ICH Q2 Guidelines Research Articles

Analytical Methods for the Quantification of Ribavirin in Pharmaceutical Preparations, A Comparative Study

Ribavirin is a broad-spectrum antiviral drug and is used to treat diseases caused by many viral infections. In this study, high-pressure liquid chromatographic and UV-Vis spectrophotometric methods were developed, validated and statistically compared to determine the amount of ribavirin in pharmaceutical preparations. HPLC analyses were performed by UV detection at 207 nm using a mobile phase consisting of 20mm KH2P04 with a flow rate of 0.8 mL min-1 on a C18 column. Ultrapure water was used as a solvent in spectrophotometric analyses. The UV spectrum of the standard and sample solutions was recorded between 200 and 400 nm and absorbance values were measured at 207 nm. Both methods were validated according to the procedures described in ICH guidelines Q2(R1) for verification of analytical methods. The results showed that spectrophotometric and liquid chromatographic methods were linear, precise, accurate, rugged, and robust with RSD values less than 1.00%, and the recovery percentage was within standard limits (98-102%). Then a statistical comparison of these two analytical methods was performed, and the results showed no significant difference. As a result, the developed analytical methods were determined to be accurate, highly effective, reliable, fast, simple, and could be used for routine quality control analysis of ribavirin in pharmaceutical formulations.

Surveillance of Stability Under Different Stress Conditions in Desloratadine and Study of Degradation Kinetics.

A stability-indicating method (SIM) stipulates the testing of the drug product and drug substances under the stressed conditions that will imply a clear notion about the stress conditions that would affect the drug in its finished dosage form. Furthermore, manufactures can clearly define the state at which the drug is unstable and present its storage conditions. The present article deals with the stability testing and degradation kinetic studies of desloratadine (DL). The method of analysis was UV visible spectroscopy, which is a most convenient and reliable method for the analyst. A method was developed and validated according to the ICH Q2 guidelines along with the amendment in 2018 (ICH Q14). The study denotes that the drug is extremely unstable in the presence of dry heat and then follows the oxidative and basic degradations. The acidic, neutral, and photolysis did not show notable degradations. The chemical kinetic studies were carried out to more clearly understand the mechanism of degradation and to present the order of reaction, rate of reaction, and reaction half-time. The application of a spectrophotometric method in the development of a SIM and study of degradation kinetics is much handier and cost-effective.

Synthesis, characterization and control of eight process-related and two genotoxic fingolimod impurities by a validated RP-UPLC method.

An HPLC method has been described in the European Pharmacopoeia and United States Pharmacopeia for the determination of nine organic impurities (imp A-I) in fingolimod hydrochloride, a synthetic sphingosine-1-phosphate receptor modulator. The manufacturing process of fingolimod hydrochloride consists of multistep chemical synthesis wherein controls of precursors, intermediates and process steps should be performed to assure the final quality of the drug substance. We synthesized and isolated eight process-related impurities (FINI imp A-H) of fingolimod, which were different from the pharmacopoeial impurities. One unknown process-related impurity was found as a key intermediate (FINI) and was identified by LC-MS. Characterization of all of the impurities were done using spectroscopic techniques (1 H and 13 C NMR, FTIR, MS), and the mechanistic pathways to the formation of these impurities were also discussed. Two of these impurities were evaluated as potential genotoxic impurities owing to their alerting structures and alkylating properties (alkyl sulfonates and alkyl halides, class 3, ICH M7). We also developed and validated an RP-UPLC method in line with ICH Q2 guidelines for control these impurities (FINI imp A-H) and to assure the pharmacopoeial quality drug substance.

VALIDATED NOVEL SPECTROSCOPIC METHOD FOR ESTIMATION OF TRIAMCINOLONE ACETONIDE IN NASAL SPRAY

A simple, sensitive, accurate, precise and cost-effective pH independent spectrophotometric method has been developed for the estimation of triamcinolone acetonide in nasal sprays. Estimation was carried out at the isosbestic point of drug solutions, Isosbestic point was measured by scanning equimolar solution of triamcinolone acetonide separately in phosphate buffer pH 3.0, phosphate buffer pH 7.0 and phosphate buffer pH 10.0 at a concentration of 10 µg mL-1. They were scanned in the wavelength range of 200-400 nm. The isosbestic point was found to be 230.0 nm in acidic, basic and neutral condition; hence pH independent wavelength 230 nm was used for estimation of triamcinolone acetonide. The method observed linear response in the concentration range of 3-18 µg mL-1 at 230 nm. Developed method has been validated in accordance with ICH guideline Q2 R1 for several parameters like linearity, precision, accuracy, limit of detection and limit of quantification. The developed and validated analytical method was successfully applied to the estimation of triamcinolone acetonide in marketed nasal spray formulations.

Chromatographic Method for Quantification of Tenofovir in Pharmaceutical Formulations: Comparison with Spectrophotometric Method

Aims: An efficient therapeutic drug or antiviral agent to treat Covid-19 is still not available. But, previously licensed pharmaceuticals to treat other virus infections are usedon an off-label basis either alone or in combination. Tenofovir emerged as a promising treatment for Covid-19 in September 2020. It is an antiretroviral drug that is active against many viruses. This study explains the development and asessment of liquid chromatographic and UV spectrophotometric methods to quantify tenofovir in raw materials and tablets.
 Methods: HPLC analyzes were performed using a C18 column and a mobile phase composed of 20 mM KH2PO4, with a flow rate of 0.8 mLmin-1 and UV detection at 260 nm. For the spectrophotometric analyzes, ultra-pure water was used as a solvent. UV spectrum of standard and sample solution were recorded between 200 and400 nm and Tenofovir was detected using a wavelength of 260 nm. Both methods have been validated according to the procedures described in ICH guidelines Q2(R1) for the validation of analytical methods.
 Results: The results showed that both spectrophotometric and liquid chromatographic methods were linear, precise, accurate, rugged and robust with relative standard deviation R.S.D.% values less than 1%, and the recovery percentage was within standard limits (98-102%). Then a statistical comparison of these two analytical methods was performed, and the results of both methods showed no significant difference. It was found that both methods were not statistically significant with respect to each other in the 95% confidence interval (p<0.05). As a result, the proposed methods were found to be highly effective and could be used for routine analysis of tenofovir in pharmaceutical formulations.

Development and Validation of A Reverse-Phase High Performance Liquid Chromatography Analysis for Paracetamol in Tablets Formulations

A simple yet sensitive high performance liquid chromatography method was developed and validated for the determination of paracetamol (PCM) content in different tablets dosage form. The mobile phase consisted of a mixture of methanol: water (80:20 v/v). The HPLC analysis was performed at a flow rate of 1 mL/min using a Thermo Synchronise C18 (150 × 4.6mm, 5μM) and an UV detection wavelength of 254 nm was used. The method was validated for selectivity, linearity, precision, accuracy, limit of quantification (LOQ), limit of detection (LOD), robustness and solution stability. The calibration curve was linear over a concentration range of 20 to 70 µg/mL (r2 = 0.9997) with LOD and LOQ of 0.01 and 0.03 μg/mL, respectively. The retention time for paracetamol was 1.5 min. The relative standard deviation percent (%RSD) and the relative error percent (%RE) of calibration curve were between 0.03-1.03% and 0.05-1.11%, respectively. The intra- and inter-day precision and accuracy were between 0.02-1.55% and 0.10-0.78%, respectively. In addition, the %RSD of short term stability and the method robustness were between 0.4-1.20% and 0.09-1.43%, respectively. All results followed the ICH guideline Q2 (R1) which indicates the precision and accuracy of the developed method. The method was successfully applied to assess PCM content in different marketed tablets in Iraq.

Determination of progesterone (steroid drug) in the semi-solid dosage form (vaginal gel) using a stability-indicating method by RP-HPLC/PDA detector.

A simple stability-indicating method was developed and validated for the determination of progesterone (a steroid drug) in the semi-solid dosage form. All the impurities were separated from the main compound with a simple stationary phase (Eclipse XDB, C8, 150 × 4.6 mm, 5 μm). The mobile phase A contained phosphate buffer and acetonitrile in the ratio of 90:10,v/v, and mobile phase B contained purified water and acetonitrile in the ratio of 10:90,v/v. The optimized chromatographic conditions were as follows: flow rate, 1.0 mL min-1 ; UV detection, 241 nm; injection volume, 10 μL; and the column temperature, 30°C. The method was validated as per the current ICH Q2 guidelines. The recovery study and linearity ranges were established from 50 to 300% optimal concentrations. The method validation results were found between 98 and 102% for accuracy and r2 = 0.999 for linearity. Forced degradation in hydrolytic, oxidative, thermolytic, and photostability conditions was performed, and the stability indicating nature of the method was proved. Based on the validation and forced degradation results, the current method was found to be specific, precise, accurate, linear, robust, and stability-indicating method. The developed method was cost effective and easy to handle for quality control analysis.

Stability Indicating Photodiode Array Detector Based Estimation of Dapagliflozin Propanediol Monohydrate in API and Tablet Dosage Form by RP-UPLC

Stability indicating RP-UPLC photo diode array detector based method for determination of Dapagliflozin propanediol monohydrate (DPM) in active pharmaceutical ingredient (API) and in tablet dosage form (5mg dapagliflozin) has been developed and validated on Bridge Ethylene Hybride (BEH) C18 column (50mm × 2.1 mm, 1.7µm). Mobile phase composition was water: acetonitrile (60:40 v/v), flow rate 0.5ml/min and detection carried out at 223nm at column temperature 30ºC. Chromatographic separation achieved within 2 min with retention time 0.77 min. Linearity of the method was found over the concentration range of 25-75µg/ml (R2 = 0.9977). The degradation was carried out in five different stress conditions. The developed method was able to resolve peak of API from all generated peaks. Sufficient degradation was achieved in the range of 5.25 to 12.31%. The peak purity is acceptable, Method validation was performed as per ICH guideline Q2(R1).

Design of experiment-driven stability-indicating RP-HPLC method for the determination of tofacitinib in nanoparticles and skin matrix

BackgroundTofacitinib—an oral JAK inhibitor—has been recently approved by US FDA to treat moderate to severe RA. The delivery of tofacitinib to specific inflammation site at joint via topical route using nanoformulations helps in managing the potential adverse effects. The objective is to develop and validate a simple, specific, and sensitive stability-indicating HPLC method for quantification of tofacitinib in topical nanoformulations and different matrices (adhesive tape, and skin layers, i.e., stratum corneum, viable epidermis, and dermis). The major objective was to avoid use of instruments like LC–MS/MS and to ensure a widespread application of the method.ResultA 32 factorial ‘design of experiments’ was applied to optimize process variables, to understand the effect of variables on peak properties. The calibration curve showed regression coefficient (R2) 0.9999 and linearity in the concentration range of 50 to 15,000 ng/mL, which is suitable for the analysis of conventional dosage forms and nanoformulations. Method validation was performed as per ICH guideline Q2 (R1). The accuracy by recovery studies ranged between 98.09 and 100.82%. The % relative standard deviations in intraday and interday precisions were in the range of 1.16–1.72 and 1.22–1.80%, respectively. Forced degradation studies indicated the specificity of method and showed stability-indicating potential for tofacitinib peak.ConclusionThe validated method provides a quantification method of tofacitinib in the presence of formulation excipients, dissolution media, and skin tissues in detail. In addition, the method was successfully utilized for determination of various dermatokinetics profile of tofacitinib.

Quantitative structure retention relationship (QSRR) approach for assessment of chromatographic behavior of antiviral drugs in the development of liquid chromatographic method

A quantitative structure-retention relationship (QSRR) model has been developed and experimentally verified for the prediction of retention behavior of antiviral drugs in the reversed-phased liquid chromatography technique. The multiple linear regression (MLR) based QSRR model was derived using molecular descriptors. The molecular descriptors were calculated using HyperChem, ChemAxon, and ACD/labs software, whilst the QSRR-Automator was used to correlate the variables. The cross/external and experimental validations were done for the QSRR model. The model predicted the retention time (tR ) for five antiviral drugs, viz. darunavir, emtricitabine, efavirenz, lamivudine, and tenofovir disoproxil fumarate (TDF), then verified on C18 column. The model significantly correlated (r 2 > 0.9) the retention time (log10tR) to hydrophobic fragmental constant, Geary autocorrelation of lag 1, 3 D-MoRSE descriptors (Mor27m and MATS1dv), and JGI4 descriptors. The coefficient of determination value between the predicted and experimental retention time was 0.9782, thus the developed QSRR model was trusty enough for retention simulation of antiviral drugs in RP-HPLC method development. In the end, the QSRR based methods for a single component (emtricitabine) and multicomponent (lamivudine and TDF) analyses were validated as per ICH Q2 guidelines. Thus, the QSRR approach in chromatographic method development reduces the obscurity of experimental trials, cost, and time.

Integrated Quality by Design (QbD) Approach for Stability Indicating RPHPLC Method for the Estimation of Tadalafil Hydrochloride in Bulk Drug and Pharmaceutical Formulations

Background: In view of the current FDA standardization and product quality control criteria, Quality by design approach for analytical methods is gaining importance to develop a robust analytical method. A new Quality by Design approach by RP-HPLC was developed and validated for the quantification and purification of Tadalafil hydrochloride and its tablet formulations. Objective: The objective of the study was to develop and validate a simple, robust, and accurate method by QbD approach for detection Tadalafil hydrochloride and its degradation products in bulk drug and tablet formulation. Methods: The chromatographic separation was performed on JASCO Crest Pack RPC18 column (250mm×4.6mm, 5μm) with mobile phase A consisting of a mixture of Acetonitrile: Methanol (40:20 v/v); and mobile phase B: 0.01M Ammonium acetate in water pH adjusted to 3.50± 0.05 with glacial acetic acid with 1.0ml/min flow rate at 285nm. Box-Behnken's three-level, 3-factorial design was employed to create and analyze a "Design Space" (DoE). This design was statistically analyzed by ANOVA, counter-plot, and 3D response surfaces plots, which demonstrated that the model was statistically significant. The developed method was validated as per the ICH guidelines Q2 (R1). Results and Discussion: The tadalafil hydrochloride showed good regression (R2>0.9995) within tested ranges, and the percent recovery was found to be 98% in the marketed formulation. Conclusion: The method was found to be highly specific without the interference of impurities and degradation products of tadalafil hydrochloride. For quantification and routine quality control of tadalafil and its marketed formulation, the stability-indicating the RP-HPLC method could thus be extended.

Development, Validation and Application of an ICP-SFMS Method for the Determination of Metals in Protein Powder Samples, Sourced in Ireland, with Risk Assessment for Irish Consumers.

A method has been developed, optimised and validated to analyse protein powder supplements on an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS), with reference to ICH Guideline Q2 Validation of Analytical Procedures: Text and Methodology. This method was used in the assessment of twenty-one (n = 21) elements (Al, Au, Ba, Be, Bi, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Pb, Pt, Sn, Ti, Tl, V) to evaluate the safety of thirty-six (n = 36) protein powder samples that were commercially available in the Irish marketplace in 2016/2017. Using the determined concentrations of elements in samples (µg·kg−1), a human health risk assessment was carried out to evaluate the potential carcinogenic and other risks to consumers of these products. While the concentrations of potentially toxic elements were found to be at acceptable levels, the results suggest that excessive and prolonged use of some of these products may place consumers at a slightly elevated risk for developing cancer or other negative health impacts throughout their lifetimes. Thus, the excessive use of these products is to be cautioned, and consumers are encouraged to follow manufacturer serving recommendations.

Enhancement of teprenone isomer separation by subcritical fluid chromatography using porous graphitic carbon column

Teprenone is a therapeutic anti-ulcer agent developed in Japan. As described in the Japanese Pharmacopoeia (JP) 17th Edition, gas chromatography/hydrogen flame ionization detection (GC/FID) and high-performance liquid chromatography/ultraviolet detection (HPLC/UV) have been used for the assay of active pharmaceutical ingredients (APIs) and teprenone capsules, respectively. The critical aspect of the assay is a separation of the structural isomers (mono-cis and all-trans) of teprenone. Herein, we propose an improved quantitative method for the quality control of teprenone in APIs and capsules via subcritical fluid chromatography/photo diode array detection (SubFC/PDA) using a porous graphitic carbon column. SubFC conditions, i.e., type and content of the organic modifier in the mobile phase, column temperature, injection volume, and flow rate, were optimized. The developed SubFC/PDA method was validated according to ICH guidelines Q2(R1) in terms of accuracy, precision (repeatability and intermediate precision), specificity, linearity, quantification range, robustness, and stability. Comparison of SubFC/PDA method with the GC/FID or HPLC/UV method (described in JP) revealed that the SubFC/PDA method gave better resolution and run time than the JP methods. The developed SubFC/PDA method is expected to be useful for pharmaceutical analysis or quality control of teprenone isomers.

Development and validation of a reporter gene assay for bioactivity determination of Anti-CGRP monoclonal antibodies

Calcitonin gene-related peptide (CGRP) is critical for the pathophysiology of migraine, and four therapeutic antibodies targeting CGRP and its corresponding receptors have been approved by the Food and Drug Administration (FDA), while many others are in the different stages of clinical trials. Bioactivity determination is essential for the quality control and clinical application of therapeutic monoclonal antibodies (mAbs). However, no bioassay has been reported to date. In this study, we developed a reporter gene assay (RGA) based on SK-N-MC cells stably expressing firefly luciferase driven by cAMP response element (CRE). The key assay parameters were optimized according to signal-to-noise (SNR), the response value, and the fitted dose-response curve. Validation of the RGA in accordance with ICH-Q2 guidelines showed that the method had good specificity, accuracy, linearity, and precision. The established RGA can be utilized as a reference method for release testing and stability studies of relevant antibodies.

Influence of the mobile phase composition on hyphenated ultraviolet and charged aerosol detection for the impurity profiling of vigabatrin

Recently, charged aerosol detection (CAD), a universal detection technique in liquid chromatography, has been introduced into monographs of the European Pharmacopoeia (Ph. Eur.), which now employs HPLC-UV-CAD for assessing the impurities of the drug vigabatrin. The separation of vigabatrin and its impurities is facilitated by ion pair chromatography (IPC) in the compendial method using tridecafluoroheptanoic acid (TDFHA) as ion-pairing reagent. However, the subsequent detection of the impurities by UV-CAD is considerably impaired due to the substantial amount of ion-pairing reagent applied in the method generating high levels of background noise.In this study, the influence of the mobile phase composition on the background noise of the CAD was evaluated applying response surface methodology. The model’s results indicated that the chain length of the ion-pairing reagent is a predominant factor for noise generation. Thus, an alternative method for the impurity analysis of vigabatrin using mixed-mode chromatography (MMC) instead of IPC was developed. The dual separation mechanism of the MMC column enabled the choice of a mobile phase better suited for the individual requirements of the UV-CAD detectors, while maintaining excellent selectivity. The MMC method does not require the addition of a post-column solution to reduce the TDFHA concentration in the mobile phase, and, therefore, needs less instrumentation. Moreover, the sample concentration could be halved due to the improved LOQs of the impurities (<50 ng on column) and the analysis time could be shortened (30 to 20 min) due to improved separation efficiency. The MMC method was validated with respect to ICH guideline Q2(R1).

Fabric phase sorptive extraction combined with high-performance liquid chromatography-photodiode array detection for the determination of tazarotene in gel dosage forms

Tazarotene, a member of the acetylenic class of retinoids, is a third-generation prescription topical retinoid sold as a cream, gel, or foam. In its gel or cream formulations, tazarotene is at a concentration of either 0.1 % or 0.05 %. Fabric phase sorptive extraction membranes are used to selectively isolate and preconcentrate target analytes from different sample matrices. In this study, the tazarotene gel formulation was directly applied to fabric phase sorptive extraction membranes and extracted through a mixture of acetonitrile and water (80:20, v/v) to obtain a clean product free of colloidal suspension of tazarotene gel formulation. The final solutions were injected into an HPLC system equipped with a Zorbax 5 μm Phenyl-Hexyl LC Column (250 × 4.6 mm). Injection volume was 50 μL and UV detection was performed at 326 nm. The flow rate was 1.0 mL min−1 while using an isocratic elution with a mixture of ammonium acetate (50 mM) and methanol (15:85, v/v) as the mobile phase. The method was validated according to ICH guideline Q2 (R1) and successfully applied to gel formulations including 0.01 % tazarotene. This is the first reported application of fabric phase sorptive extraction in the analyses of gel formulations. The capability of fabric phase sorptive extraction membranes to clean up the sample matrix and prepare active pharmaceutical ingredients to be analyzed with acceptable recovery (>98.0 %) and reproducibility may encourage quality control laboratories to use fabric phase sorptive extraction in routine applications.

Novel stability-indicating RP-UPLC method for simultaneous estimation of sitagliptin and ertugliflozin in bulk and pharmaceutical formulations

BackgroundThe present investigation was proposed to develop a simple, sensitive, rapid, accurate, precise stability-indicating RP-UPLC method for simultaneous estimation of sitagliptin and ertugliflozin. Chromatographic separation was performed with Waters Hibar C8 [100×2.1mm, 2μ] column and mobile phase acetonitrile: water (pH 3.5) [50:50%, v/v], pumped at a flow rate 0.2ml/min. The separated analytes were detected with a UV detector at a wavelength of 218nm.ResultsThe separation of sitagliptin and ertugliflozin was done at a retention time of 0.859min and 1.570min, respectively. The present method was validated according to the ICH guidelines Q2 R1, and stability-indicating studies were carried out as per ICH guidelines Q1A R2. Intra-day and inter-day precision were found to be within acceptable limits. The linearity of the proposed method was in the concentration range of 25–125μg/ml and 3.75–22.5μg/ml for sitagliptin and ertugliflozin, respectively. Limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.5μg/ml and 1.53μg/ml for sitagliptin and 0.13μg/ml and 0.38μg/ml for ertugliflozin, respectively. The recovery of the method was found in between 99.7% and 100.7%.ConclusionThe proposed method was able to distinguish the analytes from by-products. Hence, the method was successfully implied for stability-indicating studies and for routine examination of sitagliptin and ertugliflozin in pharmaceutical formulation.

Identification and characterization of new degradation products of belinostat using UHPLC-Q-TOF-MS/MS and in silico toxicity prediction

Belinostat is a histone deacetylase inhibitor used for the management of hematological malignancies and solid tumors. In the current study, stability-indicating ultra-performance liquid chromatography (RP-UPLC) and developed highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) for identification, quantification and characterization of belinostat and its degradation products. The separation was carried out with Acquity BEH C-18 (100 mm × 2.1 mm, 1.7 µm) column using mobile phase comprising of 0.05% ortho-phosphoric acid in water and acetonitrile with gradient elution with 5 µL injection volume at 220 nm detection wavelength. The analysis was carried out at a 0.25 mL/min flow rate along with 45 °C column temperature throughout the analysis. The optimized method was completely validated as per ICH guideline Q2 (R1). Drug substance was treated with different stress conditions for a different time period as indicated by ICH recommended guidelines. Belinostat was sensitive to acidic, basic and oxidative degradation conditions. High-resolution mass spectrometry coupled with QTOF was used to identify and characterize degradation products of belinostat. The most plausible mechanism for all degradation products was established for the first time. TOPKAT (TOxicity Prediction by Komputer Assisted Technology) software was used to evaluate the in silico toxicity of belinostat and its degradation products.

HPLC method development/validation and skin diffusion study of caffeine, methyl paraben and butyl paraben as skin-diffusing model drugs.

The focus of this research was to develop and validate a suitable HPLC method, which allows simultaneous determination of three proposed skin model penetrants to investigate the percutaneous diffusion behavior of their combination: caffeine, methyl paraben and butyl paraben. These penetrants were selected because they represent a wide range of lipophilicities. This model highlights the effect of combining penetrants of different molecular properties on their diffusion behavior through skin. The proposed method employed a gradient system that was systematically optimized for separation and quantification of the penetrants. The effect of the stationary phase (C18, C4 and cyano (CN)) was assessed with CN proven to be superior in terms of peak shape, retentivity and dynamic linear range. Significant differences in retention time, peak broadening, and quantifiability between different stationary phases could be demonstrated. The method was validated as per ICH guidelines Q2 (R1) with a satisfactory outcome. The method was successfully applied for real diffusion experiments, and revealed notable differences between the individual penetrants and their ternary mixture on transdermal permeation. The method could potentially be extended to determine these analytes in other related skin permeation investigations.

Development and Validation of a Robust HPLC Method for Simultaneous Estimation of 5-Fluorouracil and Resveratrol and its Application in the Engineered Nanostructured Lipid Carrier

Background: 5-fluorouracil and resveratrol are the two most effective anticancer drugs. Combination therapy with these two drugs has shown promising results in cancer. The formulation containing 5-fluorouracil and resveratrol has been prepared, but no analytical method is available in the literature for their simultaneous estimation. However, several analytical methods are there for estimation of either 5-fluorouracil or resveratrol alone. Therefore, the present article is designed for the simultaneous estimation of 5-fluorouracil and resveratrol by HPLC and its application in the quantification of the drugs present in the formulated nanostructured lipid carrier (NLC). Methods: The method was developed using a C18 column (Purospher® STAR RP-18 endcapped (5 μm) Hibar® RT 250-4.6) with acetonitrile and water as the mobile phase (25:75 v/v) and estimated at 272 nm. The currently developed method was further validated by the ICH guideline Q2 (R1). Combinatorial NLC of 5-fluorouracil and resveratrol was also prepared and characterized. Results: The LOD and LOQ were 8.22 and 24.91 μg mL-1 and 6.58 and 19.93 μg mL-1, respectively. The precision was under the acceptable limits of &lt;2% RSD. The content of 5-fluorouracil and resveratrol in NLC were found to be 99.452% and 96.421%, respectively. Conclusion: The findings showed that the developed and validated method was simple, fast, costeffective, and reproducible for the simultaneous estimation of both the drugs in the same formulation.