkar
Spectrophotometric titrations use changes in absorbance of light to determine the endpoint of a titration. There are two main types of spectrophotometric titration - acid-base and precipitation titrations. Acid-base titrations involve a color change of an indicator and precipitation titrations involve monitoring the formation of a precipitate.
bio analytical method validation usfda guidlineschandu chatla
This document provides guidance from the US FDA on bioanalytical method validation. It discusses key principles such as method development, validation parameters for chromatographic and ligand binding assays, calibration curves, quality controls, selectivity, sensitivity, accuracy, precision, recovery, and stability. The guidance is intended to help sponsors validate analytical methods used in human and animal studies to quantify drug, metabolite, protein, and biomarker levels in biological matrices like blood, serum and tissue.
This document discusses various concepts related to high performance liquid chromatography (HPLC) peak analysis including:
1. It describes factors that influence peak shape such as column packing, mobile phase composition, pH, and buffers which can improve peak symmetry and resolution.
2. Key parameters for characterizing chromatographic performance are discussed including retention factor (k), selectivity factor (α), plate number (N), and height equivalent of a theoretical plate (HETP).
3. Optimizing these parameters through adjusting mobile phase or column properties can enhance separation and analysis of chromatographic runs.
This document outlines the five main steps for developing an analytical HPLC method: 1) selecting the initial HPLC method and conditions, 2) selecting the initial chromatographic conditions, 3) optimizing selectivity, 4) optimizing system parameters, and 5) validating the method. Key aspects of each step are discussed, including selecting the type of chromatography, column, detector, and mobile phase based on the analytes. The goal is to develop a validated method that provides adequate resolution and selectivity within a desired analysis time.
Human liver microsomes & rat liver microsomesgaurav sharma
Human and rat liver microsomes are subcellular fractions containing cytochrome P450 enzymes and other drug-metabolizing enzymes. They are commonly used in vitro to study drug metabolism and interactions. Human liver microsomes are obtained from human liver tissue through differential centrifugation and contain enzymes for phase I and phase II drug metabolism. They are useful for identifying drug metabolites, evaluating interspecies differences, predicting in vivo clearance, and studying interindividual variability in drug metabolism. Rat liver microsomes were also discussed as an experimental model. Incubation methods and analytical techniques like HPLC were described for evaluating metabolism using liver microsomes.
High performance thin layer chromatographySravani Ganti
This document provides an overview of high performance thin layer chromatography (HPTLC). It describes the advantages of HPTLC over HPLC and traditional TLC, such as its ability to process multiple samples simultaneously. The key steps of HPTLC are outlined, including plate selection, sample preparation, development, detection, and applications. HPTLC allows for enhanced separation resolution and automation compared to TLC. It is commonly used in pharmaceutical analysis and clinical testing due to its low cost, simplicity, and reproducibility.
Shraddha roll no- 7 -m. pharm final presentation ---rbvrr college of pharmacysaathiyaa
The document describes the development and validation of an analytical method for ziprasidone using reverse phase high performance liquid chromatography. The method utilizes a Sunsil C18 column with a mobile phase of water and methanol (55:45) at a flow rate of 1 mL/min. Ziprasidone was detected at 261 nm with a retention time of 3.082 minutes. The method was validated for specificity, precision, linearity, accuracy, limit of detection, limit of quantification and robustness as per ICH guidelines. The developed and validated method can be used for the analysis of ziprasidone in bulk and pharmaceutical formulations.
This document discusses the validation of bioanalytical methods. It defines validation as demonstrating the performance and reliability of an analytical method. There are three types of validation: full validation for new methods, partial validation for modifications to existing methods, and cross-validation to compare two methods. Key validation characteristics discussed include selectivity, accuracy, precision, linearity, stability, carryover, matrix effects, and recovery. Proper validation of bioanalytical methods is necessary to ensure reliable results.
This document describes the development and validation of a UV spectrophotometric method for the estimation of methocarbamol in bulk and pharmaceutical dosage forms. The method was developed using acetone and 0.1N sodium hydroxide solution as solvents, in which methocarbamol is soluble. The drug has maximum absorbance at 267 nm. The method was validated as per ICH guidelines and was found to be linear, precise, accurate and specific. The developed method can be used for the quantitative analysis of methocarbamol in bulk and pharmaceutical formulations.
Related Substances-Method Validation-PPT_slideBhanu Prakash N
This document provides an overview of analytical method validation. It defines validation as demonstrating a method is suitable for its intended purpose. Key validation characteristics discussed include precision, accuracy, specificity, linearity, range, detection limit, quantitation limit, ruggedness and robustness. The document describes the methodology for evaluating each characteristic, such as spiking known concentrations of analytes and establishing acceptance criteria. It emphasizes that validation confirms a method consistently produces results meeting pre-defined standards of quality.
Stability indicating rp hplc method development and validation for simultaneo...Rajasekhar
The document describes the development and validation of a stability-indicating RP-HPLC method for the simultaneous estimation of levodropropizine and chlorpheniramine maleate in bulk and pharmaceutical dosage forms. The method utilizes a C18 column with a mobile phase of 45% 0.1% orthophosphoric acid and 55% acetonitrile at a flow rate of 1 mL/min. The method was validated for parameters such as linearity, accuracy, precision, specificity, limit of detection, limit of quantification and robustness according to ICH guidelines. Forced degradation studies were also performed to demonstrate the stability-indicating ability of the developed method.
This document discusses various reagents used in pharmaceutical analysis including PDAB, Folin Ciocalteau, and MBTH reagents. It provides details on the principles, mechanisms, procedures, examples, and applications of each reagent. PDAB is used to detect primary amine groups via a colorimetric reaction. Folin Ciocalteau is used to detect phenols and reduces tungstate-molybdate to form a blue complex. MBTH forms colored complexes with aldehydes, phenols, and amines through oxidative coupling and can be used to analyze samples containing these functional groups. The document concludes that optimizing reagent volume and concentration allows these reagents to be successfully used to quantify drugs in pharmaceutical formulations.
USFDA guidelines for bioanalytical method validationbhatiaji123
The document discusses guidelines for bioanalytical method validation from the USFDA. It describes key parameters that must be validated for a bioanalytical method, including selectivity, accuracy, precision, recovery, calibration curves, sensitivity, reproducibility and stability. Accuracy and precision are determined by analyzing quality control samples in replicates across multiple runs. Recovery experiments compare extracted samples to unextracted standards. A calibration curve consisting of multiple concentrations over the expected range must be precise and reproducible.
The document discusses hyphenated techniques, specifically liquid chromatography-nuclear magnetic resonance (LC-NMR). It begins by introducing hyphenated techniques as the combination of two analytical methods, usually a separation technique coupled with a spectroscopic technique. It then describes LC-NMR in more detail, explaining how it works to separate components with liquid chromatography and then uses NMR for identification. Key aspects covered include instrumentation, modes of data acquisition (continuous flow, stopped flow, time sliced), and advantages such as automation, reproducibility, and simultaneous separation and quantification.
This document discusses HPLC columns used for separation of compounds. It begins with an introduction describing typical HPLC column dimensions and materials. It then covers different types of columns based on scale of preparation such as analytical and preparative columns. The main types of columns based on mode of operation are reversed phase and normal phase. The document explains the chemistry and mechanisms involved in reversed phase and normal phase HPLC columns. It also discusses column efficiency, the Van Deemter equation, and the Lx table in USP which provides standards for HPLC columns based on particle size and chemical compounds.
This document presents information on preparative high pressure liquid chromatography. It begins with an introduction to chromatography and classification of column chromatographic methods. It then discusses the differences between analytical and preparative HPLC and the objectives, instrumentation, method development, applications, and commercially available instruments for preparative HPLC. The instrumentation section describes the major components of a preparative HPLC system including the solvent reservoir, pump, injector, columns, detectors, fraction collector and more. Method development and optimization factors like mobile phase selection, temperature, retention, selectivity, and column overloading techniques are also covered.
This document provides an overview of high performance liquid chromatography (HPLC). It begins by defining HPLC and explaining that it uses high pressure to pump the mobile phase, yielding faster separation than traditional column chromatography. The document then discusses the basic principles of chromatography and liquid chromatography. It provides details on the types of HPLC based on mode of separation, principle of separation, elution technique, scale of operation, and type of analysis. The key components of an HPLC instrument are described including the solvent reservoir, pump, injector, column, detectors, and data recording system. Various columns, stationary phases, and pumps used in HPLC are also outlined.
1) Ion pair chromatography is a type of column chromatography that uses ion pairing agents to neutralize charged analytes and allow their separation on a reversed-phase column.
2) By adding counter ions with the opposite charge to the mobile phase, ion pairs form between the counter ions and analytes, neutralizing their charge and increasing their hydrophobicity.
3) The use of ion-pairing reagents as mobile phase additives allows the separation of ionic and highly polar substances that cannot otherwise be separated by reversed-phase chromatography.
The document discusses bioanalytical sample preparation. It begins with an introduction to sample preparation as an essential step in the bioanalytical process. Sample preparation techniques discussed include protein precipitation, liquid-liquid extraction, and solid phase extraction. Protein precipitation involves denaturing proteins to isolate analytes. Liquid-liquid extraction uses differential solubility to separate analytes between immiscible liquid phases. Solid phase extraction selectively retains analytes on a solid sorbent under specific conditions. The document provides details on each technique's principles, steps involved, advantages, and disadvantages.
The document discusses the formulation and evaluation of gastro-retentive mucoadhesive microballoons of nizatidine for the management of peptic ulcer. It provides background information on peptic ulcers, drug delivery systems, gastroretentive drug delivery systems, and mechanisms of bioadhesion. The objective is to develop mucoadhesive microballoons of nizatidine to increase its retention time in the stomach and improve the treatment of peptic ulcers.
Spectrophotometric titration by Mr. Pradeep SwarnkarPradeep Swarnkar
Spectrophotometric titrations use changes in absorbance of light to determine the endpoint of a titration. There are two main types of spectrophotometric titration - acid-base and precipitation titrations. Acid-base titrations involve a color change of an indicator and precipitation titrations involve monitoring the formation of a precipitate.
bio analytical method validation usfda guidlineschandu chatla
This document provides guidance from the US FDA on bioanalytical method validation. It discusses key principles such as method development, validation parameters for chromatographic and ligand binding assays, calibration curves, quality controls, selectivity, sensitivity, accuracy, precision, recovery, and stability. The guidance is intended to help sponsors validate analytical methods used in human and animal studies to quantify drug, metabolite, protein, and biomarker levels in biological matrices like blood, serum and tissue.
This document discusses various concepts related to high performance liquid chromatography (HPLC) peak analysis including:
1. It describes factors that influence peak shape such as column packing, mobile phase composition, pH, and buffers which can improve peak symmetry and resolution.
2. Key parameters for characterizing chromatographic performance are discussed including retention factor (k), selectivity factor (α), plate number (N), and height equivalent of a theoretical plate (HETP).
3. Optimizing these parameters through adjusting mobile phase or column properties can enhance separation and analysis of chromatographic runs.
This document outlines the five main steps for developing an analytical HPLC method: 1) selecting the initial HPLC method and conditions, 2) selecting the initial chromatographic conditions, 3) optimizing selectivity, 4) optimizing system parameters, and 5) validating the method. Key aspects of each step are discussed, including selecting the type of chromatography, column, detector, and mobile phase based on the analytes. The goal is to develop a validated method that provides adequate resolution and selectivity within a desired analysis time.
Human liver microsomes & rat liver microsomesgaurav sharma
Human and rat liver microsomes are subcellular fractions containing cytochrome P450 enzymes and other drug-metabolizing enzymes. They are commonly used in vitro to study drug metabolism and interactions. Human liver microsomes are obtained from human liver tissue through differential centrifugation and contain enzymes for phase I and phase II drug metabolism. They are useful for identifying drug metabolites, evaluating interspecies differences, predicting in vivo clearance, and studying interindividual variability in drug metabolism. Rat liver microsomes were also discussed as an experimental model. Incubation methods and analytical techniques like HPLC were described for evaluating metabolism using liver microsomes.
High performance thin layer chromatographySravani Ganti
This document provides an overview of high performance thin layer chromatography (HPTLC). It describes the advantages of HPTLC over HPLC and traditional TLC, such as its ability to process multiple samples simultaneously. The key steps of HPTLC are outlined, including plate selection, sample preparation, development, detection, and applications. HPTLC allows for enhanced separation resolution and automation compared to TLC. It is commonly used in pharmaceutical analysis and clinical testing due to its low cost, simplicity, and reproducibility.
Shraddha roll no- 7 -m. pharm final presentation ---rbvrr college of pharmacysaathiyaa
The document describes the development and validation of an analytical method for ziprasidone using reverse phase high performance liquid chromatography. The method utilizes a Sunsil C18 column with a mobile phase of water and methanol (55:45) at a flow rate of 1 mL/min. Ziprasidone was detected at 261 nm with a retention time of 3.082 minutes. The method was validated for specificity, precision, linearity, accuracy, limit of detection, limit of quantification and robustness as per ICH guidelines. The developed and validated method can be used for the analysis of ziprasidone in bulk and pharmaceutical formulations.
This document discusses the validation of bioanalytical methods. It defines validation as demonstrating the performance and reliability of an analytical method. There are three types of validation: full validation for new methods, partial validation for modifications to existing methods, and cross-validation to compare two methods. Key validation characteristics discussed include selectivity, accuracy, precision, linearity, stability, carryover, matrix effects, and recovery. Proper validation of bioanalytical methods is necessary to ensure reliable results.
This document describes the development and validation of a UV spectrophotometric method for the estimation of methocarbamol in bulk and pharmaceutical dosage forms. The method was developed using acetone and 0.1N sodium hydroxide solution as solvents, in which methocarbamol is soluble. The drug has maximum absorbance at 267 nm. The method was validated as per ICH guidelines and was found to be linear, precise, accurate and specific. The developed method can be used for the quantitative analysis of methocarbamol in bulk and pharmaceutical formulations.
This document presents the development and validation of an RP-HPLC method for the simultaneous estimation of a muscle relaxant drug and analgesic drug in pure form and pharmaceutical dosage forms. It discusses conducting a literature review on existing methods, determining the physicochemical properties of the drugs, optimizing the analytical method, and validating the developed method as per ICH guidelines. The expected outcomes are an accurate, precise, simple, cost-effective and fast method for simultaneously analyzing the two drugs.
Here are the key IR frequencies identified in the sample that match the reference standard of propafenone:
- C-C stretch at 1186 cm-1
- C=C stretch at 1651 cm-1
- C-H stretch (symmetric) at 2939 cm-1
- C-H bend at 1328 cm-1
- CH2 stretch (symmetric) at 1369 cm-1
- CH2 bend at 1485 cm-1
- CH3 bend at 1398 cm-1
- C-O stretch at 1100 cm-1
- C=O stretch at 1695 cm-1
- N-H stretch at 3417 cm-1
The IR spectrum
This document presents a comparative study of three research papers on RP-HPLC method development and validation for the estimation of diclofenac sodium from tablet dosage forms. The objective is to determine the most effective and cost-efficient method. The materials and methods from each paper are compared based on preparation of standard and sample solutions, run time, and validation parameters. The results show that Paper 1 has the fastest run time of 10 minutes and is more accurate, precise, sensitive and cost-effective than Papers 2 and 3. Paper 1 uses a more optimal column dimension. In conclusion, the RP-HPLC method from Paper 1 is deemed superior for the quantitative analysis of diclofenac sodium in tablet dosage forms.
HPLC Method Development & Method Validation (mr.s)22suresh
This document describes the development and validation of an HPLC method for estimating drugs. It discusses the principles of HPLC, steps in method development including selecting the method, column, mobile phase and detector. Method validation parameters like accuracy, precision, specificity, linearity and robustness are also summarized. The document provides details on the optimization process and validation procedures to ensure the method is suitable for its intended use.
To perform Analytical method validation of Paracetamol Tablets by UV-spectrop...Aakashdeep Raval
This document outlines the validation of an analytical method for the quantification of paracetamol using UV spectrophotometry. It describes the validation parameters that will be tested which include accuracy, precision, linearity, range, limit of detection and limit of quantification, selectivity and specificity, and robustness and ruggedness. The procedure involves preparing calibration standards of paracetamol to generate a linear curve and then testing the method's accuracy by spiking samples. Precision will be evaluated by repeatability, intraday, and interday testing. The document provides the theory and equations needed to calculate the validation parameters.
This document discusses instrumental techniques of analysis, specifically visible and ultraviolet spectroscopy. It covers the basic theory of spectroscopy and how matter interacts with electromagnetic radiation. Key points include:
1. Spectroscopy involves studying the interaction of matter with electromagnetic radiation like light.
2. UV-visible spectroscopy measures absorption of samples when electrons are excited from ground state to excited state.
3. Beer's law and Lambert's law describe the relationship between absorbance, concentration, and path length of samples.
4. The combined Beer-Lambert law states absorbance is directly proportional to concentration and path length of the absorbing species.
This document provides an overview of HPLC methodology and validation requirements. It discusses the key components of an HPLC test procedure including system suitability testing, relative response factors, and the validation parameters of specificity, linearity, accuracy, precision, LOD/LOQ, and robustness. Validation requirements depend on whether the method is compendial or non-compendial, with full validation needed for non-compendial methods. System suitability criteria and validation acceptance limits are outlined for various analytical techniques like assay, impurities testing, and dissolution.
Process Validation is Key important factor for the Pharmaceutical Industry to maintain Consistent Quality in product which claimed by the manufacturer.
The document describes planar chromatography techniques, specifically thin layer chromatography (TLC). It explains that TLC separates mixtures by using a thin stationary phase like silica gel coated on a plate and a mobile phase liquid solvent. The steps of TLC are described as sample application, development where separation occurs, visualization under UV light, and interpretation by calculating Rf values. Applications for separating lipids, carbohydrates and other compounds are outlined.
This document discusses various anti-tubercular drugs used to treat tuberculosis. It describes the classification of first-line drugs which include isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin. It also discusses second-line drugs including para-amino salicylic acid, ethionamide, cycloserine, thiacetazone, fluoroquinolones and macrolides. For each drug, it provides information on mechanisms of action, pharmacokinetics, dosing, adverse effects and drug interactions. The document is intended as an educational reference on anti-tubercular medications.
Introduction and Principle of IR spectroscopyRajaram Kshetri
This document provides an introduction to infrared (IR) spectrophotometry. It discusses how IR spectroscopy analyzes molecular vibrations when molecules absorb IR radiation that matches their natural vibrational frequencies. The document outlines the principle of IR spectroscopy and describes the different types of molecular vibrations observed in IR spectra, including stretching and bending vibrations. It also discusses the criteria for a molecule to absorb IR radiation, such as having a change in dipole moment when vibrations occur.
HPLC Principle,Instrumentation and ApplicationAlakesh Pradhan
HPLC Chromatography and its principle
Liquid chromatography
High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph (HPLC).
The separation process.
The chromatogram
Validation of analytical method for Diabetes Mellitus DhruvkumarPatel25
This document describes the development and validation of an analytical method for the simultaneous estimation of teneligliptin hydrobromide hydrate and metformin hydrochloride in a combined dosage form. It provides background on the drugs, reviews relevant literature, and outlines the methodology that will be used including the stationary and mobile phases, flow rate, detection wavelength, and other analytical conditions. The objective is to develop a stability-indicating method that can accurately quantify both drugs simultaneously.
This document describes the development and validation of a reverse phase high performance liquid chromatography (RP-HPLC) method for the estimation of the drug Zidovudine. The method was developed using a C18 column with a mobile phase of acetonitrile and water at a pH of 4.8. The method was validated according to ICH guidelines and showed good linearity, precision, accuracy, specificity, robustness and recovery. The proposed RP-HPLC method can be used for the routine analysis of Zidovudine in pharmaceutical formulations.
Haemolysis effect of Mefenamic Acid 250 mg Capsule in Bio analysis by liquid ...IOSR Journals
A rapid, simple and specific method for estimation of Mefenamic acid in human plasma was validated using Indomethacin as internal standard. The analyte and internal standard were extracted from plasma using simple solid phase extraction. The compound were separated on a reverse-phase column with an isocratic mobile phase consisting of 2 mM Ammonium Acetate in Water and acetonitrile (20:80, v/v) and detected by tandem mass spectrometry in negative ion mode. The ion transition recorded in multiple reaction monitoring mode were m/z 240.1 196.0 for Mefenamic acid and m/z 356.1312.0 for internal standard. Linearity in plasma was observed over the concentration range 35.000 – 7000.000 ng/mL for Mefenamic acid. The cv of the assay was 4.89 % to 5.98 % and accuracy was 99.36 to 102.20 % Intra and Interday respectively at LLOQ level. The validated method was applied to bioequivalence study of 250 mg Mefenamic acid in 28 healthy human volunteers. Total 50 samples from individual volunteers identified as Haemolyzed which were analyze initial and repeat again to cross check the method reproducibity for Haeamolysis effect and compared which found acceptable range
This document describes the development and validation of a quantitative method for determining penbutolol and its metabolite 4-hydroxy penbutolol in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method involves solid phase extraction of the analytes from plasma followed by separation using liquid chromatography with mass spectrometric detection in multiple reaction monitoring mode. The method was validated according to FDA guidelines and showed good linearity, accuracy, precision, recovery, selectivity and stability. The developed and validated LC-MS/MS method was found to be suitable for pharmacokinetic studies of penbutolol in human volunteers.
In-vitro evaluation techniques of anticancer, anti oxidant, anti microbial ZakiyaUsmani
This document discusses various in vitro methods used to evaluate potential anti-cancer and antioxidant compounds, as well as antimicrobial activity. It describes cytotoxicity assays such as MTT, SRB, clonogenic assays and dye exclusion tests that are used to study anti-cancer activity against cell lines. Methods to evaluate antioxidant activity in vitro include DPPH radical scavenging, hydrogen peroxide and superoxide radical scavenging assays. Diffusion and dilution methods are discussed for determining antimicrobial activity of compounds in vitro prior to animal studies.
RP-HPLC method development and validation for simultaneous determination of d...BRNSSPublicationHubI
This article describes the development and validation of an RP-HPLC method for the simultaneous quantification of decitabine and cedazuridine in bulk and pharmaceutical formulations. Various mobile phase compositions were trialled until a mixture of 65% 0.01N KH2PO4 and 35% acetonitrile was found to adequately separate the two drugs, eluting decitabine and cedazuridine at 2.263 and 3.001 minutes respectively. The method demonstrated good linearity and recovery within the range of 50-150%. The developed method is accurate, precise, robust and simple for the simultaneous analysis of decitabine and cedazuridine.
Conceição et al, 2006. orpotrin a novel vasoconstrictor peptide from the veno...pryloock
1. Researchers isolated and characterized a novel peptide from the venom of the Brazilian stingray Potamotrygon gr. orbignyi.
2. Using RP-HPLC and mass spectrometry techniques, they purified a peptide from the venom which was shown to strongly constrict blood vessels when applied to mice cremaster muscle tissue during intravital microscopy experiments.
3. Through de novo amino acid sequencing with mass spectrometry, the researchers determined the peptide's sequence to be HGGYKPTDK, which does not match any known bioactive peptides but aligns with residues 97-105 of creatine kinase, suggesting it may be produced from limited proteolysis of creatine kinase.
ANALYTICAL METHOD DEVELOPMENT AND VALIDATION HPLC UVVenkatesh Mantha
The document describes the development and validation of a reverse phase high performance liquid chromatography (RP-HPLC) method for the estimation of drugs in bulk and pharmaceutical dosage forms. It discusses the need for analytical methods in pharmaceutical analysis and introduces RP-HPLC as a commonly used technique. The document provides details of various studies done to develop and validate RP-HPLC methods for estimating specific drugs and drug combinations in research papers. It also provides chemical and pharmacological profiles of drugs that were estimated using RP-HPLC methods discussed in the literature review section. The aim is to develop a validated RP-HPLC method for estimation of a drug in bulk and marketed dosage form.
Simultaneous estimation of meclizine and nicotinic acid by using RP-HPLCpharmaindexing
This document describes the development and validation of a reverse phase high performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of Meclizine and Nicotinic acid. The method utilizes a mobile phase of acetonitrile and potassium dihydrogen phosphate buffer with a flow rate of 1.0 mL/min. The retention times were 3.01 minutes for Meclizine and 6.07 minutes for Nicotinic acid. The method was validated and found to be accurate, precise, specific, linear, robust, sensitive and able to quantify the drugs in tablet dosage forms without interference from other components.
Validation and method development of Apixaban A research project.Bhavana Gundavarapu
The document presents the development and validation of an HPLC-MS method for the estimation of Apixaban in human plasma. Key points:
- The aim was to develop a simple, precise and accurate HPLC-MS method for quantifying Apixaban levels in plasma using Apixaban-13C D3 as an internal standard.
- The method involved protein precipitation followed by HPLC-MS analysis on a C18 column with mobile phase containing acetonitrile, methanol and ammonium formate buffer at a flow rate of 0.4 mL/min and detection at m/z 459.3 and 462.3 for Apixaban and internal standard.
- The method was
1. A new RP-HPLC method was developed and validated for the estimation of Clopidogrel bisulphate in bulk drug and pharmaceutical formulations.
2. The method involved using a C18 column, mobile phase of acetonitrile and phosphate buffer at a ratio of 60:40, and detection at 224 nm.
3. The method was found to be linear, precise, accurate, specific, robust and suitable for the routine analysis of Clopidogrel bisulphate in quality control.
Method Development and Validation of Clopidogrel Bisulphate by Reverse Phase-...SriramNagarajan15
A new, simple sensitive, rapid, accurate and precise RP-HPLC method was developed for the estimation of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Clopidogrel bisulphate was chromatographed on a reverse phase C18column (150 mm x 4.5 mm, i.d 5μm) in a mobile phase consisting of acetonitrile and phosphate buffer (pH: 3.0) in the ratio of 60:40 % v/v. The mobile phase was pumped at a flow rate of 1 ml/min with detection at 224 nm. The detector response was linear in the concentration of 50-150 μg /ml. The limit of detection and limit of quantitation was found to be 1.3 and 4.2 µg/ml, respectively. The intra and inter day variation was found to be less than 2%. The mean recovery of the drug from the solution was 99.79%. The proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for routine quality control analysis of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Key words: Clopidogrel bisulphate, RP-HPLC, Validation, Accuracy, Precision.
Method Development and Validation of Clopidogrel Bisulphate by Reverse Phase-...SriramNagarajan15
A new, simple sensitive, rapid, accurate and precise RP-HPLC method was developed for the estimation of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Clopidogrel bisulphate was chromatographed on a reverse phase C18column (150 mm x 4.5 mm, i.d 5μm) in a mobile phase consisting of acetonitrile and phosphate buffer (pH: 3.0) in the ratio of 60:40 % v/v. The mobile phase was pumped at a flow rate of 1 ml/min with detection at 224 nm. The detector response was linear in the concentration of 50-150 μg /ml. The limit of detection and limit of quantitation was found to be 1.3 and 4.2 µg/ml, respectively. The intra and inter day variation was found to be less than 2%. The mean recovery of the drug from the solution was 99.79%. The proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for routine quality control analysis of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Key words: Clopidogrel bisulphate, RP-HPLC, Validation, Accuracy, Precision.
1. A new RP-HPLC method was developed and validated for the estimation of Clopidogrel bisulphate in bulk drug and pharmaceutical formulations.
2. The method involved using a C18 column, mobile phase of acetonitrile and phosphate buffer at a ratio of 60:40, and detection at 224 nm.
3. The method was found to be linear, precise, accurate, specific, robust and suitable for the routine analysis of Clopidogrel bisulphate in quality control labs.
This document describes the development and validation of a new reverse phase high performance liquid chromatography (RP-HPLC) method for the estimation of paracetamol in pharmaceutical dosage forms. Some key points:
- An isocratic RP-HPLC method was developed using a mobile phase of acetonitrile and potassium dihydrogen orthophosphate buffer at a ratio of 15:85, pH 2.5.
- The method was validated for parameters such as linearity, accuracy, precision, limit of detection, limit of quantification, and robustness as per ICH guidelines.
- The method showed good linearity in the range of 25-60 μg/ml with a correlation coefficient of 0.999
This document summarizes a dissertation submitted for a PharmD degree. It describes the development and validation of an analytical method for the quantitative analysis of metoclopramide hydrochloride using HPLC and UV spectroscopy. The objectives are to develop a simple, sensitive, accurate and economic RP-HPLC method and validate it according to ICH guidelines. A literature review provided background on previous related studies and informed the methodology. The method was developed using an HPLC system with a C8 column, gradient elution and UV detection. The method will be validated for accuracy, precision and other parameters and applied to analyze metoclopramide in samples.
Structural elucidation, Identification, quantization of process related impur...IOSR Journals
Major process related unknown impurity associated with the synthesis of Hydralazine hydrochloride bulk drug was detected by high performance liquid chromatography (HPLC) and was subjected to high resolution accurate liquid chromatography mass spectroscopy (HR/AM-LCMS) for identification. The proposed impurity was isolated from Hydralazine hydrochloride active pharmaceutical ingredient (API) by preparative chromatographic method and was injected on HPLC for comparison of retention time with that of the unknown process related impurity in Hydralazine hydrochloride. The molecular ion peak of preparatively isolated impurity and that of unknown process related impurity in Hydralazine hydrochloride were compared for confirmation. The postulated structure was unambiguously confirmed with the help of HR/AM- LC MS/MS, NMR and FTIR data proposed to be 1-(2-phthalazin-1-ylhydrazino)phthalazine (Hazh Dimer). This impurity of Hydralazine hydrochloride is not been previously reported. A rapid Acquity H-class gradient method with runtime of 15.0min was developed for Quantitation on Unisphere Cyno column and validated for parameters such as accuracy, precision, linearity and range, robustness. The LOD and LOQ of method were 0081% and 0.0246% respectively.
Multi-residue pesticide analysis of food samples using acetonitrile extractio...Kate?ina Svobodov
This document describes a method for multi-residue pesticide analysis of food samples using two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS). The method involves acetonitrile extraction of samples followed by separation using reverse phase and HILIC columns connected by a switching valve. The method showed improved peak shape and sensitivity for polar pesticides compared to single column methods. Recoveries of 79.67% of analytes were between 60-100% and reporting limits were 1 ppb or lower for most pesticides tested, demonstrating this 2D-LC-MS/MS method is effective for broad-scope pesticide residue testing in foods.
Multi-residue pesticide analysis of food samples using acetonitrile extractio...
ANAND PPT
1. “RP-HPLC METHOD DEVELOPMENT & ITS VALIDATION FOR
SIMULTANEOUS ESTIMATION OF ANTI-TUBERCULOSIS DRUG IN
HUMAN PLASMA”
MASTER OF PHARMACY
(PHARMACEUTICAL CHEMISTRY)
SUPERVISED BY : SUBMITTED BY:
Dr. DEEPTI JAIN ANAND SHRIVASTAVA
SCHOOL OF PHARMACEUTICAL SCIENCES
RAJIV GANDHI PROUDYOGIKI VISHWAVIDYALAYA
BHOPAL
2016
1
3. INTRODUCTION
Tuberculosis is an infectious bacterial disease characterized by the
growth of nodules (tubercles) in the tissues, especially the lungs.
Tuberculosis, or TB caused by Mycobacterium tuberculosis. It is
transmitted from person to person via droplets from the throat and
lungs of people with the active respiratory disease.
The symptoms of active TB of the lung are coughing, sometimes
with sputum or blood, chest pains, weakness, weight loss, fever and
night sweats.
Tuberculosis is treatable with a six-month course of antibiotics.
3
4. Global situation and trends:
Tuberculosis (TB) is contagious and airborne. It ranks alongside HIV
as a leading cause of death worldwide. 9.6 million people are
estimated to have fallen ill with TB in 2014: (5.4 million men, 3.2
million women and 1.0 million children). An estimated 1.2 million
people living with HIV developed TB in 2014. There have been major
advances in prevention, diagnosis and treatment of TB: mortality has
fallen 47% since 1990. Effective diagnosis and treatment of TB saved
an estimated 43 million lives between 2000 and 2014. TB remains one
of the world’s biggest threats. In 2014, TB killed some 1.5 million
people (1.1 million HIV-negative and 0.4 million HIV-positive).
4
5. Most common TB drugs
o Isoniazid
o Rifampin
o Ethambutol
o Pyrazinamide
For drug-resistant TB, a combination of antibiotics called
fluoroquinolones and injectable medications, such as amikacin,
kanamycin or capreomycin, are generally used.
5
6. BIOANALYSIS
Bioanalytical methods are used for the quantitative analysis of drugs
and their metabolites in the biological media like saliva, urine,
plasma, serum.
Development and validation of bioanalytical method is important to
understand the pharmacokinetics of any drug and/or its metabolites.
Bioanalytical method development consists of three essential inter
related parts sample preparation, chromatographic separation and
detection by using proper analytical method.
Validation of a Bioanalytical method is the process by which it is
established that the performance characteristics of the method meet
the requirements for the intended Bioanalytical application.
6
7. BIOANALYTICAL METHOD VALIDATION
Bioanalytical method validation is the approach employed to indicate
that the analytical method used to assess an analyte in biological matrix
is reliable and also reproducible.
Accuracy
Selectivity
Precision
Specificity
Detection limit
Quantitation limit
Linearity
Range
Robustness.
7
8. LITERATURE REVIEW
S. NO. METHOD DESCRIPTION
1 A simple and rapid
HPLC/UV method for
simultaneous quantification
for four constituents in Anti-
Tuberculosis 4-fdc tablets by
pre-column derivatization
Mobile Phase : Mobile phase gradient consisting of Acetonitrile- Phosphate Buffer
(8 mM, pH – 6.8)
Column : Phenomenex Luna C18 (250 X 4.6 mm I.D.)
Mode of Method: Gradient Mode
UV Wavelength : 210 nm
Injection Volume : 20 µL
Flow Rate : 1 ml/min
Retention Time (in min.) : PYP (4.65), INH (13.14), RIF (18.01), EMB (21.66)
2 Development and
validation of HPLC
method for the
determination of
pyrizanamide in human
plasma
Mobile Phase : Mixture of 15:85 % v/v Methanol and 0mM potassium
dihydrogen phosphate (pH adjust to 7.4)
Column: Phenomenox ODC2 C18 Column (150 X 4.6 mm I.D.) 5µm
Mode of Method : Isocratic Mode
UV Wavelength : 268 nm
Flow Rate : 1 ml/min
Retention Time (in min.) :PZA (6.80)
8
9. S. NO. METHOD DESCRIPTION
3 Development and
validation of RP-HPLC
method for simultaneous
estimation of Rifampicin,
Isoniazid, pyrizinamide in
human plasma
Mobile Phase: Mixture of ACN, Methanol and Water in the ratio of 30:5:65 v/v
pH adjust to 5.2
Column : Phenomenox ODC2 C18 Column (150 X 4.6 mm I.D.) 5µm
Mode of Method : Isocratic Mode
UV Wavelength : 242 nm
Flow Rate : 1 ml/min
Retention Time (in min.) : RIF (5.99), INH (5.11), PZA (10.97)
4 Development and
validation of HPLC
method for determination
of Rifampicin in human
plasma
Mobile Phase : Mixture of 42:60 & v/v Acetonitrile and 10mM potassium
dihydrogen phosphate (pH adjust to 3.2)
Column : Phenomenox ODS2 C18 Column (150 X 4.6 mm I.D.) 5µm
Mode of Method : Isocratic Mode
UV Wavelength : 335 nm
Flow Rate : 1 ml/min
9
10. S. NO. METHOD DESCRIPTION
5 Development and
validation of RP-HPLC
method for quantitative
estimation of Pyrazinamide
in bulk and
pharmaceutical dosage
forms.
Mobile Phase : Phosphate buffer (pH 4.4): Methanol 80:20 (v/v)
Column : Hypersil C8 (4.6 x 250mm, 3.5 μm) column
UV Wavelength : 269 nm
Injection Volume : 20 µL
Flow Rate : 1 ml/min
Retention Time (in min.) :PYP (3.62)
6 Method deveopment and
validation of anti-
tubercular drugs in fixed
dose formulation by RP-
HPLC technique”
Mobile Phase : Methanol : ammonium acetate buffer (pH-7.03) in the ratio of
(50:50).
Column : C18 Thermo Hypersil ODS, (250 X5.4 mm X 4.5μ) column
UV Wavelength : 276 nm
Injection Volume : 10 µL
Flow Rate : 1.3 ml/min
Retention Time (in min.) : ETH and INH were 2.01 min and7.0 min
respectively.
10
11. S. NO. METHOD DESCRIPTION
7 Development and
validation of RP-HPLC
method for the
determination of
pyridoxine hydrochloride,
Isoniazid, Pyrazinamide
and Rifampicin in
pharmaceutical
formulation
Mobile Phase : Acetonitrile and potassium dihydrogen phosphate buffer 11:89
v/v (pH adjusted to 4.0 ± 0.1)
Column : Phenomenex Luna C18 (250 X 4.6 mm I.D.)
Mode of Method : Gradient Mode
UV Wavelength : 235 nm
Injection Volume : 20 µL
Flow Rate : 1 ml/min
Retention Time (in min.) : pyridoxine hydrochloride, Isoniazid, Pyrazinamide
and Rifampicin were 3.687, 4.113, 5.041 and 12.829 min, respectively.
8 Method development and
validation for simultaneous
estimation of Isoniazid and
Ethambutol by using RP-
HPLC in bulk and
pharmaceutical dosage
form
Mobile Phase :Acetonitrile-Phosphate Buffer 65:35 v/v (pH – 4.6)
Column: Inertsil C18 (4.6 x250mm, 5μm
Mode of Method : Isocratic Mode
UV Wavelength : 255 nm
Injection Volume : 10µL
Flow Rate : 1 ml/min
Retention Time (in min.) : Isoniazid and Ethambutol was found to be 2.325
and 4.322nm respectively.
11
12. S. NO. METHOD DESCRIPTION
9 Simultaneous HPLC
determination of Isoniazid
and acetylIsoniazid in
plasma
Mobile Phase : 1-hexanesulfonic acid sodium salt solution (20 mM, pH 3,
adjusted with phosphoric acid) and acetonitrile
Column : Waters X-terra RP18 column
Mode of Method : Gradient Mode
UV Wavelength : 290 nm
Injection Volume : 20 µL
Flow Rate : 0.4 ml/min
Retention Time (in min.) : 4.5 and 7.4 min for INH and AcINH
10 Contribution to the
development and
validation of a high
performance liquid
chromatography by the
UV detection method for
Isoniazid and omeprazole
determination
Mobile Phase : Triethylamine (with a pH value of 10.5): acetonitrile (67:33,
v/v)
Column: C8 column Octasilil (Purospher RP8) 250mm x 4.6 mm i.d. 5μm
Mode of Method : Isocratic Mode
UV Wavelength : 260 nm
Injection Volume : 5 µL
Flow Rate : 1 ml/min
Retention Time (in min.): 2.323 min for Isoniazid; 3.497 min for 2-
pyridylamine; 4.013 min for omeprazole and 6.837 min, respectively.
12
13. S. NO. METHOD DESCRIPTION
11 Validation of HPLC
methods for determination
of Isoniazid, Rifampicin,
Pyrazinamide, and
Ethambutol in a fixed-dose
combination
antituberculosis
Mobile Phase : Mobile phase gradient consisting of Acetonitrile- Phosphate
Buffer (8 mM, pH – 6.8)
Column : Phenomenex Luna C18 (250 X 4.6 mm I.D.)
Mode of Method : Gradient Mode
UV Wavelength : 210 nm
Injection Volumev : 20 µL
Flow Rate : 1 ml/min
Retention Time (in min.) : PYP (4.65), INH (13.14), RIF (18.01), EMB (21.66)
12 A validated high
performance liquid
chromatography method
for the determination of
Rifampicin and desacetyl
Rifampicin in plasma and
urine
Mobile Phase : Mobile phase was 0.05 M potassium dihydrogen phosphate
buffer (pH 2.6): acetonitrile (55:45 v/v)
Column: Phenomenex Luna C18 (250 X 4.6 mm I.D.)
UV Wavelength : 254 nm
Injection Volume : 20 µL
Flow Rate : 1.2 ml/min
Retention Time (in min.) : DRIF, RIF and Rifapentine (RPN), the internal
standard were 2.9, 4.8 and 10.5 min respectively.
13
14. DRUG PROFILE
ETHAMBUTOL
Molecular Structure :
Molecular Formula : C10H24N2O2
Molecular Weight : 204.31 g·mol−1
Solubility : Soluble in water, DMSO; sparingly soluble in
ethanol; difficult to dissolve in acetone and
chloroform
Pka : 6.6 & 9.5
Dosage : Available as 100 and 400 mg tablets. 15 mg/kg
daily or up to 25 mg/kg but risk of ocular toxicity.
Weekly dose, 30 mg/kg three times/week.
Plasma Half Life(t½) : 3–4 hours
Protein Binding : 20 – 30%
Mechanism of Action : Ethambutol inhibits arabinosyl transferases which
is involved in cell wall biosynthesis. By inhibiting this enzyme, the bacterial cell
wall complex production inhibited. This leads to an increase in cell wall
permeability.
14
15. ISONIAZID
Molecular Structure :
Molecular Formula : C6H7N3O
Molecular Weight : 137.13928 g/mol
Solubility : Solubility in water, ethanol, Chloroform, Practically insoluble
in ether, benzene
Pka : 1.82(at 200C)
Dosage : Adult dosing generally 300 mg capsule administered orally,
once daily; or 15 mg/kg up to 900 mg/day, two or three
times/week, ideally dose administered one hour before or two
hours after a meal.
Plasma Half Life(t½) : Fast acetylators: 0.5 to 1.6 hr. Slow acetylators: 2 to 5 hr.
Protein Binding : 0 – 10%
Mechanism of Action : Isoniazid is a prodrug and must be activated by bacterial
catalase. Specficially, activation is associated with reduction of the mycobacterial ferric
KatG catalase peroxidase to form an oxyferrous enzyme complex. Once activated, isoniazid
inhibits the synthesis of mycoloic acids, an essential component of the bacterial cell wall.
actively growing intracellular and extracellular Mycobacterium tuberculosis organisms.
Specifically isoniazid inhibits InhA, the enoyl reductase from the NAD cofactor. It is the
INHNAD. adduct that acts as a slow, tight binding competitive inhibitor of InhA.
15
16. PYRAZINAMIDE
Molecular Structure :
Molecular Formula : C5H5N3O
Molecular Weight : 123.11 g/mol
Solubility : Soluble in chloroform, methylene chloride; less soluble in
benzene; sparingly soluble in water
Pka : -0.5
Dosage : Dose 20- 25 mg/kg daily, or 50-70 mg/kg three times in a
week.
Plasma Half Life(t½) : 9-10 hrs
Protein Binding : ~10%
Mechanism of Action : Pyrazinamide diffuses into M. tuberculosis, where the
enzyme pyrazinamidase converts pyrazinamide to the active form pyrazinoic acid. Under
acidic conditions, the pyrazinoic acid that slowly leaks out converts to the protonated
conjugate acid, which is thought to diffuse easily back into the bacilli and accumulate.
The net effect is that more pyrazinoic acid accumulates inside the bacillus at acid pH
than at neutral pH. Pyrazinoic acid was thought to inhibit the enzyme fatty acid synthase
(FAS) I, which is required by the bacterium to synthesise fatty acids.
16
17. RIFAMPICIN
• Molecular Structure :
• Molecular Formula : C43H58N4O12
• Molecular Weight : 822.94 g/mol
• Solubility : Freely soluble in chloroform and DMSO; soluble in
ethyl acetate, methanol, tetrahydrofuran; slightly
soluble in acetone, water, carbon tetrachloride
• Pka : 1.7
• Dosage : Dose 10 mg/kg, in a single daily administration, not
to exceed 600 mg/day, oral or i.v.
• Plasma Half Life(t½) : 3.35 (±0.66) hours
• Protein Binding : 89%
• Mechanism of Action : Rifampin inhibits the DNA dependent RNA
polymerase, leading to a suppression of RNA synthesis and cell death.
17
18. RESEARCH ENVISAGED
Bioanalytical method employed for the quantitative
determination of drugs and their metabolites in
biological fluids plays a significant role in the evaluation
and interpretation of bioavailability, bioequivalence,
pharmacokinetic, and toxicokinetic study data.
Monitoring the concentration of the drug in the blood
and plasma ascertains that the calculated dose actually
delivers the plasma level required for the therapeutic
effect.
18
19. To develop selective and sensitive analytical method for
the quantitative evaluation of drug and their metabolism
are critical for the successful conduct of
biopharmaceutics and clinical pharmacology studies.
The manuscript could be used as a guide in some
Therapeutic Drug Monitoring, Bioavailability and
Bioequivalence studies of drug candidates.
Literature survey reveals that as such no UV and HPLC
method has yet been reported for simultaneous
estimation of Isoniazid, Rifampin, Ethambutol and
Pyrazinamide.
19
20. PLAN OF WORK
Literature Survey
Development of Method
• Selection of suitable detecting wavelength.
• Selection of optimization of method for extracting drug for plasma.
• Determination of appropriate working concentration range.
• Validation of proposed method as per ICH guidelines.
• Analysis of biological samples with the developed method,
• Recovery study of statistical evaluation of developed method.
Validation of Developed Method
20
21. REFERENCES
Tuberculosis Fact Sheet No. 104, World Health Organization November
2010. Retrieved 26 July 2011.
Robertson Brian D, Brennan Patrick J, Young Douglas B, Handbook of
Anti-Tuberculosis Agents, 2008 88(2): 85–170
Wong EB, et al. Rising to the challenge: new therapies for tuberculosis.
Trends in Microbiology. 2013,21:493
Tripathi K.D, Essentials of Medical Pharmacology, 4th Edition, Jaypee
Brothers Medical Publishers (P) Ltd, New Delhi Page No. 475
US Food and Drug Administration, Guidance for industry- Bioanalytical
method validation, Center for Drug Evaluation and Research, Rockville,
MD, 2001.
"Ethambutol” (CHEBI:4877) Chemical Entities of Biological Interest. UK:
European Bioinformatics Institute. 18 August 2010. Main. Retrieved 26
April 2012. (Accessed December 24,2015.)
http://www.drugbank.ca/drug/DB00951 (Accessed December 24,2015.)
http://www.drugbank.ca/drug/DB00339 (Accessed December 24,2015.)
21
22. ICH, Specifications test procedures and acceptance criteria for new drug
substances and new drug products: chemical substances. International
Conference on Hormonisation, IFPMA, Geneva 1999
Chung Chow Chan, Analytical Method Validation: Principle and Practices.
(727-742).
U.S. Food and Drug Administration. Guidance for Industry,Bioanalytical
Method Validation, September 2013:1
22