1,2,3 Dr. Naikwadi College of D pharmacy, Jamgaon, Sinnar, Nashik, Maharashtra-422103 India.
4 SMBT College of Pharmacy, Affiliated to Savitribai Phule Pune University, Dhamangaon, Nashik, Maharashtra-422403 India.
5 MVP’s College of Pharmacy, Gangapur Road, Nashik-422002, Maharashtra, India.
High-Performance Liquid Chromatography (HPLC) remains a cornerstone analytical technique widely applied in pharmaceutical, biological, food, and environmental sciences due to its exceptional sensitivity, accuracy, and versatility. Recent technological advancements have significantly enhanced its performance, with innovations such as ultra-high-performance liquid chromatography (UHPLC), improved instrumentation enabling faster analysis, greater resolution, and reduced solvent consumption. The emergence of multidimensional and hyphenated techniques including comprehensive two-dimensional liquid chromatography (2D-LC), LC–MS/MS, and high-resolution, structural elucidation, and trace-level quantification essential for complex matrices like biopharmaceuticals, natural products, and metabolomics. Parallel to these advancements, green analytical chemistry has driven the adoption of eco-friendly solvents, solvent-minimized approaches, nano-LC formats, and microextraction techniques, supporting sustainability without compromising analytical performance. Additionally, Analytical Quality by Design (AQbD) principles and automation, supported by statistical tools such as Design of Experiments (DoE), ensure robust method development and improved method reliability. The integration of artificial intelligence (AI) and machine learning has further accelerated chromatographic optimization, enabling automated gradient prediction, peak deconvolution, retention-time modelling, and efficient method transfer across instruments. Despite these innovations, regulatory-compliant method validation remains essential to confirm that HPLC procedures are fit for purpose, with parameters such as accuracy, precision, specificity, linearity, range, LOD, LOQ, and robustness defined by international guidelines from ICH, WHO, and FDA. Collectively, these technological, methodological, and regulatory advancements highlight the continuing evolution of HPLC as a powerful, future-ready analytical platform supporting high-quality research, quality control, and regulatory compliance.
Drugs are an essential part of our daily lives. We take the medicines from the time the child is born until it dies. The quality of the medications is critical since it has a direct impact on the lives of customers. Analysing any product or substance is the greatest way to determine its quality. Analysis is a field of science that deals with determining the qualitative and quantitative properties of any substance. Analysis can give us the answer what (qualitative analysis) is present and how much (quantitative analysis) is present in the matter. Quality cannot be testing into product but appropriate testing using appropriate technique and instruments can aid in the development of quality into a medicinal product. Chemical stability is a major concern for pharmaceutical molecules since it has a direct impact on the drug's safety and efficacy. The USFDA and ICH recommendations have recommended that stability testing data be required in order to evaluate the impact of various environmental conditions on the quality of a drug substance and drug product over time(1,2).
The stability of the molecule influences the selection of an appropriate formulation and package, as well as the provision of adequate storage conditions and shelf life, which is required for regulatory compliance. A process that involves degradation of drug products and drug substances at conditions more severe than accelerated conditions and thus generates degradation products that can be studied to determine the stability of the molecule is known as forced degradation. It is essential to understand potential degradation reaction that may occur in the formulated product under various stress conditions that might be encountered during storage and in shipment of final package.
Unwanted chemicals that can develop during synthesis or with aging of active pharmaceutical ingredient (API) are the impurities in pharmaceuticals(3). Even a small amount of impurity can affect the efficacy and safety of pharmaceutical products, hence there is increasing interest in impurities present in API. According to ICH guideline stress testing is intended to identify the likely degradation products which further helps in determination of the intrinsic stability of the molecule and establishing degradation pathways and to validate the stability indicating procedures used(4,5).
Forced degradation studies are a regulatory requirement and scientific necessity(2). Conducting stability studies for any newly developed drug has now become a compulsory requirement prior to submitting the registration dossier. Forced degradation studies can be used to develop the stability indicating method which can be applied latter for the analysis of samples generated from accelerated and long-term stability studies. Titrimetric, spectrophotometric and chromatographic have been commonly employed in analysis of stability samples.
1.1. ANALYTICAL METHODS
Analytical methods which are measure of quality of the drugs play a very comprehensive role in drug development and follow up activities to assure that a drug product meets the established standard is stable and will continue to meet purported quality through its shelf life. Analysis includes a wide range of Simple and instrumental analytical methods but the most widely used analytical methods for quality assurance are spectroscopy and chromatography based(6).
Analytical methods can be divided into two areas:
There are various methods used in quantitative analyses which are broadly classified as:
1.1.1 Chemical/ Classical methods
These methods depend upon quantitative performance of a suitable chemical reaction and measuring the number of reagents needed to complete the reaction or ascertaining the amount of reaction product obtained, e.g., Titrimetric (acid base, redox, non-aqueous and complexometric titrations), gravimetric and volumetric methods(7).
1.1.2 Instrumental methods
These methods are based upon the measurement of physical properties of a substance such as electrical or optical and to correlate them for determination of concentration of analyte(8). These properties are being exploited for developments of analytical methods such as spectrophotometry, high pressure liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC)(9), gas chromatography (GC)(10), etc.
Presently instrumental methods of analysis are widely accepted over the classical methods. These methods are extremely sensitive, providing precise and detailed information even from a very small amount of sample materials. Instrumental methods are usually much faster than chemical methods and are applicable at wide range of concentration and find wide application in industry.
Following is advantage of instrumental methods over classical methods:
1.2. Chromatography:
To separate coloured compounds like plant pigments and dyestuffs. But it can now be used on colourless compounds as well. M. Tswett created chromatography in 1906. The chromatography (from the Greek chromatos – colour, graphos – writing) Michel Tswett discovered these rules. Chromatography is an analytical technique used to purify and separate organic and inorganic compounds. It can also be used to fractionate complex mixtures, separate closely related compounds like isomers, and isolate unstable chemicals(11).
1.2.1 Principles of chromatographic separation:
1.2.2 Adsorption chromatography:
Adsorption chromatography separates solutes by passing the mobile phase containing the dissolved solutes over the surface of the stationary phase. In normal phase, the mobile phase is non-polar, while in reverse phase, the mobile phase is polar(13).
1.2.3 Partition chromatography:
The partitioning behaviour of a chemical between two immiscible liquids is well understood in partition chromatography. Few compounds entirely partition into one of two immiscible liquids when shaken. Instead, most substances partition across liquids so that the partition coefficient (the ratio of concentrations in each phase) stays constant regard less of the total amount, provided neither phase is saturated.
Figure 1. Classification of Chromatography
1.2.4 Mode of chromatographic operations:
There are three modes of chromatographic operation they are as follows:
Elution techniques
Types of chromatography techniques:
Types of liquid chromatography:
1.3. High performance liquid chromatography:
Modern liquid chromatography is characterized by high performance, high pressure, high resolution, and fast speed. HPLC is a type of column chromatography where the mobile phase is pumped through the column quickly. Due to the lower particle size of the adsorbent or support, the analysis time is reduced by 1-2 orders of magnitude.
HPLC has the following benefits:
1.3.1 Normal phase chromatography:
In this the solvent system is non-polar and the solid phase is polar. As a result, the polar analyte is retained in the station phase(14). The adsorption capacity and elution duration are both increased by increasing the polarity of solute molecules. In this chromatography, a stationary phase made of chemically modified silica (cyanopropyl, aminopropyl, and diol) is utilized(15). An illustration the internal diameter of a standard column is about 4.6 mm, and its length is typically between 150- and 250-mm. Polar silica in the column will retain polar chemicals in the mixture for a longer period of time than non-polar substances. The non-polar chemical remove from column very fatly(16).
1.3.2 Reversed phase HPLC:
While the mobile phase of RP-HPLC is either polar or slightly polar, the stationary phase is non- polar. The water-phobic interaction theory serves as the basis for RP-HPLC. Analyte with a combination of components that are relatively less polar hold substantially in the non-polar stationary phase for a longer amount of time than analytes with a combination of components that are significantly more polar. Because of this, the most polar component will elute first(17).
1.4 Instrumentation:
Pumps, injectors, columns, detectors, integrators, and display systems are all components of HPLC apparatus. The elements are:
The solvent system components are contained in a glass bottle. In HPLC, the mobile phase, sometimes referred to as the solvent, is made up of a polar and non-polar liquid ingredient together. Different polar and non-polar solvents will be utilized, depending on the characteristics of the sample. Pump: Prior to transferring it to the detector, the pump forces the mobile phase out of the solvent reservoir and into the column. 42000 KPa is the pump's operating pressure.
A computerized or a manual could be the injector. This framework able to infuse fluid samples at high pressures and with great repeatability in volumes between 0.1 and 100 ml. (up to 4000 psi).
Standard column lengths range from 50 to 300 mm, and they are made of polished stainless-steel. and have an inside diameter between 2 and 5 mm. They typically contain a stationary phase with molecules that range in size from 3 to 10µ.
The HPLC detector, which is located towards the column's end, helps identify the sample of substances as they remove from a chromatographic column. Electrochemical identification, fluorescence, mass spectrometry, and UV spectroscopy detectors are frequently used.
The ability to handle, store, and reprocess chromatographic data might differ greatly between graph recorders and electronic integrators, on which signals from the detector may be captured. The PC coordinates the indicator's response to Each ingredient and loads through a chromatograph that is incredibly simple to comprehend(18).
Figure 2. Instrumentation of HPLC System
1.5 Method development and optimization:
The goal of the HPLC methods is try to separate and quantify the main active drug, any reaction impurities, all available synthetic inter-mediates and any degradants. The number of pharmaceutical products released each year continues to rise, consisting of both completely new drug molecules and modified versions of existing compounds. In many cases, there is a significant delay between the commercial launch of a drug and its official listing in pharmacopoeias. This delay occurs due to limited long-term data on safety and effectiveness, the emergence of previously unknown adverse effects that may even lead to market withdrawal, the possibility of developing resistance, and competition introducing more effective alternatives. As a result, official standards and validated analytical methods for such drugs are often not immediately available in pharmacopoeial references. Thus, it becomes necessary, to develop newer analytical methods for such drugs and their combination(19).
Need for Study:
Method development in chromatography is the setting up of an analytical procedure that will be appropriate for the analysis of a particular sample. In industries, new measurement technologies can only be adopted if a sound scientific rationale for the application has been developed, proven, justified and the developed method has been approved by internal company. Novel analytical techniques are designed for these drugs or their combinations due to several key reasons:
Initial conditions such as resolution and peak shape, plate counts asymmetry, capacity, elution duration, detection limits, limit of quantification, and overall ability to quantify the specific analyte of interest are enhanced or maximized during optimization.
An experimental variable is varied manually while the others are kept constant, and the reaction is recorded. Flow rates, mobile or stationary phase composition, temperature, detecting wavelength, and pH are examples. This single-variable system optimization method is slow, time-consuming, and costly. However, it may help grasp the underlying ideas and theories, as well as the variables' interactions.
A computer-driven automated method development methodology maximisers efficiency while minimizing experimental input. Many applications can benefit from computer-aided automation. They can also considerably reduce the time, energy, and expense of developing practically all instrumental approaches
The various parameter s that includes to be optimized during method development.
Selection of stationary phase / column: The choosing of separating column is the first and most significant stage in method development(20).
Selection of separation system:
The particle size and the nature of the column packing.
The physical parameters of the column i.e., the length and the diameter Considerations for selecting chromatographic columns include:
The most retentive reversed phase chromatography columns include dimethyl silane, butylsilane, octylsilane, oct decylsilane, base deactivated silane BDS phenyl, cyanopropyl,nitro, amino. This sort of column facilitates sample modification.
Selection of mobile phase:
The basic goal of mobile phase selection and optimization is to maximise impurity and degradant separation from each other and from the analyte peak. Solvent selectivity and elution strength must be optimised in the mobile (solute separation) To achieve low solute retention in normal phase and high solute retention in reverse phase, a polar mobile phase is required. The solvent strength is a measure of its capacity to draw analytes off the column. It is normally controlled by the strongest solvent's concentration.
The following are the parameters, which shall be taken into consideration while selecting and optimizing the mobile phase.
Buffer and its strength determine peak symmetries and separations. Acetate, phosphoric acid and format buffers are among the most widely used. The retention times depend on the buffer's molar strengths. Molar strength increases with retention time. The buffer's strength can be increased to achieve the desired separations. Analytes are pulled from the column by the solvent strength. It is normally controlled by the strongest solvent's concentration.
pH of the buffer: Most columns cannot handle pH values outside of 2.0 to 8.0, so keeping the mobile phase pH within this range is critical. The siloxane linkage region cleaves below pH 2.0, while silica dissolves above pH 8.0.
Mobile phase composition:
In most cases, the optimal mobile phase composition may be reached. This is because selecting the qualitative and quantitative composition of aqueous and organic components allows for a high degree of selectivity. Methanol and acetonitrile are the most commonly utilized reverse phase solvents. The best separations between pollutants were tested using mobile phases with varying pH buffers and organic phases. A mobile phase that separates all impurities and degradants from each other and from the Analyte peak and is tough enough to change the aqueous and organic phases by at least 0.2%.
Selection of detector: The detector was chosen based on the analyte's UV absorbance, fluorescence, conductance, oxidation, reduction, etc.
The following features must be present in an HPLC detector:
In addition, the detecting wavelength must be chosen carefully. Understanding how organic contaminants and the active pharmaceutical substance absorb UV light is particularly useful. Use max for maximum sensitivity. UV wavelengths below 200 nm should be avoided due to detector noise. Less selectivity at higher wavelengths.
Figure 3. Steps of analytical method development
In order to have an efficient method development process, the following steps have to be followed:
Step 1: Establish the purpose of the method and gain a clear understanding of the chemical properties involved
Determine the goals for method development (e.g., what is the intended use of the method?). and to develop a thorough understanding of the chemical characteristics of the analytes and the final drug formulation.
Step 2: Initial chromatographic conditions
Establish initial chromatographic parameters that provide at least a basic level of acceptable separation between components. These HPLC conditions will be used for all subsequent method development experiments.
Step 3: Sample preparation procedure
Develop a suitable sample preparation scheme for the drug product.
Step 4: Standardization
Identify the most suitable method for standardization and decide whether relative response factors should be applied in the calculations.
Step 5: Final method optimization/robustness
Recognize the limitations or potential shortcomings of the method and improve it using systematic experimental optimization. Understand the method performance with different conditions, different instrument set ups and different samples.
2. Parameters of Suitability Testing
The resolution and repeatability of the chromatographic system are being checked using system suitability tests to ensure that they are suitable for analysis once more(21)(22).
Figure 4. Fundamental parameters of HPLCs
Where,
1) Resolution:
Calculations used to determine how to separate out two or more components from a mixture are as follows:
R = 2 × (TR1 – TR2) / (W1 + W2)
Whereas,
R1 & R2 = The two component retention times
W1 & W2 = The bases of the peaks have the corresponding widths when the nearly straight peak sides are extrapolated to the baseline.
2) Retention Times (RT):
RT in LC and GC is defined as the amount of time that has elapsed between the introduction of the sample and the appearance of the analyte specimen area's greatest peak response. R.T is another parameter that may be utilised for recognition. Chromatographic retention durations are not only generic but also symbolic of the substances they represent. Although it might not be enough to establish identity on its own, the similarity of specimen substance retention. durations can also be employed as a partial criterion in the construction of an identifying characteristic. A compound's absolute retention durations may vary amongst chromatograms.
3) Asymmetry or Tailing factor:
The ideal situation should result in a Gauss-shaped chromatographic peak. Additionally, a deviation from a normally distributed distribution always persists in practice, signifying a non-uniform manner of mobility and transmission. Regulatory bodies like the USP Pharmacopoeia and EP have advised that it be included as one of the system suitability characteristics as a result. Although they are frequently inaccurate and unequal, the asymmetry factor and tailing factor are about the same for almost the whole era. Values should typically range from 1.0 to 1.5, with values larger than 2 being inappropriate.
As= B/A
Whereas,
4) Theoretical plate count:
Efficiency in a particular column is determined by peak dispersion, but it has to have column characteristics. Another way to symbolize efficacy is through the multiplicity of theoretical plates.
Figure 5. The Half – height method is used to calculate N.
Analytical methods are used for estimation of multiple components for HPLC are mentioned below:
1. Simultaneous equations method:
The simultaneous equation technique (Vierordt's method) will be used to compute all of these medicines if the conditions are met if a specimen contains two absorption drugs (x and y), each of which absorbs just at the maximum of the other.
The following requirement must be fulfilled for that are:
The concentrations of medications x and y are recalculated using the following formula –
???????? = ????2????????1 − ????1????????2/????????2????????1 − ????????1????????2
???????? = ????1????????2 − ????2????????1/????????2????????1 − ????????1????????2
3. Method validation:
A technique's suitability for the intended function is demonstrated through the process of method verification. Such a foundation for establishing this sort of validation for pharmaceutical techniques is provided by the US Pharmacopeia (USP), International Council on Harmonisation (ICH), World Health Organization (WHO), and Food and Drug Administration (FDA) recommendations.
Accuracy:
An analytical protocol's accuracy indicates the degree of agreement between the values that are regarded as either a conventional true value or a recognised reference value discovered. The percentage of recovery is used to express how accurate the results of research.
Precision:
The degree of dispersion between numerous samples taken from the same population under the prescribed conditions is known as the precision of an analytical method. A percentage of RSD is often used to express it. To be considered are three levels of accuracy.
Specificity:
Analyse your capacity for making an accurate evaluation of the analyte when components associated to presence start to show up. Examples of these include matrices, contaminants, and degradants.
DL:
The lowest concentration of analyte in a sample that can still be identified but is not always quantified as an accurate number is known as the detection limit of a certain analytical process. DL is defined as a composition at a given signal-to-noise ratio. The amount injected in chromatography that results in a peak with a height at least two to three times that of the baseline noise level is known as the detection limit.
S/N = 2/1 or 3/1
Where,
It may be calculated as
???????? = ????. ???? × ????????/S
Where,
QL:
The smallest unit of analyte in a sample that can be evaluated with sufficient precision and accuracy is the quantitation limit of a certain test technique. A composition at a particular signal to-noise ratio is the definition of the limit of quantitation (QL). The quantitation limit in chromatography is the amount poured that yields a peak that is 10 times higher than the background noise level(23).
Linearity:
Linearity is the ability of an analytical technique (within a certain range) to produce results that are really directly proportional to the concentration of an analyte in the sample. (The correlation coefficient must really be larger than 0.999 for linearity investigations(24).
Range:
The vast range of analyte concentrations in a specimen for which the test technique has been shown to have an appropriate level of precision, accuracy, and linearity is truly the variety of an analytical procedure.
Robustness:
An analytical technique's robustness is a measurement of its propensity to remain unaffected by small but deliberate variations in method parameters, and it provides proof of its dependability in typical circumstances(25).
4. Applications:
Pharmaceutical analysis: quantification of active pharmaceutical ingredients (APIs), impurities, degradation products; stability studies; assay of finished dosage forms; content uniformity; impurity profiling; quality control(26).
Method development & validation: for drug substances optimizing separation, sensitivity, robustness. HPLC remains central in regulatory and QC laboratories(27).
Biological and clinical research: analysis of biomolecules, metabolites, vitamins, small endogenous compounds; therapeutic drug monitoring; bio-analytical assays(26).
Environmental & food safety analysis: detection/quantification of pollutants, pesticide residues, contaminants, and quality control of food / environmental samples(26).
Preparative (semi-prep) HPLC: isolation and purification of compounds — for example, separation of stereoisomers, purification of synthetic products, natural products purification, peptides, etc(28).
5. Strengths and Limitations:
Strengths:
Limitations / Challenges:
6. Recent Trends & Research Directions
Recent advances in liquid chromatography have converged on three parallel fronts: (32) hardware and column innovations (UHPLC, core–shell and monolithic columns) that increase throughput and separation efficiency;(33) multidimensional and hyphenated techniques (2D-LC, LC-MS/HRMS) that enable resolution and structural elucidation of highly complex pharmaceutical matrices; and (34) sustainability and automation strategies (green solvents, miniaturized LC, online sample preparation, QbD and AI-assisted method development) that reduce environmental footprint while improving reproducibility and speed. Together these developments expand the analytical capability for drug discovery, impurity profiling, and biopharmaceutical characterization, though practical adoption requires attention to system compatibility, validation and regulatory expectations(35,36).
7. Multidimensional and Hyphenated LC Techniques:
2D-LC combines orthogonal separation mechanisms to achieve high peak capacity, ideal for complex mixtures such as:
Recent developments include automated modulation and intelligent data processing(31).
Hyphenation improves:
High-resolution MS (Orbitrap, Q-TOF) combined with HPLC provides unprecedented analytical specificity(37).
8. Emerging Trends: Green Chromatography, Automation & AI
Recent focus areas include:
Analytical Quality by Design (AQbD) incorporates statistical tools (DoE) to optimize:
Tome et al. (2019) emphasize AQbD as essential in modern chromatographic method development(40).
AI enables:
This reduces development time and improves robustness in regulated environments.
CONCLUSION:
HPLC continues to be an indispensable analytical technique due to its precision, versatility, and regulatory acceptance. The integration of UHPLC, advanced column technologies, multidimensional separations, and MS-based detection has dramatically expanded its capabilities. Future trends involving green chromatography, automation, AI-driven method development, and real-time analytical technologies promise to make chromatographic analysis even more efficient, sustainable, and powerful. These advances collectively strengthen HPLC’s position as the central analytical method for pharmaceutical research, quality control, biopharmaceutical analysis, and environmental monitoring.
Relevant conflicts of interest/financial disclosures: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Competing interest: The authors state that they have no conflicts of interest to disclose.
Consent for publication: Not applicable
Funding: No any kind of financial support from National or International Agency was received for the present review work.
Availability of data and materials: The dataset used and analysed during current study available from the corresponding author on reasonable request.
Ethics approval and consent to participate: Not applicable
REFERENCES
Ganesh Tupe, Monali Khatake, Kanchan Vetal, Archana Tupe, Ashwini Amrutkar, Recent Advances in High- Performance Liquid Chromatography (HPLC): Principles, Method Development Strategies, Modern Innovations and Applications on Pharmaceutical Analysis, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 12, 880-896. https://doi.org/10.5281/zenodo.17829954
10.5281/zenodo.17829954
