High Performance Liquid Chromatography (HPLC) method development, validation, and analysis is one of the most widely used techniques for drug testing in formulations and biological fluids. Usually, the first step in HPLC method development is to review existing information given substantial research has already been published on this topic. Next, it is imperative to select a proper HPLC column that can provide a sharp peak with a reasonable retention time. In this step, a neat solution of the analyte is chromatographed using an appropriate column. There are countless choices of HPLC columns from different vendors, and the selection of an adequate column can itself be challenging. Afterward, we should select a suitable mobile phase for sound separation of analyte from impurities or endogenous material. Closer to the end, we deliberate over the detection technique for HPLC method development. This step, however, has become somewhat routine given the mass spectrometer has emerged as a popular choice for HPLC analysis due to its highly selective nature. Alternative detection modes require revisiting method development and further troubleshooting. Finally, careful sample preparation is also essential and must be reviewed thoroughly during HPLC method development. Often analysts take a shortcut for HPLC sample preparation and rely on the mass spectrometer for selective analyte detection. This practice, however, sometimes does not work out successfully due to ion suppression and a variety of other reasons. In summary, High-performance liquid chromatography (HPLC) Analysis is an analytical chemistry technique to separate, detect, and quantitate different drugs and metabolites within various formulations, vehicles, and biological matrices. For assay reproducibility, HPLC Method Development relies on the right combination of pumps, analytical columns and pre-columns, mobile phase, and autosampler conditions. HPLC Method Validation ensures proper assay selectivity, sensitivity, and reproducibility.
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Our projects with NorthEast BioLab include successful method development, validation, stability studies during Clinical Phase I – IV studies.
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HPLC method development is required at each phase of drug R&D, starting from the early discovery phase. During this stage, the HPLC method can be developed utilizing a generic protocol as the drug is being analyzed at a higher concentration and only semi-quantitative values are needed. Naturally, this fit-for-purpose approach helps lower the HPLC test cost significantly during early discovery studies. As the drug advances in the next phase, however, it becomes crucial to optimize the method and make it more reproducible. HPLC method validation becomes necessary when Tox studies are planned for preparing IND application filing. These methods developed properly can be promptly validated. Therefore, we must prioritize robust HPLC method development and analysis even during the earlier stages. This way, the methods used in preclinical phase can be seamlessly transferred to the clinical programs and would not require extensive HPLC method development and validation.
Mass spectrometry is a widely used procedure observed as having outstanding sensitivity. Triple-quadrupole mass spectrometry (MS/MS) offers additional benefits due to its selectivity. In mass spectrometry analysis, it is essential to isolate….
NorthEast BioLab provides HPLC method development, validation, and Analysis services for various stages – from early discovery to clinicaldevelopment phase.
During the drug discovery phase, our expert scientists collaborate to select and optimize the HPLC method that would be utilized for your analysis. HPLC methods help analyze drug concentration in biological fluids to evaluate ADME (absorption, distribution, metabolism, and excretion) profile of the drug. The drug discovery phase also involves pharmacology and toxicology assessments of the drug compounds.
HPLC method development and validation are essential in this phase as the preclinical studies aim to generate and document the safety, pharmacokinetics, toxicokinetics, and potency parameters for the drug compounds.
Upon successful IND filing, the clinical phase of drug development is initiated and the HPLC method developed completed earlier is further refined for the analysis of drugs and metabolites in biofluids as per the pharmacokinetic studies.
NorthEast BioLab has state-of-the-art PerkinElmer equipment coupled to spectrophotometric or mass spectrometry detection as well as a deep bench of talent skilled at swift and dependable HPLC Method Development, Validation, and Analysis.
Our client-centric approach along with 20+ years rich experience in HPLC method development and analysis enables us to contribute expert guidance for your drug development program. We constantly realign ourselves to your evolving drug discovery and development goals – flexibility for iterative research or expectation for a swifter turnaround – as your drug compounds progress onwards. Therefore, our HPLC method development, validation, and sample analysis protocols are designed to cater to your dynamic needs.
Driven by founding values, our primary focus is making your drug product a great success through our outstanding HPLC method development offering. Our scientists and analysts are genuinely centered on productive long-term collaboration with your team. Finally, we play for the long term and do it right by ensuring the full integrity of our in-house processes, even as we continue trying to lower the resource intensity and HPLC test cost of your studies at every stage.
Answers to additional HPLC Method Development, Validation, and Analysis questions popular among our potential clients.
High-performance liquid chromatography is the technique that can separate, identify, and quantify components in a mixture. HPLC testing is the most powerful chromatography technique essential to most laboratories across the globe. There are many applications for HPLC analysis but not limited to water purification, detection of impurities in pharmaceutical industries, Pre-concentration of trace components, ligand-exchange chromatography, Ion-exchange chromatography of proteins, and high pH anion-exchange chromatography of carbohydrates. HPCL assay is also used in method development, method validation, and study testing.
There are many steps involved, the hplc assay is largely automated and the process is therefore highly reproducible and precise. The limitations of hplc include lower sensitivity and possible intereference from other peaks. Because of the speed of HPLC analysis and its dependence on different polarities of the compounds, multiple compounds with similar structures and polarities can elute off the chromatography apparatus at the same time or nearly the same time. HPLC analysis has a high operating cost and increased run time as compared to UPLC.
HPLC method validation is a recognized and efficient way to determine the suitability of the assay method to provide relevant data to confirm that the process or the HPLC testing gives acceptable and reliable results within the scope of the process. The most widely applied HPLC method validation characteristics are accuracy, precision (repeatability and intermediate precision), specificity, detection limit, quantitation limit, linearity, range, robustness, and stability of analytical resolutions.
High-Performance Liquid Chromatography (HPLC) and Ultra Performance Liquid Chromatography (UPLC) are both liquid chromatography procedures used to separate and detect the different components found in complex mixtures. However, UPLC operates at higher pressures (~15,000 psi) and permits for smaller particle sizes in columns, while HPLC operates at lower pressures (max <6000 psi). UPLC shows higher analyte resolution and sensitivity, and uses less solvents, and shorter run times. In contrast, HPLC analysis consumes more solvent with less sample throughput, but the hardware costs less.
The HPLC analysis of small molecules is most often performed in reversed-phase separation mode. Hydrophilic interaction chromatography (HILIC) and normal-phase chromatography are also suitable for separating polar compounds, with HILIC being the preferred method. Ion chromatography can also separate ionic compounds, ion-exchange separation modes, and inorganic anions or cations.
High-performance liquid chromatography (HPLC) testing is an analytical chemistry procedure to separate, detect, and quantify drugs and their metabolites within various formulations, vehicles, and biological matrices. HPLC lab offered actives quantitation, additive analysis, extractable and leachable, assay and impurity analysis, identification, including contaminant analysis and quantitation.
The System Suitability Test (SST) is used to verify that an analytical method was suitable for its intended purpose of the day for the analysis was done. It is an essential parameter to ensure the quality of the process for correct measurements. Each SST is specific to a method with predefined acceptance criteria for certain parameters, e.g., absorbance values between 0.2 and 1.0 for a photometric content determination method or some resolution factors for chromatographic techniques. System suitability proves that the system is working flawlessly before the analysis on HPLC or any other system, and it is required to be done before every sample analysis.
The applicable procedures of ultra−high-performance liquid chromatography (UHPLC) are the analysis of assay and organic impurities, related substances, drug products, or raw material of pharmaceuticals or degradation products of a drug substance or intermediate. A suitable method is developed only after assessing chromatography’s main and critical separation parameters (examples for UPLC-HPLC are wavelength, a choice of diluent, stationary phase, detector, flow rate, column temperature, solvent system, injection volume, and elution mode, Etc.). The development of the analytical method shows that the developed analytical approach is appropriate for its proposed use for the quantitative assessment of the targeted analyte present in therapeutic drugs. And it typically plays a vital part in the development and making of pharmaceutical medications.
Dose confirmation study for formulation samples is a significant part of any compelling GLP study and is mandatory by the United States Food and Drug Administration (USFDA) for formulations dosed in toxicology studies. We provide high-quality, preclinical dose formulation analysis for GLP and non-GLP studies to ensure your study starts on the right path. NorthEast Biolab (NEBA) has significant experience in all aspects of dose formulation analysis. Methods utilize either UV/Vis or Fluorescence detectors, appropriate for your protein or small molecule. In addition, NEBA has substantial infrastructure and decades of expertise in qualitative and quantitative separations utilizing HPLC mass spectrometry detection of peptide drugs. Solid, semisolid, and liquid formulations of pharmaceutical leads are one essential step in drug development. Methods are optimized to confirm the API concentration in your dosing formulation, as well as solution uniformity and stability. In addition, if stereoisomers are part of the formulation, we have years of chiral experience at your service.
In HPLC testing, determination of dosing vehicle stability of the product for its formulation, the impact of its pharmaceutical components, the manufacturing and processing conditions, the impact of the container and closure, packaging components, storage environments, estimated conditions of shipping, light, temperature, and humidity. Stability testing is utilized to control if the quality of a drug substance or drug product is transformed over time by numerous environmental factors, such as temperature, light, and humidity. Stability testing is determined to indicate how the quality of a drug substance or drug product differs with time under the influence of various environmental factors such as humidity, light, and temperature, and also determines a re-test period for the drug material or shelf life for the drug.
HPLC method development shows that the developed chromatography method is suitable for its intended use in the development and manufacturing of pharmaceutical drug ingredients and drug products. The fundamental separation techniques and principles involved in the analytical method development using the HPLC and UPLC are a selection of chromatography mode, detector, column (stationary phase), mobile phase, organic modifiers, ion-pair reagents, flow rate, solvent delivery system (elution mode), diluent, methods of extraction, samples to be used, experimentation to finalize the procedure, selection of test concentration and injection volume, forced degradation studies (stress resting), evaluation of stress testing Mass balance study, finalization of wavelengths, stability of the solution, system suitability, robustness of the method, relative response factor, quantification methods.
Quantitative results in high-performance liquid chromatography (HPLC) are usually based on calibration with standards and peak-area measurement. A distinctive method to determine the absolute number of moles of a compound from peak area measurements using HPLC with a UV–vis detector has been proposed.
