Stability-indicating Ultra-performance Liquid Chromatography Method Research Articles

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.

Quality by design tool-evaluated stability-indicating ultra-performance liquid chromatography method for the determination of drugs (ritonavir and darunavir) used to treat the human immunodeficiency virus/acquired immunodeficiency syndrome.

Ritonavir and darunavir were examined using a ultra-performance liquid chromatography (UPLC) approach in pharmaceutical dosage forms. The small number of analytical studies that are currently available do not demonstrate the method's stability or nature. The study sought to assess both chemicals using a stability-indicating approach with a relatively short run time. The HSS C18 (100 × 2.1 mm), 2-mm column was used for the chromatographic separation, and isocratic elution was used to achieve this. In the mobile phase, methanol and 0.01 M phosphate buffer (pH 4.0) were included in a 60:40 (v/v) ratio. Throughout the analysis, the flow rate was kept at 0.2 mL min-1 , and a photodiode array detector set to 266 nm was used to find the major components. The proposed method showed a linear response (r2 > 0.999), and the accuracy was between 98.0% and 102.0%. The precision data showed relative standard deviation ≤1.0%. The UPLC method for quantification of ritonavir and darunavir in pharmaceutical dosage forms using a very short run time of under a minute is the subject of the proposed article. To meet current regulatory criteria, the quality by design idea was used in the method performance verification.

A Simple Stability-Indicating UPLC Method for the Concurrent Assessment of Paracetamol and Caffeine in Pharmaceutical Formulations

A fixed-dose combination of paracetamol (PCM) and caffeine (CAF) tablets/capsules is the most frequently used over-the-counter medicine for fever and headache. In this paper, a simple, reliable, sensitive, rapid, and stability-indicating ultra-performance liquid chromatography (UPLC) analytical method was proposed for simultaneously assessing PCM and CAF in pharmaceutical formulations. The UPLC method was developed on an Acquity UPLC® CSHTM C18 column, and the column oven temperature was maintained at 35 ± 5 °C with isocratic elution by using a solution of methanol and water (30:70, v/v). The maximum absorbance of PCM and CAF was observed at 272.5 nm. The flow rate was 0.2 mL/min, and the injection volume was 1 µL, with the total run time of 2 min for the separation of PCM and CAF. The proposed UPLC method was validated according to the ICH guidelines, and it demonstrated excellent linearity, with correlation coefficients of 0.9995 and 0.9999 over the concentration ranges of 40–400 and 7–70 ng/mL for PCM and CAF, respectively. The mean retention times of 0.82 ± 0.0 and 1.16 ± 0.02 were observed for PCM and CAF, respectively. The limits of detection and quantification were 16.62 and 3.86 for PCM, respectively, and 50.37 and 11.70 for CAF, respectively. PCM and CAF were subjected to acidic, alkali, oxidative, phytochemical, dry-heat, and wet-heat degradation. The method was found to well separate the analytes’ peaks from degradation peaks, with no alterations in retention times. The proposed method is linear, precise, accurate, specific, and robust, and it can indicate stability and be used for the quantitative assessment of pharmaceutical formulations comprising PCM and CAF within a short period of time.

Development and validation of stability-indicating UPLC method for the determination of gliclazide and its impurities in pharmaceutical dosage forms

BackgroundFor the determination of gliclazide and its three potential impurities quantitatively, the development of a stability-indicating, accurate, simple, and fast, Ultra-Performance Liquid Chromatography (UPLC) method was done.ResultsOn Acquity CSH 18 column (50 mm×2.1 mm, 1.7 μ) separation was achieved by the isocratic elution mode using mobile phase (5 mM ammonium acetate buffer of pH 4 and 10% ammonium acetate buffer + 90% acetonitrile, 65/35 v/v). In total, 0.7 mL−1 was the chosen flow rate and UV detection was carried out at 227 nm.ConclusionBy analyzing forced degradation products of the sample, the stability-indicating characteristic of the developed method was proved where the separation of the products of degradation from analyte peak was seen along with spectral purity of gliclazide. Validation of the developed UPLC method was done as per the guidelines of the International Conference on Harmonization in terms of system suitability, precision, accuracy, specificity, sensitivity, linearity, and robustness.

Oxidative degradation profile studies of tavaborole by a validated stability indicating RP-UPLC method: Isolation and characterization of novel degradant using 2D-NMR and HRMS.

The current research work reports a study on the degradation profile of tavaborole, which is an oxaborole antifungal drug used to treat infections in the toenails. This work also reports the chemical stability of tavaborole in different stress conditions along with the isolation and characterization of degradation products by high-resolution mass spectrometry and two-dimensional nuclear magnetic resonance techniques. A sensitive and reproducible stability-indicating ultra-performance liquid chromatography method was developed and validated for quantification of tavaborole bulk drug in the presence of degradation products. Significant degradation was observed during oxidative stress conditions using H2 O2 . It was observed that the drug was highly unstable under oxidation stress conditions and thus degradation profiles with various oxidizing reagents were studied. One unknown impurity (DP-1) was formed during peroxide degradation, which was isolated by reverse-phase preparative chromatography. The structure of this degradant was characterized by high-resolution mass spectrometry and multidimensional nuclear magnetic resonance techniques. The structure of this novel impurity DP-1 was identified as [4-fluoro-2-(hydroxymethyl)phenol], which was not reported as a degradant in the literature. An Acquity BEH C18 , 100 × 2.1 mm, 1.7 μm column was used to achieve the desired separation within a shorter runtime of 4.0 min. The method was validated for specificity, precision, linearity and accuracy over the concentration range of 5.0-400 μg ml-1 (r2 -0.9999) and limit of quantitation 5.0 μg ml-1 . This method is compatible with LCMS analysis which enables to identify the unknown impurities formed in the process.

A stability-indicating UPLC method for the determination of curcumin diethyl disuccinate, an ester prodrug of curcumin, in raw materials

A stability-indicating reversed-phase ultra-performance liquid chromatographic (UPLC) method for quantitative analysis of curcumin diethyl disuccinate (CDD) in raw materials was developed for applications in product development and quality control. Chromatographic separation was performed using the Waters ACQUITY UPLC® H-Class system consisting of an ACQUITY UPLC® BEH C18 (1.7 μm, 2.1 × 50 mm) column and a photodiode array detector set at a wavelength of 400 nm. The mobile phase consisting of 2%v/v acetic acid in water and acetonitrile was delivered at a flow rate of 0.3 mL/min under gradient elution program. The method was validated according to the ICH Q2(R1) guideline for the validation of analytical procedures. The method was found to be linear over the concentration range of 8–12 μg/mL with the coefficient of determination >0.995. The accuracy of the method established as %recovery ranged from 98.3 – 100.8%. The precision of the method expressed as %CV was found to be <1%. The coelution of degradation products from six stress test conditions was not observed. The method was robust under the variation of chromatographic parameters. The method was successfully applied in the determination of CDD content in raw materials.

Development and Validation of Novel and Highly Sensitive Stability-Indicating Reverse Phase Ultra Performance Liquid Chromatography Method for Quantification of Ibrutinib and its ten Degradation Products

In present study, a novel, simple, robust, stability-indicating, highly sensitive and specific reverse phase ultra-performance liquid chromatographic method was established and validated for the determination of ibrutinib using acquity charged surface hybrid C-18 (100 mm×2.1 mm, 1.7 μm) column with a mobile phase consisting of mixture of Eluent-A phosphate buffer, 0.1 % triethylamine, (pH 6.0) and Eluent-B acetonitrile with flow rate 0.3 ml min-1. The detection was monitored at 215 nm using photodiode array detector. Linearity was observed from 25 to 250 ng ml-1 (correlation of coefficient was 0.9999) with equation, y=100.1880x-130.8966. The observed limit of detection and limit of quantitation were 25 ng ml-1 and 50 ng ml-1 respectively. Ibrutinib was subjected to stress conditions comprising hydrolytic (acid, alkali and neutral), oxidative, photolytic and thermal degradation as per International council for harmonisation Q1 guidelines. The results showed that substantial degradation were observed in acid, base and peroxide degradation condition and resistant to neutral, photolytic and thermal degradation. This is first highly sensitive stability-indicating ultra-performance liquid chromatography method capable of separating ibrutinib and its ten degradation products at nano gram (ng) level. The method was validated as stated by International council for harmonisation guidelines.

A Stability-Indicating UPLC Method for the Determination of Potential Impurities and Its Mass by a New QDa Mass Detector in Daclatasvir Drug Used to Treat Hepatitis C Infection.

A stability-indicating ultraperformance liquid chromatographic method has been developed for the quantitative determination of degradation products and process-related impurities of daclatasvir in a pharmaceutical dosage form. Chromatographic separation was achieved on a polar Waters ACQUITY BEH phenyl 100 × 2.1 mm, 1.7-μm column using the gradient program consisting of mobile phase A: 0.03 M sodium perchlorate with 0.002 M of 1-octanesulfonic acid sodium salt (pH 2.5 buffer) and mobile phase B: 0.03 M sodium perchlorate with 0.02 M of 1-octanesulfonic acid sodium salt (pH 2.5 buffer) with acetonitrile in the ratio of 20:80% v/v. A flow rate of 0.4 mL/min is maintained under ultraviolet detection at 305 nm. The run time was 15 min, within which daclatasvir, its related impurities and unknown degradants were well resolved. The method was found to produce symmetric and sharp peaks with good separation between process-related impurities and degradation impurities. Samples were subjected to hydrolysis (acid and base), oxidative, photolytic and thermal stress conditions to prove the stability-indicating nature of the method. The unknown degradation products were identified by PDA/QDa mass detector. This mass spectrum reveals protonated molecular ion peaks [M + H]+ at m/z DP1-582.4 in acid and base hydrolyses and m/z DP2-778.5 in peroxide hydrolysis. The method was validated in terms of specificity, precision, linearity, accuracy, limit of detection, limit of quantification and robustness as per ICH guidelines.

Development of a stability-indicating UPLC method for determination of isotretinoin in bulk drug

A highly sensitive and rapid stability indicating ultra-performance liquid chromatographic (UPLC) method was developed for the quantification and identification of isotretinoin in bulk. Chromatographic separation was developed using a gradient elution in a reversed-phase system at flow rate of 0.5 ml/min with 12 min run time. The mobile phase was a gradient mixture of mobile phase A (contained a 30:70:0.5 mixture solution of methanol/purified water/glacial acetic acid) and mobile phase B (contained a 70:25:4.5:0.5 mixture solution of methanol/acetonitrile/purified water/glacial acetic acid). Eluents were monitored at 355 nm. The analytical method was validated for accuracy, precision, robustness, linearity, and forced degradation in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) topic Q2 (R1) ‘Validation of Analytical Procedures: Text and Methodology’. The method was linear over a concentration range of (1–7 µg/ml) with correlation coefficient of (r2 > 0.9999). The accuracy was confirmed by calculating the % recovery which was found to be 100.0–101.6%. The RSD values obtained for repeatability and intermediate precision experiments were less than 2%. The limit of detection (LOD) was 0.12 µg/ml, while the limit of quantification (LOQ) was 0.38 µg/ml. The drug samples were exposed to different stressed conditions and the results showed that all degradation products were satisfactorily separated from each other and from the peak of the drug using the developed method. The proposed method can be used for the quantitative determination of isotretinoin with confidence.

Quality-by-design-based development and validation of a stability-indicating UPLC method for quantification of teriflunomide in the presence of degradation products and its application to in-vitro dissolution

ABSTRACTA systematic design-of-experiments was performed by applying quality-by-design concepts to determine design space for rapid quantification of teriflunomide by the ultraperformance liquid chromatography (UPLC) method in the presence of degradation products. Response surface and central composite quadratic were used for statistical evaluation of experimental data using a Design-Expert software. The response variables such as resolution, retention time, and peak tailing were analyzed statistically for the screening of suitable chromatographic conditions. During this process, various plots such as perturbation, contour, 3D, and design space were studied. The method was developed through UPLC BEH C18 2.1 × 100 mm, 1.7-µ column, mobile phase comprised of buffer (5 mM K2HPO4 containing 0.1% triethylamine, pH 6.8), and acetonitrile (40:60 v/v), the flow rate of 0.5 mL min−1 and UV detection at 250 nm. The method was developed with a short run time of 1 min. Forced degradation studies revealed that the method was stability-indicating, suitable for both assay and in-vitro dissolution of a drug product. The method was found to be linear in the range of 28–84 µg mL−1, 2.8–22.7 µg mL−1 with a correlation coefficient of 0.9999 and 1.000 for assay and dissolution, respectively. The recovery values were found in the range of 100.1–101.7%. The method was validated according to ICH guidelines.

Development and validation of RP-UPLC method for simultaneous estimation of Cobicistat and Darunavir

A new, simple, precise and stability-indicating UPLC (Ultra Performance Liquid Chromatography) method was developed and validated for the simultaneous determination of anti-viral drugs Cobicistat and Darunavir. The method was developed using Acquity UPLC HSS C18 x 1.8 μ.with isocratic elution. 0.1% orthophosphoric acid buffer and acetonitrile (35: 65 v/v) were used as mobile phase with 0.4 ml/min flow rate at 30°C temperature. The detection wavelength was fixed at 250 nm, the run time was within 2 min. The Retention time of Cobicistat and Darunavir were 1.06 and 0.79 min respectively. The method was validated in terms of linearity, precision, accuracy and robustness. Calibration plots were linear over the range of 7.5–45 μg/ml for Cobicistat and 40–240 μg/ml for Darunavir. Recovery was in the range of 98–102% with the relative standard deviation of less than 2% for both drugs. The limit of detection and the limit of quantification for the Cobicistat were found to be 0.161 and 0.488 μg/ml, respectively, and for Darunavir 0.55 and 1.67 μg/ml respectively. The specificity of the method was analyzed by the stress degradation study in acidic, alkaline, oxidative, thermal, UV and hydrolytic conditions. The suggested method was suitable for determination of Cobicistat in bulk and pharmaceutical dosage forms.

Stability-indicating UPLC-MS/UV Method for Simultaneous Determination of Sildenafil Citrate and Dapoxetine Hydrochloride from Bulk and Formulation

The present work was focused on development of rapid, specific and novel stability-indicating ultra-performance liquid chromatographic method for simultaneous determination of sildenafil citrate and dapoxetine hydrochloride in bulk and formulation. The drugs were subjected to hydrolysis (acidic, alkaline and neutral), photolytic and thermal stress, as per International Conference on Harmonisation guidelines Q1A (R2). The identification of major stressed degradation products was performed using qudrupole electrospray ionization mass spectroscopy. Separation of drugs from their degradation products was achieved on acquity ultra-performance liquid chromatographic I class equipped with photodiode array detector. Acquity BEH Shield RP18 column was used with the gradient elution and detection wavelength of 294 nm. The method was found to be linear in the concentration range of 2.5-100 µg/ml for both sildenafil and dapoxetine hydrochloride. Limit of detection and quantitation was found to be 0.15-0.45 μg/ml and 0.1-0.3 μg/ml for sildenafil and dapoxetine, respectively. Moreover, the fragmentation pattern of both drugs was studied using ultra-high pressure liquid chromatography-tandem mass spectrometry. Sildenafil was found to be unstable under acidic condition while dapoxetine was found to be unstable under alkaline conditions.

Development and validation of a stability-indicating ultra-performance liquid chromatographic method for the determination of rifampicin in bulk drug and capsules

Development and validation of a stability-indicating ultra-performance liquid chromatographic method for the determination of rifampicin in bulk drug and capsules

Development and Validation of a Simple, Sensitive, Selective and Stability-Indicating RP-UPLC Method for the Quantitative Determination of Ritonavir and Its Related Compounds

A simple, sensitive, selective and reproducible stability-indicating ultra-performance liquid chromatographic method was developed for the quantitative determination of degradation products and process-related impurities of Ritonavir in a pharmaceutical dosage form. Chromatographic separation was achieved on a polar embedded Waters Acquity BEH Shield RP18 (100 × 2.1 mm, 1.7 μm) column thermostated at 50°C under gradient elution by using a binary mixture of potassium dihydrogen phosphate (0.01 M, pH 3.5) and acetonitrile at a flow rate of 0.5 mL/min. Chromatogram was monitored at 240 nm using a photodiode array detector. The drug and its related impurities are eluted within 20 min. To prove the stability-indicating power of the method, the drug was subjected to hydrolytic (acid, alkaline and water), oxidative, photolytic and thermal stress conditions. The unknown degradants were identified by the LC-MS-MS method, which revealed protonated molecular ion peaks [M + H](+) at m/z 551.40 for hydrolytic degradants, and m/z 737.60 and m/z 753.40 for photolytic degradants. A plausible mechanism for the formation of degradation and process impurities was proposed. The performance of the method was validated according to the International Conference on Harmonization guidelines.

A validated stability-indicating UPLC method for determination of diclofenac sodium in its pure form and matrix formulations

The aim of this work is to develop a validated stability indicating reverse phase ultra performance liquid chromatography (UPLC) method for the rapid and accurate determination of diclofenac sodium in its pure form and in matrix formulations. This UPLC method is composed of isocratic mobile phase, 0.05M ammonium acetate buffer (pH=2.5) and acetonitrile (50:50), with flow rate 0.5ml/min, column BEH C18 (2.1×50mm, 1.7μm). The method is rapid (1.2min run), selective with well resoluted diclofenac peak with retention time 0.94min and sensitive (LOD=2ppm and LLOQ=6ppm) with UV detection at 254nm. The drug was subjected to acidic, alkaline media, boiling and oxidizing agent to apply stress conditions. The developed method was able to separate degradation product generated under forced degradation studies. The developed method was validated as per the FDA guidelines for specificity, linearity, accuracy, precision, LOD, LLOQ and found to be satisfactory. The study suggests that the developed UPLC method can be used for the assessment of drug purity and stability. It can be also used to monitor the drug content and release from different formulations without any interference of excipients and/or degradation products.

Development and validation of stability-indicating UPLC method for the determination of lafutidine and its impurities in bulk and pharmaceutical dosage form

Abstract Background A simple and rapid stability-indicating, reversed phase ultra-performance liquid chromatography (UPLC) method was developed for the quantitative determination of lafutidine and its four potential impurities. Results Separation was achieved on Acquity BEH-shield RP18 UPLC column (3.0 mm × 100 mm, 1.7 μm) under the gradient mode of elution by using mobile phase A (0.02M diammonium hydrogen phosphate/acetonitrile, 80:20 v/v) and mobile phase B (0.02M diammonium hydrogen phosphate/acetonitrile, 30:70 v/v). The flow rate was maintained at 0.5 mL min−1. UV detection was carried out at 276 nm. Conclusions Stability-indicating capability of the developed method is established by analyzing forced degradation samples in which the spectral purity of lafutidine is ascertained along with the separation of degradation products from analyte peak. The developed UPLC method is validated as per International Conference on Harmonization guidelines with respect to system suitability, specificity, precision, sensitivity, accuracy, linearity, and robustness.

UPLC Method for Simultaneous Determination of Valsartan &amp; Hydrochlorothiazide in Drug Products

A new, simple, precise and stability-indicating UPLC (Ultra Performance Liquid Chromatography) method was developed and validated for the simultaneous determination of anti-hypertensive drug Valsartan (VAL) and Hydrochlorothiazide (HCTZ) in combined dosage forms. The method was developed using Kromasil eternity C-18 column (50 mm×2.1 mm, 3.5 μm) with isocratic elution. Triethylamine buffer (0.1% v/v) and methanol (75:25 v/v) were used as mobile phase with 0.6 mL min-1 flow rate at room temperature. The detection wavelength was fixed at 225 nm; the run time was within 2 min. The method was validated in terms of linearity, accuracy and reproducibility. Calibration plots were linear over the range of 7-13 μg/ml for HCTZ and 56-104 μg/ml for VAL. Recovery was in the range of 98-102% with the relative standard deviation of less than 2% for both drugs. The limit of detection and the limit of quantification for the valsartan were found to be 0.8 and 2.4 μg/ml respectively and for hydrochlorothiazide 0.12 and 0.36 μg/ml respectively. The proposed method was also suitable for determination of VAL & HCTZ in bulk and pharmaceutical dosage forms.

Stability-Indicating UPLC Method for Tramadol HCl Impurities in Tramadol Injection After Dilution by Infusion Fluids (5% Dextrose and 0.9% Sodium Chloride)

A novel, rapid, and sensitive ultra-performance liquid chromatography (UPLC) method has been developed and validated as per ICH guidelines for the determination of tramadol HCl impurities in the tramadol HCl injection after reconstitution by infusion fluids (5% dextrose and 0.9% sodium chloride). The tramadol HCl injection is for the treatment of patients with moderate-to-severe pain. The stability of the reconstituted solution is critical before intravenous injection. The literature search resulted in few published articles on assays of tramadol in infusion fluids by conventional HPLC. No attempts have yet been made to determine the impurities in infusion fluids, as the concentration of tramadol after reconstitution is extremely low (0.4 mg/mL) and that of impurities is even lower. The proposed method is novel as it allows the quantitation of the impurities of tramadol HCl and is based on modern chromatographic techniques like UPLC. The method was developed using the Waters Acquity BEH C18 column with a mobile phase consisting of a gradient mixture of solvent A (trifluroacetic acid buffer) and solvent B (methanol: acetonitrile). The model stability study was designed by diluting the tramadol HCl injection in the 5% dextrose injection and 0.9% sodium chloride injection. Each mixture was kept under storage at room temperature (25 ± 2°C) for testing at initial, 2, 4, 8, 12, 18 & 24 hours. The validation study illustrates that the proposed method is suitable for the determination of tramadol and its impurities. The proposed method makes use of the LC-MS-compatible mobile phase. It can be useful for the determination of tramadol HCl and its impurities in plasma samples and other pharmaceutical dosage forms.

Development and Validation of Stability-Indicating Assay Method by UPLC for a Fixed Dose Combination of Atorvastatin and Ezetimibe

A stability-indicating ultra-performance liquid chromatography method was developed and validated for the simultaneous determination of a fixed dose combination of atorvastatin and ezetimibe in bulk drugs. The developed method was successfully applied to the simultaneous quantitative analysis of the combination drugs in tablet. The chromatographic separation was performed on a Kromasil Eternity C18 UHPLC column (2.5 µm, 2.1 × 50 mm) using a gradient elution of acetonitrile and ammonium acetate buffer (pH 6.70; 0.01M) as the mobile phase at a flow rate of 0.2 mL/min with column oven temperature of 40°C. Ultraviolet detection was performed at 245 nm. Total run time was 5 min, within which the primary compounds and their degradation products were separated. The method was validated for accuracy, repeatability, reproducibility and robustness. Linearity, limit of detection and limit of quantitation were established for atorvastatin and ezetimibe.

UPLC and LC-MS Studies on Degradation Behavior of Irinotecan Hydrochloride and Development of a Validated Stability-Indicating Ultra-Performance Liquid Chromatographic Method for Determination of Irinotecan Hydrochloride and its Impurities in Pharmaceutical Dosage Forms

The objective of the current investigation was to study the degradation behavior of irinotecan hydrochloride under different International Conference on Harmonization (ICH) recommended stress conditions using ultra-performance liquid chromatography and liquid chromatography-mass spectrometry and to establish a validated stability-indicating reverse-phase ultra-performance liquid chromatographic method for the quantitative determination of irinotecan hydrochloride and its seven impurities and degradation products in pharmaceutical dosage forms. Irinotecan hydrochloride was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Irinotecan hydrochloride was found to degrade significantly in oxidative and base hydrolysis and photolytic degradation conditions. The degradation products were well resolved from the main peak and its impurities, thus proving the stability-indicating power of the method. Chromatographic separation was achieved on a Waters Acquity BEH C8 (100 × 2.1 mm) 1.7-µm column with a mobile phase containing a gradient mixture of solvent A (0.02M KH(2)PO(4) buffer, pH 3.4) and solvent B (a mixture of acetonitrile and methanol in the ratio of 62:38 v/v). The mobile phase was delivered at a flow rate of 0.3 mL/min with ultraviolet detection at 220 nm. The run time was 8 min, within which irinotecan and its seven impurities and degradation products were satisfactorily separated. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also suitable for the assay determination of irinotecan hydrochloride in pharmaceutical dosage forms.