Stability-indicating Liquid Chromatographic Method Research Articles

A validated stability-indicating liquid chromatographic method for determination of process related impurities and degradation behavior of Irbesartan in solid oral dosage.

The present work describes the development and validation of a stability-indicating RP-HPLC method for the estimation of degradation and process related impurities of Irbesartan, namely Impurity-1, Impurity-2, Impurity-3 and Impurity-4. The developed LC method was validated with respect to specificity, limit of detection and quantification, linearity, precision, accuracy and robustness. The chromatographic separation was achieved on Hypersil Octadecylsilyl (4.6 mm × 150 mm, 3 μm) column by using mobile phase containing a gradient mixture of solvent A (0.55% v/v ortho-phosphoric acid, pH adjusted to 3.2 with triethyl amine) and B (95:5 v/v mixture of acetonitrile and solvent A) at a flow rate of 1.2 mL/min. The detection was carried out at a wavelength of 220 nm. During method validation parameter such as precision, linearity, accuracy, specificity, limit of detection and quantification were evaluated, which remained within acceptable limits. HPLC analytical method is linear, accurate, precise, robust and specific, being able to separate the main drug from its degradation products. The degradation products were well-resolved from the main peak and its impurities, thus proving the stability-indicating power of the method. The method is stability-indicating in nature and can be used for routine analysis of production samples and to check the stability of the Irbesartan HCl tablets.

Stability-Indicating LC Method for the Simultaneous Determination of Lisinopril and Hydrochlorothiazide

A simple and rapid stability-indicating liquid chromatographic method was developed and validated for the simultaneous determination of lisinopril and hydrochlorotiazide (HCTZ) in drug substances and dosage forms in the presence of degradation products. Forced degradation studies were conducted on the pure drugs under hydrolytic, oxidative, thermal and photolytic conditions. A chromatographic separation of the two drugs and its degradation products was achieved with an RP-18 column, using methanol, acetonitrile and phosphate buffer (pH 7.1; 0.05 M) (15:15:70, v/v/v) as mobile phase at a flow rate of 0.8 mL min(-1) and UV detection at 210 nm. Lisinopril and HCTZ were well resolved from its degradation products showing the stability-indicating capability of the method. The described method was linear over a range of 40-200 µg mL(-1) for lisinopril and 25-175 µg mL(-1) for HCTZ. The assay was also selective, accurate and precise for lisinopril and HCTZ determination. This method represents an alternative to the United States Pharmacopeia (USP) method showing shorter retention time. The method was successfully applied for determination of lisinopril and HCTZ in combined commercial tablets. The results showed that the proposed method was found to be suitable for quantitative determination and the stability study of lisinopril and HCTZ in pharmaceutical samples.

STABILITY INDICATING HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR ESTIMATION OF ADAPALENE IN TABLET FORMULATION

Stability-indicating high performance liquid chromatographic (HPLC) method for estimation of Adapalene was developed and validated. Adapalene was separated and quantitated on Nucleosil C8 column (250 mm length, 4.6 mm internal diameter, 5 µm particle size) using a blend of methanol-ammonium acetate buffer pH 4.0 (80:20 v/v) as a mobile phase and at a flow rate of 1.3 mL/min. Quantification was achieved with ultraviolet (UV) detector at 270 nm over the concentration range 10–60 µg/mL. The applied HPLC method allowed separation and quantification of Adapalene with good linearity (r2 = 0.999) in the studied concentration range. Limit of detection and limit of quantification were found to be 1.43 µg/mL and 4.34 µg/mL, respectively. The method was validated as per the International Conference on Harmonization (ICH) guidelines. Adapalene stock solution was subjected to different stress conditions. The degraded product peaks were well-resolved from the pure drug peak with significant difference in their retention time values. Stressed samples were assayed using developed HPLC method. Statistical analysis of the data showed that the method is precise, accurate, reproducible, and selective for the analysis of Adapalene. The method was successfully applied to the estimation of Adapalene in tablet dosage forms.

A Validated Specific Stability-Indicating RP-HPLC Assay Method for Ambrisentan and Its Related Substances

A validated specific stability-indicating reverse-phase liquid chromatographic method was developed for the quantitative determination of Ambrisentan as well as its related substances in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its related impurities. Forced degradation studies were performed on bulk samples of Ambrisentan as per the ICH-prescribed stress conditions using acid, base, oxidative, thermal stress and photolytic degradation to show the stability-indicating power of the LC method. Significant degradation in acidic, basic stress conditions was observed and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from the forced degradation studies and the impurity-spiked solution. Good resolution between the peaks corresponds to Ambrisentan-related impurities and degradation products from the analyte were achieved on a SunFire C18 column using a mobile phase consisting of a mixture of potassium dihydrogen orthophosphate at a pH adjusted to 2.5 with ortho-phosphoric acid in water and a mixture of acetonitrile:methanol using a simple linear gradient. The detection was carried out at 225 nm. The limit of detection and the limit of quantification for the Ambrisentan and its related impurities were established. The stressed test solutions were assayed against the qualified working standard of Ambrisentan and the mass balance in each case was between 98.9 and 100.3%, indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per the ICH requirements. The developed method was found to be suitable to check the quality of bulk samples of Ambrisentan at the time of batch release and also during its storage (long-term and accelerated stability).

Stability-Indicating HPLC-DAD Determination of Ribavirin in Capsules and Plasma

A simple, selective and stability-indicating high-pressure liquid chromatographic method was developed for the analysis of ribavirin. Chromatographic separation was achieved by using a CPS Hypersil cyano column (4.6 × 250 mm, 5 µm particle size) with isocratic elution of the mobile phase, which was composed of 50 mM phosphate buffer, adjusted at pH 4 with phosphoric acid. The mobile phase was pumped at a flow rate of 0.8 mL/min. The detector was set at 240 nm and quantification of the analyte was based on peak area measurement. The method was validated with respect to linearity, range, precision, accuracy, selectivity, robustness, limit of detection and limit of quantitation. The calibration curve was linear in the range of 5-200 µg/mL with correlation coefficient > 0.999. Ribavirin was subjected to forced degradation studies under two conditions: mild and extensive stress testing. These studies included the effects of hydrolysis (neutral, acidic and alkaline) and oxidation, photolysis and dry heat). The proposed method was proved to be stability-indicating by the resolution of the drug from its forced degradation products, making use of the diode array detector as a tool for confirmation of peak identity and purity. Moreover, the kinetics of alkaline degradation of ribavirin were investigated, an Arrhenius plot was constructed and the activation energy was calculated. The developed method was also extended to analyze ribavirin in capsules and in human plasma with good recovery values.

Development of Novel Stability-Indicating Method for the Determination of Dimethindene Maleate and Its Impurities

A novel and selective stability-indicating liquid chromatographic method has been developed and validated for the analysis of dimethindene maleate, the related substance 2-ethylpyridine, and three degradation products. Dimethindene maleate was subjected to forced degradation study by acid and basic hydrolysis, oxidation, and thermal decomposition. Three degradation products that were formed during the forced degradation study were separated from dimethindene using a Zorbax SB CN column (150 × 4.6 mm; 5 μm); cyanopropyl-bonded stationary phase was applied for the first time for the separation of dimethindene and its impurities. The proposed method was validated and was found suitable for quality control and stability tests of pharmaceuticals containing dimethindene maleate.

A Validated Stability-Indicating Liquid Chromatographic Method for the Determination of Retapamulin in Topical Dosage Form

A sensitive, stability-indicating reversed-phase high-performance liquid chromatographic method is developed and validated for the quantitative determination of retapamulin in topical dosage form. The chromatographic separation is achieved by using a C18 column (XTerra RP 18 250 × 4.6 mm, 5 µm) at 30°C. The mobile phase comprises a mixture of 0.05M potassium dihydrogen phosphate buffer (pH 6.1), acetonitrile and methanol in the ratio of 35:50:15 (v/v/v). The flow rate is set at 1.0 mL/min and chromatograms are extracted at 243 nm using a photodiode array detector. The method is validated with respect to linearity, accuracy, precision, robustness and forced degradation studies, which further prove the stability-indicating supremacy of the method. During forced degradation studies, retapamulin is observed to be labile to oxidative and base hydrolysis stress and stable in thermal, photolytic and acid hydrolysis stress. The degradation products are well separated from the retapamulin peak, thus proving the stability-indicating superiority of the method. The method is found to be sensitive for retapamulin, with a detection limit of 25 ng/mL and a quantification limit of 80 ng/mL. The proposed method is found to be very sensitive and accurate for the determination of retapamulin in topical dosage form. The method is also demonstrated to be robust, because it is resistant to small variations of chromatographic variables such as pH, mobile phase composition, flow rate and column temperature.

STABILITY-INDICATING HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF FLUCONAZOLE IN THE PRESENCE OF ITS OXIDATIVE DEGRADATION PRODUCT - KINETIC AND STRESS STUDY

A simple, specific, accurate, and stability-indicating high performance liquid chromatographic method (HPLC) method has been established for analysis of fluconazole (FLZ) in the presence of its degradation products generated in the stress degradation study. FLZ was subjected to stress conditions of acid, alkali, and neutral hydrolysis, oxidation, photolysis, and thermal decomposition. Extensive degradation was found to occur in oxidative medium under thermal stress. Successful separation of drug from degradation products was achieved on a C-18 column using phosphoric acid 0.5% v/v: acetonitrile (80:20% v/v) as the mobile phase. The flow rate was 1.5 mL min−1 and the detector was set at 261 nm. The retention times of FLZ and its main oxidative degradation product were found to be 5.389 min and 2.729 min, respectively. Linearity was established for fluconazole in the range of 0.5–50 µg/ml. The percentage recovery of fluconazole was found to be 99.91 ± 0.74. Because the method effectively separates fluconazole from its oxidative degradation products, it can be used as stability-indicating method. The proposed method was also used to study the kinetics of fluconazole oxidative degradation that was found to follow a zero-order reaction. The t1/2 was 21.66 min while k (reaction rate constant) was 2.91 × 10−8 mole/min.

Validation of a Stability-Indicating Hydrophilic Interaction Liquid Chromatographic Method for the Quantitative Determination of Vitamin K3 (Menadione Sodium Bisulfite) in Injectable Solution Formulation

A simple, specific, accurate, and stability-indicating method was developed and validated for the quantitative determination of menadione sodium bisulfite in the injectable solution formulation. The method is based on zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) coupled with a photodiode array detector. The desired separation was achieved on the ZIC-HILIC column (250 mm × 4.6 mm, 5 μm) at 25°C temperature. The optimized mobile phase consisted of an isocratic solvent mixture of 200mM ammonium acetate (NH4AC) solution and acetonitrile (ACN) (20:80; v/v) pH-adjusted to 5.7 by glacial acetic acid. The mobile phase was fixed at 0.5 ml/min and the analytes were monitored at 261 nm using a photodiode array detector. The effects of the chromatographic conditions on the peak retention, peak USP tailing factor, and column efficiency were systematically optimized. Forced degradation experiments were carried out by exposing menadione sodium bisulfite standard and the injectable solution formulation to thermal, photolytic, oxidative, and acid-base hydrolytic stress conditions. The degradation products were well-resolved from the main peak and the excipients, thus proving that the method is a reliable, stability-indicating tool. The method was validated as per ICH and USP guidelines (USP34/NF29) and found to be adequate for the routine quantitative estimation of menadione sodium bisulfite in commercially available menadione sodium bisulfite injectable solution dosage forms.

Stability-Indicating HPLC Method for the Determination of Mometasone Furoate, Oxymetazoline, Phenyl Ethanol and Benzalkonium Chloride in Nasal Spray Solution

A sensitive stability-indicating reversed-phase high performance liquid chromatographic method was developed for the simultaneous determination of mometasone furoate (MON), oxymetazoline (OXY), phenyl ethanol (PEL) and benzalkonium chloride analogs (BKC1and BKC2) in nasal spray solution. The chromatographic separation was achieved on Cosmosil CN 150 mm x 4.6mm, 5 um column Using a mobile phase consisting of 50 mM KH2PO4 and Acetonitrile in the ratio of 60:40 (v/v), at a flow rate of 2.0 ml/min. The column compartment temperature was set at 40°C. The typical HPLC chromatograms were extracted at 215 nm using a photodiode-array detector (PDA). The inter-day and intra-day precision values are found within 2% of relative standard deviation. The described method gives LOQ values of 21 ng/mL for MON, 23 ng/mL for OXY, 25 ng/mL for PEL, 10 ng/mL for BKC1 and 9 ng/mL for BKC2. The method shows accuracy of 99.6% for MON, 99.8% for OXY, 99.8% for PEL, 99.0% for BKC1 and 99.5% for BKC2. The described method shows excellent linearity over a range of LOQ to 150% of test concentration. The correlation coefficient for MON, OXY, PEL, BKC1 and BKC2 was 0.9998, 0.9999, 0.9998, 0.9999 and 0.9996 respectively.

Development and Validation of a Stability-Indicating Liquid Chromatography Method for the Determination of Dabigatran Etexilate in Capsules

An RP-LC method was developed and validated for the determination of dabigatran etexilate in a capsule formulation. The LC method was carried out on a Zorbax C18 column (250 x 4.6 mm id). The mobile phase consisted of acetonitrile and a solution of triethylamine 0.1%, pH 6.0 adjusted with phosphoric acid (65 + 35, v/v) at a flow rate of 1.0 mL/min. The diode array detector was set at 225 nm. The chromatographic separation was obtained with retention time of 6.31 min, and linearity was in the range of 10-70 microg/mL (R2 = 0.9991). The specificity and stability-indicating capability of the method was proven through degradation studies and showing that there was no interference from the excipients. The accuracy was 100.23%, with an RSD of 1.34%. The LOD and LOQ were 0.04 and 10 microg/mL, respectively. Moreover, method validation demonstrated acceptable results for robustness and a system suitability test. The proposed method was applied for the analysis of the capsules to ensure their therapeutic efficacy.

Photodegradation kinetics of lodenafil carbonate, structure elucidation of two major degradation products using UPLC-MS/MS and in vitro cytotoxicity

The photostability of lodenafil carbonate was studied and some degradation products were observed. A stability-indicating liquid chromatography method for the determination of lodenafil carbonate was used to determine the kinetics of photodegradation. The identification of two major photodegradation products was performed by an isocratic ultra performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS). UPLC-MS/MS was carried out on a Waters® Acquity Ultra Performance LC system coupled to a Micromass® Quadrupole Time of Flight tandem mass spectrometer equipped with an electrospray ionization interface in positive ion mode. The column applied was Acquity UPLC® BEH C18; the mobile phase consisted of a mixture of methanol–formic acid 0.1% pH 4.0 (55 : 45, v/v) at a flow-rate of 0.4 mL min−1 and UV detection at 290 nm. The photodegradation of lodenafil carbonate followed first-order reaction kinetics and the kinetic parameters of degradation rate constant and t90% were calculated. Under photodegradation conditions, ion products were detected at m/z 393 (DP-1) and at m/z 377 (DP-2). The product DP-1 is 4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo [4,3-d]pyrimidin-5-yl)-benzenesulfonic acid and DP-2 probably is 4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo [4,3-d]pyrimidin-5-yl) benzenesulfinic acid. The degraded samples of lodenafil carbonate were also evaluated in order to determine the in vitro cytotoxicity against mononuclear cells.

Stability of azacitidine in sterile water for injection.

The product monograph for azacitidine states that once reconstituted, the drug may be held for only 30 min at room temperature or 8 h at 4°C. Standard doses result in wastage of a portion of each vial, and the cost of this wastage is significant, adding about $156 000 to annual drug expenditures at the authors' institution. To evaluate the stability of azacitidine after reconstitution. Vials of azacitidine were reconstituted with sterile water for injection. At the time of reconstitution, the temperature of the diluent was 4°C for samples to be stored at 4°C or -20°C and room temperature for samples to be stored at 23°C. Solutions of azacitidine (10 or 25 mg/mL) were stored in polypropylene syringes and glass vials at room temperature (23°C), 4°C, or -20°C. The concentration of azacitidine was determined by a validated, stability-indicating liquid chromatographic method in serial samples over 9.6 h at room temperature, over 4 days at 4°C, and over 23 days at -20°C. The recommended expiry date was determined on the basis of time to reach 90% of the initial concentration according to the fastest observed degradation rates (i.e., lower limit of 95% confidence interval). Azacitidine degradation was very sensitive to temperature but not storage container (glass vial or polypropylene syringe). Reconstitution with cold sterile water reduced degradation. At 23°C, 15% of the initial concentration was lost after 9.6 h; at 4°C, 32% was lost after 4 days; and at -20°C, less than 5% was lost after 23 days. More than 90% of the initial azacitidine concentration will be retained, with 97.5% confidence, if, during the life of the product, storage at 23°C does not exceed 2 h, storage at 4°C does not exceed 8 h, and storage at -20°C does not exceed 4 days. These expiry dates could substantially reduce wastage and cost where the time between doses does not exceed 4 days.

Degradation Pathway for Eplerenone by Validated Stability Indicating UP-LC Method.

Degradation pathway for eplerenone is established as per ICH recommendations by validated and stability-indicating reverse phase liquid chromatographic method. Eplerenone is subjected to stress conditions of acid, base, oxidation, and thermal and photolysis. Significant degradation is observed in acid and base stress conditions. Four impurities are studied and the major degradant (RRT about 0.31) was identified by LC-MS and spectral analysis. The stress samples are assayed against a qualified reference standard and the mass balance is found close to 99.5%. Efficient chromatographic separation is achieved on a Waters symmetry C18 stationary phase with simple mobile phase combination delivered in gradient mode and quantification is carried at 240 nm at a flow rate of 1.0 mL min−1. In the developed LC method the resolution between eplerenone and four potential impurities (imp-1, imp-2, imp-3, and imp-4) is found to be greater than 4.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.999 for eplerenone and four potential impurities. This method is capable to detect the impurities of eplerenone at a level of 0.020% with respect to test concentration of 1.0 mg mL−1 for a 20 μL injection volume. The developed UPLC method is validated with respect to specificity, linearity and range, accuracy, precision, and robustness for impurities and assay determination.

Stability-indicating RP-HPLC method for analysis of the antibiotic doripenem in pharmaceutical formulation—comparison to UV spectrophotometry and microbiological assay

Summary A stability-indicating liquid chromatographic (LC) method with UV detection was developed for the determination of doripenem in the marketed formulation (Doribax® 500 mg, powder for injection). A forced degradation study was conducted according to available guidelines and main references. Thermal, oxidizing, acidic and basic stress conditions were assayed to show the stability-indicating power of the method. Chromatographic separation was achieved using an isocratic elution method in a reversed-phase system using a mobile phase prepared from phosphate buffer and acetonitrile. Extensive degradation was observed under thermal, oxidative and basic treatment, and the products formed were detected without interference in the analysis of doripenem. To verify the efficiency of chromatographic run, the system suitability was studied. The theoretical plates (N = 5498.3) and tailing factor (tf = 0.951) were constant during repeated injections. The retention time of doripenem was 7.35 min and the method was validated within the concentration range 5–50 μg mL−1 (r = 0.999). Adequate results were obtained that indicate repeatability (RSD % = 1.03–1.37), inter-day precision (RSD % = 0.51) and accuracy. In comparison to spectrophotometric and microbiological methods, statistical analysis showed no significant difference between the obtained results. The proposed method was successfully applied to doripenem quantification, showing it is applicable to determine the antibiotic in the presence of degradation products and also that is a reliable method for routine analysis.

A SINGLE COMMON STABILITY INDICATING ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR ESTIMATION OF IMPURITIES IN FOUR ANGIOTENSIN II RECEPTOR BLOCKERS

A novel single common stability-indicating gradient reverse phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the determination of purity of four angiotensin II receptor blockers namely valsartan, losartan potassium, irbesartan, and telmisartan from respective finished dosage forms in the presence of their impurities and forced degradation products. The method was developed using Waters Aquity BEH C18 column with mobile phase containing a gradient mixture of solvent A and B. The eluted compounds were monitored at 230 nm. The run time was 8.2 min within which each four actives and their impurities were well-separated. Valsartan, losartan potassium, irbesartan, and telmisartan were subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. For each drug the degradation products were well-resolved from main peak and its impurities, thus proving the stability indicating power of the method. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision, and robustness.

A sensitive and specific high-performance thin-layer chromatography-densitometry method for determination of Rebamipide in the bulk material and in pharmaceutical formulation

Rebamipide (RBP), α((4-chlorobenzoyl)-amino)-1,2-dihydro-2-oxo-4-quinoline propanoic acid is antiulcer (gastric cytoprotectant). Literature survey revealed many reverse-phase high-performance liquid chromatography (RP-HPLC) methods for determination of RBP in human plasma, rat plasma, and coionic tissue. Stability-indicating liquid chromatographic assay method and first-order derivative spectrophotometric methods have been established for the determination of RBP in bulk and tablet dosage form. Nowadays, high-performance thin-layer chromatography (HPTLC) is becoming a routine analytical technique due to its low operating cost, high sample throughput, and need for minimum sample clean up. The major advantage of HPTLC is that several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC, thus lowering analysis time and cost per analysis. The intention of the present work is to establish simple, precise, and accurate stability-indicating densitometric thin-layer chromatography (TLC)-densitometry method for analysis of RBP in the bulk material and in tablets. Further the developed method has to be validated as per the International Conference on Harmonisation (ICH) guidelines.

A Novel and Rapid Validated Stability-Indicating UPLC Method of Related Substances for Dorzolamide Hydrochloride and Timolol Maleate in Ophthalmic Dosage Form

A novel stability-indicating gradient reversed-phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the determination of purity of dorzolamide hydrochloride and timalol maleate in presence of their impurities, and forced degradation products and placebo. The method was developed using a Waters UPLC BEH C18, 100 × 2.1mm, 1.7 µm column with mobile phase containing a gradient mixture of solvents A and B. Phosphate buffer (0.04M), pH 2.6 was used as buffer. Buffer pH 2.6 was used as solvent A and Milli-Q water, methanol and acetonitrile in 200:300:600, v/v/v ratios were used as solvent B. The gradient program was set as 0/5, 8/8, 10/15, 16/45, 20/55, 24/80, 25/5 and 30/5. The eluted compound dorzolamide hydrochloride and its impurities were monitored at 254 nm, and timalol maleate and its impurities were monitored at 295 nm. The run time was 30 min, within which dorzolamide hydrochloride and its five impurities as well as timalol maleate and its three impurities were well separated, with resolution more than 2.0. Dorzolamide hydrochloride and timalol maleate were subjected to the stress conditions of oxidative, acid, base, photolytic and thermal degradation. The peak purity of dorzolamide hydrochloride, timalol maleate and their related compounds did not show any flag, thus proved the stability-indicating power of the method. The developed method was validated as per International Conference of Harmonization guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness.

IDENTIFICATION, CHARACTERIZATION OF DEGRADATION COMPONENT IN DESOXIMETASONE PHARMACEUTICAL DOSAGE FORMS AND ITS QUANTIFICATION IN THE PRESENCE OF PROCESS RELATED IMPURITIES

A single, short, and stability-indicating reverse phase liquid chromatographic method has been developed for the simultaneous determination of desoximetasone as well as its related substances in its three pharmaceutical dosage forms. A major impurity was enhanced during accelerated and long term stability studies of desoximetasone gel and cream dosages. Forced degradation studies (or stress studies) under various conditions recommended by International Conference on Harmonization (ICH) were carried out to elucidate the inherent stability characteristics of active substance. The major degradant was identified by using LC-MS, FTIR, and 1H/13C NMR spectral analysis. A novel degradation mechanism was proposed, which should ease the pharmaceutical development of drug products containing desoximetasone. The chromatographic conditions were optimized using the impurity spiked solution and the samples generated from forced degradation studies. The chromatographic separation was achieved on a C18, 75 mm × 4.6 mm, 3.5 µm column with a linear gradient elution. The detection wavelength was at 240 nm. The stress samples were assayed against a qualified reference standard and the mass balance was found to be close to 99.6%. The developed RP-LC method was validated with respect to linearity, accuracy, precision, and robustness.

A VALIDATED STABILITY-INDICATING HPLC-PDA METHOD FOR ANALYSIS OF DESMODIUM GANGETICUM: AN IMPORTANT INGREDIENT OF AYURVEDIC DRUG “DASHMOOL”

A stability-indicating reversed-phase liquid chromatographic (RPLC) method has been established for analysis of rutin (1), kaempferol-3-O-robinobioside (2), and nicotiflorin (3) in Desmodium gangeticum. The study was performed in the presence of the degradation products generated in the study of forced degradation. Marker compounds were subjected to stress by hydrolysis (acidic and basic), oxidation, photolysis, and thermal treatment. Under the optimized conditions, well-resolved separation of pure compounds from the degradation products with significantly different Rt values was achieved on a Spherisorb ODS2 column (250 mm × 4.6 mm, 10 µm) using isocratic elution of methanol and water (0.5% acetic acid); with acceptable validation results such as linearity, sensitivity, and recovery in terms of RSD (%). The calibration curves were linear in the concentration range of 20–100 µg/mL. Method was validated as per ICH guidelines. The reproducible and robust method may be applied for assays and stability tests of D. gangeticum and phytopreparations containing D. gangeticum.