Stability-indicating HPLC Assay Method Research Articles

Develop and Validate a Stability-Indicating HPLC Assay Method for Paracetamol, and parabens in Pharmaceutical Dosages

The drug's stability, which is a prerequisite for good drug quality, cannot be simply established unless a reliable and trustworthy method for demonstrating stability is applied. Creating such a method is a challenging task. In this work, we developed and validated a reversed-phase stability indicating liquid chromatographic technique with photo diode-array detection to analyze paracetamol (PCM), methylparaben (MP), propylparaben (PP), and their degradants in commercial oral suspension. The compounds and their degradants were separated using a Phenomenex C8 column (250 mm x 4.6 mm, 5 µm) and a mobile phase of pH 4 consisting acetonitrile, potassium di-hydrogen ortho-phosphate, tri-ethylamine, and glacial acetic acid in a 40:58:1.5:0.5 ratio, at 1.2 mL/min. All chemicals separated in within 12 min. The developed method followed the guidelines of the International Conference on Harmonization (ICH) and fulfilled all its requirements in terms of linearity, recovery, reproducibility, detection, and quantitation limits. The dependability of the developed method was shown by recoveries of 98.88% to 101.80% in 102 samples from 34 batches, indicating that it can be used in quality control step for paracetamol, methylparaben, and propylparaben in oral suspension products.

Stability-indicating HPLC assay method of zomepirac

An HPLC assay method for determining the degradation of zomepirac was developed and validated under acidic, basic, and photo-irradiated conditions. The HPLC system consisted of an Inertsil 5 ODS-3V column (4.6 × 250 mm i.d.), and a guard column of Inertsil 7 ODS-3V (4.6 × 50 mm i.d.) using a mobile phase of CH3CN: CH3OH: 1%HOAc (2:64:34, v/v/v) with UV detection at 254 nm. The developed method satisfies the system suitability criteria, peak integrity, and resolution for the parent drug and its degradants. The results indicate that the established assay method shows good selectivity and specificity suitable for stability measurements of zomepirac. From the intra- and interday tests of 6 replicates, the coefficients of variations (CVs) were between 0.12% and 2.24% for the former and 0.15% and 3.93% for the latter. Recoveries were found to be between 97.14% and 101.58%. From the stress treatments, zomepirac was determined to be more sensitive to the light and acidic conditions, but it was stable in basic medium. A preliminary kinetic study of the photodegradation of zomepirac in methanol showed that it followed an apparent first-order reaction.

High-Performance Liquid Chromatography and Liquid Chromatography/Mass Spectrometry Studies on Stress Degradation Behavior of Sulfapyridine and Development of a Validated, Specific, Stability-Indicating HPLC Assay Method.

The objective of the current investigation was to develop a simple, rapid, and stability-indicating high-performance liquid chromatography method and to study the degradation behavior of sulfapyridine (SP) under different International Conference on Harmonization (ICH)-recommended conditions. The chromatographic method was developed using C18 (250 × 4.6 mm, 5 μ) column, and mobile phase consisting of acetonitrile-0.1% formic acid (30:70 v/v) at ambient temperature, at a flow rate of 1 mL/min. The elution was monitored at 265 nm using a photodiode array detector. The developed method was subsequently validated as per ICH Q2 (R1) guidelines. The retention time of SP was observed as 4.56 min with the linearity range between 2 to 10 μg/mL. Limit of detection and limit of quantitation for SP were 0.115 and 0.35 μg/mL, respectively. Forced degradation studies were carried out on bulk samples of SP using prescribed acidic, basic, oxidative, thermal, and photolytic conditions. Extent of degradation in 0.1 M hydrochloric acid and under photolytic conditions was found to be 21.56% and 28.57%, respectively. The degradation products formed in stress conditions were identified by liquid chromatography-mass spectrometry (LC-MS). The utility of the method was verified by quantification of SP in different laboratory-made pharmaceutical preparations. The proposed method could be successfully used to quantify SP in different pharmaceutical dosage forms.

Development and validation of a stability-indicating HPLC assay method for determination of ethacrynic acid in solution formulation. HPLC-MS identification of hydrolytic and oxidative degradation products

An isocratic stability-indicating HPLC assay method was developed to quantitate ethacrynic acid in solution formulations. Utilizing the method, the main hydrolytic, and one of the oxidative degradation products could be separated. The structure of the separated degradation products was confirmed by negative ion-mode HPLC-MS. The hydrolytic results confirmed the previously reported sequence of the formation of the dimeric Michael adduct. One new oxidative metabolite has been identified. The validated analytical method can be used to quantitate ethacrynic acid in a solution formulation in the 0.5-500 µg/ml concentration range.

A Validated Stability-Indicating HPLC Assay Method for Flucytosine in Bulk Drug

A Validated Stability-Indicating HPLC Assay Method for Flucytosine in Bulk Drug

STABILITY INDICATING RP-HPLC ASSAY METHOD FOR ESTIMATION OF MIDODRINE HYDROCHLORIDE IN BULK AND TABLETS

<p><strong>Objective: </strong>To develop an accurate, simple, sensitive and precise stability indicating reverse phase-high performance liquid chromatographic (RP-HPLC) assay method for estimation of Midodrine hydrochloride (MD) in bulk and tablets.</p><p><strong>Methods: </strong>The chromatographic separation was performed on enable C<sub>18</sub>, (250 mm X 4.6 mm, 5 μm) column. The mobile phase consists of triethylamine buffer 0.02%, pH-3: acetonitrile (38:62 v/v) was delivered at a flow rate of 0.6 ml/min and UV detection at 289 nm. The method was validated with forced degradation studies as per ICH guidelines.</p><p><strong>Results: </strong>The retention time of the drug was found to be 3.56 min. The developed method was found to be linear in a concentration range of 19.98-99.9μg/ml of the drug (r<sup>2</sup>= 0.9998). The low value of % RSD indicates reproducibility of the method. The low value of LOD and LOQ suggests the sensitivity of the method. The results of forced degradation studies indicated that the drug was stable in acidic condition and degraded in basic, oxidative and hydrolytic conditions.</p><strong>Conclusion: </strong>The present study represents first stability-indicating HPLC assay method that deals with the estimation of midodrine hydrochloride. It can be concluded from the results that the developed method is simple, rapid, accurate, specific, sensitive and precise. Thus, this method can be used for routine analysis of midodrine hydrochloride formulation and to check the stability of bulk samples.<p> </p>

Study of Doxofylline and its Degradation Products in Bulk Drug by a Newly Developed Stability-Indicating HPLC Method

The purpose of this work was to study the stability behavior of doxofylline under different stress conditions and to develop a sensitive stability-indicating HPLC assay method. The stress conditions applied included heat, moisture, acid-base hydrolysis, oxidation, and UV light. The drug was particularly labile under oxidative and thermal stress conditions, with 58.40 and 53.90% degradation, respectively. Good resolution of drug from degradation products formed under stress conditions was achieved on a C18 column using 10 mM KH2PO4 buffer solution (pH 6) methanol (40 + 60, v/v) as the mobile phase (pH 7.2). The flow rate was 1 mLlmin, and the detection wavelength was 273 nm. The method was validated for linearity, range, precision, accuracy, LOD, and LOQ. The RSD was found to be <2%. Since the method effectively separates the drug from its degradation products, it can be used as a stability-indicating method.

Development and Validation of a Stability-Indicating HPLC Assay Method for Simultaneous Determination of Spironolactone and Furosemide in Tablet Formulation

The objective of the current study was to develop and validate a simple, precise and accurate isocratic stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) assay method for the determination of spironolactone and furosemide in solid pharmaceutical dosage forms. Isocratic RP-HPLC separation was achieved on an SGE 150 × 4.6 mm SS Wakosil II 5C8RS 5-μm column using a mobile phase of acetonitrile-ammonium acetate buffer (50:50, v/v) at a flow rate of 1.0 mL/min. The detection was carried out at 254 nm using a photodiode array detector. The drug was subject to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The method was validated for specificity, linearity, precision, accuracy, robustness and solution stability. The method was found to be linear in the drug concentration range of 40-160 µg/mL with correlation coefficients of 0.9977 and 0.9953 for spironolactone and furosemide, respectively. The precision (relative standard deviation; RSD) among a six-sample preparation was 0.87% and 1.1% for spironolactone and furosemide, respectively. Repeatability and intermediate precision (RSD) among a six-sample preparation were 0.46% and 0.20% for spironolactone and furosemide, respectively. The accuracy (recovery) was between 98.05 and 100.17% and 99.07 and 100.58% for spironolactone and furosemide, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of spironolactone and furosemide; therefore, the assay can be considered to be stability-indicating.

A Chromatographic Determination of Aripiprazole using HPLC and UPLC: A Comparative Validation Study.

A simple, precise, and accurate isocratic reversed-phase (RP) stability-indicating HPLC assay method was developed and validated for determination of Aripiprazole in bulk and solid pharmaceutical dosage form. A reversed-phase C8 (250×4.0 mm, 5 μm particle size) column for HPLC and C8 (50×2.1mm, 1.7 μm particle size) for UPLC method in isocratic mode was used. The mobile phase consists of acetonitrile: 20 mM ammonium acetate (90:10, v/v), flow rate was set at 1.0 ml/min and 0.250 ml/min for HPLC and UPLC, respectively and the detection was performed for both methods were at 240 nm. Further the validation of both developed method was performed and subsequently compared to prove its better applicability.

Initial characterization of D-cycloserine for future formulation development for anxiety disorders

The purpose of this study is to characterize D-cycloserine (DCS) physicochemical properties to facilitate future formulation development of DCS for anxiety disorders. A stability-indicating HPLC assay method for the quantitation of DCS was developed and calibrated to be used for this study. The partition coefficient was determined and compared with the predicted value. The solution stability of DCS was studied under various pH (2.0-11.5) and ionic strengths of 10 and 20 mM at physiological temperature of 37°C. The 250 mg capsule was compounded to the nominal strength of 50 mg used for anxiety disorders. These capsules were then put under stability. The in vitro dissolution was also carried out at 37°C as per the United States Pharmacopeia (USP) guidelines. The partition coefficient value (Kp) determined for the DCS was log Kp = -2.89 ± 0.06 (n = 6). The pH-solution stability profile shows that DCS has maximum stability under alkaline conditions. The maximum rate of degradation was seen at pH of 4.7. The mean percent recovery of DCS from the capsules compounded to strength of 50 mg was 100.3 ± 1.4. The stability study of the reformulated capsules concluded that reformulated DCS is stable for at least one year at room temperature. The in vitro dissolution illustrates that all the DCS is released from the capsules in 10 min. The present characterization of DCS study will serve as guidance for the future directions regarding the reformulation of DCS in order to be used in anxiety disorders.

Stress Degradation Studies and Kinetic Determinations of Duloxetine Enteric-Coated Pellets by HPLC

A stability-indicating HPLC assay method was developed for the quantitative determination of duloxetine (DLX) in a pharmaceutical dosage form in the presence of its degradation products, and kinetic determinations were evaluated in acid conditions and UV-C radiation exposure. Chromatographic separation was achieved by use of an ACE C18 column (250 x 4.0 mm id, 5 microm particle size). The mobile phase was prepared by mixing aqueous 50 mM potassium phosphate buffer (pH 6.0 containing 0.3% triethylamine) and acetonitrile (60 + 40, v/v). DLX was rapidly degraded in an acid medium and in the presence of hydrogen peroxide and UV-C radiation; it was more stable in alkaline medium. The described method was linear over a range of 4.0-14.0 microg/mL for determination of DLX (r = 0.9998). The precision was demonstrated by the RSD of intraday (0.79-1.07%) and interday (0.85%) studies. The mean recovery was found to be 100.56%. The acid degradation of DLX in 0.1 M HCI solution showed an apparent zero-order kinetics (k = 0.177 microg/mL/min), and the photodegradation demonstrated an apparent first-order kinetics (k = 0.082 microg/mL/min). The developed method was found to be simple, specific, robust, linear, precise, and accurate for the determination of DLX in enteric-coated pellets.

Development and Validation of a Specific Stability Indicating High Performance Liquid Chromatographic Method for Valsartan

A stability-indicating HPLC assay method has been developed and validated for valsartan in bulk drug and pharmaceutical dosage forms. An isocratic RP-HPLC was achieved on Waters 2695 using Symmetry C18 (250mm × 4.6mm × 5μ) column with the mobile phase consisting of 0.02 mM sodium dihydrogen ortho-phosphate, pH adjusted to 2.5 using ortho-phosphoric acid (solvent A), and acetonitrile (solvent B) in the ratio of 58:42 %v/v. The stress testing of valsartan was carried out under acidic, alkaline, oxidative, thermal, and photolytic conditions. Valsartan was well resolved from its degradation products. The proposed method was validated as per ICH guidelines. The method was found to be suitable for the quality control of valsartan in bulk and pharmaceutical dosage forms as well as the stability-indicating studies.

Stability-Indicating Reverse Phase HPLC Method for the Determination of Cefazolin

Purpose: The aim of the present study was to establish the inherent stability of cefazolin through stress studies under a variety of ICH recommended test conditions and, also to develop a stability indicatingassay. Methods: A stability-indicating HPLC assay method was developed and validated for cefazolin using an isocratic RP-HPLC method which employed an SS Wakosil II- C18 column (250 mm × 4.6 mm i.d., 5 μm) with a mobile phase consisting of phosphate buffer (pH 6.8) and methanol (5:2 v/v), and UV detection at 254 nm at a flow rate of 1 ml/min. The stress testing of cefazolin was carried out under acidic, alkaline, neutral, oxidation and thermal conditions. Results: The drug peak was well resolved from the peaks of the degradation products. The proposed method was validated for sensitivity, selectivity, linearity, accuracy, precision and solution stability. From the degradation studies it was found that the drug was thermally stable but unstable in acidic, alkaline, neutral and oxidative conditions. The response of drug was linear in the concentration Range of 1 - 50 μg/ml with the number of theoretical plates, and tailing factor being 1341 and 1, respectively. Limit of detection and limit of quantification were 0.1 and 0.2, μg/ml respectively while recovery ranged from 95 - 100%. Method precision and precision of the system were within the limits of acceptance criteria. Conclusion: This study presents a simple and validated stability-indicating HPLC method for the estimation of cefazolin in the presence of degradation products. The developed method is specific, accurate, precise and robust. All the degradation products formed during forced degradation studies were well separated from the analyte peak.Keywords: Cefazolin; Stability-indicating assay; Reversed-phase HPLC

A Validated Stability-indicating Reverse Phase HPLC Assay Method for the Determination of Memantine Hydrochloride Drug Substance with UV-Detection Using Precolumn Derivatization Technique

This present paper deals with the development and validation of a stability indicating high performance liquid chromatographic method for the quantitative determination of Memantine hydrochloride. Memantine hydrochloride was derivatized with 0.015 M 9-fluorenylmethyl chloroformate (FMOC) and 0.5 M borate buffer solution by keeping it at room temperature for about 20 minutes and the chromatographic separation achieved by injecting 10 muL of the derivatized mixture into a Waters HPLC system with photodiode array detector using a kromasil C18 column (150 x 4.6 mm), 5 mu. The mobile phase consisting of 80% acetonitrile and 20% phosphate buffer solution and a flow rate of 2 milliliter/minute. The Memantine was eluted at approximately 7.5 minutes. The volume of FMOC used in derivatization, concentration of FMOC and derivatization time was optimized and used. Forced degradation studies were performed on bulk sample of Memantine hydrochloride using acid (5.0 Normal (N) hydrochloric acid), base (1.0 N sodium hydroxide), oxidation (30% hydrogen peroxide), thermal (105 degrees C), photolytic and humidity conditions. The developed LC method was validated with respect to specificity, precision (% RSD about 0.70%), linearity (linearity of range about 70-130 mug/mL), ruggedness (Overall % RSD about 0.35%), stability in analytical solution (Cumulative % RSD about 0.11% after 1450 min.) and robustness.

Stress Degradation Studies on Dutasteride and Development of a Stability-Indicating HPLC Assay Method for Bulk Drug and Pharmaceutical Dosage Form

A simple stability-indicating LC method has been developed for the quantitative determination of dutasteride in bulk drug samples and in pharmaceutical dosage forms in the presence of degradation products. The retention time of dutasteride is about 7 min. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Degradation was found to occur under hydrolysis and to a lesser extent under oxidation conditions but the compound was stable to photolytic and thermal stress. The assay of stress samples was calculated against a reference standard and the mass balance was found close to 99.3%. The developed method was validated with respect to linearity, accuracy, precision and ruggedness.

Stress Degradation Studies on Tadalafil and Development of a Validated Stability-Indicating LC Assay for Bulk Drug and Pharmaceutical Dosage Form

A stability-indicating HPLC assay method was developed for the quantitative determination of tadalafil in bulk samples and in pharmaceutical dosage forms in the presence of the degradation products. It involved a 250 mm × 4.6 mm, 5 μm C-18 column. The gradient LC method employs solution A and B as mobile phase. Solution A contains a mixture of buffer (phosphate buffer and tetra-n-butyl ammonium hydrogen sulfate) pH 2.5: acetonitrile (80:20, v/v) and solution B contains a mixture of water: acetonitrile (20:80, v/v). The flow rate was 1.0 mL min−1 and the detection wavelength was 220 nm. The retention time of tadalafil is about 17 min. Tadalafil was subjected to different ICH prescribed stress conditions. Degradation was found to occur in hydrolytic and to some extent in oxidative stress conditions, while the drug was stable to photolytic and thermal stress. The drug was particularly labile under alkaline hydrolytic conditions. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The assay of stress samples was calculated against a qualified reference standard and the mass balance was close to 99.5%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and ruggedness.

A stability-indicating HPLC assay method for docetaxel

A novel stability-indicating high-performance liquid chromatographic assay method was developed and validated for docetaxel in the presence of degradation products generated from forced decomposition studies. A gradient HPLC method was developed to separate the drug from the degradation products, using a Hichrom RPB HPLC column. Mixture of water and acetonitrile was used as mobile phase. The flow rate was 1.0 ml/min and the detection was done at 230 nm. Using the above method one can carry out the quantitative estimation of impurity namely DCT-1 and docetaxel. The developed gradient LC method was subsequently validated.

A stability-indicating HPLC method for medroxyprogesterone acetate in bulk drug and injection formulation

A stability-indicating HPLC assay method has been developed and validated for medroxyprogesterone acetate (MPA) in bulk drug and injectable suspension. An isocratic RP-HPLC was achieved on a Hichrom C 18 column (150 mm × 4.6 mm i.d., 5 μm) utilizing a mobile phase of methanol 0.020 M acetate buffer pH 5 (65:35, v/v) and a photodiode array detector at 245 nm. The stress testing of MPA was carried out under acidic and alkaline hydrolysis, and oxidation conditions. MPA was well resolved from its degradation products, a main related substance (megestrol acetate) and two preservatives (methyl paraben and propyl paraben) with the resolution ≥2. The proposed method was validated for selectivity, linearity, accuracy, precision and solution stability. The method was found to be suitable for the quality control of MPA in bulk drug and injections as well as the stability-indicating studies.

Establishment of inherent stability of stavudine and development of a validated stability-indicating HPLC assay method

The present study describes degradation of stavudine under different stress conditions (hydrolysis, oxidation, photolysis and thermal stress), and establishment of a stability-indicating reversed-phase HPLC assay method. The drug was found to hydrolyse in acidic, neutral and alkaline conditions and also under oxidative stress. The major degradation product formed under various conditions was thymine, as evidenced through comparison with the standard and spectral studies (NMR, IR and MS) on the isolated product. Separation of drug, thymine and another minor degradation product was successfully achieved on a C-18 column utilising water–methanol in the ratio of 90:10. The detection wavelength was 265 nm. The method was validated with respect to linearity, precision (including intermediate precision), accuracy and specificity. The response was linear in the drug concentration range of 25–500 μg ml −1. The mean values (±R.S.D.) of slope and correlation coefficient were 24256 (±0.679) and 0.9994 (±0.0265), respectively. The R.S.D. values for intra- and inter-day precision studies were <0.210 and <1%, respectively. The recovery of the drug ranged between 99.7 and 101.5% from a mixture of degraded samples. The method even proved to be affective on application to a stressed marketed capsule formulation.

HPLC and LC–MS studies on stress degradation behaviour of tinidazole and development of a validated specific stability-indicating HPLC assay method

The objective of the current investigation was to study the degradation behaviour of tinidazole under different ICH recommended stress conditions by HPLC and LC–MS, and to establish a validated stability-indicating HPLC method. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal decomposition. Extensive degradation was found to occur in alkaline medium, under oxidative stress and in the photolytic conditions. Mild degradation was observed in acidic and neutral conditions. The drug was stable to thermal stress. Successful separation of drug from degradation products formed under stress conditions was achieved on a C-18 column using water–acetonitrile (88:12) as the mobile phase. The flow rate was 0.8 ml min −1 and the detection wavelength was 310 nm. The method was validated with respect to linearity, precision, accuracy, specificity and robustness. The utility of the procedure was verified by its application to marketed formulations that were subjected to accelerated stability studies. The method well separated the drug and degradation products even in actual samples. The products formed in marketed liquid infusions were similar to those formed during stress studies.