1Associate Professor, Department of Pharmaceutical Chemistry, Priyadarshini J. L. College of Pharmacy, Electronic Zone MIDC, Hingna Road, Nagpur 440016, Maharashtra.
2M. pharm 2nd year student, Department of Pharmaceutical Quality Assurance, New Montfort Institute of Pharmacy, Ashti 442202 Dist. Wardha, Maharashtra.
3Assistance Professor, Department of Pharmaceutics, New Montfort Institute of Pharmacy, Ashti 442202 Dist. Wardha, Maharashtra
High Performance thin-layer liquid chromatography refers to a feasible, sophisticated analytical method established upon thin-layer chromatography (TLC) concepts that offers several important advantages, including better material application, quicker and more efficient separation and lower mobile phase usage. The intent of this investigation had been to provide a quick as well as easy HPTLC approach to Piroxicam identification. The suggested technique can be applied to detect the substances under analysis at extremely low quantities. The suggested approach is straightforward, eco-friendly, stable, sensitive, and economical. It could be applied to regular quality control procedures or research. The primary purpose for HPLC validation is essential to ensure the process stays within predetermined bounds under expected circumstances. Examine deviations from predetermined parameters, take corrective action, think about if revalidation was required, and make adjustments to the process or product. Then, assess the impact of the changes by evaluating the equipment, personnel, environment, process, and product.
The renowned analgesics and nonsteroidal anti-inflammatory medicament 4-hydroxy-2-methyl-N-(pyridine-2-yl)-2H-1,2-benzothiazine-3-carboxamide-1,2-dioxide, often known as piroxicam is prescribed for the Chronic or temporary treatment of swelling associated with joints and diseases affecting the muscles, such as ankylosing spondylitis, rheumatoid arthritis, and osteoarthritis (AS).11. These are analgesics, or nonsteroidal anti-inflammatory medications (NSAIDs). Although they can be used to treat other painful illnesses like injuries, cancer surgery, and oral infections, they are typically used to treat osteoarthritis and rheumatoid arthritis. In addition to being used to treat painful disorders, piroxicam has lately been employed in a number of protocols to treat hemangiosarcoma and squamous cell carcinoma. 5-hydroxy-prioxicam is the most prevalent of several physiologically inactive metabolites produced by the metabolism of piroxicam via a cytochrome P450 2C isoform.2
The most widely used class of painkillers and inflammatory-reducing medicine worldwide are nonsteroidal anti-inflammatory medicines, its main purpose is for treating fever, inflammation, as well as pain in people. Along with pK values ranging from 3 to 7, these medications are weakly acidic due to their carboxylic groups or ketoenol tautomeric structure. Although the majority of NSAIDs are chiral, they are frequently given as racemates.3 The appropriate use of NSAIDs will probably continue to be a top priority during the treatment of all domesticated animals for painful inflammation-related conditions as a result of increasing public knowledge of animal welfare. To guarantee these drugs are used safely and effectively, it’s going to be necessary to understand the pharmacokinetic of NSAIDs in the particular species of interest.4 In accordance with the ideas of High Performance thin-layer chromatography (HPTLC) and thin-layer chromatography (TLC) are practical modern analytical techniques.5,6 Essential advantages of HPTLC over classical TLC include better sample implementation, more efficiency of separation, lower usage of mobile phases, automation of the drying process for plates, shorter analysis times, less exposure to hazardous solvents, and fewer potential environmental contaminations.7,8 8. HPTLC techniques may provide further advantages even if there are several analytical techniques accessible today for the identification, isolation, and quantification of substances. These include removing the need for before a solvent manipulation (like filtering or degassing), easy sampling, being able to analyze a number of samples simultaneously, lower solvent expenses, and the removal of evidence from previous searches (new stationary phase for every evaluation). TLC is the most straightforward and affordable chromatography technique when compared to other techniques like gas chromatography and HPLC.9,10 In addition, there are many different application areas for both TLC and HPTLC, including biotechnology, chemistry, phytochemistry, the food and pharmaceutical industries, cosmetics, and many more.11-16
TLC is still crucial to the pharmaceutical sector even with the wide range of analytical methods available for characterization. The European Pharmacopoeia and U.S. Pharmacopoeia monographs contain numerous TLC tests for the detection of drugs or contaminants. The intent of this study had been to provide a quick as well as easy HPTLC technique for meloxicam and piroxicam identification. Both of meloxicam and piroxicam are significant non-steroidal anti-inflammatory medications that are administered globally to treat osteoarthritis, rheumatoid arthritis, and chronic as well as acute pain of various causes.17
Fig no. 1: Chemical Structure of Piroxicam
Concept of validation
The process of demonstrating and recording that a method, activity, or process consistently produced the desired outcomes is known as validation.
History Of Validation:
FDA officials, Ted Byers and Bud Loftus, first presented idea of validation in an effort to raise the caliber of medications in the mid-1970s. (Agalloco 1995). It is suggested as a direct solution to a number of issues with the sterility of the high-volume parenteral market. The procedures utilized to make these items were the main focus of the first validation efforts, but they soon expanded to related processes Process validation was initially advocated by the U.S. Food and Drug Administration. However, definition of process validation was not included in any U.S.F.D.A. publications up till September 29, 1978. No cGMP regulation made reference to procedure validation. People who obtained biological products from techniques that were not adequately vetted have been killed, injured, or put in danger in a number of situations. Examples of situations where insufficient raw material management or insufficient viral clearance during processing resulted in compromised products include the contamination of biological systems with viruses. Other products manufactured from human blood or plasma have been tainted by the hepatitis B virus as well as the human immunodeficiency virus. The exact origin of the term "process validation" is up for debate because the ideas behind it are not new, and the terms "verification" and "confirmation" seem to have been used before the term "validation." This phrase first arose in U.S.F.D.A. documents. There was no legal definition for this. "A validated manufacturing process is one that has been proven to do what it purports to or is represented to do," based on an FDA Compliance program from June 1978 called "Drug Process Inspections" (prior to the release of the updated cGMP Regulations). In addition to method qualification—the qualifying of constructions, staff, infrastructure, machinery, and materials—validation also encompasses process oversight for recurring batch as well as run. Through gathering and analyzing data, ideally starting with the stage of method development as well as extending during the fabrication phase, the proof of validation is being gathered.18
None of the yearly revisions of that specific adherence plan included this specific term. However, it remained the sole recognized Validation of processes concept up till the 29th of March 1983. The "Guidelines on General Principles of Process Validation" draft was released on March 29, 1983, and it was finalized in May 1987. The final definition has been a written program that provides high level of trust that certain technique will always provide goods that satisfies its specifications as well as superior features. This definition, which has been widely used in FDA presentations since 1978 and continues to be utilized today, was included in the initial drafts of the May 1987.19
Benefits of validation:
The principles of quality assurance are extended by validation since strict process control is required to guarantee product quality. It is hard to continuously manufacture high-quality products without processes that are validated and managed. When end product testing is done without validation, it provides minimal quality assurance for a number of reasons, including
The restricted quantity of tests that are conducted on a sample.
3. The test's decreased sensitivity.
a) Expenses of prevention.
b) Costs of appraisals
c) Expenses related to internal failure.
d) External failures expenses
E.g.: internal failure costs: Any validated and controlled process were result in fewer internal failures like
1. Fewer rejects
2. Reworks
3. Re-tests
4. Re-inspection Process validation makes it possible to do the job right the first time. Also, a scientifically studied and controlled process makes it unlikely that defective products were dispatched to market thus no recalls or market complaints.20, 21
3. Optimization of the Process:
As a result of validation, the procedure was optimized for optimum efficiency while upholding quality standards. The word "optimize" literally means "to make as efficient, perfect, or helpful as possible."22
4. Safety
Moreover, validation may lead to improved operational safety. For instance, gauges on machinery intended to function at specific temperatures and pressures need to be dependable, meaning they need to be calibrated.23
In order to show that dependability is upheld Validation entailed checking the good's quality elements to ensure that the product's correctness, preciseness, resilience, pureness, and description hadn't been affected during the course of its lifespan.
Types of Qualification:
Every crucial component of a process must be qualified in order for it to be validated. Below are a few of the components:
1. Design Qualification (DQ): The formal confirmation that the equipment, system, and facility design being suggested is appropriate for the intended use.
2. Installation Qualification (IQ): Creating assurance that machinery, processes, and auxiliary systems can function reliably or within predetermined tolerances and limits. It is a written confirmation that The premises, the structure, and technology as installed or altered adhere to the manufacturer's suggestion and the authorized design.
3. Documentation proving that the machinery, structures, and infrastructure that have been built or updated operate as planned within the anticipated operational limits is known as operational qualification, or OQ.
4. Performance Qualification (PQ): The recorded confirmation that, in accordance with an authorized process technique and product specification, the facilities, system, and equipment are connected to perform efficiently and consistently. 24,25,26,27
“Determining documented evidence that offers a high degree of reassurance that a particular process were consistently produce a product that met its predetermined specifications and quality attributes” is the definition. FDA Manual, 1987. The FDA have been revised its recommendations, while the new definition of validation of processes is "the collection and evaluation of data, via the design phase all through fabrication, which the primary goal of dosage form development is to accomplish a predictable therapeutic reaction to a drug within a formulation which is able to undergo massive the production with reproducible product quality.28 The term validation means "Confirmation, via the supply of verified proof, that the specifications for a specific intended purpose or implementation have been satisfied". Generally, depending on the intricate structure of today's medicines, the manufacturer must make confident that the products continue to be of satisfactory quality suitable for the purpose for which they are intended. Validation is the overall expression for a series of activities that show and record that an item in question can be accurately manufactured by the designed processes. To accomplish this with confidence, process control and final product evaluation alone are insufficient to guarantee product quality; all components, including services, It might affect the overall quality of the item in question. must be properly designed, proven to function efficiently, consistently, and have their performance routinely adhered to in order to produce consistently high-quality products. A verified manufacturing procedure is one that "has been proven to do what it purport or it represented to do," according to its working norms. Simply put, "validation" refers to the evaluation of validity or the process of demonstrating efficacy. Validation is the process of demonstrating that a procedure is effective, and it must be carried out according to statistical and scientific criteria. Validation of processes is not just required by the FDA or the US government. In addition to those of the European Union, the Pharmaceutical Inspection Co-operation Scheme, Australia, Canada, Japan, as well as other international organizations, the World Health Organization (WHO) all have comparable standards.
Steps in validation process:
1.Create a validation procedure
2.Perform installation qualification
3.Carry out functioning certification
4.erform performance evaluation
5.Examine findings and draw conclusions
6. Process monitoring and control
Purpose: to guarantee that, in expected circumstances, the process stays inside predetermined bounds.
Examine variations from predetermined parameters.
2.Implement remedial measures
3.Examine if revalidation was required.
4. Modifications to the product or process
To ascertain the impact of a change, assess modifications to the environment, staff, equipment, processes, products, and so on.29,30
In biological chemistry as well as analysis, one type of column chromatography is one which is commonly used for separation, recognize, as well as measure the active ingredients Commonly known as high-pressure liquid chromatography, it is additionally known as high-performance liquid chromatography. This serves as a widely used analytical approach for determining, quantifying, as well as separating every single ingredient. One advanced column liquid chromatography technique is HPLC.31 In order to separate the sample into different elements according to differences in comparative affinities, the HPLC pushes the solvent up to 400 atmospheric pressure. Usually, gravity causes solvent to travel throughout the opening in the column. Typical HPLC elements include a column that contains stationary phase., a pump which forces the mobile phase as well as phases along with the column the detector which detect retention period of molecule.32 The period frame for retention is influenced by connections among the stationary phase, analyzed molecules, as well as the type of solvents being used. The examined substances are mixed in the little amounts to the streams from the mobile phase, also they get slowed down by specific physical or chemical reactions involving the stationary phase.33 Level of attenuation depends on the kind of analyte versus the concentration of the mobile and stationary stages. The retention period is amount of time required by analyte to be eluted.34 Any dispersed mixture of organic substances constitutes a prevalent solvent. The gradient elution technique was successfully utilized to vary the mobile phase's concentration throughout the course of the study. The gradient separates sample mixture on the basis of sample's attraction to the mobile phase. The properties of the analyte and the phase that is stationary have an impact on the choice of solvents, ingredients, and gradients.35
Fig no. 2: flow chart of HPLC
Types of HPLC:
HPLC falls under one of the following categories:
Affinity chromatography, Adsorption chromatography, Size Exclusion Chromatography, Chiral Phase Chromatography, and Ion-exchange Chromatography.37
• In accordance with the elution method are Isocratic separation and gradient separation
• In accordance with operating modes chromatography in both reverse and normal phases.38
1. Normal phase chromatography: This kind of chromatography requires a polar stationary phase as well as a mobile phase that is non-polar. Consequently, the station phase retains the polar component..39 A longer time for elution is the result of the solute molecules' enhanced adsorption ability due to their higher polarity. This chromatography uses chemically modified silica (cyanopropyl, aminopropyl, and diol) as a stationary phase.40 A regular one column, for example, Its within 4.6 mm of its interior diameter & is 150–250 mm long. Following passage down the tube, the polar chemicals in the combination will adhere to polar silicon more persistently than non-polar ones. The non-polar ones will therefore descend a column more quickly.41
2. RP-HPLC (Reversed-phase HPLC): In RP-HPLC, the fixed phase is non-polar, whereas either polar or somewhat polar is the mobile phase. The concept of hydrophobic interaction is the foundation of high-performance liquid42 In a combination of elements, the non-polar in nature stationary phase is going to hang on to analytical substances that are comparatively less strongly polar for an extended amount duration than analytical substances which are much polar. The most polar element thus elutes first.43
Fig no.3: Steps involved in HPLC Method development
The following steps are involved in method development:
1. Understanding the Physicochemical Properties of the drug molecule.
2. Selection of chromatographic conditions.
3. Developing the approach of analysis.
4. Sample preparations.
5. Method optimization.
6. Method validation.44
1. Knowing the drug molecule's physicochemical characteristics:
Physical and chemical features of a pharmaceutical compound are vital to its development. Before creating a method, it is necessary to evaluate the drug molecule's physical properties, such as its ability to dissolve, polarization, pKa value and pH value. The polarity of a compound refers to its physical characteristic.45 this aids analyst in determining the mobile phase as well as solvent compositions. The polar nature between the different molecules explains molecule solubility. Nonpolar solvents like benzene and polar solvents like water don't mix. Generally speaking, materials with similar polarities dissolve in one another because "like dissolves like." Diluents are chosen on the basis of solubility of the analytical substance.46The acidic or basic nature of a material is frequently determined by its pH level. Sharp, symmetrical peaks in HPLC are often the consequence of selecting the appropriate pH for ionisable analytes. The pH level is the amount of hydrogen ions represented as a negative logarithmic of base Ten.47 pH = - log10 [H3O+]. Sharp, symmetrical peaks in HPLC are often the consequence of choosing the right pH for ionisable analytes. Sharp, symmetrical peak forms be present in quantitative evaluation to provide small detecting limits, minimal standard deviations in relation across administering medication, and reliable retention periods.48
2. Chromatographic condition selection:
Previously in the development procedure, an assortment of prerequisites are chosen in order to produce the initial specimen "scouting" chromatograms and these usually depend on reversed phase separates on a column of C18 in addition to Ultraviolet identification. A decision between an isocratic and a gradient method ought to be made at this point in time.49 Column selection the most important and initial step in developing a technique is selecting the stationary phase as well as column. It is challenging to produce a dependable as well as reproducible process lacking a stable as well as high-performance column. The columns should be robust as well as reliable to prevent issues due to non-reproducible sample preservation through the creation of methods. A C8 or C18 column, made of carefully cleansed, lower-acid silicon and intended for separating groups from basic chemicals, will usually be suitable for all specimens.50
The three most significant elements involve column dimensions, silica substrates qualities as well as bound stationary phase parameters. Because of its numerous physical characteristics, silica based packaging is used in the majority of contemporary HPLC segments. The three main parts of an HPLC column are the stationary phase, matrix, and matrix.51 The matrix supporting the stationary phase include polymers, silica, zirconium, and alumina. The most commonly utilized matrices for columns in HPLC are silica. Matrix silica is robust, readily derivatized, consistently sized spheres, and does not compress when compressed. In acidic liquids and almost all organic solvents, silica is chemically unaffected. The fact that silica solid supports disintegrate above pH 7 is one of its drawbacks.52
Chromatographic mode selection:
The analyte's polarity and molecular mass dictate the chromatography phases. Reversed-phase chromatography (RPC), the most widely used method for small chemical compounds, will be the focus of all examples. Ion-pairing chemicals and buffering mobile phases are commonly used in RPC to separate ionisable substances by preventing their analytes from ionizing.53
Buffer Selection
The entire chromatography efficiency and compliance of acetate, potassium phosphate, sodium phosphate, and other buffers had been investigated. General factors to take into account while choosing a buffer Compared to acetonitrile or THF, phosphate dissolves more readily in methanol and water. Certain hygroscopic salt buffers might result in chromatographic changes includes possibly inconsistent selection and enhanced following of fundamental chemicals. In organic/water mobile phases, ammonium salts frequently dissolves more readily. Over time, Trifluoro acetic acid breaks down. It absorbs at low UV wavelengths and is a volatile chemical. In mobile phases that are buffered and have little to no organic modifier, microbial growth can happen rapidly. The growth might affect chromatographic performance because it accumulates on the column inlets. Phosphate buffer speeds up silica dissolution and drastically shortens the lifespan of silica-based HPLC columns at pH values higher than 7. Organic buffers ought to be employed at pH levels higher than seven, if it's possible. Ammonium bicarbonate buffers can be affected by pH changes and are frequently troublesome lasting just 24 to 48 hours. Over time, the pH of this mobile phase keeps getting more basic due to the quantities of carbon dioxide emissions. Once those buffers are ready, A 0.2-m filter should be used to screen them. It is necessary to purify the mobile stages.54
Concentration of buffer:
For tiny compounds, a buffer concentration of 10-50 mm is usually acceptable, and a buffer material should not include more than fifty percent organic matter. This is contingent on the sort and quantity of the buffer. The most frequently used buffering solutions of reversed-phase HPLC refers to phosphoric acid and its potassium or sodium salt equivalents. When assessing organic phosphate compounds, sulfonate solutions might be used rather than phosphoric buffers.55
Selection of Mobile Phase:
The mobile phase affects accuracy, specificity, and efficacy. The composition of the mobile phase or the potency of the solvent used are important factors in a RP- extractionAcetonitrile (ACN), methanol (MeOH), and tetrahydrofuran (THF) are regularly used solvents in reverse phase HPLC having UV thresholds of 190, 205, and 212 nm, correspondingly. Such solvents are mixable with water. When creating a technique, the ideal initial choice for the phase of mobility is a mixture of acetonitrile and water.56
Selection of detectors:
The detection device is a crucial component of HPLC. The choice of detector is influenced by the chemical composition of the study, potential interference, the required detecting limit, the detector's level of accessibility, and/or its cost. Commercial detectors used in liquid chromatography include mass spectrometry (MS), UV, fluorescence, electrochemical, and refractive index (RI). The detector employed depends on the sample and the objective of the investigation.57
3. Developing the approach of analysis:
The first step in establishing an analytical technique for RPHPLC is to choose many chromatographic variables, including the mobile phase, mobile phase columns, mobile phase flow velocity, and mobile phase acidity.. Trials are used to choose each of these attributes, which are subsequently contrasted with the system appropriateness factors. Typical system suitable variables include a following factor that is fewer than two, a spatial resolution of at least five, a retention period of at least five minutes, a hypothetical plate count for more than two thousand, and a percentage R.S.D. for the region of the substance increases in typical the chromatograms of not more than two percent. When two parts have been determined at the same time, a measured wavelengths is frequently an isosbestic spot. By examining the experimental combo, the viability of the suggested method for simultaneously estimating is also evaluated. After that, the commercial item has been diluted to the desired concentration range for uniformity testing.58
4. Sample preparation:
Getting samples ready is a crucial step in the creation of methods which the analyst must investigate. For example, an analyst ought to look at if centrifuged (figuring out the optimal rpm and duration), if there are recalcitrant elements in the sample, shaking and filtration will be needed. The goal is to demonstrate whether leachable sorption or extraction render sample filtering ineffective in influencing the analytical result. The ability of filters for syringes to draw out pollutants as well as insoluble substances Their efficacy is determined by their ability to prevent the release of undesirable artifacts (i.e., extractable) into the filtrate. The sample collecting approach should be thoroughly documented in the appropriate analytical method when applied to an actual in-process sample or dosage form for subsequent HPLC analysis.59 The kind, producer as well as pore dimension of the medium for filtering should all be stated in the analytical process. Twelve providing a treated sample that produces better analytical findings than the raw sample is the intent of sample development. An ounce of the produced sample should be compatible with the HPLC process, fairly free of interferences, and safe for the column. Despite compromising sample retention or resolution, the specimen's solvent needs to dissolve in the phase that is mobile aliquot must be compatible with the intended HPLC process, relatively devoid of interferences, and safe for the column. Collecting samples is the first step in the preparation of samples, which then moves on to injection of sample in the HPLC.60
5. Optimization of method:
Specify the techniques "weaknesses" as well as make improvements via experimental design. Understand how the technique functions using various instrument configurations, samples, as well as environments. The optimization of HPLC settings has been the main focus of HPLC technology development optimization.61The chemical makeup of both the stationary and mobile stages should be considered. Mobile phase parameter tuning is always priority even though it is far simpler and less unpleasant than stationary phase parameter tweaking.62 Only those elements that are anticipated to have a major influence on selectivity throughout the optimization procedure need to be looked in order to minimize the number of test chromatograms needed. The various constituents of the mobile phase that specify acidity, solvent, gradient, flow rate, temperature, sample quantities, injecting volume, and dilution solvent kind are the key control parameters in the optimizing of liquid chromatography (LC) procedures.63 Once enough selectivity is achieved, it is used to compute the ideal resolution for the analysis duration ratio. The factors involved include flow rate, column dimensions, and the size of the column-packing particles. These variables can be altered without affecting selectivity or the capacity factor.64
6. Method Validation
The technique of evaluating and providing objective evidence that the conditions for a certain planned usage are met is known as validating. a way to assess a technique's effectiveness and demonstrate how it satisfies specific requirements. Put differently, it recognizes the potential of your process, particularly when used at low quantities.65
Analytical techniques require validation or revalidation. Prior to their regular implementation; anytime the circumstances moment the method has been successfully proven to work; or time the method is altered.66
Accuracy67
Precision68
Linearity69
Detection and quantification limits70
LOQ is 10 × S/SD, while LOD is 3.3 × S/SD.71
Specificity72
Robustness73
Range74
CONCLUSION:
An outline of the creation and validation of the RP-HPLC Technique is given on this page. Technique creation and verification are ongoing and related procedures which are necessary to evaluate an attribute, designed as well as determine the measurement's effectiveness. The buffering agent as well as mobile phases organic as well as pH compositions have a huge influence on separating efficiency. The advantages of HPLC technology were its great selection, sensitivity, less detecting limit, as well as affordability. Lastly, temperature of the gradient, velocity of flow, slope, and amount of mobile phase enhancers may all can be adjusted. The enhanced method is validated using different specifications in accordance with ICH requirements. The method's linearity, and accuracy, LOD and LOQ, accuracy, and specificity have all been demonstrated to be valid.
REFERENCES
D. Kawade*, Aishwarya Shrirao, Irshad Ahmad Mohd Salim, Comprehensive Review on Analytical Method Development and Validation: Focus on HPLC Techniques, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 2, 1444-1458. https://doi.org/10.5281/zenodo.14883977
10.5281/zenodo.14883977
