A validated stability indicating LC method for oxcarbazepine

The present paper describes the development of a stability indicating reversed phase liquid chromatographic (RPLC) method for oxcarbazepine in the presence of its impurities and degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The degradation of oxcarbazepine was observed under base hydrolysis. The drug was found to be stable to other stress conditions attempted. Successful separation of the drug from the synthetic impurities and degradation product formed under stress conditions was achieved on a C18 column using mixture of aqueous 0.02 M potassium dihydrogen phosphate–acetonitrile–methanol (45:35:20, v/v/v) as mobile phase. The developed HPLC method was validated with respect to linearity, accuracy, precision, specificity and robustness. The developed HPLC method to determine the related substances and assay determination of oxcarbazepine can be used to evaluate the quality of regular production samples. It can be also used to test the stability samples of oxcarbazepine.     

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 x 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.     

Rapid liquid chromatographic method for the determination of roflumilast in the presence of degradation products

A forced degradation study on roflumilast drug substance was conducted under the conditions of hydrolysis, oxidation, thermal and photolysis. The method was developed and optimized by analyzing forcefully degraded samples. The best separation was achieved on a Zorbax SB C18 1.8 μm column with 0.005 M ammonium formate buffer pH 3.5 and acetonitrile as mobile phase in a 13 min run time. The proposed method was able to resolve all the possible degradation products formed during stress study. The drug was stable to neutral, thermal and photolytic conditions but unstable to acidic, alkaline and oxidative conditions at 80° for 24 h. The degradation products resulting from stress study did not interfere in assay and related substances of roflumilast and thus the method can be regarded as stability indicating. An alternate method was also developed on a conventional 250×4.6 mm, 5 μm column wherein runtime was 38 min. Thus rapid resolution high throughput column was able to reduce the run time from 38 min to 13 min.     

ICH guidance in practice: establishment of inherent stability of secnidazole and development of a validated stability-indicating high-performance liquid chromatographic assay method

The degradation behaviour of secnidazole was investigated under different stress degradation (hydrolytic, oxidative, photolytic and thermal) conditions recommended by International Conference on Harmonisation (ICH) using HPLC and LC-MS. A stability-indicating HPLC method was developed that could separate drug from degradation products formed under various conditions. Secnidazole was found to degrade significantly in alkaline conditions, oxidative stress, and also in the presence of light. Mild degradation of the drug occurred in acidic and neutral conditions. The drug was stable to dry heat. Resolution of drug and the degradation products formed under different stress studies were successfully achieved on a C-18 column utilizing water-methanol in the ratio of 85:15 and at the detection wavelength of 310 nm. The method was validated with respect to linearity, precision (including intermediate precision), accuracy, selectivity and specificity.     

A validated HPLC stability-indicating method for the determination of diacerhein in bulk drug substance

A novel stability-indicating high-performance liquid chromatographic (HPLC) method was developed and validated for the assay of diacerhein in bulk forms. Diacerhein was found to degrade in alkaline and acidic conditions and also under oxidative stress. The drug was stable to dry heat and in presence of light. Resolution of drug, its potential impurities and degradation products were achieved on a RP18 (endcapped) column utilizing 0.1 M phosphoric acid and methanol (40:60, v/v) as eluent at the detection wavelength of 254 nm. The validation studies were carried out fulfilling International Conference on Harmonisation (ICH) requirements. The procedure was found to be specific, linear, precise (including intermediate precision), accurate and robust.     

The ICH guidance in practice: stress degradation studies on ornidazole and development of a validated stability-indicating assay

The present study describes degradation of ornidazole under different ICH prescribed stress conditions (hydrolysis, oxidation and photolysis), and establishment of a stability-indicating reversed-phase HPLC assay. Degradation was found to occur in alkaline medium, under high acidic conditions, under oxidative stress, and also in the presence of light in acid conditions. Previously the drug is only known to decompose under alkaline conditions. Separation of drug and the degradation products under various conditions was successfully achieved on a C-18 column utilising water-acetonitrile in the ratio of 86:14. The detection wavelength was 310 nm. The method was validated with respect to linearity, precision, accuracy, selectivity, specificity and ruggedness. The response was linear in the drug concentration range of 5–500 μg ml⁻¹. The mean values (±RSD) of slope, intercept and correlation coefficient were 45251 (±1.59), 104418 (±2.49) and 0.9996 (±0.03), respectively. The RSD values for intra- and inter-day precision studies were <1 and <2.6%, respectively. The recovery of the drug ranged between 100–103% from a mixture of degradation products. The method was specific to drug and also selective to degradation products.     

Identification of new impurities of enalapril maleate on oxidation in the presence of magnesium monoperoxyphthalate

Stress stability testing and forced degradation were used to determine the stability of enalapril maleate (EM) and to find a degradation pathway for the drug. The degradation impurities, formed under different stressed conditions, were investigated by HPLC and UPLC–MS methods. HPLC analysis showed several degradation impurities of which several were already determined, but on oxidation in the presence of magnesium monoperoxyphthalate (MMPP) several impurities of EM were observed which were not yet characterized. The HPLC methods for determination of EM were validated. The linearity of HPLC method was established in the concentration range between 0.5 and 10 μg/mL with correlation coefficient greater than 0.99. The LOD of EM was 0.2 μg/mL and LOQ was 0.5 μg/mL. The validated HPLC method was used to determine the degradation impurities in samples after stress stability testing and forced degradation of EM. In order to identify new degradation impurities of EM after forced degradation UPLC–MS/MSⁿ, Orbitrap has been used. It was found that new impurities are oxidation products: (S)-1-((S)-2-((S)-1-ethoxy-4-(o,m,p-hydroxyphenyl)-1-oxobutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid, (2S)-1-((2S)-2-((2S)-1-ethoxy-4-hydroxy-1-oxo-4-phenylbutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid. (S)-2-(3-phenylpropylamino)-1-(pyrrolidin-1-yl)propan-1-one was identified as a new degradation impurity.     

A validated stability-indicating normal phase LC method for clopidogrel bisulfate and its impurities in bulk drug and pharmaceutical dosage form

A novel stability-indicating normal phase liquid chromatographic (NP-LC) method was developed for the determination of purity of clopidogrel drug substance and drug products in bulk samples and pharmaceutical dosage forms in the presence of its impurities and degradation products. This method is capable of separating all the related substances of clopidogrel along with the chiral impurities. This method can be also be used for the estimation of assay of clopidogrel in drug substance as well as in drug product. The method was developed using Chiralcel OJ-H (250 mm × 4.6 mm, 5 μm) column. n-Hexane, ethanol and diethyl amine in 95:5:0.05 (v/v/v) ratio was used as a mobile phase. The eluted compounds were monitored at 240 nm. Clopidogrel bisulfate was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. The degradation products were well resolved from main peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to specificity, limit of detection, limit of quantification, precision, linearity, accuracy, robustness and system suitability.     

Stress degradation studies on lornoxicam using LC, LC-MS/TOF and LC-MSn

Lornoxicam was subjected to forced degradation studies under hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermal stress conditions, as defined under ICH guideline Q1A (R2). The drug degraded significantly in hydrolytic, oxidative and photoneutral conditions, leading to the formation of eight degradation products in total. It was stable on exposure to light and dry heat in the solid state. The stressed samples in which degradation was observed were mixed together and used to develop a stability-indicating HPLC method wherein degradation products were separated from the drug and also from each other. To characterize the degradation products, a complete mass fragmentation pathway of the drug was first established with the help of MS/TOF, MSⁿ and H/D exchange mass studies. The same was followed by LC-MS/TOF and on-line H/D exchange experiments on the degradation products. The degradation pathway of the drug was outlined, justified by the mechanisms of formation of the degradation products.     

Development and validation of a stability indicating HPLC assay method for cyclosporine in cyclosporine oral solution USP

The suitability of the United States Pharmacopeia (USP) assay method for analysing stressed samples of cyclosporine oral solution USP was evaluated for stability samples by analyzing cyclosporine oral solution after acid, alkali, hydrogen peroxide, heat and light treatment. Some of the degradants generated during stress testing, as well as dihydrocyclosporine A, which is a known degradant of cyclosporine A, were not adequately resolved from the cyclosporine peak and mobile phase adjustments did not improve the resolution. In addition, isocyclosporine A, another known degradant of cyclosporine, could not be quantitated as it was eluting too early with the system peaks. Therefore, a binary gradient, reverse phase, stability indicating, HPLC method for the assay of cyclosporine in cyclosporine oral solution USP has been developed and validated. Analysis of degraded samples showed that the cyclosporine A eluted as a spectrally pure peak resolved from its degradation products.     

Liquid chromatographic analysis of ethacrynic acid and degradation products in pharmaceutical systems

An accurate, reproducible, and specific reversed-phase high-performance liquid chromatographic (HPLC) system was developed for the determination of ethacrynic acid and its degradation products. The method was used in stability studies of the drug in the solid state, in solution, and in dosage forms. Three degradation products were isolated by preparative chromatography and identified by several techniques, principally NMR and MS. TLC Rt and HPLC response factors are quoted. A degradation scheme consistent with the observed stability profiles is proposed.     

Identification of Forced Degradation Products of Itopride by LC-PDA and LC-MS

Degradation products of itopride formed under different forced conditions have been identified using LC-PDA and LC-MS techniques. Itopride was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, dry and wet heat, in accordance with the International Conference on Harmonization. The stress solutions were chromatographed on reversed phase C18 (250×4.6 mm, 5 μm) column with a mobile phase methanol:water (55:45, v/v) at a detection wavelength of 215 nm. Itopride degraded in acid, alkali and oxidative stress conditions. The stability indicating method was developed and validated. The degradation pathway of the drug to products II-VIII is proposed.     

HPTLC method for guggulsterone. II. Stress degradation studies on guggulsterone

Stress degradation studies were carried out on guggulsterone (the hypolipidemic agent in the gum-resin exudates of Commiphora mukul) following the conditions prescribed in the parent drug stability testing guideline (Q1AR) issued by International Conference on Harmonization (ICH). The present study describes degradation of guggulsterone under different ICH prescribed stress conditions (acid and base hydrolysis, oxidation, dry and wet heat degradation and photodegradation) and establishment of a stability indicating HPTLC assay. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene-acetone (9:1, v/v). Densitometric analysis of guggulsterone was carried out in the absorbance mode at 250 nm. This system was found to give compact spots for E- and Z-guggulsterone, (Rf value of 0.38 +/- 0.02 and 0.46 +/- 0.02, respectively) following double development of chromatoplates with the same mobile phase. The drug undergoes degradation under acidic and basic conditions, oxidation, dry and wet heat treatment and photodegradation. All the peaks of degraded products were resolved from the standard guggulsterone with significantly different Rf values. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.     

Development and validation of a specific stability indicating high performance liquid chromatographic method for rizatriptan benzoate

A gradient, reversed-phase liquid chromatographic (RP-LC) method was developed for the quantitative determination of rizatriptan benzoate, used to treat relieves migraine headache symptoms. The developed method can be also employed for the related substance determination in bulk samples. Forced degradation studies were performed on bulk sample of rizatriptan benzoate using acid (0.5 N hydrochloric acid), base (0.1 N sodium hydroxide), oxidation (3.0% hydrogen peroxide), water hydrolysis, heat (60 degrees C) and photolytic degradation. Mild degradation of the drug substance was observed in base hydrolysis and considerable degradation observed during oxidative stress. The chromatographic method was fine tuned using the samples generated from forced degradation studies. Good resolution between the peaks corresponds to degradation products and the analyte was achieved on Agilent Zorbax SB-CN (250 mm x 4.6 mm, 5 microm) column. The mobile phase consists of a mixture of aqueous potassium di hydrogen ortho phosphate (pH 3.4), acetonitrile and methanol. The stress sample solutions were assayed against the qualified reference standard of rizatriptan benzoate and the mass balance in each case was close to 99.7% indicating that the developed method is stability indicating. Validation of the developed method was carried out as per ICH requirements.     

A validated stability-indicating LC method for acetazolamide in the presence of degradation products and its process-related impurities

The objective of the current study was to develop a validated, specific and stability-indicating reverse phase liquid chromatographic method for the quantitative determination of acetazolamide and its related substances. The determination was done for an active pharmaceutical ingredient, its pharmaceutical dosage form in the presence of degradation products, and its process-related impurities. The drug was subjected to stress conditions of hydrolysis (acid and base), oxidation, photolysis and thermal degradation as per International Conference on Harmonization (ICH) prescribed stress conditions to show the stability-indicating power of the method. Significant degradation was observed during acid and base hydrolysis, and the major degradant was identified by LC–MS, FTIR and ¹H/¹³C NMR spectral analysis. The chromatographic conditions were optimized using an impurity-spiked solution and the generated samples were used for forced degradation studies. In the developed HPLC method, the resolution between acetazolamide and, its process-related impurities (namely imp-1, imp-2, imp-3, imp-4 and its degradation products) was found to be greater than 2. The chromatographic separation was achieved on a C18, 250 mm × 4.6 mm, 5 μm column. The LC method employed a linear gradient elution, and the detection wavelength was set at 254 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.     

Combined application of high resolution and tandem mass spectrometers to characterize methionine oxidation in a parathyroid hormone formulation

Identification and monitoring of degradation products is a critical aspect of drug product stability programs. This process can present unique challenges when working with complex biopharmaceutical formulations that do not readily lend themselves to straightforward HPLC analysis. The therapeutic 34 amino acid parathyroid hormone fragment (PTH1–34) contains methionine (Met) residues at positions 8 and 18. Oxidation of these Met residues results in reduced biological activity and thus efficacy of the potential drug product. Here, we present an effective approach for the identification of PTH1–34 oxidation products in a drug product formulation in which the stability indicating method used non‐MS compatible HPLC conditions to separate excipients, drug substance and degradation products. High resolution and tandem mass spectrometers were used in conjunction with cyanogen bromide (CNBr) mediated digestion to accurately identify the oxidation products observed in an alternative MS compatible HPLC method used for drug substance analysis. All anticipated CNBr digested peptide fragments, including both oxidized and nonoxidized peptide fragments, were positively identified using TOF MS without the need for additional enzymatic digestion. Once identified, the oxidation products generated were injected onto the original non‐MS compatible HPLC drug product stability indicating method and the respective retention times were confirmed. This allowed the oxidative stability of different formulations to be effectively monitored during the solid state stability program and during variant selection. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1169–1179, 2010     

Study of forced degradation behavior of enalapril maleate by LC and LC-MS and development of a validated stability-indicating assay method

In the present study, comprehensive stress testing of enalapril maleate was carried out according to ICH guideline Q1A(R2). The drug was subjected to acid (0.1N HCl), neutral and alkaline (0.1N NaOH) hydrolytic conditions at 80 degrees C, as well as to oxidative decomposition at room temperature. Photolysis was carried out in 0.1N HCl, water and 0.1N NaOH at 40 degrees C. Additionally, the solid drug was subjected to 50 degrees C for 60 days in a dri-bath, and to the combined effect of temperature and humidity, with and without light, at 40 degrees C/75% RH. The products formed under different stress conditions were investigated by LC and LC-MS. The LC method that could separate all degradation products formed under various stress conditions involved a C18 column and a mobile phase comprising of ACN and phosphate buffer (pH 3). The flow rate and detection wavelength were 1 ml min(-1) and 210 nm, respectively. The developed method was found to be precise, accurate, specific and selective. It was suitably modified for LC-MS studies by replacing phosphate buffer with water, where pH was adjusted to 3.0 with formic acid. The drug showed instability in solution state (under acidic, neutral, alkaline and photolytic stress conditions), but was relatively stable in the solid-state, except formation of minor products under accelerated conditions. Primarily, maximum degradation products were formed in acid conditions, though the same were also produced variably under other stress conditions. The LC-MS m/z values and fragmentation patterns of two of the five products matched with enalaprilat and diketopiperazine derivative, previously known degradation products of enalapril. Also, m/z value of another product matched with an impurity listed in the drug monograph in European Pharmacopoeia. Rest two were hitherto unknown degradation products. The products were characterized through LC-MS fragmentation studies. Based on the results, a more complete degradation pathway for the drug could be proposed.     

ICH guidance in practice: validated stability-indicating HPLC method for simultaneous determination of ampicillin and cloxacillin in combination drug products

Ampicillin and cloxacillin were degraded together under different stress test conditions prescribed by International Conference on Harmonization. The samples so generated were used to develop a stability-indicating high performance liquid chromatographic (HPLC) method for the two drugs. The drugs were well separated from degradation products using a reversed-phase (C-18) column and a mobile phase comprising of acetonitrile:phosphate buffer (pH 5.0), which was delivered initially in the ratio of 15:85 (v/v) for 1 min, then changed to 30:70 (v/v) for next 14 min, and finally equilibrated back to 15:85 (v/v) from 15 to 20 min. Other HPLC parameters were: flow rate, 1 ml/min; detection wavelength, 225 nm; and injection volume, 5 microl. The method was validated for linearity, precision, accuracy, specificity and selectivity. It was also compared with the assay procedures given in British Pharmacopoeia for individual drugs. Similar results were obtained, indicating that the proposed single method allowed selective analysis of both ampicillin and cloxacillin, in the presence of their degradation products formed under a variety of stress conditions. The developed procedure was also applicable to the determination of instability of the drugs in commercial products.     

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.     

Stability-indicating UPLC method for determination of Valsartan and their degradation products in active pharmaceutical ingredient and pharmaceutical dosage forms

A simple, precise, accurate stability-indicating gradient reverse phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the quantitative determination of purity of Valsartan drug substance and drug products in bulk samples and pharmaceutical dosage forms in the presence of its impurities and degradation products. The method was developed using Waters Aquity BEH C18 (100 mm × 2.1 mm, 1.7 μm) column with mobile phase containing a gradient mixture of solvents A and B. The eluted compounds were monitored at 225 nm, the run time was within 9.5 min, which Valsartan and its seven impurities were well separated. Valsartan was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Valsartan was found to degrade significantly in acid and oxidative stress conditions and stable in base, hydrolytic and photolytic degradation conditions. The degradation products were well resolved from main peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per international conference on harmonization (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 Valsartan in pharmaceutical dosage forms.