Chapter 14 - Method development in liquid chromatography

The development and validation of a high performance liquid chromatography method to determine the radiochemical purity of [177Lu]Lu-HA-DOTA-TATE in pharmaceutical preparations

Background: Fluconazole is a triazole antifungal agent widely employed in the treat ment of systemic and superficial fungal infections. Accurate, precise, and reliable quantification of the active pharmaceutical ingredient in dosage forms is of critical importance for both quality control and therapeutic efficacy. Methods: In this study, a reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantitative determina tion of fluconazole in pharmaceutical preparations. Chromatographic separation was performed using a Zorbax C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phase consisted of a methanol–water mixture (70 : 30, v/v) in an isocratic system at a flow rate of 1.0 mL/min. The injection volume was set at 5 μL, the detection wavelength at 210 nm, and the column temperature at 30°C. The system suitability parameters of the method were found to be within the acceptable limits. Results: The retention time for fluconazole was determined as 1.13 ± 0.03 minutes. The method demonstrated linearity within the concentration range of 3.0 × 10−7 M to 9.0 × 10−6 M, with a correlation coefficient (r2) of 0.9999. The limit of detection (LOD) and limit of quantification (LOQ) were calculated as 6.0 × 10−8 M and 1.8 × 10−7 M, respectively. Accuracy and precision values were within acceptable ranges for both intra-day and inter-day analyses. Recovery values ranged from 96.2% to 101.3%, while the relative standard deviation values remained below 2%. Conclusion: The developed RP-HPLC method is a simple, accurate, precise, and robust approach that can be employed for the routine quality control of fluconazolen in pharmaceutical preparations. Moreover, the high sensitivity and reproducibility of the method indicate its potential applicability for the determination of fluconazole in biological samples in future studies. Cite this article as: Dal Poçan AG. Development and validation of a rapid reverse-phase high-performance liquid chromatography method for quantification of fluconazole in pharmaceutical preparations. Trends in Pharmacy, 2025, 2, 0011, doi: 10.5152/TrendsPharm.2025.25011

Recent development in liquid chromatography stationary phases for separation of Traditional Chinese Medicine components

The current studies entail systematic quality by design (QbD)-based development of a simple, rapid, sensitive and cost-effective stability-indicating method for the estimation of olmesartan medoxomil. Quality target method profile was defined and critical analytical attributes (CAAs) for the reverse-phase liquid chromatography method earmarked. Chromatographic separation accomplished on a C18 column using acetonitrile and water (containing 0.1% orthophosphoric acid, pH 3.5) in 40 : 60 (v/v) as mobile phase at a flow rate of 1.0 mL/min with UV detection at 243 nm. Risk assessment studies and screening studies facilitated comprehensive understanding of the factors affecting CAAs. The mobile phase ratio and flow rate were identified as critical method parameters (CMPs) and were systematically optimized using face-centered cubic design, evaluating for CAAs, namely peak area, retention time, theoretical plates and peak tailing. Statistical modelization was accomplished followed by response surface analysis for comprehending plausible interaction(s) among CMPs. Search for optimum solution was conducted through numerical and graphical optimization for demarcating the design space. Analytical method validation and subsequent forced degradation studies corroborated the method to be highly efficient for routine analysis of drug and its degradation products. The studies successfully demonstrate the utility of QbD approach for developing the highly sensitive liquid chromatographic method with enhanced method performance.

The rapid and direct determination of ATPase activity by ion exchange chromatography and the application to the activity of heat shock protein-90

In the present investigation, a simple, rapid and sensitive liquid chromatography and mass spectrometry (LC/MS) method was developed for quantification of Upadacitinib (UCB) in spiked human plasma. The drug UCB was extracted by liquid –liquid extraction using diethyl ether and chloroform in the ratio of 75:25 (v/v). The drug Leflunomide (LFM) was used as an internal standard. The extracted drug mixture was followed by the liquid chromatography mass spectrometry (LC/MS) analysis and electrospray ionization interface. The chromatographic separation of UCB was carried out on Phenomenex Luna C18 column (100mm X 2.0 mm X 5μm) at ambient temperature followed by isocratic elution of 0.05% formic acid in methanol and acetonitrile in the ratio of 65:30(v/v). The method is concluded at a flow rate of 0.8 mL/min, 262 nm of UV detector wavelength. Protonated ions formed by electrospray ionization were recorded in the positive mode and were used to detect the analyte (UCB) with internal standard (LFM). The mass detection was made by monitoring the fragmentation of m/z 381.0 for UCB m/z 271.0 for LCM on mass spectrometer. The method was validated for accuracy, precision, linearity and recovery. The assay was linear over the entire range of calibration standards i.e 2.5-500ng/mL. The recoveries of the UCB after liquid-liquid extraction at 5, 20, 35 ng/mL were 100.7, 99.4 and 99.8. The lowest limit of the analytical method of UCB was 2 ng/mL in spiked human plasma. The developed method was successfully validated and can be used for determination of the pharmacokinetic parameters of the UCB in biological samples.

The primary objective of the present investigation is to develop and validate a simple, robust, and cost-effective isocratic reverse phase-high-performance liquid chromatography (RP-HPLC) method for determining isoliquiritigenin (ISL) in both bulk and biological samples using an analytical quality-by-design (AQbD) approach. The central composite design was employed for method optimization using Design Expert® software, by taking mobile phase ratio and flow rate as independent variables and peak area, retention time, tailing factor, and theoretical plates as dependent variables. The design suggested the use of a mobile phase consisting of acetonitrile:0.2% ortho-phosphoric acid (75:25, v/v) and a flow rate of 0.9 mL/min as optimal chromatographic conditions. The detection of ISL was performed at 364 nm. The optimized method was validated in accordance with International Conference on Harmonization (ICH) Q2(R1) guidelines. The method showed excellent linearity, limit of detection, limit of quantification, accuracy, precision, robustness, and system suitability. All validation parameters fell within the acceptable limits set by ICH. Additionally, the applicability of the method in biological samples were analyzed. In conclusion, the results suggest that the developed and validated AQbD-based RP-HPLC method was well-suited for the estimation of ISL in bulk and biological sample.

The implementation of 'Analytical Quality by Design' (AQbD) is currently recognised as a methodical and scientific approach to liquid chromatographic (LC) method development. It begins with defining the Analytical Target Profile (ATP), identifying Critical Method Parameters (CMPs) and Critical Quality Attributes (CQAs) or responses. This process also includes risk assessment study by Design of Experiment (DoE) and to optimise LC effectively. The next step is to create an analytical design space and implement a control strategy that will allow for continuous method improvement over the life of the method. Understanding the AQbD principles, methodologies, techniques and applications towards the high-performance liquid chromatography (HPLC) method lifecycle is considered as essential in today's pharmaceutical landscape. As industry demands for quality assurance and regulatory compliance, this review paper briefly discusses the AQbD principles and methodology in HPLC method lifecycle. Also, the paper covers AQbD components such as identification of ATP, CQA, MODR (method operable design region), control strategies and continuous method monitoring. In addition, it also covers regulatory perspectives on AQbD, obstacles and potential outcomes in the development of pharmaceutical analytical methods. These aspects provide valuable insights into the application of AQbD in the field of LC.

Amphotericin B (AmB) is one of the most potent antifungal agents and the drug of choice in the treatment of serious fungal infections. A liquid chromatographic (LC) method was developed to determine AmB in pharmaceutical formulations for injection, tissue culture, cream, and lotion. pBondapak C18 reversed-phase column and a simple mobile phase consisting of acetonitrile-water-acetic acid (40 + 54 + 6, v/v) was used. The flow rate was 1.8 mL/min and the effluent was monitored at 405 nm. The developed LC method uses piroxicam as an internal standard and has a limit of detection of 10 ng/mL, a limit of quantitation of 30 ng/mL, and the assay is linear from 0.01 to 100 microg/mL. AmB and piroxicam elute with retention times of 12.4 and 4.0 min, respectively, and the resolution between AmB and piroxicam was 10.6. In comparison with the official United States Pharmacopeia microbial assay for AmB, this LC method is more rapid, selective, sensitive, and offers positive identification.

Selectivity differences for C 18 and C 8 reversed-phase columns as a function of temperature and gradient steepness : II. Minimizing column reproducibility problems

Sensitive, accurate and efficient Liquid Chromatographic Method is developed for the quantitative determination of Nitrofuran residues in fish samples. It was found that the coefficient of variation for all four test substances of the Nitrofuran group is from 0.03 ìg / ml less than 2%, respectively the detection minimum was considered to be 0.03ìg / ml; 376 nm wavelength is specific for Nitrofurans; For the quantification of nitrofuran residues in a fish sample validation of the developed Liquid Chromatographic Method revealed complete compliance of the method (Guidance for Industry Bioanalytical Method Validation U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Veterinary Medicine (CVM) May 2001) with the following validation characteristics of the requirements: detection minimum, specificity, accuracy, correctness and linearity.

Objective: The objective of this study was to develop a new and simple reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of riluzole (RZ) and levodopa (LD) in the marketed tablet dosage form. Materials and Methods: The chromatographic separation was achieved on a RP hypercil C18 column (250 × 4.6 mm i.d., 5 μm) using a mobile phase consisting of methanol and water (75:25 v/v) at a flow rate of 1.0 ml/min with the ultraviolet detection wavelength of 273 nm at ambient temperature. The developed method was validated as per the International Council on Harmonization guideline for linearity, accuracy, precision, robustness, ruggedness, limit of detection (LOD), limit of quantitation (LOQ), and specificity. Results: Results of validation studies were found satisfactory with % relative standard deviation values of <2% indicating good specificity, validity, and reliability of the method. The method showed good linearity over the concentration range of 20–80 μg/ml with correlation coefficient (r2) values of 0.990and 0.999 for RZ and LD, respectively. The mean percentage recoveries were between 99.46–99.80% and 100.15–101.24% for RZ and LD, respectively. The LOD and LOQ values were found to be 0.036and 0.012 μg/ml, and 0.110and 0.036 μg/ml for RZ and LD, respectively. The assay of RZ and LD in the marketed tablet formulation was found to be 99.67and 98.95%, respectively. Conclusion: The RP-HPLC method is reported to be simple, specific, accurate, and precise. The proposed method can be successfully applied for the routine analysis of RZ and LD in the bulk drugs as well as in combined pharmaceutical dosage forms.

The current research involved the development and validation of an easy, cost-effective, and sensitive bioanalytical reverse-phase high-performance liquid chromatography method for the assessment of palbociclib (PAL) in rat plasma and kidney, liver, spleen and heart. A response surface methodology-based Box-Behnken design was used to optimize critical chromatographic conditions such as buffer pH, organic phase concentration and flow rate to attain good sensitivity, tailing factor and retention time. The conditions were: pH of buffer, 4.5; organic phase concentration, 40%; Shimpac column with 1.0ml/min flow rate. The responses were: tailing factor, 1.29 ± 0.03, sensitivity, 366,593 ± 8,592; and retention time, 4.5 ± 0.05 min. The samples were extracted by a protein precipitation method, and absolute recoveries were in the range of 88.99-95.08%. The linearity of the developed method was validated over the range 100-2,000 ng/ml (r2 ≥ 0.994) in all tested matrices. The developed bioanalytical method showed greater accuracy (0.98 and 4.01%) and precision (<4.88%). The method was optimized for the sensitive analysis of the PAL in rat plasma, and the kidney, liver, spleen and heart were effectively applied to pharmacokinetic studies.

The aim of this research was the development and validation of a high performance liquid chromatography (HPLC) method for the simultaneous and quantitative determination of benzophenone-3, octyl methoxycinnamate and octyl salicylate contained in sunscreen emulsions. The separation and quantitative determination was achieved using a LiChrospher((R)) 100 RP-18 (5 microm) Merck column, a mobile phase constituted of methanol/water (85/15, v/v), a flow-rate of 1.0 mL min(-1) and UV detection at 310 nm. The correlation coefficients and percentage of recovery for benzophenone-3, octyl methoxycinnamate and octyl salicylate were 0.9999 and 99.46%; 0.9995 and 98.85%; 0.9998 and 98.84%, respectively. The relative standard deviations (RSD) for commercial samples were between 0.50 and 0.70%.

Development of a simple liquid chromatographic method with UV and mass spectrometric detection for the separation of substances related to amoxicillin sodium