Rajgad Dnyanpeeth's College of Pharmacy, Bhor, Pune-412206, India
RP-HPLC or Reverse Phase High-Performance Liquid Chromatography is easy to use by highly trained specialists, with applications in pharmaceutical analysis, food technology, and even environmental science. It is a technology that combines two techniques, high chromatography and special compound separation. In this review, I will discuss stationary and mobile phase matters, optimization parameters for RP-HPLC systems, multi component multiseries advanced detectors, and the challenges of multi component detection using modern detectors and zeotropic chromatographic techniques. Central to new technology is improvement in productivity and efficiency and decreases in the amount of energy used and the effect on the environment with increased sustainability, both aspects important in contemporary medicine and science. The approach ‘Quality by Design’ employs the development of green mobile phases along with novel stationary and hybrid systems. Sharper analysis, stronger sensitivity, and increasing the number of separable complex components in one CRAM allow for more versatile methods. Different practices of RP-HPLC in pharmaceutical, environmental, food and beverage industries, as well as biological and biomedical research, highlight the product’s quality, safety, and efficacy. This review analysis highlights the significance of method validation, methods to solve specific problems, and best practices for positive outcomes for results reliability and reproduction. Potential future directions and advancements in RP-HPLC, which include miniaturization, automation, and sustainability are also covered, showing the prospects of more compact, efficient, and eco-friendly systems for chromatography.
Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely accepted technique of analysis for separation and quantitation of substances in different disciplines like pharmaceutical analysis, food science, and environmental monitoring. It employs the use of non-polar stationary phase and polar mobile phase to separate analytes by hydrophobicity (Marie et al, 2023; Shamim et al, 2023). Real-time HPLC (RP-HLC): High resolution, high sensitivity, and very versatile. This method can be used for simultaneous determination of multiple compounds in complex matrices in the examples of drug combinations including \[\alpha\]- and \[\beta\]-metformin, pioglitazone and glimepiride ozenoxacin and benzoic acid (Ramireddy & Behara, 2023). This technique can also be used on other instruments for example UV visible spectrophotometer, fluorescence and mass spectrometer. More recent developments in RP-HPLC have focused on reducing time and resource consumption, minimizing environmental impact, and increasing method robustness. In spectrometric method development, the introduction of Quality by Design (QbD) principles was implemented for method parameters optimization with an aim to establish design spaces for reproducible and robust outcomes (Marie et al., 2023; Shamim et al., 2023; Sukumar et al., 2023) In addition, the development of a method with glycerol as a mobile phase component (Habib et al., 2023) indicates the need for more environmentally friendly RP-HPLC methods that reduce the adverse effect of the analysis.
3. RP-HPLC Principles and Components
Chromatographic Principles (stationary phase, mobile phase, column, and detector):
Interaction between stationary and mobile phase are the principle of chromatographic separation. The stationary phase is typically a solid or liquid that is held in a column, while the mobile phase is a liquid or gas that moves through that column (Buthmann et al., 2024; Westerbeek et al., 2023). As for the liquid chromatographic side, the solid phase may be silica-based C18 Columns (Elsheikh et al., 2023; Ramireddy & Behara, 2023), polymer monolith (Zhou et al., 2023) and functionalized substrates such as polycaprolactone with guanidinium ionic Liquid (Ba et al., 2023) The composition of the mobile phase is crucial as it will influence separation efficiency and could be a combination of diverse solvents, buffers, and additives. Ethanol and water mixtures (Elsheikh et al., 2023) and acetonitrile with phosphate buffer and ionic liquids (Axente et al., 2023) have all been used as mobile phases. 2: Columns and their role in chromatographic separation According to their size, materials and concept design, they can be used as capillary columns (Santos et al., 2023), open-tubular channels (Westerbeek et al., 2023) or packed columns (Zhou et al., 2023). Column types create distinction in resolution and efficacy of separation. are utilized in detecting and quantifying the purified compounds (e.g., UV detectors (Elsheikh et al., 2023; Ramireddy & Behara, 2023), mass spectrometers (Guimaraes et al., 2023; Santos et al., 2023)). We will also introduce some novel chromatographic strategies that utilize unique principles to enhance separation efficiency, such as centrifugal partition chromatography (Buthmann et al., 2024) and vortex chromatography (Westerbeek et al., 2023). Furthermore, the application of green analytical chemistry principles throughout the method development process (Elsheikh et al., 2023) has further highlighted the importance of sustainability in chromatographic approaches.
The Role of Reverse Phase:
Reversed-phase liquid chromatography (RPLC) is the most widely used mode of liquid chromatography for separation and characterization of wide range of compounds in pharmaceutical and proteomic applications. In reversed-phase approaches, a stationary phase is most frequently non-polar (commonly alkyl chain-linked silica particles (Dobó et al., 2024; Lardeux et al., 2023)), taking advantage of hydrophobic interaction with analytes. Unlike normal-phase, ion-exchange, and other modes of chromatographic separation, RPLC is fundamentally different. Normal-phase chromatography (NPC) uses a polar stationary phase and non-polar mobile phase; reversed-phase liquid chromatography (RPLC) uses a non-polar stationary phase and polar mobile phase (Chapel et al. 2023). This change in the polarity enables a greater separation of moderately polar to non-polar compounds. On the contrary, ion-exchange chromatography separates molecules by the strength of their charge interactions with the stationary phase. RPLC can also be coupled with other modes (8) to improve separation performance. An example of this approach is reversed hydrophilic interaction chromatography (revHILIC), which employs a highly polar stationary phase under reversed gradient conditions, thus providing orthogonal selectivity to both RPLC and HILIC (Chapel et al., 2023). However, advanced liquid chromatography techniques such as two-dimensional chromatography (2D-LC) based on RPLC × RPLC or high pH RPLC × revHILIC can achieve remarkable performance in peak capacity and sensitivity (Chapel et al., 2023; Kaulich et al., 2023).
Components in RP-HPLC:
Instrumentation: RP-HPLC instruments generally consist on pumps system, injector, column and detector. For example, (Ramireddy & Behara, 2023) describes an RP-HPLC instrument with a C8 column (150 mm × 4.6 mm, 5 μm particle size) and a UV detector (Ramireddy & Behara, 2023). The system may also have other components, e.g., a column oven to control temperature using the same approach as that of the column oven temperature (45 °C) in (Ramireddy & Behara, 2023).
RP-HPLC uses different detectors: e.g. UV, fluorescence, mass spectrometry. UV stage detectors are often used as illustrated in (Ramireddy & Behara, 2023), which placed a UV detector at a wavelength of 235 nm. Fluorescence detectors are very sensitive and specific; as an example, a fluorescence detector was set up at 365 nm for excitation and 450 nm for emission (Almutairi et al., 2023). (Mahdavijalal et al., 2024) also use mass spectrometry for greater sensitivity and structural information. The RP-HPLC columns are usually packed with any of the silica-based stationary phases like C8, C18, etc. For example, (Shamim et al., 2023) used a Kromasil-C18 (4.6 × 150 mm, 5 μm) column to separate the ciprofloxacin hydrochloride and rutin (Shamim et al., 2023).
Solvents: The mobile phases used in RP-HPLC in most cases consist of a mixture of water and organic solvents, like acetonitrile or methanol. Curcumin AOP (54:46 v/v, 0.1 mM), an acetonitrile-phosphate buffer-based mobile phase, has been reported for the separation of curcuminoids (Mohammed et al., 2023). HPLC-based fluorescence detectors have been commercially available for decades; however, new trends include miniaturized smartphone-based fluorescence detectors designed as alternatives to perform analysis in the field with low investment costs (Shamsaei et al. 2023). Moreover, uptaking these approaches to RP-HPLC method development in consideration of sustainability and efficiency is indispensable, such as green solvents (Abdallah et al., 2023), and experimental design strategies in development such as Quality by Design (QbD) (Azhakesan & Kuppusamy, 2023).
4. Advanced RP-HPLC Method Development
Step-by-step Method Development
Choosing the Right Stationary Phase
Choosing the appropriate stationary phase is a crucial aspect of HPLC method development depending on the sample properties and analysis needs. There are a few things to consider in this process: Think about the properties of the sample and the goals of the analysis. As an example, in (Ferencz et al., 2023) the authors searched two types of chiral stationary phase (CSP) to separate ezetimibe and its relevant substances. While Ferencz et al. (2023) reported BPR of six impurities using the cellulose-based column, the cyclodextrin-based CSP displayed good separation capacity. In a similar study, (Mallik et al., 2023) proposed the preparation of an amino acid-derived stationary phase (Sil-Ala-C12) exhibiting coordinated hydrophobicity and hydrophilicity, which allows separation of a variety of analytes in reversed-phase HPLC and in HILIC modes (Mallik et al., 2023). You already examined the chemical properties for an analyte. As an example, according to (Sukumar et al., 2023), authors separated Metronidazole, Lidocaine, and Miconazole in oral dosage, according to their chemical properties using a Zorbax C18 column (Sukumar et al., 2023). In (Carotti et al., 2023), octyl and octadecyl stationary phases have been compared to measure the hydrophobicity of pharmaceutical fragments (Carotti et al., 2023). Use a different or specific type of stationary phase. This has been covered in (Santos et al., 2023), a report on wall open tubular columns (WCOT) in liquid chromatography, where the stationary phase composition and the column dimensions were respectively shown to influence the efficiency (Santos et al., 2023). (Hao et al., 2023) reported the use of polyamide-6 powder as a solid-phase extraction (SPE) stationary phase for the determination of trace permanganate in water (Hao et al., 2023). Selection of stationary phase The selection of appropriate stationary phase is complex process which requires consideration of the sample to be tested, the characteristics of the analytes, and the desired separation outcome. Considerations include selectivity, efficiency and compatibility with the mobile phase and detection method. Jose et al. demonstrate the application of design of experiments approaches that can be useful for optimizing separation conditions and to explore multiple stationary phases (Mohammed et al., 2023).
Mobile Phase Composition
Detailed knowledge of mobile phase composition is crucial for successful compound separation and analytical performance in HPLC. The above papers may yield a few rules of thumb regarding solvent selection, pH adjustment, buffers, and additives: The solvent selection is usually made up of a mixture of organic solvents and aqueous buffers. Acetonitrile and methanol represent widely used organic solvents in these experiments (Axente et al., 2023; Mohammed et al., 2023; Nekkalapudi et al., 2023), whereas phosphate buffers are commonly applied for pH adjustment. 30:70 to 54:46 v/v (Enginar et al., 2024; Mohammed et al., 2023) pH adjustment for ionizable compounds is essential. Mobile phase pH should be adjusted as 2 units below the pKa of the anlytes, so they remain in protonated form (Kechagia et al., 2023). Stabilizing pH conditions are relied upon with buffers such as acetate (pH 4.7) and phosphate (pH 1.0-5.2) (Axente et al., 2023; Enginar et al., 2024; Nekkalapudi et al., 2023). These additives can greatly enhance chromatographic performance. Mobile phase modifiers, such as 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM[BF4]) as an ionic liquid, had shown some promise particularly for polar basic molecules (Axente et al., 2023). Even deep eutectic solvents could serve as effective additives, as for example choline chloride: glycerol has shown good ability to separate phenolic acids when it was added in 0.25% to the mobile phase (Pérez-López et al., 2023).
Optimization Parameters:
Innovating methods led to optimization of chromatographic methods by varying parameters such as temperature, flow rate, gradient elution, and run time.
Operation Method (4) Temperature optimization– Temperature is one of the important parameters for method optimization; for example, in (Neumann et al., 2024), gradient time and ternary composition, along with the temperature of the column, were varied in order to use retention times of peptides in supercritical fluid chromatography to create predictions (Neumann et al., 2024). Flow rate is another important parameter, as gradient profiles were shown to be unique to each (solvent combination, flowrate, gradient duration - (Niezen et al., 2023)).
This is a vital optimization variable: Gradient elution, as shown in multiple studies. In their chatper, Niezen et al., 2023 describe the application of empirical modeling to describe and predict analyte retention and peak width as a function of time and solvent composition in gradient-elution liquid chromatography (Niezen et al., 2023). Such an optimization of gradient adjustments to enhance the separation of bacitracin fingerprints is presented in (Neumann et al., 2024).
Resolution and Sensitivity
Key steps in making RP-HPLC method with good resolution and sensitivity As a typical method development strategy, this process begins with choosing the chromatographic system under investigation, such as the stationary phase, the mobile phase composition, and the detection method. Additionally, (Y A Alanazi et al., 2023) reports using a Hypersil BDS C18 column via a mobile phase consisting of acidic water: acetonitrile in the ratio of (85:15; v: v) at pH 4.5 for cephalexin and cefixime separation (Y A Alanazi et al., 2023). The same is true for (Shamim et al., 2023), which used a Kromasil C18 column with the mobile phase npH phosphoric acid buffer and acetonitrile to separate ciprofloxacin and rutin (Shamim et al., 2023). In fact, most of the studies seen utilized Quality by Design (QbD) principles to optimize chromatographic conditions. ÅFlexibility of Box-Behnken Design Modelå After literature survey, Box-Behnken design was selected as method of choice to determine the influence of various parameters on resolution and retention time (Alanazi et al., 2023; Marie et al., 2023; Shamim et al., 2023). In this way, we can help researchers determine their critical method parameters (CMPs) and define a method operable design region (MODR) for robust and reproducible separations (Marie et al., 2023).
Techniques for Speeding Up Development: (Fast HPLC, Ultra-Fast HPLC (UF-HPLC), and other high-throughput methods) The provided papers describe various methods, described as a series of different approaches, to increase the development and analysis rate by HPLC: Short columns can yield a considerable reduction in the time analysis. An example of a short cation-exchange column (50 mm x 4 mm) for iron speciation, i.e., with a total analysis time of ∼5 minutes (with a low flow rate of 0.5 mL/min) can be found in (Or?owska et al., 2023). This shows how shallow columns can allow for fast separations. A different approach is to optimize the composition and flow rate of the mobile phase. Dealing with other pharmaceutical products has been shown to be effective; for example, Siddique et al. (2023) reported separation of two drugs in 5 min with an optimized ratio of mobile phase and flow at 1 mL/min (Siddique et al., 2023). Likewise, the rapid HPLC method for pterostilbene (retention time 2.54 min) was established by optimizing mobile phase components, and utilizing a 1.0 mL/min flow rate (Haq et al., 2023). Researchers solved this complex problem by using high pH conditions combined with isocratic elution to simplify and speed up the separations. A well-known example included (Attia et al., 2023), which used isocratic conditions coupled with optimized mobile phase to achieve the simultaneous analysis of two compounds (Attia et al., 2023). This may decrease equilibration times between runs in relation to gradient methods. One interesting approach which has been gaining popularity is combining HPLC with other techniques for targeted analyses. At the same time, Lu et al (2023) report an affinity-based ultrafiltration (UF) coupled with HPLC for rapid screening of inhibitor present in the extracts of plants (Lu et al., 2023). This enables the selective isolation of compounds of interest before HPLC analysis. Although these methods are often not specifically referred to as "ultra-fast", they often attain very short analysis times that are comparable to UF HPLC performance. A recently developed LM1010 HPLC instrument mentioned in (Akamine et al., 2023) can achieve a 7-minute total run time for therapeutic drug monitoring, showing how newer instrumentation can allow for rapid analysis (Akamine et al., 2023).
5. Multicomponent Analysis in RP-HPLC
Consequently, a reversed-phase high-performance liquid chromatography (RP-HPLC) appears to be a suitable technique for multicomponent analysis in many domains. Since the method enables concurrent evaluation of multiple analytes, it assumes immense significance in considering such mixtures and formulations (Shamim et al., 2023; Zhang et al., 2024). This approach has been previously used for drug, natural products and food as the compounds. RP-HPLC has been used in pharmaceutical analysis for the simultaneous determination of a number of drugs in combined dosage forms. For example, a simultaneous quantification approach for metformin hydrochloride, pioglitazone, and glimepiride in binary dosage forms and fortified human plasma had been devised (Marie et al., 2023). Some other work published in this year is the simultaneous determination of Ozenoxacin and Benzoic Acid in a pharmaceutical cream formulation (Ramireddy & Behara, 2023). In addition, RP-HPLC is not just a pharmaceutical analysis. It was used in food analysis such as free tryptophan determination in vegetable oils (Raži? et al., 2023) and strobilurin fungicides detection in water, juice and vinegar (Wang et al., 2023). Furthermore, the method has scope for screening of complex natural product mixtures as evidenced by the simultaneous quantification of curcuminoids in extracts of Curcuma longa and dietary supplements (Mohammed et al., 2023)
Chalenges Multicomponent Analysis
RP-HPLC is quite often complicated when analyzing multicomponent substances due to complex sample matrices and the analyte coexistence. Among those, coelution and overlaps are the key bottlenecks for the accurate identification and quantification of each contributing compound (Jiang et al., 2023; Pérez-López et al., 2023). This is a specific challenge when the analysis of complex natural product mixtures, such as traditional Chinese medicines, implicates a high number of components. For example, 430 compounds were detected or tentatively characterized in the analysis of Jiawei Fangji Huangqi decoction (Jiang et al., 2023), which reflects the demonstrating complexity of such samples. Moreover, various experiments for extracts of Curcuma longa and pharmaceutical formulations where the simultaneous determination of curcuminoids and their forced degradants was obtained (Mohammed et al., 2023). Various approaches have been used to address these problems. Gradient elution with controlled composition of the mobile phase and temperature control of the column are aspects that have been shown to improve separation of complex mixtures (Mohammed et al, 2023; Ramireddy & Behara, 2023; Zhang et al, 2024). In addition, strong data acquisition and processing approaches such as data-independent acquisition (DIA) coupled to chemometric techniques (region of interest) multivariate curve resolution (ROIMCR) have been established to analyze such large data collected in multicomponent analysis case (Pérez-López et al., 2023). These strategies facilitate deconvolution of co-eluting species and overlapping chromatographic peaks without the requirement for extensive data pre-treatment.
Optimization Strategies:
A wide application of the high-performance liquid chromatography (HPLC) separation has posed an efficacious approach for the multicomponent analysis and diverse strategies for the optimization bolster the prowess of HPLC separation. Novel multidimensional chromatographic approaches that integrate liquid chromatography into the ion mobility–mass spectrometry paradigm (Figure 1B) have further enhanced our capabilities to profile a range of metabolites (Pena et al., 2023). By applying the other algorithms such as Peak Decoder, this technique enables accurate identification and quantification of metabolites in complex biological samples. In this respect, advanced detectors are a key aspect of multicomponent analysis optimization. This contribution relies on tandem mass spectrometry (MS/MS), using ion trap analyzers, which are suitable for differentiating isomers and assigning signals (Pallecchi et al., 2023). Simultaneous detection of multiple compounds at different wavelengths has already been established using PDA detectors as exemplified for the analysis of ciprofloxacin hydrochloride and rutin (Shamim et al. 2023). Abstract: Fluorescence detection presents a high specificity and sensitivity, and more recently, we developed a smartphone-based fluorescence detector that serves as a low-cost and portable solution compared to the traditional HPLC (Shamsaei et al., 2023).
Case Studies/Examples:
Multicomponent analysis is an essential approach, second only to drugs, and environmental sample testing is one of the most common areas of application. Biological assays using linear and nonlinear regression models are important for assessing complex biological systems and establishing relationships between variables in pharmaceutical research (Jarantow et al., 2023). These analytical techniques play a crucial role in quantifying responses to drug or other stimuli, underscoring the concept of multicomponent analysis, which is essential to drug discovery and development. As established in multicomponent pharmaceutical salification, the formation of multicomponent molecular salts with metformin and rhein for drug analysis points out promising areas for future studies in the field of drug analysis. This was successful with both a combination of drugs that provided complementary benefits of both drugs and improved hypoglycemic activity and bioavailability (Yu et al., 2023). In the same way, the formation of drug-drug salts between a couple of the NSAIDs and ciprofloxacin illustrates how multicomponent analysis can improve drug solubility without diminishing the antibiotic (Acebedo-Martínez et al., 2023). Multicomponent analysis has also been adopted in environmental testing. Detection of pharmaceutical and personal care products as emerging environmental contaminants requires advanced analytical techniques. An example of application of multicomponent analysis in environment is the development of a Dysprosium manganite/carbon nanofiber composite sensitive for mefenamic acid, an anti-inflammatory drug considered a chemical pollutant (Alagarsamy et al., 2023).
6. Applications of Advanced RP-HPLC
Pharmaceutical Industry
It has a wide range of applications in the pharmaceutical sectors for designing new multi-drug formulations, stability testing, and impurity profiling. Its effect in these areas has been shown in several studies: RP-HPLC methods have been reported for simultaneous estimation of multicomponent drug formulations. For example, a single method was developed for determination of ozenoxacin and benzoic acid from pharmaceutical cream-formulated product (Ramireddy & Behara, 2023). Likewise, Siddique et al. (2023) provided one method for the simultaneous determination of Eletriptan hydrobromide and Itopride hydrochloride in combination. September 20 to October 24, 2023 IN-NANOVAC - Nanoparticulate Techniques for Cancer Treatment and Diagnostics I in European Journal of Pharmacology. RP-HPLC has been an attractive method in stability testing and in impurity profiling or profiling of the unknowns. An HPLC stability-indicating method for simultaneous separation of curcuminoids in dosage from and examination of forced degradants, which was developed (Mohammed et al., 2023). An alternative one employed RP-HPLC and charged aerosol detection (CAD) for the same purpose to detect nine potential impurities in ursodeoxycholic acid, exhibiting enhanced sensitivity and compatibility with LC-MS for identifying impurities (Huang et al., 2023). Indeed, RP-HPLC methodology in some work has been applied not just to standard pharmaceutical analysis. For instance, one study performed an RP-HPLC method for obtaining MIC and MBC of cefoperazone toward bacteria strains (Al-Hakkani et al., 2023). RP-HPLC combined with multivariate analysis was also used for the quality control of herbal products (Nguyen et al., 2023).
Environmental Monitoring
With the development of advanced RP-HPLC techniques, these analytical methods have been developed as invaluable tools for environmental monitoring of pollutants, pesticides, and toxins in water, soil, and air samples. As an example of quantifying multi-class pesticides and metabolites in medicine extracts by HPLC-MS/MS analysis, a modified method under QuEChERS conditions has been applied for the detection of 39 pollutants (34 multi-class pesticides and 5 metabolites) in the medlar matrix (Zhuang et al., 2023). This approach showed great accuracy and efficiency with average recoveries within 70%−119% and relative standard deviations of 1.0%−19.9%. This study provides a validated method for fast and reliable screening of multi-class multi-pesticide residues in food products, and thus promotes food security. Although HPLC-MS proved to be the most efficient method for pesticide quantification, it is not available for real-time monitoring as it requires sophisticated laboratory pre-treatment of samples (Berkal & Nardin, 2023). To overcome this limitation, alternative methods are currently being developed such as biosensors which can achieve analyte detection, using selectivity and sensitivity, without sample pre-treatment. Hydrophilic interaction chromatography has been employed in drinking water analysis to develop a high-resolution tandem-mass spectrometry platform hyphenated to an anion exchange for non-targeted and suspect screening of anionic and polar compounds (Egede Frøkjær et al., 2023). The method successfully detected anionic pesticide residues and other environmental contaminants in environmental samples from drinking water, emphasizing improving RP-HPLC techniques for effective environmental monitoring.
Food and Beverages
RP-HPLC methods have been advanced and these have expanded their usage within the food and beverage industry for QCA, pesticide residue, and flavour compounds analysis.
Introduction: Medlar, a type of small fruit, is rich in nutrients and has therapeutic properties and as a result is widely used. However, the presence of pesticide residues in medlar is often associated with potentially adverse health outcomes, such as cancer, endocrine disruption, and reproductive toxicity. To understand the distribution of pesticide residues in medlar, a modified QuEChERS method coupled with HPLC-MS/MS was established for 39 pollutants (34 pesticides and 5 metabolites) analysis in medlar matrix. Their method showed high recoveries (70–119%) and low relative standard deviations (1.0–19.9%), indicating that it is suitable for fast and effective multi-class screening of pesticide residue in medlar products (Zhuang et al., 2023). Likewise, UPLC-MS/MS was also applied to simultaneously detect the 31 pesticide residues as well as six mycotoxins in Pu-erh tea in 11 min, with acceptable recoveries (62.0-130.3%) and precision (Chau et al., 2023). In terms of flavor compound analysis, HPLC-MS was used to analyze taste components in different grades of green tea infusion. Statistical analyses enabled the identification of specific compounds as taste markers of high-grade baked green tea, which were obtained through hierarchical clustering methods (Zou et al., 2023). In this regard, Zhang et al. (2023) recently performed high-performance liquid chromatography (HPLC) to determine the physicochemical quality and volatile components in fermented rape stalks, which could contribute to a deeper understanding of the flavor development of fermented products. Explains the flexibility and possibilities of advanced RP-HPLC techniques for ensuring food safety and quality in the food and beverage industry. These approaches are robust tools to tackle pesticide residue analysis and flavor compound characterization in complex food matrices that demand high sensitivity, selectivity, and rapid analysis.
Biological and Biomedical Studies
Mass spectrometry (MS)-based proteomics and metabolomics have emerged as essential technologies in biological and biomedical analysis, predominantly in the fields of metabolomics, proteomics and biomarker research. These novel approaches offer significant advantages compared to traditional approaches regarding multiplexing capacity, analytical specificity/sensitivity, and turnaround time (Birhanu, 2023). In the realm of metabolomics, untargeted MS-based methods have displayed significant promise for exploring health and disease conditions, discovering new biomarkers, and elucidating metabolic pathways (Kaczmarek et al., 2023). Pang & Hu, 2023 demonstrate that metabolomics applied to drug research and development significantly accelerates the process by predicting pharmacokinetics, pharmacodynamics, and drug responses forcing drug repositioning and personalized treatment strategies. From the proteomics side, the progress in identifying potential biomarkers is on-going, moving from the discovery phase to validation thanks to an emerging peptide library technology of Pep Quant that was developed that enables a more robust transition from discovery phase to validation. For instance, this tactic has resulted in discovering and validating new biomarkers for diseases like breast cancer (Kim et al., 2023). Moreover, the application of MS-based metabolomics and lipidomics for the analysis of extracellular vehicles (EVs) has demonstrated potential in cancer diagnosis, prognosis, and prediction of treatment responses (Bai et al., 2023). Processing and analytics however face challenges still. In recent years, the methods applied to instrument drift and batch effects have also been extended to new areas, such as large high-throughput metabolomics studies (Märtens et al., 2023). Integration of ion mobility with LC-MS has further advanced metabolomics analysis by expanding its coverage, sensitivity, and resolving power for metabolite isomers (Luo et al., 2023).
Forensic and Clinical Analysis
Advanced RP-HPLC technique, forensic and clinical analysis can be an important in drug abuse testing and biomarker detection applicable in disease diagnosis. Other RP-HPLC methods are also described for the detection of commonly abused drugs including morphine, methamphetamine, and ketamine in human hair samples (Xu et al., 2023), and thus forensic toxicology studies have also reported RP-HPLC methods. These techniques have high sensitivity, with detection limits as low as 0.079 ng/mL for ketamine. As drug abuse is the major public safety issue and criminal investigation challenge it is an essential tool for fast and effective drug abuse examination (Rosendo et al., 2023). RP-HPLC is extremely common to assess clinical biomarkers for diseases. As another example, a novel HPLC method was developed for simultaneous determination of free mannose and glucose in serum, utilized for diagnosis of ovarian cancer (Chen et al., 2023). In advanced-stage ovarian cancer patients, both serum mannose levels as well as the ratio of glucose to mannose were significantly elevated relative to the controls and the ratio is a potential biomarker with high sensitivity and specificity [17].
7. Validation of RP-HPLC Methods
RP-HPLC Method Validation — 1 min read Post October 2023Read More Method validation is one of the most critical steps of analytical method development and especially in case of RP-HPLC methods. Various guidelines have been framed by the regulators such as the Food and Drug Administration (FDA), International Conference on Harmonisation (ICH) and United States Pharmacopeia (USP) to ensure reliability, accuracy and reproducibility of the analytical methods (Chiarentin et al., 2023; Ramireddy & Behara, 2023; Siddique et al., 2023). Method validation is the process that is vital to establishing that an analytical process is fit for its intended use. This includes parameters such as accuracy, precision, specificity, linearity, range and robustness. Example of well-developed and validated RP-HPLC method according to ICH guidelines is in (Ramireddy & Behara, 2023), where a new RP-HPLC method for simultaneous estimation of Ozenoxacin and Benzoic Acid in a pharmaceutical cream formulation was developed by the authors. In the similar heads (Siddique et al., 2023) elucidates the work of the validation of RP-HPLC method for Eletriptan hydrobromide and Itopride hydrochloride according to the USP and ICH guidelines. Intriguingly, advanced methodologies for method validation have been employed in some studies. A recent example is the paper by Chiarentin et al., which presents the "Analytical Quality by Design" (AQbD) approach to the development and validation of rheology methods used for topical creams (Chiarentin et al., 2023). The method used for risk assessment in this approach is Ishikawa diagrams and failure mode, effects, and criticality analysis (FMECA), which explains for a better framework for method development and validation to be accomplished (Chiarentin et al., 2023; Marie et al., 2023).
Accuracy
Various studies used for validation of RP-HPLC methods were showing validity in accuracy. Accuracy is generally measured with recovery experiments, where known quantities of analytes are spiked into samples and recovery percentages are determined. The RP-HPLC method for ciprofloxacin and rutin was validated according to the ICH instructions and the findings fell in an acceptable range (Shamim et al., 2023) (Shamim et al., 2023). Likewise, (Siddique et al., 2023) measured accuracy values ranging from 98.44% to 99.96% for eletriptan hydrobromide and itopride hydrochloride with varying concentration levels (Siddique et al., 2023). Using a microwell spectrofluorimetric and HPLC-fluorescence detection methods, alectinib quantitation was demonstrated with high accuracy, where the mean recovery values were ?86.90% and ?95.45%, respectively (Almutairi et al., 2023). Oddly enough, some studies had provided more detailed accuracy data. They reported an accuracy system with the following values for quercetin analysis varying between 88.6% and 110.7% (Carvalho et al., 2023) An analytical quality by design (AQbD) approach for canagliflozin has been established demonstrating its accuracy and other validation parameters (Azhakesan & Kuppusamy, 2023).
Precision
Precision is one of the important parameters in the validation of RP-HPLC methods. Repeatability studies assess precision within a single run, while intermediate precision studies assess precision over multiple runs. Some papers described their precision results for their RP-HPLC methods. For example, the method was found to be repeatable with variation coefficients ranging from 2.4% to 6.7%, and intermediate precision with variation coefficients ranging from 7.2% to 9.4% (Carvalho et al., 2023). Precision with %RSD < 1.67% was also reported for their method of curcuminoid analysis (Mohammed et al., 2023). Other papers, interestingly, took different approaches to assessing precision. For the first method of analysis of Eletriptan hydrobromide and Itopride hydrochloride, (Siddique et al., 2023) also performed intra-day and inter-day precision studies for their method (Siddique et al., 2023). They validated precision in accordance with the ICH guideline for their alectinib quantitation methods (Almutairi et al., 2023).
Linearity
The method is linear for RP-HPLC methods, and linearity is a veryimportant aspect of validation, as it ensures that the method canproduced results directly proportional to the analyte concentration over a specified range. Results of linearity of the developed RP-HPLC methods have been reported in few studies as follows: For ciprofloxacin hydrochloride and rutin, acceptable linearity were observed, but its specific values are not available (Shamim et al., 2023). The coefficient of correlation for Eletriptan hydrobromide and Itopride hydrochloride was found to be 0.9993 and 0.9965, respectively, and demonstrates excellent linearity (Siddique et al., 2023). Curcuminoids analysis method had excellent R2 ≥ 0.999 linearity for all analytes (Mohammed et al., 2023). Mannose and glucose were determined free in serum with a linear range of 0.5-500 μg/mL for mannose and 0.5-1500 μg/mL for glucose (Chen et al., 2023). Notably, linearity was established by different routes. For example, ten different concentrations from 25 to 250 μg mL-1 were tested with the juçara crude extracts method, obtaining an R2 of 0.994 (Tunin et al., 2023). Canagliflozin method was linear over the range 12.6–37.9 μg/mL (Azhakesan & Kuppusamy, 2023)
Specificity
Specificity of RP-HPLC methods has been validated in several studies as the identification and quantification of target analytes. The reviewed papers illustrate specificity in a number of ways. As shown in (Shamim et al., 2023), the developed RP-HPLC method separates ciprofloxacin hydrochloride and rutin very well, showing that the developed method is highly specific for these compounds. Adulteration in pharmaceutical cream formulations is a major drawback, mainly due to its higher accessibility, leading to sodium benzoate in cream formulations. (Ramireddy & Behara, 2023) has published a method for the simultaneous estimation of Ozenoxacin and Benzoic Acid, a method used for the detection of ozenoxacin and benzoic acid using high-performance thin-layer chromatography (HPTLC). Forced degradation studies were considered to evaluate the potential of the method in stability-indicating (Ramireddy & Behara, 2023). A few studies even conducted more sophisticated tests of specificity. Chen et al. 2023 describes a method for simultaneous determination of free mannose and glucose in serum with highlights on its specificity in complex biological matrices. A stability-indicating HPLC method was developed for curcuminoids (Mohammed et al., 2023) that displayed specificity not just for the assay compounds but also its forced degradants produced by variation in experimental conditions (Mohammed et al., 2023).
Sensitivity
Sensitivity is an important validation parameter usually included in methods with RP-HPLC, generally defined as LOD (limit of detection) and LOQ (limit of quantification). These parameters showed the capability of the method to detect and quantify an analyte at very low concentrations. Many studies published sensitivity values for their established RP-HPLC methods. A previous example includes the validation of a method for the analysis of ciprofloxacin hydrochloride and rutin showing good sensitivity (Shamim et al., 2023). For thiopental sodium analysis, the method demonstrated an LOD of 14.4 μg/mL and LOQ of 43.6 μg/mL (Al-Hakkani, Ahmed, Hassan, et al., 2023). Imperfectly low LOD of 0.003 both for cephalexin and for cefixime were reported while LOQ values reached 0.008 and 0.013 ppm, respectively (Y A Alanazi et al., 2023). For short-chain fatty acids, this method had LOD and LOQ values of 0.14 mg/mL and 0.43-0.45 mg/mL respectively for acetic, propionic and butyric acids (Díaz-Corona et al., 2023). In fact, an HPLC-ECD method for the analysis of L-ascorbic acid evidenced an impressively low sensitivity of 0.0043 µg mL-1 LOD (Wu et al., 2023), demonstrating that electrochemical detection can improve sensitivity in specific applications. These results highlight the significance of sensitivity as validation parameter in RP-HPLC methods, with values dependent on used analytes and detection methods.
Robustness
Robustness validation is an important parameter in RP-HPLC methods which checks for a method reliability when subjected to modifications. The significance of robustness in method validation has been well-established in multiple studies. The developed RP-HPLC method for simultaneous estimation of ciprofloxacin hydrochloride and rutin was validated in accordance with ICH Q2 R(1) guidelines in (Shamim et al., 2023), where the robustness was one of the validation parameters. The robustness of the method suggested its insensitivity to small changes in method parameters (Shamim et al., 2023).
Observation: (Mohammed et al., 2023) used a Quality by Design (QbD) approach for developing a stability indicating HPLC method for curcuminoids. Method development, validation, and robustness assessment were carried out using factorial experimental designs. This enabled a wide-ranging analysis of the method robustness taking into account different critical method parameters (CMPs), including mobile phase composition, pH and column temperature (Mohammed et al., 2023).
System Suitability Testing
Simpson, G., & Noble, S. (2008). The Role of System Suitability Testing in HPLC Method Validation for RP-HPLC. This has been covered in various studies: System Suitability Testing Experimental conditions differ from analysis to analysis. The developed RP-HPLC method for the simultaneous determination of ciprofloxacin hydrochloride and rutin was validated in accordance with ICH Q2 R(1) guidelines, including system suitability testing and so forth (Shamim et al., 2023). Likewise, the thiopental sodium analysis method proved its efficiency by achieving system suitability in robustness and ruggedness (Al-Hakkani et al., 2023). In fact, some studies have taken more than a half-baked system suitability test. For example, factorial experimental designs were utilized to systematically optimize critical parameters in an RP-HPLC method under analytical quality by design (AQbD) approach for Canagliflozin (Azhakesan & Kuppusamy, 2023). This means a wider view of the performance of the system in different situations.
8. Troubleshooting in RP-HPLC
More specifically, reverse-phase high-performance liquid chromatography (RP-HPLC) is one of the most utilized analytical tools, but a multitude of issues can arise here. Examples of these problems include:
Low peak resolution: This can be due to improper choice of column, poor mobile phase composition, poor chromatographic condition. For example, in a case of ciprofloxacin hydrochloride and rutin study, researchers optimize the mobile phase composition and flow rate, it had been reported a good separation on a Kromasil C18 column (Shamim et al., 2023).
Sensitivity issues: Low sensitivity might be a problem, both in cases of compounds with weak chromophores. This issue was solved with the analysis of triterpenoids with chemical derivatization techniques to improve sensitivity detection in HPLC (Huang et al., 2023). Likewise, for alectinib quantitation, researchers have employed fluorescence detection to enhance sensitivity (Almutairi et al., 2023).
Matrix effects: Complex sample matrices can suppress and/or enhance the detection and quantification of the analyte. The detection of trace levels of contaminants is challenging, which may hinder its usage, thus several sample preparation methods (e.g. solid-phase extraction (SPE), solid-phase micro-extraction (SPME), etc.) have been developed to overcome this challenge. To improve selectivity and minimize matrix effects, advanced materials such as molecularly imprinted polymers (MIPs) and stimuli-responsive adsorbents have also been used (Mahdavijalal et al., 2024).
Root Cause Analysis
RP-HPLC root cause analysis involves a systematic problem-solving approach that caters to identifying problems with different parts of the RP-HPLC system. These are the types of issues you can identify with these key elements: At the same time, mobile phase problems can be identified by changes in retention time, peak shape and/or baseline stability. Poor Peak Resolution or Tailing – For instance, the improper composition of the mobile phase or pH could result in poor peak resolution or tailing. For example, in another study the mobile phase composition of acetonitrile and phosphate buffer (54:46 v/v, 0.1 mM) was optimized for optimal separation of curcuminoids (Mohammed et al., 2023). Tracking these parameters helps recognize mobile phase issue easily. Problems with the column usually present as changes in the peak shape or retention time, or increase in backpressure. For instance, column degradation or contamination results in peak broadening or splitting. The right column is the first step for a successful chromatographic method, as we can see in a dedicated research where four different polysaccharide columns were tested for the analysis of DE ketoprofen impurities (Dobó et al., 2024). RP-HPLC performance is greatly impacted by temperature. Changes in column temperature can impact retention time and peak resolution. Temperature was shown to play a dual role in determining the resolution and the enantiomer elution order separately in one study (Dobó et al., 2024). It is important to monitor the stability of temperature and its impact on chromatographic parameters as this may indicate potential temperature related issues. System Suitability Testing can identify instrumentation problems by analyzing such parameters as retention time, peak area, and theoretical plates. For example, one study performed system suitability, accuracy, precision, and robustness to validate their method (Shamim et al., 2023). Performing routine system suitability tests can allow for the early detection of instrumentation problems. It is also important to realize that these factors do not act in isolation, and troubleshooting will require a systematic approach. As an example, gradient elution with specific column type and controlled temperature to optimal separation was demonstrated in a study for simultaneous determination of Ozenoxacin and Benzoic Acid (Ramireddy & Behara, 2023). That is the importance of multiple factor in root cause analysis.
Solutions and Best Practices
RP-HPLC: Reverse-Phase High Performance Liquid Chromatography used for analysis and purification of varions compounds. However, it has to do with some parts that should be troubleshoot. Below some issues might happen and their solutions: One of the big concerns of RP-HPLC remains the correct separation of the compounds with similar structure. This is mostly illustrated by the analysis of triterpenoids in botanical samples (e.g. Huang et al., 2023). Thus, chemical derivatization has proved to be an important technique to allow better HPLC performance. For this reason, the derivatization of the triterpenoids has been applied with several reagents such as acid chlorides, acid anhydrides, rhodamines, isocyanates, sulfonic esters or amines to allow greater detection and separation. The poor sensitivity is another common issue, particularly for compounds that lack UV absorption groups, chromophores, or have low ionization efficiency in mass spectrometry. Related for triterpenoid classification (Huang et al., 2023) Overcoming this challenge, novel approaches like fresh reversed-phase dispersive liquid-liquid microextraction (RP-DLLME)-HPLC coupling with deep eutectic solvents (DES) have been developed (Raži? et al., 2023). It was developed as a sustainable and efficient method, particularly for challenging materials, such as oily food samples. Researchers often apply quality by design (QbD) approaches and design of experiments (DoE) to improve RP-HPLC methods and troubleshoot issues. For instance (Shamim et al., 2023), reported that Box–Behnken design was applied for the development of a new RP-HPLC method for simultaneous determination of ciprofloxacin hydrochloride and rutin. Thus, leading the analysis to give the factor vs responses statistically significant values & also the quality of analysis.
9. Future Trends and Innovations in RP-HPLC
In addition, miniaturization is a crucial trend for sensor technology due to the increasing need for sensitivity, enhanced features, small footprint (miniaturization) and reduced power consumption. Just as silicon nanowires are being used in the manufacture of sensors within MEMS (microelectromechanical systems), similar hybrid architectures are created for various physical sensor types (Karimzadehkhouei et al., 2023). RP-HPLC systems will necessarily follow this trend by having compact, effective chromatography systems. Automation and high-throughput systems are increasingly being adopted by the industry. To enhance the functionality of RPA, technologies such as Natural Language Processing, Machine Learning, and Deep Learning are thus becoming part of that (Siderska et al., 2023), making RPA as Intelligent Process Automation [IPA]. In RP-HPLC, this may result in smarter automation of sample preparation, data interpretation, and system maintenance. Technological development must be done with an eye towards sustainability. Themes explored include, among others, the intersection of artificial intelligence and circular economy Maisel et al. (Danish & Senjyu, 2023). For RP-HPLC, this may result in (a) the invention of instruments that require less energy, (b) green mobile phases, and (c) low solvent consumption. Having discussed how the work involved touches upon organic synthesis and biomolecular techniques like proteomics, it is logical as well to put the pieces together and note that RP-HPLC is indirectly addressed. Such improvements may eventually lead to smaller, fully automated and eco-friendlier RP-HPLC settings, thus enhancing laboratory analytical throughput while increasing the environmental sustainability of HPLC.
SUMMARY
The technique used for separation of compounds in this research was Reversed-phase high-performance liquid chromatography (RP-HPLC), an analytical technique applied in many fields for separation and quantification of compounds. It has high resolution, sensitivity, and versatility, allowing for the simultaneous detection of different compounds in complex matrices. The latest developments are mainly addressing the area of efficiency, and how to minimize the ecological impact of the separation processes while making the method discipline more robust by incorporating Quality by Design (QbD) principles, as well as greener mobile phases. These methods design progressive steps of stationary phase selection, mobile phase composition, column temperature and flow rate optimization. An important application is multicomponent analysis, which has been studied using multidimensional chromatography and advanced detectors. RP-HPLC finds broad application in the pharmaceutical industry for multi-drug formulations, stability studies, and impurity profiling. Other usages include environmental monitoring, food and beverage analysis, and biological and biomedical studies. It is paramount for the processes of validation to be followed by regulatory guidelines to ensure reliability, accuracy, and reproducibility. Common issues such as poor peak resolution, sensitivity, and matrix effects can often be detected by performing root cause analysis and implementing best practices. The future of RP-HPLC will therefore follow the path of further reduction, automation, and sustainability to furnish compact, efficient and green chromatography systems.
REFERENCES
Dattatray Yadav*, Dr. Deepak Kardile, Dr. Vishwas Bhagat, Tushar Shinde, Dr. Rajkumar Shete, Review on Advanced RP-HPLC Method Development for Multicomponent Analysis: Techniques, Applications, and Validation Protocols, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 5, 4780-4803. https://doi.org/10.5281/zenodo.15546583
10.5281/zenodo.15546583
