Chromatographic Method Research Articles

Modern Chromatographic Methods for Determination Flavonoids

Flavonoids are a prominent group of plant phenolics (polyphenol group)extensively present in the human diet and associated with health promotion. Flavonoids are essential in various pharmaceutical, nutraceutical, and cosmetic applications. To collect and categorize these compounds, we comprehensively assessedHigh-performance liquid chromatography (HPLC) techniques published to analyze flavonoids in various matrices. Mostdocumented methods employed liquid chromatography with ultraviolet detection (HPLC-UV) and tandem mass spectrometry (LC-MS/MS) methods. Among chromatographic techniques, reversed-phaseliquidchromatographyremains the predominant and efficient method for separating various analytes. This review explores the primary chromatographic methods used to separate and quantify flavonoids in herbs, fruits, and biological samples. This article exam inesrecent literature on applying liquid chromatography with different detection methods for analyzing flavonoids. To examine recent literature on applying liquid chromatography with different detection methods for analyzing flavonoids. It offers valuable insights to researchers as they prepare to develop quantification methods.

A Robust Chromatographic Method for Drug Release profiling of liposomal doxorubicin HCl

Liposomes are excellent drug delivery vehicles for chemotherapeutics as they may change the pharmacokinetics of therapeutic compounds, resulting in altered tissues distribution, and in some cases, reduced cytotoxicity and enhanced distribution and efficacy of the active pharmaceutical ingredient (API) at target tissues. Drug release profiles of liposomal formulations are crucial to support equivalence evaluation and quality control in pre- and post-approval stages. We developed an automated chromatographic method for quantifying the drug release profile of liposomal formulations containing doxorubicin to overcome the shortcomings of currently available methods. The newly developed method employs nanoparticle exclusion chromatography (nPEC), using a monolithic silica column coated with polyvinylpyrrolidone to separate the released drug from liposomal encapsulated drug. We evaluated the effects of pH, temperature, and ammonium formate concentration on the drug release rate. The optimized release buffer consisting of 5 % sucrose, 20 mM l-histidine, and 200 mM ammonium formate was selected for the drug release profiling of five liposomal formulations at 47 °C. The drug release profiles of five liposomal doxorubicin formulations were similar. Our automated method requires very small amounts of the sample and provides release profiles with high sensitivity and accuracy. In addition, this method can be applied to other liposomal products to allow for simple, fast, and accurate analysis of in vitro drug release profiling.

Analytical strategies to identify multicomponent quality markers for commercial Hua-ju-hong using multidimensional chemical analysis.

Hua-ju-hong (HJH) is a Chinese medicinal material obtained from Citrus grandis 'Tomentosa' (CGT) and Citrus grandis (L.) Osbeck (CG) with various commercial specifications. It is known for relieving cough and dispelling phlegm. To reveal the quality marker for distinguishing the various HJH, 215 batches of commercial HJH were studied systematically using multidimensional chemical analysis. Ten major components were identified by high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry and quantified via high-performance liquid chromatography coupled with diode array detection. In this study, a rapid, efficient, and low-cost chromatographic method was established. Total coumarin-hemiterpene and total coumarin-monoterpene were first classified and analyzed in HJH. The result indicated that the main component, naringin, was not the quality marker for differentiating CGT from CG. For reflecting the unique medicinal and food value of HJH, coumarins should be the more potential quality markers. Flavonoids were the possible quality markers for distinguishing two growth stages of fruit-exocarp and young fruit. For the first time, two chemotypes of HJH were identified in CG. This study provides a convenient yet reliant chromatographic method and novel yet systematic strategies for overall quality control of commercial HJH.

Rapid and reliable electrochemical detection of bisphenol S in thermal paper

Bisphenol S (BPS) is a common, persistent, and mobile chemical found in everyday products such as thermal paper. BPS can easily enter the body by migrating from the paper to the fingers, disrupting the endocrine system by mimicking the oestrogen hormone, thus negatively influencing human health. Assessing BPS levels in daily life is of great importance. This study introduces a rapid and reliable approach for detecting BPS in thermal paper and tap water by developing an electrochemical analytical method. This method allows for in-situ, real-time measurements. We present a simple, low-cost electrochemical sensor for detecting BPS using screen-printed electrodes based on carbon (SPEC) and single-wall carbon-nanotube (SPE-SWCNT) working electrodes. BPS was detected over a wide linear range from 1 to 400 μM. The detection limits were 0.73 μM and 0.87 μM for the SPE-C and SPE-SWCNT electrodes, respectively. Good repeatability was observed for both sensors when using one electrode 16 times, which demonstrates its potential for real-time environmental monitoring. Additionally, traditional chromatographic methods, high-performance liquid chromatography with a diode-array detector (HPLC-DAD), and liquid chromatography-mass spectrometry triple quadrupole (LCMS), were incorporated to enhance analytical capabilities. HPLC-DAD achieved a detection limit of 3 nM after solid-phase extraction preconcentration, while LCMS triple quadrupole demonstrated a detection limit of 10 pM without preconcentration. Electrochemical screen-printed electrodes can be employed for on-site analysis and health-risk assessments in everyday settings, such as shops. However, for detecting very low concentrations where time is not a constraint, LCMS quadrupole remains the preferred technique.

In vitro evaluation of the percutaneous absorption of progesterone in anhydrous permeation-enhancing base using the Franz skin finite dose model and mass spectrometry

Progesterone is used for hormone replacement therapy through various routes of administration. This study was conducted to (a) evaluate the stability of progesterone in a proprietary anhydrous permeation-enhancing base (APEB) and the efficiency of its skin permeation, and (b) determine the appropriateness of mass spectrometry as a method of analysis for permeated progesterone. Using a proven stability-indicating ultra-performance liquid chromatographic method, the compounded hormone (100 mg progesterone/g APEB gel) was determined to be physically and chemically stable at room temperature for six months. Skin permeation analysis using the Franz skin finite dose model and mass spectrometry imaging showed an optical density of 1699 for the permeated progesterone compounded in APEB and 550 for the permeated progesterone in a water containing VBC, which is a statistically significant different (P = 0.029). The study suggests that APEB can be used as a compounding base for effective skin permeation of progesterone, and mass spectrometry is a reliable method for visualization and quantitative analysis of permeated progesterone.

Enhanced HPLC Method for Boar Taint Quantification.

Boar taint is an unpleasant odour found in the carcasses of entire male pigs, resulting from androstenone and skatole accumulation during pubertal development, and impacting pork quality. This study proposes the validation of an adapted chromatographic method for quantifying skatole and androstenone in the pigs' liquid fat using fluorescence detection. A good chromatographic separation was achieved, with skatole (SKA) and androstenone (AND) elution at 4.4 and 9.9 min., respectively. An external calibration method was applied, with calibration curves correlation coefficient of 0.9999 for both analytes. Detection limit values were 1.53 and 16.02 ng/g for SKA and AND, respectively. SKA recovery was 99.72±2.34 % (2.34 % RSD) and 102.84±1.62 % (1.57 % RSD) for AND. Results showed good precision values (repeatability <2.46 % RSD for SKA, <6.85 % RSD for AND; intermediate precision <2.87 % RSD for SKA, <6.98 % RSD for AND). The method's robustness was tested and the values were within the reference ranges. The validation results proved that the adaptation of an existing method resulted in good assessments of robustness, reliability and accuracy.

Chromatographic approaches to food quality control by chemical compo-sition

The article provides an overview of chromatographic methods, which, when used comprehensively, ensure food quality control and safety. It considers gas chromatography methods that allow analysing volatile components in products using a flame ionization detector, an electron capture detector, and mass-selective detectors (GC-FID, GC-ECD, GC-MS, and GC-MS/MS). The article provides a list of analytes that can be detected by high performance liquid chromatography in combination with refractometric, spectrometric, and mass selective detectors (HPLC-RMD, HPLC-UV, HPLC-SPD, HPLC-MS, and HPLC-MS/MS). The study showed how ion chromatography and capillary electrophoresis with electrochemical detectors (IC-ECD, CE-ECD) can be used to detect ionogenic compounds. It highlighted the importance of thin layer chromatography (TLC) in combination with optical digital colour recording devices in the qualitative and quantitative analysis of contaminants in food products. Since it has been established that micro- and nanoplastics have a negative impact on human health, it is very important to detect these particles in food products. This task can be solved by using gel permeation chromatography (GPC) and hydrodynamic chromatography (HDC), as well as the method of field-flaw fractionation (FFF). It was shown that the GC-MS method is now becoming a priority instrumental method used in accredited analytical laboratories to identify impurities of contaminants in agricultural products. The pyrolytic GC-MS method is promising in this regard. The pyrolytic cell does not require the transfer of a solid sample into a solution for its analysis. The paper considers extraction and sorption methods for the preparation of samples for chromatographic analysis. A variety of liquid-liquid extraction (LLE) and solid-phase extraction (SPE.) methods are most commonly used. Recently, the QuEChERS method has been developed and widely implemented. The overview is primarily based on the papers dedicated to chromatography and related separation methods published by researchers belonging to Voronezh scientific schools.

Development and Validation of High-Performance Thin Layer Chromatographic Method for Estimation of Acyclovir

Development and Validation of High-Performance Thin Layer Chromatographic Method for Estimation of Acyclovir

Simultaneous quantification of terpenes and cannabinoids by reversed-phase LC-APCI-MS/MS in Cannabis sativa L. samples combined with a subsequent chemometric analysis

Cannabis sativa L. has been the most discussed medicinal plant in recent years. In particular, the dynamic shift from a formerly illicit and tightly controlled substance to a plant recognized for both medicinal and recreational purposes has brought C. sativa into the global spotlight. Due to the ongoing international legalization processes, fast and convenient analytical methods for the quality control of C. sativa flowers for medicinal and recreational purposes are of tremendous interest. In this study, we report the development and validation of a liquid chromatography-tandem mass spectrometry (LC–MS/MS)-based method applying atmospheric pressure chemical ionization (APCI) to fully quantify 16 terpenes and 7 cannabinoids including their acidic forms by a single chromatographic method. The method presented here is unique and simple, as it eliminates the need for derivatization reactions and includes the unconventional analysis of volatile compounds by liquid chromatography. Samples were prepared by a simple and fast ethanolic extraction. Separation was accomplished within 25 min on a reversed-phase C18 column. Method validation was conducted according to international guidelines regarding selectivity, accuracy, precision, robustness, and linearity. Detection was done in multiple reaction monitoring, which allowed the simultaneous quantification of co-eluting analytes applying two selective mass transitions. In addition, due to reproducible in-source decarboxylation, the acidic forms of cannabinoids were reliably quantified using mass transitions of the neutral forms. The accuracy given as the bias was below 15% for all analytes. Matrix effects for cannabinoids were studied by spiking Humulus lupulus extracts with the analytes at varying concentrations. APCI did not show susceptibility toward ion suppression or enhancement. In addition, the recovery effect after spiking was between 80 and 120% for terpenes. Further, 55 authentic C. sativa extracts were fully quantified, and the obtained results for the terpene profiles were compared to state-of-the-art gas chromatography coupled to flame ionization detection. Comparable results were achieved, emphasizing the method’s applicability for cannabinoids and terpenes. Further, acquired metabolite patterns for C. sativa samples were studied, identifying a relationship between cannabinoid and terpene patterns, as well as the abundance of myrcene in CBD-dominant C. sativa strains.Graphical

Current analytical methods and applications used in the insight of serum proteins interactions with various food additives, pesticides, and contaminants

In recent years, many societies have expressed increasing apprehension regarding the potential negative impacts of food additives, pesticides, and environmental contaminants on human health. Environmental or occupational exposure to these compounds can cause significant adverse effects on human health by causing temporary or permanent changes in the immune system. There is supporting evidence linking pesticides/food ingredients/contaminants-induced immune alterations to the prevalence of diseases associated with changes in immune responses. Hence, it is essential to comprehensively understand the key mechanisms contributing to immune dysregulation induced by these substances, including direct immunotoxicity, endocrine disruption, and antigenicity. The impact of pesticides/food ingredients and contaminants on the human body ranges from mild to severe, depending on their affinity for blood components. These compounds form complexes with blood serum proteins, influencing their metabolism, transport, absorption, and overall toxicity. Numerous studies in the literature have explored the interactions between serum proteins and various molecules, including pesticides, drugs, and food dyes. These investigations employed a range of techniques, including spectroscopy, electrochemical and chromatographic methods as well as molecular modeling and molecular dynamics simulations analyses. This recent review, spanning from 2020 to the present, has been employed to investigate the binding characteristics, mechanisms, and attributes of different food additives, pesticides, and contaminants with serum proteins by using various techniques such as steady-state fluorescence, circular dichroism and ultra-violet spectroscopies, and computational docking methods. The review provides insights into these compounds’ positions and affinities to proteins and possible effects on human health through detailed research studies.

Fast quantitative determination of monoclonal antibody in infusion bags using protein A nano liquid chromatography

AbstractStaphylococcus aureus Protein A was immobilized on an in‐house made neutravidin poly (glycidyl methacrylate‐co‐ethylene dimethacrylate) capillary column with a 25 µm internal diameter, a length of 30 mm and a mass loadability of 60 ng. The immobilized quantity of protein A on the organic monolith was very low, in the pico mole range (1.80 pmol). This was of significance importance when working with less available or expensive purified enzyme. This capillary column was integrated into a nano liquid chromatographic system and used for the fast determination without dilution of the doses of therapeutic monoclonal antibodies (mAbs) in standardized infusion bags prepared in advance in a pharmacy department. This chromatographic method was linear in the studied concentration range with good precision and accuracy. Heat stressed studies indicated that the protein A affinity capillary column was able to monitor degraded mAbs. As well, the high specificity of this column to capture immunoglobulin G2 in cell culture supernatant was visualized. As the mAbs are produced through genetic engineering of animal cells this last result demonstrated that this novel protein A column could be used in the feature for rapid screening of immunoglobulin G concentration in cell culture.

Oak Leaves as a Raw Material for the Production of Alcoholic Fermented Beverages.

This study aimed to point out the possible use of oak leaves (Q. petraea) in the production of fermented alcoholic beverages. Parameters such as antioxidant capacity, total phenolic content, phenolics and sugars were determined using spectrophotometric and chromatographic methods. pH values were also determined, and in the final product with a fermentation length of 85 days, the alcohol content was determined and sensory analysis performed. The antioxidant capacity of the beverage was lower compared to the infusions before fermentation, and its highest values were recorded in the leaf samples, in which the highest values of phenolic compounds and the total phenolic content were also recorded. A decrease in the content of total phenolics was recorded with the increasing length of fermentation in beverage samples. However, the fermentation process had a positive effect on the contents of some phenolic substances such as catechin, gallic acid and gallocatechin. Sensory analysis showed a higher acceptability of the fermented beverage without the addition of orange, which could be caused by the higher sugar content in these samples. Oak leaves therefore represent a suitable raw material for the production of a fermented alcoholic beverage, without the need to enrich the taste with other ingredients.

Stability of Bioactive Compounds and Antioxidant Activity in Rosehip Juice (Rosa spp.).

Rosehip fruits, characterized by their high concentrations of bioactive compounds and antioxidant activity (AA), have been traditionally used to make jams, infusions, and juices. Thus, the objective of this research was to evaluate the stability of rosehip juice by determining the concentrations of bioactive compounds and total phenols and the AA using chromatographic and spectroscopic methods. The stability of the juice was evaluated with three treatments and different storage conditions, namely, unpasteurized-refrigerated, pasteurized-room temperature, and pasteurized-refrigerated, and measurements were taken for eight months. Individual and total phenolic compounds, evaluated by chromatographic methods, reported differences until the end of this study. The total phenolic compounds by Folin-Ciocalteu method presented an average decrease of 57% in the three treatments in relation to the initial conditions. On the other hand, the ascorbic acid content decreased considerably, disappearing at week six. Furthermore, for the unpasteurized-refrigerated and pasteurized-refrigerated samples, a correlation was found between flavonols, total phenols, ascorbic acid, and antioxidant activity determined by the TEAC method. For the pasteurized-room temperature samples, correlations were found between the levels of several flavonols, hydroxycinnamic acid, total phenols, and ascorbic acid and the antioxidant activity determined by the CUPRAC method. The stability of the compounds was mainly correlated with the storage conditions of the juice and not with pasteurization. The highest stability was observed for the unpasteurized-refrigerated and pasteurized-refrigerated samples. Although the concentrations of the compounds evaluated decreased during this study, significant levels of AA persisted, providing beneficial characteristics for consumer health.

Enhancing simultaneous determination of some angiotensin II receptor antagonists and amlodipine in plasma using HPTLC with fluorescence densitometry: Independent fluorescence detection of the co-administrative drugs in the mixture across various pH conditions

A novel and highly sensitive high-performance thin-layer chromatographic (HPTLC) method was developed and validated to quantify a combination of five pharmaceutical mixtures spiked to human plasma. The compounds comprised Amlodipine (AML) along with five angiotensin II receptor antagonist drugs (AIIRAs), namely Olmesartan (OLM), Telmisartan (TLM), Candesartan (CAN), Losartan (LOS), and Irbesartan (IRB). HPTLC was performed on silica gel 60 F254 plates using a mobile phase of Toluene: ethyl acetate: methanol: acetone: acetic acid (6:1.5:1:0.5:1, v/v/v/v/v). In a pioneering move, a reflectance/fluorescence detection mode was employed to identify two concurrently administered drugs at different pH levels for the first time. This method utilized the same chromatographic system, incorporating a specific measurement for AML at a neutral medium to achieve its maximum fluorescence at a 360 nm excitation wavelength, and measuring emission using a 540 nm optical filter. The process involved obtaining a very low fluorescence response from AIIRA. Subsequently, to enhance AIIRA’s fluorescence, the plate was sprayed with perchloric acid to transition to a strong acidic medium, ultimately attaining the maximum fluorescence of AIIRA using various excitation wavelengths and a 400 nm emission filter. Through this strategic process, we could optimize the fluorescence signals of both drugs, thereby elevating the sensitivity of detection for this drug combination. AML demonstrated a linear range of 18–300 ng/band, while AIIRAs drugs exhibited a linear range of 6–150 ng/band. The method satisfied the International Conference on Harmonization (ICH) criteria for recovery, precision, repeatability, and robustness, showcasing exceptional sensitivity. The approach was successfully applied to quantify AML and AIIRAs drugs in both bulk drug and plasma samples, achieving high recovery percentages and minimal standard deviations.

Adulteration in dietary supplements: HPTLC profiling of Withania somnifera and Terminalia bellirica

In the realm of traditional medicine, (Withania somnifera), commonly known as Ashwagandha, has long been revered for its multifaceted benefits, earning it the moniker "Indian Ginseng" or winter cherry within Unani systems. Its reputation precedes it, boasting a spectrum of advantageous properties such as antiviral, antioxidant, and anti-inflammatory effects. Moreover, research suggests its potential efficacy against various cancers including breast, colon, prostate, ovarian, and lung cancer. Similarly, Terminalia bellirica, referred to as Behra, holds its own array of therapeutic potential, addressing maladies ranging from headaches to liver disorders, gastrointestinal discomforts, common colds, and constipation. However, the widespread popularity of these medicinal plants has led to concerns regarding their sustainability. Overexploitation poses a significant risk, potentially depleting these valuable resources. Compounded with this risk is the issue of adulteration or contamination, where other plant parts or species resembling the authentic herbs are utilized, compromising the safety and efficacy of the end the product. In a recent investigation, two samples each of authentic and commercial Withania somnifera and Terminalia bellirica were subjected to rigorous assessment through physiochemical, phytochemical, TLC, and HPLC chromatographic methods. Alarming findings surfaced, revealing instances of adulteration or contamination in both the Ashwagandha and Behra products. Phytochemical analyses and HPTLC profiles of various extracts unveiled the intentional or unintentional addition of undesired herbs or substitutions. These adulterants not only jeopardize the expected therapeutic outcomes but also dilute the potency and safety of the herbal medicines flooding the local markets. This study serves as a clarion call for stringent authentication measures in the procurement and utilization of herbal medicines. High-Performance Thin-Layer Chromatography (HPTLC) emerges as a reliable tool for confirming adulteration, urging stakeholders to adopt thorough authentication protocols to safeguard public health and preserve the integrity of traditional herbal remedies.

Contamination and pesticide multiresidue analysis in cotton production systems in Benin

High quantity of chemical pesticides spraying in conventional cotton production systems may affect negatively natural ecosystems components. This study examined the contamination levels of soil in Benin's cotton production system in West Africa by residues of pesticides used in cotton farming. One hundred and fourteen soil samples from six conventional and organic cotton production systems were collected and analysed. Multi-class pesticide residues were extracted from the soil samples, and analysed using respectively modified QuEChERS extraction and LC–MS/MS chromatography methods. Results showed an overall soil contamination to emamectin benzoate (18.5 μg/kg), imidacloprid (116.3 μg/kg), profenofos (12.7 μg/kg), acetamiprid (10.8 μg/kg), triasophos (12.6 μg/kg), abamectin (14.3 μg/kg), and deltamethrin (10.4 μg/kg). Especially, emamectin benzoate was detected with a high contamination in Banikoara district (18.5 μg/kg). Nevertheless, these contaminations were not deemed threatening from a toxicological perspective. Soils in conventional cotton cultivation displayed lower organic matter levels but higher concentrations of pesticides, whereas soils in organic cotton cultivation were less degraded and contaminated. Consequently, it is imperative to conduct environmental risk assessments and monitor key pesticide metabolites to establish sustainable cotton production systems in Benin.

The importance of choosing the appropriate cholesterol quantification method: enzymatic assay versus gas chromatography

Cholesterol is a major lipid of the animal realm with many biological roles. It is an important component of cellular membranes and a precursor of steroid hormones and bile acids. It is particularly abundant in nervous tissues, and dysregulation of cholesterol metabolism has been associated with neurodegenerative diseases such as Alzheimer's and Huntington's diseases. Deciphering the pathophysiological mechanisms of these disorders often involves animal models such as mice and Drosophila. Accurate quantification of cholesterol levels in the chosen models is a critical point of these studies. In the present work, we compare two common methods, gas chromatography coupled to flame-ionization detection (GC/FID) and a cholesterol oxidase-based fluorometric assay to measure cholesterol in mouse brains and Drosophila heads. Cholesterol levels measured by the two methods were similar for the mouse brain, which presents a huge majority of cholesterol in its sterol profile. On the contrary, depending on the method, measured cholesterol levels were very different for Drosophila heads, which present a complex sterol profile with a minority of cholesterol. We showed that the enzyme-based assay is not specific for cholesterol and detects other sterols as well. This method is therefore not suited for cholesterol measurement in models such as Drosophila. Alternatively, chromatographic methods, such as GC/FID, offer the required specificity for cholesterol quantification. Understanding the limitations of the quantification techniques is essential for reliable interpretation of the results in cholesterol-related research.

Development and validation of stability indicating ultra‐high‐performance liquid chromatographic method for assay and impurities of mexiletine hydrochloride and mexiletine hydrochloride capsules

AbstractMexiletine (MEX) is an antiarrhythmic drug used to treat acute and chronic ventricular arrhythmias. A simple isocratic, rapid, specific, stability‐indicating, and sensitive reverse‐phase ultra‐high‐performance liquid chromatography (UHPLC) method was developed and successfully validated to quantify the assay and impurities of MEX hydrochloride. The new UHPLC method showed the optimum separation of MEX and its seven impurities in 20 min by using sodium acetate buffer (0.14 mM) with 0.3% of glacial acetic acid (pH 4.8) and methanol in the ratio of 40:60 (v/v) as mobile phase. The method used an Acquity HSS T3 (100 × 3.0 mm, 1.8 µm) chromatographic column at a 0.2 mL/min flow rate. The injection volume is 2 µL, and 254 nm is the detection wavelength. Note that, 25°C is the column oven temperature. The method is linear over the concentration range of the quantification limit (0.03%) to 150% (0.3%) of the specification level (0.2%) for the impurities and 50%–150% for the assay method. The quality control lab of bulk drugs and finished dosage form successfully applied the new UHPLC method for quantification of assay and impurities of MEX hydrochloride.

Research Progress in the Separation of Chemical Components from Essential Oils by High-Speed Countercurrent Chromatography

Essential oils (EOs) are vital secondary metabolites in plants. They have garnered substantial attention owing to their distinct flavors and desirable attributes, including potent antioxidant, antibacterial, and antitumor properties. Nevertheless, the active constituents of EOs exhibit intricate chemical structures, and conventional separation techniques are inadequate for purifying the individual chemical components from EOs. High-speed countercurrent chromatography, based on the principles of a hydrodynamic equilibrium system, has emerged as a liquid–liquid chromatographic separation method renowned for its ability to handle substantial single injection volumes and the absence of irreversible adsorption. Consequently, in recent years, this technique has been widely employed in the isolation and refinement of natural products. In this review, a comprehensive analysis is conducted, contrasting the merits and demerits of high-speed countercurrent chromatography with conventional separation methods. The solvent systems, elution modes, commonly employed detectors, and practical applications are reviewed in the context of high-speed countercurrent chromatography for essential oil separation and purification. Furthermore, this review offers a glimpse into the potential prospects of applying this technique, with the intention of serving as a valuable reference for the use of high-speed countercurrent chromatography in the purification of EOs.

STABILITY BASED HPLC METHOD FOR CYCLOPHOSPHAMIDE RELATED SUBSTANCES IN FINISHED DRUG PRODUCTS: DEVELOPMENT AND VALIDATION

Objective: The current study aimed to develop a simple, sensitive, and precise high-performance liquid chromatographic (HPLC) method for estimating cyclophosphamide and its related substances, as well as to implement the developed method in a capsule product. Methods: Method development was performed using various solvent and buffer-solvent ratios at different flow rates for better resolution and to decrease the run time. The developed method was validated in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The developed method was implemented to estimate the amount of cyclophosphamide and its related substances. Results: Chromatographical conditions were optimised, and the best chromatographical conditions with adequate resolution for cyclophosphamide and its related substances were achieved using the enable X-Bridge C18 column, using a mobile phase combination of phosphate buffer pH 7.0, water, methanol, and acetonitrile at a flow rate of 0.5 ml/min. The detection was monitored at a wavelength of 200 nm. The developed method was validated for system suitability, specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity, precision, accuracy and robustness. The results indicate that the method was sensitive and could detect and quantify lower levels of cyclophosphamide and its related substances. The linearity range was from LOQ to 150 %, and a correlation coefficient ranging from 0.9987 to 0.9999 indicates that at this concentration range, substances were highly linear. This was further supported by satisfactory forced degradation studies. Conclusion: The developed analytical method is simple, precise, and reproducible and thus can be used for the stability-indicating analysis of cyclophosphamide and its related substances in pharmaceutical formulations.