File Name: hplc method development and validation .zip
A simple, sensitive, precise and accurate reversed phase high performance liquid chromatographic RP-HPLC method has been developed for the dissolution study of Bosentan in bulk and in pharmaceutical dosage forms. The method was developed using the mobile phase comprising of Triethylamine buffer pH adjusted to 2.
A new, rapid, economical and isocratic reverse phase high performance liquid chromatography RP-HPLC method was developed for the determination of eptifibatide acetate, a small synthetic antiplatelet peptide, in bulk drug substance and pharmaceutical dosage forms.
The developed method was validated as per of ICH guidelines. The chromatographic separation was achieved isocratically on C18 column x 4. Eptifibatide acetate exhibited linearity over the concentration range of 0. The intra-day and inter-day precision were between 0. The present successfully validated method with excellent selectivity, linearity, sensitivity, precision and accuracy was applicable for the assay of eptifibatide acetate in bulk drug substance and pharmaceutical dosage forms.
From the literature survey, it was revealed that few spectrometric and chromatographic analytical methods have been developed for determination of eptifibatide in pharmaceutical preparations and biological fluids 7 - Characterization of eptifibatide impurities during stability assays by using mass spectrometers coupled with a reverse phase gradient HPLC system has been performed by Wang et al.
Zhao et al. In this method, quantification of eptifibatide was achieved with UV detection at nm RT: 10 min. Kota et al. In this method, eptifibatide analysis time was 20 min. Saksena et al. The drawbacks of the reported methods were the need for using gradient LC separation method with long run time and use of mass spectrometry that might not be universally available in laboratories due to its cost implications.
Eptifibatide acetate has been approved by FDA However no compendia method has been published for its determination in bulk and pharmaceutical dosage forms. Considering the less complication and readily availability of HPLC-UV detection, the main objective of present study was to develop a rapid, economical and validated method for the assay and quality control of eptifibatide acetate in bulk drug substance and finished pharmaceutical dosage forms 15 - The validation of chromatographic parameters was performed in accordance with ICH guidelines HangZhou, China.
All other reagents were of analytical grade. Data acquisition, analysis and reporting were performed by ChemStation Software Rev. Then five working standard solutions 2, 1. All the standard stock and working solutions were daily prepared.
The quality control QC stock solution of eptifibatide independent of the standard stock solution was prepared in deionized water.
Then the QC sample solutions 1. All the QC stock and sample solutions were daily prepared. Determination of detection wavelength was based on the scanned UV spectrum of the eptifibatide acetate solution over the range of to nm. Maximum absorbance was observed at and nm. The mobile phase was composed of the mixture of Solution A 0.
The developed RP-HPLC method was validated in terms of the following parameters; specificity, linearity, sensitivity, precision, accuracy and stability of analytical solutions. The validation was carried out according to International Conference on Harmonization ICH guidelines for validation of analytical procedures The specificity and selectivity of method was investigated by injecting of blank samples deionized water and citric acid buffer as excipient to demonstrate the absence of interference with elution of eptifibatide in standard samples or pharmaceutical formulation.
To study the linearity of analytical procedure, five working standard solutions at different concentration levels 0. In order to determine that the instrumental response was directly proportional to analyte concentration, calibration curve was constructed by plotting concentration of eptifibatide on X-axis and average peak area on Y-axis.
Regression equation and value of co-relation coefficient was calculated using linear regression analysis. The sensitivity of the analytical technique was estimated in terms of limit of detection LOD and limit of quantification LOQ.
LOQ was taken as lowest concentration of eptifibatetide that could be quantitatively determined with acceptable accuracy and precision QC samples at three different concentration levels 0. The accuracy of measurement method was assessed via the methodological recovery. The percentage deviation of determined concentration of QC sample and theoretical concentration expressed the recovery of method Quantification of injections was achieved using the regression equation.
UV spectrum of eptifibatide acetate solution showed maximum absorbance at and nm Figure 2. The assay chromatograms at and nm are presented in Figure 3. The wavelength of nm was selected as suitable detection wavelength because of clear flat baseline and being prevented from interference coming from TFA along with symmetrical response peak.
Chromatograms of: A standard solution, 0. The present work was aimed at developing simple, rapid and economical assay method for eptifibatide acetate in API powder and dosage forms. Quantitative analysis was achieved with high chromatographic response peak and optimum resolution. Figure 4 shows overlay of chromatograms for standard sample and assay sample solution for injection with that of blank samples deionized water and citric acid buffer as excipient. No interference with eptifibatide peak was observed as blank has no interfering peak at retention time of eptifibatide.
Specificity of the method can be concluded based on the chromatographic peak purity observed in the chromatograms Chromatograms of: A blank sample deionized water B blank sample citric acid buffer as excepient C standard solution, 0. The calibration curve was constructed by plotting a graph of peak mean area versus concentration. The linearity of calibration curve was evaluated using linear regression analysis.
Co-relation coefficient was 0. Hence linearity of method was proved over the concentration of 0. Therefor the lowest concentration on calibration curve that can be reproducibly quantified with acceptable accuracy and precision, 0.
The intra-day and inter-day precision of method was expressed as RSD value. The results are presented in Table 1. The accuracy of method was assessed by recovery studies on QC samples. The percent recovery by the assay of QC samples ranged from The good recovery values for accuracy study ascertain that method is accurate as shown in Table 1. Stability results obtained from stability studies of QC samples stored in different conditions are summarized in Table 2.
Based on these results, QC samples are considered stable for 24 h at room temperature, up to 14 days when stored under refrigeration and after three freeze-and-thaw cycles. Short analysis time and low RSD value with acceptable accuracy indicate suitability of this method for routine analysis of eptifibatide marketed injection forms. A simple, rapid, validated and isocratic RP-HPLC method with UV detection was developed for the identification and quantification of eptifibatide acetate in pure and pharmaceutical formulation.
The method was successfully validated as per ICH guidelines and statistical data confirmed selectivity, linearity, sensitivity, precision and accuracy of proposed method. Also, the described method can be used to stability study of analytical solutions. Because of importance of short retention time in routine analysis of drug, the current method sounds to be applicable as quality control tool for assay of eptifibatide acetate in pharmaceutical industries.
We thank our managements in Shahid Beheshti School of Pharmacy for their support during this study. National Center for Biotechnology Information , U. Iran J Pharm Res. Find articles by Maryam Bavand Savadkouhi. Find articles by Hossein Vahidi. Find articles by Abdul Majid Ayatollahi.
Find articles by Shirin Hooshfar. Find articles by Farzad Kobarfard. Author information Article notes Copyright and License information Disclaimer. Received Apr; Accepted Jun. This article has been cited by other articles in PMC. Abstract A new, rapid, economical and isocratic reverse phase high performance liquid chromatography RP-HPLC method was developed for the determination of eptifibatide acetate, a small synthetic antiplatelet peptide, in bulk drug substance and pharmaceutical dosage forms.
Open in a separate window. Figure 1. Results and Discussion Selection of wavelength UV spectrum of eptifibatide acetate solution showed maximum absorbance at and nm Figure 2. Figure 2. Figure 3.
Figure 4. Figure 5. Table 2. Conclusion A simple, rapid, validated and isocratic RP-HPLC method with UV detection was developed for the identification and quantification of eptifibatide acetate in pure and pharmaceutical formulation.
Acknowledgment We thank our managements in Shahid Beheshti School of Pharmacy for their support during this study. References 1. Clinical pharmacology of eptifibatide. Platelet aggregation inhibitors. Google Patents. Scarborough RM. Development of eptifibatide. Platelet glycoprotein IIb-IIIa antagonists as prototypical integrin blockers: novel parenteral and potential oral antithrombotic agents. Pharmaceutical analysis of eptifibatide via simple, rapid, economical UV-spectrophotometric methods.
Characterization of eptifibatide during drug formulation stability assays.
Metrics details. The stability and forced degradation behaviour of bedaquiline fumarate BQF in official dissolution media 0. Mean percent recovery varied between The method was validated for other parameters like specificity, system suitability and robustness. Potential degradation of the BQF when exposed to direct sunlight in official dissolution media was
Many different strategies of high performance liquid chromatographic method development are used today. This review describes a strategy for the systematic development of High performance liquid chromatographic HPLC methods. HPLC is an analytical tool which is able to detect, separate and quantify the drug, its various impurities and drug related degradants that can form on synthesis or storage. It involves the understanding of chemistry of drug substance and facilitates the development of analytical method. A number of chromatographic parameters were evaluated in order to optimize the method. An appropriate mobile phase, column, column temperature, wavelength and gradient must be found that affords suitable compatibility and stability of drug as well as degradants and impurities.
The purpose of this work is to develop and validate stability, indicating reverse phase High-performance liquid chromatography HPLC method for the rapid and precise determination of droxidopa in its pure form and formulations. A simple, fast, accurate and economical way has been developed and validated for the quantification of droxidopa by HPLC technique. The chromato graphic system was equipped with Shimpack columnC 18 x 4. The developed HPLC technique was found to be rapid as the retention time was 2. The method was validated as per the International Conference on Harmonization ICH guidelines for specificity linearity, accuracy, precision.
Download the PDF version. Analytical method development, validation, and transfer are key elements of any pharmaceutical development program. This technical brief will focus on development and validation activities applicable to drug products. Method-related activities are interrelated. They are also iterative particularly during early drug development phases. Effective method development ensures that laboratory resources are optimized, while methods meet the objectives required at each stage of drug development. Method transfer is the formal process of assessing the suitability of methods in another laboratory.
Pharmaceutical Technology Europe. This article presents a simple and systematic approach to HPLC method development, beginning with sample preparation and finishing with practical analytical method validation. The wide variety of equipment, columns, eluent and operational parameters involved makes high performance liquid chromatography HPLC method development seem complex. The process is influenced by the nature of the analytes and generally follows the following steps:. Depending on the overall requirements and nature of the sample and analytes, some of these steps will not be necessary during HPLC analysis. For example, a satisfactory separation may be found during step 2, thus steps 3 and 4 may not be required.
HPLC Method Development and Validation for Pharmaceutical Analysis and systematic approach to HPLC method development, beginning with sample preparation and finishing 4. ebezpieczni.org
A new, rapid, economical and isocratic reverse phase high performance liquid chromatography RP-HPLC method was developed for the determination of eptifibatide acetate, a small synthetic antiplatelet peptide, in bulk drug substance and pharmaceutical dosage forms. The developed method was validated as per of ICH guidelines. The chromatographic separation was achieved isocratically on C18 column x 4. Eptifibatide acetate exhibited linearity over the concentration range of 0. The intra-day and inter-day precision were between 0.
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