INTRODUCTION:

Nintedanib, a kinase inhibitor from the oxindole class, is used to treat idiopathic pulmonary fibrosis and cancer in the form of an ethylsulfonate salt. It functions as an anti-cancer drug, tyrosine kinase inhibitor, vascular endothelial growth factor receptor antagonist, fibroblast growth factor receptor antagonist, and angiogenesis inhibitor. This compound can be classified as an aromatic ester, methyl ester, oxindole, enamine, aromatic amine, aromatic amide, or N-alkylpiperazine. This is a conjugate base of Nintedanib (1+). Nintedanib is a triple angiokinase inhibitor used to treat idiopathic pulmonary fibrosis, systemic sclerosis-associated interstitial lung disease, and non-small cell lung cancer (in conjunction with Docetaxel). Nintedanib is a small molecule kinase inhibitor used to treat pulmonary fibrosis, systemic sclerosis-related interstitial lung disease, and non-small cell lung cancer (NSCLC). It was initially approved for usage in the United States in 2014. Nintedanib is one of only two disease-modifying therapies available and indicated for idiopathic pulmonary fibrosis (the other being Pirfenidone), and as such, it is used as a first-line treatment after diagnosis to slow the progressive loss of lung function. Nintedanib is a chemotherapeutic drug for NSCLC that is used in combination with Docetaxel for patients who have tried and failed first-line chemotherapy treatments.¹

Mechanisms of action:

Nintedanib is a small-molecule tyrosine kinase inhibitor that blocks several receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases, including VEGFR, FGFR, and PDGFR. By competitively binding to the intracellular ATP-binding site of these kinases, it inhibits their autophosphorylation and downstream signaling pathways. This ultimately reduces the proliferation, migration, and survival of fibroblasts, endothelial cells, and pericytes, and attenuates angiogenesis, which helps slow the progression of fibrotic lung diseases.

Physical and chemical property:

The IUCPAC Name of Nintedanibis Methyl 2-hydroxy-3-[N-[4-[methyl-[2-(4-methyl piperazin-1-Yl) acetyl] amino] phenyl]-C-phenyl carbonimidoyl]-1H-Indole-6-carboxylate. The Chemical Structure of Nintedanib is Shown in following figure-1.

Fig.No. 1- Structure of Nintedanib

Analytical Method Development:

Analytical Chemistry is the application of advanced technology to determine composition through analytical techniques. We can get qualitative as well as quantitative outcomes. Analytical tools are crucial for producing high-quality, trustworthy data. Everyone in the analytical laboratory should be concerned about equipment quality. Analytic methods include spectroscopic, chromatographic, electrochemical, hyphenated, and other techniques. Analytical method development involves choosing an accurate assay procedure to assess the formulation's composition. This technique involves demonstrating that an analytical method is suitable for measuring the concentration of future samples in a laboratory setting. Analytical procedures must follow GMP and GLP protocols and acceptance criteria outlined in the ICH guidelines Q2(R1).²

Validation:

Validation is a notion created in the United States in 1978. Validation has evolved to encompass a variety of tasks, including analytical methods for medication quality control, computerized systems for clinical trials, process control, and labelling. Validation is a crucial aspect of cGMP^.3

Parameters (components) of method validation-

1. Accuracy

2. Precision

3. Linearity

4. Limit of detection

5. Limit of quantitation

6. Specificity

7. Range

8. Robustness

1. Accuracy: Accuracy is defined as the closeness of the test results to the true value.

2. Precision: Precision is defined as the measurement of closeness of agreement for multiple measurements on the same sample. The precision is expressed as the relative standard deviation. %RSD = Standard deviation/Mean ×100

3. Linearity: Linearity is the ability of analytical procedure to obtain a response that is directly proportional to concentration (amount) of analyte in the sample. Linearity is expressed as the confidence limit around the slope of the regression line.

4. Limit Of Detection (LOD): LOD is defined as lowest amount (con centration) of analyte in a sample that can be detected or identified, not quantified. LOD is expressed as a concentration at a specified signal: noise ratio, usually 3:1. LOD = 3.3 × S/ SD

5. Limit Of Quantitation (LOQ): LOQ is defined as lowest amount (concentration) of analyte is a sample that can be quantified. For LOQ, ICH has recommended a signal: noise ratio 10:1. LOQ = 10 × S/SD

6. Specificity: Specificity is defined as the ability of an analytical meth od to measure the analyte clearly in the presence of other components. This definition has following implications:

a. Identification

b. Purity tests

c. Assay

7. Range: The range of the method is the interval between upper level and lower level of analyte that have been determined with acceptable accuracy, precision and linearity. It is determined on either a linear or nonlinear response curve and expressed in the same unit as the test results are expressed.

8. Robustness: Robustness is defined as the measurement of capacity of analytical procedure to remain unaffected by small variations in meth od parameters⁴

Chromatographic Technique:

1. UV Vis Spectroscopy:

UV-visible spectroscopy measures the amount of light absorbed at each wavelength of the visible and UV regions of the electromagnetic spectrum. Absorption spectroscopy divides electromagnetic radiations between 200 nm and 800 nm into two regions: UV (200-400 nm) and visible (400-800 nm). UV-Visible spectroscopy uses the absorption of ultraviolet or visible light by a sample or chemical compound to produce various spectra. When a molecule absorbs UV radiation, its electrons are excited and move from a lower to a higher electronic energy level, resulting in ultraviolet emission spectrum. Common solvents for UV spectroscopy include water, methanol, ethanol, ether, chloroform, carbon tetrachloride, cyclohexane, and dichloroethane. UV spectroscopy applications include detecting functional groups, conjugation, geometric isomers, and contaminants.⁵

2. High Performance Liquid Chromatography (HPLC):

High performance liquid chromatography is a prominent technology for separating complicated mixtures of chemicals and molecules. This approach is highly successful in dealing with chemical compounds and biological components^.[6] This approach was invented in 1980, and with the adoption of HPLC, it will become the first method to evaluate bulk drug materials from the USP-1980.To ensure accuracy, precision, and a diverse range of samples, the HPLC method was used prior to drug analysis. A UV detector was utilized to estimate samples by HPLC and determine their wavelength. The UV detector procedure begins after numerous wavelength scanning programs have been applied.⁷

3. High Performance Thin Layer Chromatography (HPTLC):

This technique is widely used to identify, estimate, and evaluate the analytical profile of pharmacological compounds. This advanced technology will be widely recognized as a significant tool for drug analysis^.[8] This tool's quick separation action and flexibility make it suitable for analyzing several medication components in the pharmaceutical industry. This technique has the advantage of allowing for quick drug analysis and easy handling and cleaning of crude materials. This technique allows us to characterize chromatograms for a wide range of parameters without time constraints.⁹

4. Gas Chromatography:

Gas chromatography is a commonly used analytical technique for pharmaceutical drug analysis. This approach effectively separates volatile and organic components. Gas chromatography separates compounds for quantitative measurement of numerous medication combinations, including compound tracing and parts per trillion. Gas chromatography is essential for analyzing pharmaceutical drugs and identifying contaminants.¹⁰

Reported Method of Nintedanib:

Nintedanib is widely used as a tyrosine kinase inhibitor for the treatment of idiopathic pulmonary fibrosis, systemic sclerosis-associated interstitial lung disease, and non-small cell lung cancer. The development of reliable analytical methods is essential to ensure its quality, safety, and effectiveness in pharmaceutical formulations. In recent years, several studies have reported different analytical techniques for the determination of Nintedanib in pharmaceutical preparations. These analytical approaches mainly include UV–visible spectrophotometry, high performance liquid chromatography (HPLC), and reverse phase high performance liquid chromatography (RP-HPLC). Among these techniques, chromatographic methods, particularly HPLC-based methods, are widely employed due to their high sensitivity, accuracy, and reproducibility. These analytical methods play an important role in the quantitative estimation, quality control, and stability studies of Nintedanib in pharmaceutical analysis.

1. Mitta Chaitanya et al. (2024): developed a simple, selective, and validated isocratic RP-HPLC method for the quantitative estimation of Nintedanib. Separation was achieved on a Symmetry ODS (C18) column (250 × 4.6 mm, 5 µm) using a mobile phase of phosphate buffer (0.02 M, pH 2.8) and acetonitrile (48:52 v/v) at a flow rate of 1.0 mL/min, with UV detection at 248 nm. The method was validated as per ICH guidelines and showed excellent linearity (R² > 0.999), accuracy, precision, and robustness. The method is fast, cost-effective, and suitable for routine quality control and stability studies of Nintedanib.¹¹

Parameters	Description
Column name	Symmetry ODS (C18), 250 × 4.6 mm, 5 µm
Flow rate	1.0 mL/min
Mobile phase	Phosphate buffer (0.02 M, pH 2.8) : Acetonitrile (48:52 v/v)
Detection	248 nm
Linerality	30–70 µg/mL
Retention time	3.649 minutes

2. Sachin Kadam et al. (2024): developed a stability-indicating RP-HPLC method for the assay of Nintedanib in bulk and pharmaceutical dosage forms. The method effectively separated Nintedanib from its degradation products formed under acidic, basic, oxidative, thermal, and photolytic stress conditions. Gradient elution using a mobile phase of acetonitrile (70%) and water (30%) enabled accurate estimation of the drug even in the presence of degradation products. The method was validated as per ICH guidelines and demonstrated excellent linearity, accuracy, precision, robustness, and specificity, making it suitable for routine analysis and stability studies.¹²

Parameters	Description
Technique	RP-HPLC
Mobile phase	Acetonitrile: Water (70:30 v/v)
Detection	UV detector
Linearity range	2–10 µg/mL
Retention time	5.61 minutes
Flow rate	0.8 – 1.2 mL/min

3. Sanjay Dinkar Sawant, et.al (2024): A QbD-driven, stability-indicating RP-HPLC method was developed and validated for the estimation of Nintedanib esylate as per ICH guidelines. Method optimization was performed using a two-level, two-factorial design with Design-Expert software. Chromatographic separation was achieved in isocratic mode on a C18 column with detection at 392 nm. The method showed excellent linearity (R² = 0.9996), good precision, and suitable sensitivity. Forced degradation studies revealed that Nintedanib esylate is susceptible to acidic, oxidative, and photolytic conditions, while it remains stable under alkaline and thermal conditions. The method is suitable for routine analysis of capsule formulations.¹³

Parameters	Description
Column	C18 column
Mobile phase	Acetonitrile: Ammonium formate buffer, Ratio: 70: 30 (v/v)
Flow rate	1.0 ml/min
Detection	392 nm
Retention time	6.14 min
Linerality	10 – 60 µg/ml

4. Varalakshmi Velgacherla, et.al (2024): A rapid, sensitive, and stability-indicating RP-HPLC method was developed and validated for the estimation of Nintedanib (NTB) and its application in the quantification of nanostructured lipid carriers (NLCs). Chromatographic separation was achieved using isocratic elution on a Shimadzu C18 column with a mobile phase of 0.1% v/v triethylamine in water and acetonitrile (35:65 v/v). The method demonstrated excellent linearity (r = 0.999), high accuracy, and good precision. Forced degradation studies confirmed the stability-indicating nature of the method. The validated method is suitable for routine analysis and formulation studies.¹⁴

Parameters	Description
Column	Shimadzu C18 (250 × 4.6 mm, 5 µm)
Mobile phase	0.1% TEA in water: Acetonitrile (35:65 v/v)
Detection	390 nm
Retention time	6.77 ± 0.00 min
Flow rate	1.0 mL/min

5. Pujitha K, et.al (2023): A simple, precise, accurate, and cost-effective UV spectrophotometric method was developed and validated for the estimation of Nintedanib in pharmaceutical dosage forms. Nintedanib showed maximum absorbance at 379.5 nm. The method exhibited excellent linearity in the concentration range of 0.2–1.0 µg/mL with a correlation coefficient of 0.9999. Precision studies showed %RSD < 2, and accuracy was confirmed by high recovery values. The method was validated according to ICH Q2 (R1) guidelines and is suitable for routine quality control analysis.¹⁵

Parameters	Description
Analytical Technique	UV Spectrophotometry
Λmax	379.5 nm
Linearity Range	0.2 – 1.0 µg/mL
Correlation Coefficient (r)	0.9999

6. Yogendra B. Paemae, et.al (2021): A novel, accurate, sensitive, and rapid gradient RP-HPLC method was developed and validated for the determination of related substances (impurities) of Nintedanib esylate. Chromatographic separation was achieved on a YMC Triart C18 column using a gradient mobile phase with UV detection at 245 nm. The method demonstrated excellent sensitivity with LOD < 0.031% w/w, good linearity for all impurities, and acceptable accuracy and precision. The method was validated as per ICH Q2 (R1) guidelines and is suitable for routine impurity profiling of Nintedanib esylate.¹⁶

Parameters	Description
Column	YMC Triart C18 (250 × 4.6 mm, 3 µm)
Mobile phase	Buffer: Acetonitrile (20: 80 v/v)
Flow Rate	1.0 mL/min
Injection Volume	10 µL
Detection Wavelength	245 nm
Linearity Range	0.10 – 2.0 µg/mL
Column Temperature	35°C

7. Rohit Kumar, et.al. (2020): A simple, rapid, and validated RP-HPLC method was developed for the quantitative determination of Nintedanib in pharmaceutical formulations. Chromatographic separation was achieved using an Inertsil Sustain C18 column with an isocratic mobile phase consisting of 0.1% trifluoroacetic acid in water and acetonitrile (60:40 v/v). The method showed excellent linearity over the concentration range of 1–30 µg/mL, good specificity with no interference from excipients, and compliance with ICH guidelines, making it suitable for routine quality control analysis.¹⁷

Parameters	Description
Columns	Inertsil Sustain C18 (250 × 4.6 mm, 5 µm)
Mobile Phase	0.1% TFA in water: Acetonitrile (60:40 v/v)
Flow Rate	1.0 mL/min
Detection Wavelength	265 nm
Retention Time	5.3 min
Coloum temperature	30°C
Linerality	1–30 µg/mL

8. Snehal K. Shukla.et.al (2020): Statistical optimization and validation of a novel ultra-Performance liquid chromatography method for estimation of Nintedanib in rat and human plasma. A design of experiment and multivariate statistical approach was applied to optimize the method, which utilized protein precipitation and isocratic separation on an ACQUITY HSS T3 C18 column with potassium phosphate buffer (pH 7.5) and acetonitrile. The method was validated per US-FDA guidelines, showing linearity from 15–750 ng/mL, <15% precision (RSD), 85–115% accuracy, and >98% extraction recovery across quality control samples. This optimized method is suitable for determining pharmacokinetic parameters in preclinical studies and for the quantification of analytes in human plasma samples.^.18

Parameters	Description
Column	XSelect HSS T3 C18 (100 × 2.1 mm, 1.8 µm)
Mobile phase	25 mM potassium dihydrogen phosphate buffer + 0.1% TEA (pH 7.5) : Acetonitrile
Flow rate	0.55mL/min
Detection wavelength	385nm
Retention time	1.1min
Linerality range	15-750ng/mL

9. Dasari Purnachand, et.al (2015): Development and validation of a simple and sensitive stability Indicating RP-HPLC assay method for determination of Nintedanib And stress degradation studies. A novel stability-indicating RP-HPLC method was developed and validated for the assay of Nintedanib in the presence of its degradation products. Separation was achieved on a YMC Pack ODS-AQ (C18) column (250 × 4.6 mm, 5 µm) using gradient elution with water (pH 3.0, adjusted with orthophosphoric acid) and acetonitrile, at a flow rate of 1.0 mL/min and detection at 210 nm. Forced degradation studies (acid, base, peroxide, thermal, and photolytic) showed multiple degradation products, but none interfered with Nintedanib quantification. The method was validated as per ICH guidelines, demonstrating acceptable specificity, linearity, accuracy, precision, robustness, and ruggedness. An LOQ of 2 µg/mL and strong linearity (r² > 1.00) were obtained over the 25–150 µg/mL range. The method reliably separates Nintedanib from its major degradation impurities and is suitable for routine assay of the drug substance.¹⁹

Parameters	Description
Technique	RP-HPLC
Column	YMC pack C18(250×4.6mm,5um)
Mobile phase	Water: Acetonitrile
Flow rate	1.0mL/min
Detection wavelength	210nm
Run time	12min
Linerality	25-150ug/mL
Accuracy	99.46-100.81%

CONCLUSION:

This review comprehensively summarizes the reported RP-HPLC analytical methods developed for the estimation of Nintedanib in bulk drug and pharmaceutical dosage forms. The discussed methods demonstrate that RP-HPLC is a reliable, precise, and robust technique for routine quality control and stability assessment of Nintedanib. Various chromatographic conditions, including different stationary phases, mobile phase compositions, and detection wavelengths, have been successfully optimized to achieve accurate and reproducible results. Most of the reviewed methods comply with ICH guidelines, showing acceptable linearity, accuracy, precision, robustness, and sensitivity.

Furthermore, the inclusion of stability-indicating RP-HPLC methods highlights the importance of forced degradation studies in understanding the degradation behavior of Nintedanib under different stress conditions. These methods ensure the effective separation of the drug from its degradation products, supporting their application in stability studies and regulatory submissions. Overall, this review serves as a valuable reference for researchers and analysts by providing consolidated information on RP-HPLC method development and validation strategies for Nintedanib, and it may aid in the future development of advanced, cost-effective, and eco-friendly analytical methodologies.

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Received on 07.04.2026 Revised on 11.05.2026

Accepted on 13.06.2026 Published on 10.07.2026

Available online from July 25, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(3):233-238.

DOI: 10.52711/2231-5675.2026.00036

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.