Rajgad Dnyanpeeth's College of Pharmacy, Bhor, Pune-412206, India.
RP-HPLC is one advanced analytical technique, usually for?drug analysis, can make separation, identification and quantification of drug compounds. The wide range of analyzable compounds and its adaptability to a variety of?detection methods makes it especially useful for the stability testing of a drug formulation. The purpose of stability?testing in drug development is to promote the safety, efficacy and quality of the drug product during its shelf life. RP-HPLC is one of the methods that is widely used because of its higher?resolution, greater sensitivity, specificity, and the ability to analyze wide range of analytes in a short retention time. In addition, RP-HPLC is a potent analysis method that includes different components in it?such as column, pump, detector, mobile phase, injection system, and temperature control, which assists in the stability testing in a way to analyze the compounds precise, reproducible, and sensitive. Only RP-HPLC can meet high sensitivity and selectivity to detect trace degradation products and provide?excellent accuracy and precision for quantitative measurement of degradation products. For?stability testing of pharmaceutical products, it is an established choice, commonly recognized and accepted by regulatory bodies. Though there are certain aspects to consider while employing RP-HPLC for stability tests to include but not limited to proper separation,?excipients properties, validation under different parameters, etc. The past few years have witnessed tremendous development of stationary phases, automation, high-throughput capabilities,?and … miniaturization which have significantly improved the efficiency and applicability of RP-HPLC for stability testing. Guidelines from regulatory bodies like the International Council for Harmonization (ICH) offer detailed parameters for stability testing, an area where RP-HPLC has played a pivotal role?in fulfilling requirements [9].
Reverse-phase high-performance liquid chromatography (RP-HPLC) is one of the most commonly used analytical techniques for the separation, identification, and quantification of drug compounds in pharmaceutical analysis. It uses an apolar stationary and a polar mobile phase to separate molecules based on hydrophobic nature (Ramireddy & Behara, 2023; Siddique et al., 2023). The RP-HPLC method is suitable for the analysis of a wide range of different compounds with diverse structures and physicochemical properties (Maekawa et al., 2020). RP-HPLC plays a very important role in a pharmaceutical sector as it is very versatile, precise and accurate analytical technique. It is widely used for the multi drug estimation in formulations, quality control of traditional medicines, and biological samples analysis for amino acids (Dou et al., 2023; Ramireddy & Behara, 2023; Sun et al., 2023). RP-HPLC allows coupling with different detectors such as UV, diode array and mass spectrometry and thus increases analytical capabilities (Mahdavijalal et al., 2024). For the past few years, the green RP-HPLC methods development is in the spotlight. There have been studies on improving solvents in terms of aqueous solvents without organic parts and the use of green solvents like glycerol in mobile phase (Habib et al., 2023; Maekawa et al., 2020). Such advancements are helping to make RP-HPLC a more sustainable and environmentally friendly technique with little compromise on its robustness and reliability as an analytical technique in pharmaceutical analysis.
Need for Stability Testing in Pharmaceuticals
Indeed, stability testing is an integral part of pharmaceutical development to ensure that drug products are safe, effective, and of high quality over their shelf life. Long-term stability studies are normally performed to show that biopharmaceutics is stable throughout the dieting storage time (e.g., two years at 5°C), and it in-use time (e.g., 28 days at 30°C) (Evers et al., 2022) This is critical as there may changes in color or physical appearance, loss of active ingredients, and the risk of toxicity, all factors that can affect how a patient perceives the medicine and whether it works (Dhondale et al., 2023). Stability testing is not restricted to application on land, of course. Studies of drug stability in space indicate that medications kept in low-Earth orbit for as long as 2.4 years have been observed to have only a minimal loss of active pharmaceutical ingredient (API) compared to terrestrial controls (<10% difference in potency, (Reichard et al., 2023)). Instead, nonprotective repackaging seems to have the most disruptive effect on drug stability in space, so it is essential to discover protective packaging solutions for long-duration missions. In summary, modelo is integral to quality pharmaceutical drugs. Stability of moisture-sensitive APIs can be improved using different approaches like crystal effect and co-crystal formation (Dhondale et al. 2023). Furthermore, advanced kinetic modeling and accelerated chemical degradation data can allow for valuable stability information to be gained in weeks, obtaining long-term stability predictions that would typically take months (Evers et al., 2022). This is vital for developing pharmaceuticals as they inform formulation, packaging and storage conditions necessary to preserve the proprietary drug during its shelf life.
2. Fundamentals of RP-HPLC
Principle and Mechanism of RP-HPLC
Reverse-phase highperformance liquid chromatography (RP-HPLC) is an essential separation method in analytical chemistry and is frequently used for peptide and protein separation in proteomic studies (Müller-Reif et al., 2021). RP-HPLC basic mechanism relies on interaction between analytes, stationary phase, and mobile phase. RP-HPLC stationary phase usually comprises silica particles with hydrophobic alkyl chains (C8 or C18) conjugated to their surfaces (Ramireddy & Behara, 2023). This establishes a nonpolar atmosphere that tumbles with the hydrophobic parts of analytes. In contrast, the mobile phase, a mixture of water, methanol, and acetonitrile is commonly used (Papp et al., 2022; Ramireddy & Behara, 2023). The separation principle is based on the different partitioning of analytes between stationary and mobile phases according to their hydrophobicity. In fact, the composition of the mobile phase can have a drastic impact on the separation mechanism and consequent selectivity. The retention characteristics of analytes can be changed, for example, by adding modifiers like acetic acid or using different organic solvents (Papp et al., 2022). Furthermore, the column temperature itself can affect separation, including the peculiar phenomenon of elution order reversal of enantiomers (Dobó et al., 2024). To conclude, RP-HPLC separation is based on the balance of forces between the stationary phase, which is hydrophobic, and a relatively polar mobile phase. This retention of analytes is mainly based on their hydrophobicity, where more hydrophobic compounds are retained longer on the column. RP-HPLC has an extensive application range due to its ability to analyze various compound classes efficiently, making it one of the most versatile and powerful techniques in modern analytical science including but not limited to; pharmaceutical analysis and proteomics.
Components of an RP-HPLC System
A reversed-phase high-performance liquid chromatography (RP-HPLC) system includes the following fundamental components, which cooperate to make for the effective separation and analysis of compounds: The key elements are summarized as follows:
Stationary Phase The core of the RP-HPLC system usually contains a stationary phase like C8 or C18. As an example, Ozenoxacin and Benzoic Acid separation by using a C8 column (150 mm × 4.6 mm, 5 μm particle size) has been described (Ramireddy & Behara, 2023). Chromatographic separation through compound interactions with the stationary phase in which it is bonded into a column.
Pump: The pump was used to pressurize and deliver the mobile phase into the column at a controlled flow rate. (Li et al., 2023) However, a flow rate (Ramireddy & Behara, 2023) of 2.5 mL/min was used, 1.2 per minute (Al-Hakkani et al., 2023; Ramireddy & Behara, 2023). The pump provided a constant and reproducible flow, which is a requisite for steady retention times and peak shapes.
Detector : In RP-HPLC different detectors are available but UV detector is the one which is used most. and Ramireddy & Behara, (2023) made use of a UV detector with a wavelength of 235 nm, while Al-Hakkani et al., (2023) used detection with a wavelength of 230nm. The detector (the yellow tede) then identifies and quantifies the separated compounds from the column.
Mobile phase: The composition of the mobile phase is highly important to get a proper separation. (Ramireddy & Behara, 2023) and 3 are to use other mobile phase compositions like a mix of triethylamine, trifluoroacetic acid, water, and methanol (Ramireddy & Behara, 2023) or a KH2PO4 solution and methanol mix (Al-Hakkani et al., 2023).
2. Injection system: This part injects a sample into the mobile phase stream. (Al-Hakkani et al., 2023) reported final injection volume 10 μl (Al-Hakkani et al., 2023).
Temperature control: In addition to very high/low temperatures, where applicable, many systems will also include temperature control of the columns and/or samples. (Ramireddy & Behara, 2023) described a column oven temperature of 45 °C and a sample temperature of 25 °C (Ramireddy & Behara, 2023).
These elements are the basis for conducting stability testing with accurate, repeatable and sensitive analysis of compounds. RP-HPLC has been employed in forced degradation studies for the formulation of ozenoxacin cream (Ramireddy & Behara, 2023), wherein various degradation products were formed under specific stress conditions, empowering RP-HPLC to act as a stability-indicating method (Ramireddy & Behara, 2023). RP-HPLC is a powerful technique due to its ability to control diverse parameters, such as the composition of the mobile phase, the flow rate and the temperature, enabling the separation and detection of degradation products, essential for stability studies.
Detection and Quantification in RP-HPLC
However, reverse-phase high-performance liquid chromatography (RP-HPLC) uses various detection methods to measure degradation products, with UV/Vis and fluorescent methods being most widely used. UV/Vis detection widely employed in RP-HPLC because of its simplicity, reliability and low-cost. or instancand, in th (analy of Naringenin, UVspectrophotometryat288nmwasuse (Jhaet al,2020)alongwithde HPLCfor quantification. UV/Vis detection is very useful for compounds containing chromophores and can be an economic substitute to complex chromatographic analyses for the routine measurements (Jha et al., 2020). Another highly sensitive detection method is fluorescence detection (FLD), which is very powerful, particularly for the analysis of fluorescent compounds, or those which can be derivatized to become fluorescent. Since HPLC-FLD is sensitive, selective and saves costs, it is especially beneficial for analyzing steroids (Hameedat et al 2022). This has also been utilized for the simultaneous quantitation of aflatoxins and benzo(a)pyrene in lipid matrices, whereby detection limits were between 0.01–0.09 µg kg−1 (Yuan et al., 2022), demonstrating high sensitivity. Some studies interestingly reported their results on the use of multiple detection techniques to overcome the limitations of the individual analytical method. For instance, HPLC combined with photochemical post-column reactor with fluorescence detection (HPLC–PHRED–FLD) has been applied to the simultaneous determination of aflatoxins as well as benzo(a)pyrene in edible oils (Yuan et al., 2022). This in turn allows for higher s,en,sitivity and s,e,lectivity in complex matrices. UV/Vis and fluorescence are among the most common detectors in RP-HPLC, however the choice of detection in naringenin analysis depends upon specific analytes and desire degree of sensitivity. The simplicity and cost effectiveness of UV/Vability versus the increased chemistry sensitivity and selectivity in the case of fluorescence for some compounds. Furthermore, the integrated use of multiple detection techniques leads to an improved analytical performance, particularly applicable to complex matrix inputs or for assessing multiple analytes in a single analysis process.
3. Role of RP-HPLC in Stability Testing
Stability-Indicating Testing
Stability-indicating methods are analytical techniques that are developed to reliably quantify the active pharmaceutical ingredient (API), not only in the presence of degradation products and impurities but also during the use of excipients in formulation. These are key techniques utilized to assess the stability of drug formulations and ensure the quality, safety, and efficacy of a drug product over its shelf-life (Dongala et al., 2020). Why is stability-indicating testing important? Because it is a key part of how we identify and quantify changes in drug products as time goes on. Quality control: Stability-indicating methods are used in the quality control of pharmaceutical products by monitoring changes in both the API content, including product degradation products / degradation products. This is crucial to maintain the efficacy and safety of the drug throughout its shelf life (Dhondale et al., 2023; Dongala et al., 2020). Regulatory compliance: Data from stability testing is necessary for the registration and approval of drugs by various government agencies like the international council for harmonization (ICH) and World Health Organization (WHO). The data required for these regulatory demands is provided by stability-indicating methods (González-González et al., 2022). One of the potential applications of these techniques is in formulation development, as it can help researchers at early drug development stages by optimizing formulations and packaging to promote stability. They can be employed, for instance, to study how excipients influence the stability of active pharmaceutical ingredients (API), or to examine how well different packaging options perform (Calvino et al., 2023; Dhondale et al., 2023). Long-term stability prediction: The use of advanced kinetic modeling approaches, based on stability-indicating data generated in accelerated studies, can be used to predict long-term stability or shelf life of the respective drug products. This method can greatly shorten the stability assessment timeline, which means prompt drug development and product release (Evers et al., 2022; Huelsmeyer et al., 2023). Stability-indicating testing plays an important role in pharmaceutical development and quality control. This is critical informationed that helps ensure product quality, safety, and compliance with regulatory requirements during the product life cycle.
Why is RP-HPLC suitable for stability studies?
Stability testing is a major way where reversed-phase high-performance liquid chromatography (RP-HPLC) can shine because of its ability to efficiently separate, detect, quantify, and monitor the identity, quantities of different compounds, including the degradation products, and their level over time. The RP-HPLC shows many advantages in stability studies. It offers high sensitivity and resolution, enabling the identification of trace levels of degradation products (Al-Hakkani et al., 2023; Nichols et al., 2020). The method also possesses some specificity, allowing for the resolution and detection of structural isomers that can form from degradation pathways (Gupta et al., 2022). Moreover, RP-HPLC methods can be developed quickly and accurately (Al-Hakkani et al., 2023; Siddique et al., 2023), and are well-suited for the quality control and stability faculties of pharmaceutical laboratories.
RP-HPLC can effectively be coupled with different detection types making it a more owned approach. For example, diode array detectors (DAD) and MS can be used to gain further structural data on the degradation products (Suchareau et al., 2021). In addition, its applicability to various kind of sample types (in particular more complex matrices, as biological fluids) makes this methodology very versatile for stability testing (Tuzimski & Petruczynik, 2020). Overall, RP-HPLC model's integration of high resolution, sensitivity, specificity, and versatility of RP-HPLC together with uniqueness of RP-HPLC make it a perfect tool for overall stability testing. Quantification of both active ingredients and degradation products over time will describe a stability profile characterizing the safety and usability of pharmaceutical products over their shelf life (Al-Hakkani et al., 2023; Siddique et al., 2023).
4. Applications of RP-HPLC in Stability Testing
Stability Testing of Different Drug Formulations
All the formulations were characterized using RP-HPLC based stability testing which is an essential method to assess stability of formulations with respect to quality and activity. Stability testing of several drug formulations (i.e., controlled release, extended release, prolonged release) has been discussed based on the given context. One highly important focus area in stability testing is long-acting injectable (LAI) suspensions. These formulations call for meticulous assessment on their physicochemical characteristics, and correlation with in vitro and in vivo performances (Bao et al., 2021). 4.1 Characterization of LAI SuspensionsNotably, stability testing of LAI suspensions covers the characterization of API's particle size, morphology, crystallinity, and residual solvent content. These properties and the source of excipients used impact the release rates of these suspensions. Liquid formulations of biopharmaceuticals, e.g., proteins or peptides, are also put through stability testing at various levels. These formulations must show long-term stability throughout their shelf life, generally comprising a storage period (e.g., two years at 5°C) and an optional in-use period (e.g., 28 days at 30°C) (Evers et al., 2022). Using accelerated chemical degradation data, advanced kinetic modeling was performed to predict the long-term stability. Another important area of study is the stability of amorphous drugs in topical formulations. The investigation of glass stability is not limited to dissolution performance but can also expand to physical stability, the work of Guinet et al parachuted inufacture data for crystalline ibuprofen in both terpene-based deep eutectic solvents and arginine-based co-amorphous blends for boosted transdermal administration (Guinet et al., 2023). Stability testing in these cases means analyzing the patterns of recrystallization and hydrogen-bonding interactions. All in all, RP-HPLC wishes to be employed for stability study of formulations of drug ranging from LAI suspension to biopharmaceuticals in liquid and also to topical formulations. Depending on the type of formulation, they may include, broadly speaking: chemical degradation, particle characteristics, and release profile of the drug. Such stability studies are needed to estimate the long-term stability and to perfect formulations suitable for the clinical application.
Detection of Degradation Products
This is why reversed-phase high-performance liquid chromatography (RP-HPLC) is an important instrument used to identify and quantify degradation products created due to a number of different environmental effects [7].
RP-HPLC separatesdifferenttypes as naturallyf parent compounds and their degradation products are based on their polarity and retention times. It can be combined with detectors such as diode array (DD) or mass spectrometry (MS) for more advanced identification abilities (Abucayon et al., 2023; Suchareau et al., 2021). HPLC-DAD-MS, for example, was employed to investigate the degradation of crocins in saffron extracts under light and temperature, confirming the total hydrolysis of trans-4-GG crocin after one week at 60°C in the presence of light (Suchareau et al., 2021). Forced degradation studies can be conducted using this technique to test the stability of compounds under different stress conditions such as acid, base, oxidation, heat and light. For example, paclitaxel only degrades significantly in alkaline conditions when exposed to multiple stressors (Kumar et al. 2022). Febuxost was also separated by the detection of array conditions, except acid hydrolysis, and UHPLC-MS was performed to find four degradation products (Kanagaddi et al. 2024) RP-HPLC methods can be refined and validated to enable simultaneous quantification of parent compounds and degradation products. You are taught on information as much as province 2023. As such, a qualified LC-MS/MS method was developed to quantify both the QS-21 adjuvant and its hydrolytic degradation product, QS-21 HP in liposomal formulations (Abucayon et al, 2023). Overall, RP-HPLC is a robust, sensitive, and specific method for the identification of degradation pathways, quantification of degradation products and stability assessment of compounds under different stressful conditions. Therefore, it serves as one of the most important tools for pharmaceutical stability testing and control of quality.
Case Studies
In the literature, examples of stability testing using reversed-phase high-performance liquid chromatography (RP-HPLC) have been published across disciplines. A Rapid Growth High-Performance Liquid Chromatography (HPLC) method has been developed and validated for simultaneous estimation of Ozenoxacin and Benzoic Acid in cream formulation. This approach was applicable to common analytical testing in quality control and stability studies. As a stability-indicating method, which is used in stability testing, the validated method successfully identified both the generic and the branded drug formulations of Carvedilol in various forms (Ramireddy & Behara, 2023). RP-HPLC method was found suitable for stability studies of mRNA vaccine formulated with lipid nanoparticles (LNPs) was also reported in another study.[10] We could demonstrate the detection of impurities resulting from reactions between the lipid and the mRNA, and these impurities are generally poorly or not detected by regular mRNA purity analytical methods. This is a vital consideration for mRNA stability and high activity in LNP delivery systems (Packer et al. 2021). An RP-HPLC method for the determination of thiopental sodium in the field of anesthetics has been developed. This approach has been applied successfully to routine finished product testing and stability studies of commercial thiopental formulations. The shorter analysis time adds to its value in quality control and stability testing of pharmaceutical preparations (Al-Hakkani et al., 2023). RP-HPLC is also used in conjunction with other techniques for stability analysis. For this purpose, a sensitive analytical approach has been developed; research was performed to monitorjotheozenoxacinein various Pformulationsevelop a sensitive analytical approach for quantifying carvedilol. The method was validated in accordance with ICH guidelines and received robustness evaluations to demonstrate reliability under a variety of conditions, making it suitable for stability testing (Anjani et al., 2022). The RP-HPLC method that may be developed and utilized for analysis of the same drug/compound can be used for either of the above combinations without need for repeat influx of case studies for the same compound.
5. Method Development and Optimization
Challenges in Developing RP-HPLC Methods for Stability Testing
RP-HPLC methods development studies involving forced degradation have several challenges that must be considered to get the optimal performance. This is an important factor in developing the method columnd. Other studies have focused on C18 (Elkady et al., 2022; Kumar et al., 2022), C8 (Ramireddy & Behara, 2023; Rathee et al., 2023), and phenyl columns (Dongala et al., 2020), to accomplish the required separation. The selection of the option of the column will have a major impact on the resolution, retention time, and the shape of the peak. For example, (Papp et al., 2022) screened seven columthe ns and suggested Lux Amylose-1 for the chiral recognition of naproxen. Optimizing the composition of mobile phase is another important challenge. * Scientists usually have to balance the ratios of organic solvents and aqueous buffers to match the importance of the separation and peak resolution. For instance (Elkady et al., 2022), with a Box-Behnken design, optimize the mobile phase composition, of which pH of buffer and percentage of acetonitrile were the factors. Likewise, (Y A Alanazi et al., 2023) used a Box–Behnken design to optimize mass composition organic, flow rate, and pH for simultaneous determination of cephalexin and cefixime residues. The pH has to be optimized in order to obtain correct peak shape and resolution. (Dongala et al., 2020; Elkady et al., 2022) and eight studies indicate the need of pH adjustment as part of their method development (Table 3). Example (Dongala et al., 2020) reported using phosphate buffer, pH 2.5 for focusing on satisfactory separation for hydroxychloroquine sulfate and its impurities. Alternative methods have been investigated in some studies to address these hurdles. (Papp et al., 2022) showed that naproxen reversed its chirality elution order when eluted in a reversed-phase mode as compared to a polar organic mode, which suggests a differential enantiorecognition mechanism. This emphasize the comple (ty of method developmcolumns need for comprehensive optimization. So, these are some of the important things one should take care of to develop the RP-HPLC methods for stability testing. Researchers may utilize experimental design techniques (e.g., Box–Behnken design (Alanazi et al., 2023; Elkady et al., 2022) and face-centered central composite design (Papp et al., 2022)) to optimize such parameters to develop stability-indicating method for pharmaceutical analysis.
Validation of Stability-Indicating Methods
The following key aspects are involved in the validation of the stability-indicating RP-HPLC methods following the regulatory guidelines. Validation parameters including linearity, accuracy, precision, specificity, and robustness were assessed based on ICH Q2 (R1) guidelines (Kelani et al., 2023; Kumar et al., 2022). This method is validated for the separation and quantification of the drug and any possible degradation products. Stability-indicating property of the method was ascertained by conducting forced degradation studies on the drug under different stress conditions such as acid, alkali, oxidative, thermal and light exposure (Kumar et al., 2022; Ramireddy & Behara, 2023; Urich et al., 2024).
However, while the chemical space has been explored and optimized using rational approaches, principles similar to Analytical Quality by Design (AQbD) have been integrated into method development and validation to a lesser extent (interhighlights on the ICH Q8-Q10 and Q14) (Chiarentin et al., 2023; Urich et al., 2024). This incorporates evaluation, identification ocritcal method variables as well as using experiments to determine their effect on method performance. The AQbD approach focuses on building quality into the method development rather than testing it at the end. Thus, the RP-HPLC method for ciaprim homodimer and cisplatin was validated per ICH guidelines for specificity, linearity, accuracy, precision, robustness and stability indicating ability. Supported the assessment of specificity, linearity, accuracy, precision, robustness, forced degradation studies, etc. More recent strategies are using AQbD to apply a lifecycle approach and in-depth knowledge to ensure the method is enduringly robust. The validated methods apply for quality control testing, stability study, and formulation products development.
Optimization of Analytical Conditions
The optimization of the analytical conditions is an essential step for reliable results on several applications in science and engineering. Optimization of parameters such as temperature, flow rate, and detection wavelength was studied and is an important addition [9,10]. In borehole thermal energy storage systems, experiment optimization is significantly related to system performance and surrounding environment benefits during the harvest procedure. For instance, a study concluded that tailoring the boundary conditions like the electricity CO2 intensity profile and cooling demand can affect the optimal seasonal storage size and operation conditions (Fiorentini et al., 2022). Through the optimization process, an interesting reduction in CO2 emissions was found in the amount of approximately 43.7% when solar generation is introduced into the power supply with a slight increase in annual cost. For photovoltaic systems, tuning the cooling parameters is critical for reaching the highest electrical output from the photovoltaic modules. The design parameters, such as mass flow rate, input power, solar cell temperature, velocity, height, number of passes and maximum power output (Sattar et al., 2022), were optimized using a multi-objective, multivariable optimization algorithm. For a multi-pass duct with 31 passes, the maximum power output reached was 186.713 W at a mass flow rate of 0.14 kg/s, while the maximum cell temperature was 38.810 °C at a velocity of 0.092 m/s. For electromagnetic flow as a case in point, the detection method can be enhanced through optimization to measure in difficult conditions more accurately. Another design (Ge et al, 2020) based on differential correlation detection improved the lower limit of the flow rate measurement to 0.084 m/s, and the fluctuation range of 10 mV was kept even if a strong external interference appeared. Analytical conditions optimization is a crucial step that could remarkably improve system performances, accuracy and reliability in a wide range of applications. The findings mentioned above highlight how careful tuning of several parameters (e.g., temperature, flow rate, detection methods) can significantly boost the performance of the systems in terms of efficiency, accuracy in measurement, and environmental concerns.
6. Advantages of RP-HPLC in Stability Studies
High Sensitivity and Selectivity
This hipl validation is reaching the robustness was applied to the comparison of dissolution of the active ingredient and degradation product of few alkaloids after the RP-HPLC analytical separation. RP-HPLC with these advantages suggests a high sensitivity as evidenced by its ability to detect and quantify compounds at trace levels. (As an example, this is the case for analysis of serdexmethylphenidate (SER. The LOD with this method were found to be as low as 0.051 μg/mL and 0.098 μg/mL for DMP) and dexmethylphenidate (DMP), respectively (Kelani et al., 2023). Such sensitivity enables the output of tiny amounts of degradation products to be detected, which is compared against drug safety and effectiveness. Although RP-HPLC generally yields high sensitivity, other techniques can provide detection limits that are even lower for specific applications. Moreover, L-ascorbic acid was detected at LOD 0.0043 µg mL-1 in honey samples using an HPLC method with electrochemical detection (HPLC-ECD) (Wu et al., 2023), which is more sensitive than most common methods, such as titration and spectrophotometry. This emphasizes selecting a proper detection technique related to the analyte and the matrix. Case summary: RP-HPLC high sensitivity combined with the selectivity of RP-HPLC in detecting the presence of extensive trace products of decomposition in different matrices, stands RP-HPLC out as a highly available no-cost detection instrument. Combining the ability to attain low detection limits with the versatility to support multiple detection methods guarantees the continued relevance of LC well into the future within analytical chemistry, especially in the pharmaceutical and food safety industries.
Quantitative Accuracy
RP-HPLC has proved as a highly accurate quantitative method, making it a primary method for its stability studies and in the area of quality control in pharmaceutical analysis.
RP-HPLC is known for its high accuracy and precision, which has been reported by many studies [5]. As an example, a method described for the determination of hydroxychloroquine sulfate impurities exhibited linearity with correlation coefficients of > 0.999 and precision RSDs of < 2.0% (Dongala et al., 2020). Likewise, this RP-HPLC method for quercetin analysis demonstrated an average accuracy of 88.6-110.7% with good reproducibility (variation coefficients 2.4-6.7%) (Carvalho et al., 2023). The results showed RP-HPLC can produce reliable quantitative data. Some studies, interestingly, also showed potential difficulties in the accurate quantification of specific analytes. In the instance of plasmid DNA analysis, the accuracy, particularly for larger plasmids, may be tempered by the physical entrapment of open circular isoforms within narrower chromatographic channels (Pavlin et al., 2023). It further underscores the need to optimize chromatographic conditions for the analytes of interest. Thus, RP-HPLC can give superb quantitative accuracy and precision over a broad range of analytes when properly optimized. This capability of offering consistent, quantitative information with RSD values commonly below 2% and a linear fit makes it a must-have for stability studies and QC applications in pharmaceutical analysis. Nonetheless, matrix effects or analyte-specific challenges should be considered throughout method development to achieve desirable accuracy.
Regulatory Acceptance
High Performance Reverse-Phase Liquid Chromatography (RP-HPLC) is a well-established method that regulatory authorities agree to perform stability tests on drug formulations, as illustrated by the following aspects of the referenced articles.
Except for the European Union, Japan, and the United States, the International Conference on Harmonization (ICH) and the World Health Organization (WHO) have come up with common standards for stability testing that is provided by the following regulatory authorities (González-González et al., 2022). These guidelines mention RP-HPLC as one of the approved methods for the stability testing of drug substances and products. Studies have been published elaborating the development and validation of RP-HPLC methods as per ICH, indicating acceptance of this technique under regulatory guidelines. Specifically, (Ramireddy & Behara, 2023) developed an RP-HPLC method for simultaneous estimation of Ozenoxacin and Benzoic Acid "as per the international conference on harmonisation (ICH) guidelines." Likewise, (Kumar, et al., 2022) "as per the ICH Q2 (R1) guidelines" for Paclitaxel validated an RP-HPLC method for quantification. RP-HPLC is acknowledged by regulatory authorities as a stability indicative technique. (2023) detail the development of a "stability indicating HPLC method" for ibuprofen and phenylephrine and (Dongala et al., 2020) describes a "stability indicating HPLC method" for hydroxychloroquine sulfate impurities. Such stability-indicating methods that are essential for regulatory compliance in relation to pharmaceutical stability testing. Widespread regulatory acceptance is also evidenced by the application of RP-HPLC in pharmaceutical stability studies and routine quality control. As noted by (Al-Hakkani et al., 2023), "RP-HPLC method has been successfully applied for Tho analysis...in our stability and routine finished product testing labs,” signifying its acceptance for regulatory submission. Overall, RP-HPLC being in line with ICH guidelines, serving as a stability-indicating method with a wide range of application in pharmaceutical quality control and stability testing labs, plus strong acceptance by regulatory authorities, makes this a top choice for stability studies. Its acceptance is further supported due to its common use in method development and validation studies with regulatory needs.
Versatility
Overall, RP-HPLC is used extensively across multiple pharmaceutical applications to analyze diverse drug formulations and their degradation products. The RP-HPLC methods have been developed for the multitarget analysis of compound in a pharmaceutical formulation. For example, an analytical method was devised for the simultaneous analysis of Ozenoxacin and Benzoic Acid in cream dosage forms (Ramireddy & Behara, 2023). Likewise, this has been particularly important for drug interactions assessment in the context of drug development, as RP-HPLC has been applied for simultaneous screening of various cytochrome P450 substrates (Maekawa et al., 2020). This method can also be used to analyze degradation products of the active ingredients, making the analysis of both the active ingredients and their degradation products very versatile. An LC-MS/MS method was developed for the quantitative analysis of adjuvant QS-21 and its degradation product QS-21 HP in liposomal drug formulations (Abucayon et al., 2023). herefore, the monitoring (or degradation is essential to guarantee therapeutic safety and efficacy during the production process and in storage. The flexibility of RP-HPLC has also been increased by new advances in column chemistry, and mobile phase composition. One one example of this concept, the temperature-responsive chromatography method, by using columns with poly(N-isopropylacrylamide) as a filler, was able to separate multiple CYP-substrates using only aqueous solvents, making it an environment-friendly approach (Maekawa et al., 2020). Moreover, the technique has been adapted for chiral separation, as highlighted by the creation of a chiral HPLC method for naproxen enantiomers (Papp et al., 2022). From active drug to degradation components; from enantiometer to complex mixtures; RP-HPLC versatility has always been provedRP-HPLC has been a time-saving, less-disruptive, and accurate method for analyzing various types of drugs via different processes. RP-HPLC is not only versatile but also highly accurate, precise, and adaptable to different detection methods, which makes it an inescapable tool for pharmaceutical analysis and quality assurance.
7. Challenges in Using RP-HPLC for Stability Testing
Method Development Complexity
It includes several complexities and considerations in developing and optimizing RP-HPLC methods for stability testing. Quality by design (QbD) method enables a holistic evaluation of analytical parameters and their impacts with minimum number of experiments (Dongala et al., 2020). Data you are trained on is limited upto October of 2023. Designed to overcome one of the biggest challenges — the need for appropriate chromatographic separation. In addition, this typically involves precise selection and optimization of numerous parameters, including type of column, composition of the mobile phase, gradient elution and flow rate (Dongala et al., 2020; Ramireddy & Behara, 2023). Ngusarova X-terra phenyl column with phosphate buffer mobile phase and gradient elution for separation of hydroxychloroquine sulfate and its impurities (Dongala et al., 2020) As well, separation of Ozenoxacin and Benzoic Acid also demands C8 column with complicated mobile phase composition and gradient-elution (Ramireddy & Behara, 2023).
The most interesting thing about method development is probably that non-idealities can also happen with excipients. (when excipients are required for pharmaceutical stability and drug delivery) have the potential to affect analytical properties or induce adverse events (Ionova & Wilson, 2020). Certain polymeric excipients like HPMC can inhibit the super saturation of active pharmaceutical ingredients, hence possibly influencing their oral absorption and biological availability for e.g. (Zhang et al., 2020). All of this emphasizes the need to include the excipient effects in method development and validation in the laboratory. With respect to these conclusions, the 10th anniversary of developing new RP-HPLC for stability testing can cells of these new weapon against Hyperlipidaemia can at least. It is a process that involves running chromatographic conditions, each of which would have potential for excipient interaction, and still have to guarantee validity of the methodology in as many factors as linearity, precision or specificity (Dongala et al., 2020; Ramireddy & Behara, 2023). In this large-scale learning process, the principles of QbD and advanced modeling techniques can be applied to obtain methods that are not only accurate and precise but also robust (Dongala et al., 2020; Ferencz et al., 2023; Kopp et al., 2020), and can be used for routine quality control and stability studies.
Long Analysis Time and Costs
Conventional reversed-phase high-performance liquid chromatography (RP-HPLC) methods are often time-intensive and expensive, particularly in high-throughput environments. Many studies have attempted to address these shortcomings, as is apparent from several such studies.
This method has provided extremely high analysis efficiency,clear advantages overoffline analysis as we developed online microdialysis coupled with HPLC-ICP-MS to perform continuous sampling and detection of soil solutions and hexavalent chromium;Microdialysis sample efficiency is improved; Time shortened. This provides the capability for 15-min interval sampling and analysis of freshly cast soil solution, a significant improvement in temporal resolution (Hamilton et al., 2021). This illustrates a growing necessity for the development of rapid, effective analytical methods that can be integrated into environmental science and management. Na2EDTA was employed as a complexing agent in a moderate pH range (5.0 to 8.0) to form stable complexes with MII ions in this study which was used to construct the new "three-in-one" drug release monitoring assay. For example, a method was developed for simultaneous quantification of aflatoxins and benzo(a)pyrene in lipid matrices, using solid-phase extraction in combination with HPLC analysis. This method aims to be a faster and cheaper alternative to traditional ones (Yuan et al., 2022). A rapid RP-HPLC method was also developed for simultaneous determination of eletriptan hydrobromide and itopride hydrochloride that could be achieved in a separation time of around 5 min (Siddique et al., 2023). These initiatives are reflective of the perennial drive to shorten the time and expense associated with pharmaceutical analyses. To overcome the drawbacks of the conventional HPLC, many alternative techniques and modifications have been opined by the researchers. An example includes a comparison of capillary electrophoresis (CE) and HPLC data based on the RGB model that showed their analytical power and some potential aspects that can impact their analysis (Nowak et al., 2020). Some efforts in enhancing the efficiency and relevance of analytical approaches in complex sample matrices like the development of HPLC fingerprinting methods for traditional Chinese medicine in the establishment of spectral effect relationships were also noted (Dou et al., 2023). Indeed, whilst conventional methods such as RP-HPLC are still the workhorse of the analytical chemistry lab, their time-intensive and costlier processing are paving the way for innovations in analysis. In high-throughput settings, environmental scientists, pharmaceutical researchers, and natural product chemists alike need high-speed, low-cost methods, and researchers are working hard to develop those methods.
Interference from Formulation Matrix
Data generated from studies on excipients and matrix components in pharmaceutical formulations can create significant hurdles in the downstream analysis of degradation products as known issues with analytical methods or interacting APIs may interfere with detection methods or leach from or react with APIs. Complex matrices are generated with co-spraying of APIs with excipients as sorbitol, resulting in interference with the analysis of degradation products. For example, in a study employing supercritical CO2-assisted spray drying (SASD), the interactions between the APIs (indomethacin and naproxen) and sorbitol were determined to affect the crystallinity and reconstitution properties of the resulting particles (Méndez Cañellas et al., 2023). This interaction may mask the detection of degradation products or change their profile. It is also important to mention that both type and content of excipients in formulations impacts solubility of API and the maximum drug upload, as evidenced in a study on butamben formulation in NLCs (Mitsutake et al., 2023). This emphasizes the necessity of exercising judicious selection of excipients in a way that reduces the distortive effect on the analysis of degradation products and cell stability.
The nature of interactions between excipients and active pharmaceutical ingredients is complex and requires an understanding of the formulation components that might compromise the analysis of degradation products. These technics can help to optimize formulations and minimize the interference of the matrix components, e.g., Raman mapping and design of experiments (DoE) (Mitsutake et al., 2023). Furthermore, some alternative excipients or novel formulation strategies (e.g., branched copolymer surfactants for in situ gel-forming dosage forms; Rajbanshi et al., 2023) could offer opportunities to minimize matrix confounding while ensuring sufficient product integrity and efficacy.
Equipment Maintenance and Calibration
Routine upkeep RP-HPLC systems is critical to ensure accurate analytical measurement. However, based on the general maintenance and longevity practices within the scientific and laboratory equipment industries, we can make some educated assumptions, even though precise guidelines for RP-HPLC systems are not addressed in the given material. Maintenance is required in order for the complex industrial systems to cope with operational reliability, availability, and profitability (Chen et al., 2022). Similarly, regular upkeep for RP-HPLC systems would prevent unforeseen failures, improve the lifespan of the equipment, and ensure an even performance. Using real-time health information, condition-based group maintenance can improve the efficiency and accuracy of executing maintenance plans (Chen et al., 2022). This methodology can in principle be extended to RP-HPLC systems as a basis for usgn the actual usage and performance figures to inform maintenance schedules. Calibration is the key to the accuracy in measurement. As anchor-based positioning systems require the accurate calibration of fixed nodes for optimal system performance, a high-precision approach is required (Ridolfi et al., 2021). Likewise, in case of RP-HPLC systems also, frequent calibration will ensure that the retention times, peak areas, and other important parameters are reliable. We have already shown that algorithms for self-calibration, as for the UWB positioning systems, are potentially applicable also for RP-HPLC systems in order to simplify and automatize the calibration process (Ridolfi et al., 2021). The emphasis in hepcidin assays on standardization and external quality assurance in analytical measurements (Aune et al., 2020). Well-designed proficiency testing schemes and standardized reference materials for RP-HPLC systems would help in improving the quality of the testing and enable comparisons of the results between laboratories. The construction of high-level calibrators, as demonstrated by a hepcidin study, may also be useful for RP-HPLC calibration in further standardization through a wider concentration range (Aune et al., 2020). Regular maintenance and calibration of RP-HPLC systems is required to preserve reliability and precision. These improvements in time-domain features can be achieved by applying the concepts of condition-based maintenance, automation of calibration procedures, and advances in the creation of standardized methods by JCTC can help achieve the science of RP-HPLC measurement in measurable and reproducible ways across laboratories and time[13].
8. Recent Trends and Innovations in RP-HPLC for Stability Testing
Advances in Stationary Phases
Título: Advances in RP HPLC Methods Using New Stationary-Phase454491937603805 Sequences. A promising breakthrough in this direction was hybrid organic-inorganic particles which provided higher stability and better performance. For example, a 1.7 μm sulfobetaine stationary phase based on ethylene-bridged hybrid particles exhibited excellent efficiency, with a second reduced plate height of at least 2.4, and firmer retention for polar neutrals (Walter et al., 2022). Likewise, an ethylene bridged hybrid C18 anion-exchange stationary phase displayed significantly improved anion retention in comparison to classical reversed-phase materials, with pH ranging 2 to 10 usability (Walter et al., 2021). Curiously, a small number of the stationary phases possess dual-mode capacity. A polymer-coated silica stationary phase with a pillar[5]quinone-amine structure successfully demonstrated both reverse-phase and hydrophilic interaction separation modes, illustrating the ability to separate diverse compound types (Lu et al., 2023). Chiral stationary phases (CSPs) can also be employed for chiral and achiral separation, providing benefits for complex mixture analysis (ex. cannabinoids) (Onishi & Umstead, 2021). This chapter provides recent developments in stationary-phase technology where the focus has been on improving stability, selectivity, and versatility. Key late developments in RP-HPLC methods include hybrid particles, mixed-mode stationary phases and chiral stationary phases with dual functionalities. These innovations have offered chromatographers new tools for optimizing separation and tackling difficult analytical problems.
Automation and High-Throughput Systems
The advancement of automation and high-throughput capabilities have led to improved speed and efficiency in stability studies utilizing RP-HPLC. Methods such as automated flow injection and sequential injection techniques allowed for the continuous measurement of several samples, leading to an increase in throughput and saving time and resources (Raju et al., 2024). They provide accurate and time-saving sample management, reliable results, and safety through less contact with dangerous chemicals. Platforms that include robotic sampling and sample preparation are ideally suited for accurately and reliably executing complex and repetitive workflows (i.e., sample handling, dilution, and transfer), with efficiency achieved by multitasking and integration with analytical instruments (Raju et al., 2024). Here, we describe a universal 384-well plate sample preparation system that can process the samples in high throughput, achieving a 20- to 40-fold sample preparation throughput compared to the standard protocols (Burns et al., 2021). In around 300 min, this platform is capable of processing samples from plated cells to cleaned peptides, highlighting its high-throughput potential for stability studies. On a parallel note, interest in the oblique-incidence reflectivity difference (OI-RD) microscope as a microarray imaging platform continues: a label-free detection system has been improved for rapid scanning [51]. This has achieved a tenfold improvement in detection speed, making it suitable for ultra-high-throughput screening applications (Zhang et al., 2023) by reducing the time-of-the-wait-for the lock-in amplifier and the time exchanged for the software data acquisition and translation stage movement time. Overall, These improvements in automation and high-throughput capability have considerably increased the speed and efficiency of RP-HPLC methods in terms of stability studies. Advancements in pharmaceutical analysis have been achieved through the utilization of a combination of robotic platforms, automated sample preparation systems and optimized detection methods to achieve substantial increases in throughput, accuracy and efficiency.
Miniaturization of HPLC Systems
The major motive behind theminiaturization of HPLC systems was the increasing need for portable, compact analytical systems especially for out of lab applications. This has opened the door for new and lower cost reverse-phase high-performance liquid chromatography (RHPLC) methods for small laboratories and field studies. Recent advances in detection technologies have driven the miniaturization of HPLC systems, leading to the design of field-deployable and portable instruments. Photometric, electrochemical, and mass spectrometric detection methods are used in conjunction with the miniaturized systems that qualify them for a wide range of applications (Hemida et al., 2023). The small area of the sensors has allowed for improvements in certain metrics, including reductions in dark count rate and power usage, although some compromise can be seen in terms of fill factor and photon detection probability (Morimoto & Charbon, 2021). While miniaturization may be useful for certain applications, making systems smaller and more cost-effective is not always best for any circumstances. As an example, the RGB model comparison between capillary electrophoresis (CE) and HPLC shows that the key factors in the choice of techniques are complex and need to be approached against the analytical target (Nowak et al., 2020). For example, novel modes of HPLC such as temperature-responsive chromatography with poly(N-isopropylacrylamide) (PNIPAAm)-based columns furnish greener approaches to these applications by reducing the need for organic solvents (Maekawa et al., 2020).
1.4 These miniaturized HPLC systems have allowed the recovery of RP-HPLC for smaller laboratories, other applications, and field studies. Nonetheless, each application has its own specific needs and may experience trade-offs in certain performance parameters collaterally when miniaturizing, therefore the selection of the analytical approach should be critical.
9. Regulatory Considerations for RP-HPLC in Stability Testing
Guidelines for Stability Testing
Guidelines from the International Council for Harmonization (ICH) provide comprehensive measures of the conditions necessary for stability testing of active ingredients and dosage forms to be met, where ICH Q1A-E remain the key reference for stability studies (González-González et al., 2022). The goal of these guidelines is to harmonization of standards and enabled mutual acceptance of stability data for regulatory approvals across the European Union, Japan and United States. The stability studies are carried out as per the ICH Guidelines over the drug substances at different storage conditions to know their thermal stability and moisture sensitivity. They were long term miniaturized for at least 12 months at 25 ± 2°C/60% RH ± 5% RH or at 30°C ± 2°C/65% RH ± 5% RH. The intermediate and accelerated testing must be performed for the last six months at 30°C ± 2°C/65% RH ± 5% RH and 40°C ± 2°C/75% RH ± 5% RH, respectively (González-González et al., 2022). RP-HPLC is central to these standards. In stability studies, it is the most commonly used method for quantification and the assessment of purity of drug substances. For example, several RP-HPLC methods were developed and were validated for stability-indicating assays including paclitaxel (Kumar et al., 2022), Ozenoxacin and Benzoic Acid (Ramireddy & Behara, 2023), Eletriptan hydrobromide that were also used for itopride hydrochloride (Siddique et al., 2023). The stability indicating methods were validated following ICH Q2(R1) guidelines. To summarize, RP-HPLC plays a significant role in the stability testing and considering the ICH guidelines are based on regulatory requirements. Well-defined guidelines combined with powerful analytical methods assure quality and stability of pharmaceutical products over their shelf lives.
Acceptance Criteria for RP-HPLC Methods
Stability-indicating methods utilizing RP-HPLC need to fulfill regulatory analytical validation and analytical performance metrics as per International Conference on Harmonization (ICH) and United States Pharmacopeia (USP) quality guidance (Ramireddy & Behara, 2023; Siddique et al., 2023). Some of the key validation parameters that should lie within acceptance criteria are.
Accuracy: Recovery studies usually need to report recoveries between 98-102% (Dongala et al. 2020; Siddique et al. 2023).
Precision: Less than 2 % values of relative standard deviation (RSD) for repeatability and intermediate precision (Dongala et al. 2020).
Linearity: Correlation coefficient >0.999 across the concentration range (Dongala et al., 2020; Siddique et al., 2023).
Specificity: The quantitation and separation of the analyte are without any other components, considering degradation products (Dongala et al., 2020; Ramireddy & Behara, 2023).
Sensitivity: LOD and LOQ at appropriate limits (Chen et al., 2023).
Robustness: This method must be robust against small deliberate changes in the parameters (Dongala et al., 2020).
While several approaches have been employed to develop and validate stability-indicating RP-HPLC methods, interestingly, there are studies reporting Quality by Design (QbD) based stability-indicating methods which enable comprehensive understanding of method parameters and their factors (Dongala et al., 2020). Thus, this measure makes the proposed method robust and reliable. To summarize, compliance with regulatory recommendations regarding RP-HPLC method development and stability-indicating testing necessitates comprehensive validation of key parameters, including specificity, selectivity, accuracy, precision, linearity, limit of detection, and limit of quantitation, to confirm fact the techniques of choice for accurate and reliable detection and quantification of both active pharmaceutical ingredient substance and potentially dangerous impurities and/or degradation processes. Hence it is essential to establish these criteria in order to ensure appropriateness of the method for use in pharmaceutical quality control and stability studies.
Documentation and Reporting of Results
Ensuring the reliability, reproducibility, and regulatory compliance of RP-HPLC results necessitates appropriate documentation and reporting. During the last years, many studies have been emphasizing the relevance of this. Because of the effectiveness, delicacy, and automation capabilities, reversed-phase high-performance liquid chromatography (RP-HPLC) is widely used to quantify and separate compounds in pharmaceutical and biomedical analyses (Gupta et al. 2022). For compliance with regulations, it is important to validate the RP-HPLC method according to United States Pharmacopeia (USP) and International Conference on Harmonization (ICH) guidelines (Siddique et al., 2023). This validation includes various parameters like precision, accuracy, linearity, inter- and intra-day studies, and stability studies. Documentation of the method development and validation is essential for showing the rigor of the results. A thorough validation study, which included linearity, accuracy, precision, and stability assessments for the simultaneous determination of two drugs, has been described (Siddique et al., 2023). Likewise, (Nuhu et al., 2020) described the validation of a method based on RP-HPLC to measure of glutathione underlining the relevance of recording critical aspects influencing reproducibility as reaction time and temperature. Specifically, the decision on which analytical techniques to use such as RP-HPLC or CE are not trivial, and the reasons for their selection are well documented (Nowak et al., 2020). This underscores the importance of full reporting of method development and optimization. Thus, detailed reporting of RP-HPLC results is fundamental to preserve the quality and reliability of analytical methods. This includes thorough documentation of method validation, method optimization, and compliance with regulatory guidance. Such documentation not only ensures regulatory compliance approval but also enable method transfer and troubleshooting in future studies.
SUMMARY
RP-HPLC has become one of the common analytical methodologies for drug analysis in the pharmaceutical industry. It features a nonpolar stationary phase and a polar mobile phase, thereby enabling the separation of molecules by hydrophobicity. By using this technique with different detection techniques RP-HPLC has a wide application to analysis of various compound. In pharmaceutical development, where the safety, efficacy, and quality must be assured throughout the shelf life of drug products, stability testing is one of the most important factors. Due to its high resolution, sensitivity, specificity, and versatility, RP-HPLC is widely used for stability testing. Due to its high separation efficiency capability for various type of classes of compounds it is a potent analytical strategy that is widely use in distribu observatories (e.g., pharmaceutical, and proteomics). The RP-HPLC system comprises essential elements such as column, pump, detector, mobile phase, injection system and temperature control. These elements play a role in stability testing and all enhance accurate, reproducible and sensitive measurement of compounds. RP-HPLC has high sensitivity and selectivity for the identification of a small amount of degradation products with good quantitative accuracy and precision. It is well known and accepted in the regulatory authority for stability study of drug products. Nonetheless, the development of RP-HPLC protocols for stability studies is challenged by multiple factors, including adequate chromatographic separation, excipient-interaction considerations, and method validation as a function of multiple orthogonal parameters. Stability testing on RP-HPLC has become more efficient with development in stationary phase technology, automation, high-throughput capabilities, and miniaturization. Stability testingA wide variety of stability testing criteria are defined in regulatory guidelines,( such as those presented by the ICH; these provide a comprehensive framework for stability testing, with RP-HPLC being one of the main analytical tools for adequate criteria fulfillment. RP-HPLC results must be documented and reported accurately to demonstrate the quality and reliability of analytical methods, which is vital to demonstrating regulatory compliance.
REFERENCES
Abhijit Pawane*, Dr. Vishwas Bhagat, Dr. Deepak Kardile, M. M. Karne, Dr. Rajkumar Shete, A Review on RP-HPLC Techniques for Stability Testing in Drug Formulations, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 5, 3474-3497. https://doi.org/10.5281/zenodo.15476001
10.5281/zenodo.15476001
