Acid Stress Conditions Research Articles

Development and validation of a stability-indicating reverse phase ultra performance liquid chromatographic method for the estimation of nebivolol impurities in active pharmaceutical ingredients and pharmaceutical formulation.

A sensitive, stability-indicating gradient reverse phase ultra performance liquid chromatographic method has been developed for the quantitative estimation of nebivolol impurities in active pharmaceutical ingredient (API) and pharmaceutical formulation. Efficient chromatographic separation was achieved on an Acquity BEH C18 column (100 mm x 2.1 mm, 1.7 μm) with mobile phase of a gradient mixture. The flow rate of the mobile phase was 0.18 mL/min with column temperature of 30 degrees C and detection wavelength of 281 nm. The relative response factor values of (R*)-2-( benzylamino)-1-((S*)-6-fluorochroman-2-yl) ethanol ((R x S*) NBV-), (R)-1-((R)-6-fluorochroman-2-yl)-2-((S)-2-((S)-6-fluoro-chroman-2-yl)-2-hydroxyethyl-amino) ethanol ((RRSS) NBV-3), 1-(chroman-2-yl)-2-(2-(6-fluorochroman-2-yl)-2-hydroxyethyl amino) ethanol (monodesfluoro impurity), (S)-1-((R)-6-fluorochroman-2-yl)-2-((R)-2 (S*)-6-fluoro-chroman-2-yl)-2-hydroxyethylamino) ethanol hydrochloride ((RSRS) NBV-3) and (R*)-1-((S*)-6-fluorochroman-2-yl)-2-((S*)-2-((S*)-6-fluoro-chroman-2-yl)-2-hydroxyethylamino) ethanol ((R* S* S* S*) NBV-2) were 0.65, 0.91, 0.68, 0.92 and 0.91 respectively. Nebivolol formulation sample was subjected to the stress conditions of acid, base, oxidative, hydrolytic, thermal, humidity and photolytic degradation. Nebivolol was found to degrade significantly under peroxide stress condition. The degradation products were well resolved from nebivolol and its impurities. The peak purity test results confirmed that the nebivolol peak was homogenous and pure in all stress samples and the mass balance was found to be more than 98%, thus proving the stability-indicating power of the method. The developed method was validated according to International Conference on Hormonization (ICH) guidelines with respect to specificity, linearity, limits of detection and quantification, accuracy, precision and robustness.

Development and Validation of a Stability-Indicating RP-UPLC Method for the Estimation of Impurities in Cinacalcet Hydrochloride API and its Formulation.

A sensitive, stability-indicating, gradient reversed-phase ultra-performance liquid chromatography method has been developed for the quantitative estimation of cinacalcet hydrochloride impurities in active pharmaceutical ingredients and pharmaceutical formulations. Efficient chromatographic separation was achieved on an Acquity BEH Shield RP18, 100 × 2.1 mm, 1.7 µm column with the mobile phase containing pH 6.6 phosphate buffer and acetonitrile. The flow rate of the mobile phase was 0.3 mL min−1 with a column temperature of 35°C and detection wavelength at 223 nm. The relative response factor values of (+)-R-1-(1-Naphthyl)ethylamine, regioisomer, diastereomer isomer-1, and diastereomer isomer-2 were 1.79, 0.99, 0.89, and 0.88, respectively. The cinacalcet hydrochloride formulation sample was subjected to the stress conditions of acid, base, oxidative, hydrolytic, thermal, humidity, and photolytic degradation. Cinacalcet hydrochloride was found to degrade significantly under the peroxide stress conditions. The degradation products were well-resolved from cinacalcet hydrochloride and its impurities. The peak purity test results confirmed that the cinacalcet hydrochloride peak was homogenous in all stress samples and the mass balance was found to be more than 96%, thus proving the stability-indicating power of the method. The developed method was validated according to ICH guidelines.

Plasma membrane proteins Yro2 and Mrh1 are required for acetic acid tolerance in Saccharomyces cerevisiae.

Yro2 and its paralogous protein Mrh1 of Saccharomyces cerevisiae have seven predicted transmembrane domains and predominantly localize to the plasma membrane. Their physiological functions and regulation of gene expression have not yet been elucidated in detail. We herein demonstrated that MRH1 was constitutively expressed, whereas the expression of YRO2 was induced by acetic acid stress and entering the stationary phase. Fluorescence microscopic analysis revealed that Mrh1 and Yro2 were distributed as small foci in the plasma membrane under acetic acid stress conditions. The null mutants of these genes (mrh1∆, yro2∆, and mrh1∆yro2∆) showed delayed growth and a decrease in the productivity of ethanol in the presence of acetic acid, indicating that Yro2 and Mrh1 are involved in tolerance to acetic acid stress.

Development of a Stability-Indicating Stereoselective Method for Quantification of the Enantiomer in the Drug Substance and Pharmaceutical Dosage Form of Rosuvastatin Calcium by an Enhanced Approach.

A novel, simple, precise, and stability-indicating stereoselective method was developed and validated for the accurate quantification of the enantiomer in the drug substance and pharmaceutical dosage forms of Rosuvastatin Calcium. The method is capable of quantifying the enantiomer in the presence of other related substances. The chromatographic separation was achieved with an immobilized cellulose stationary phase (Chiralpak IB) 250 mm x 4.6 mm x 5.0 μm particle size column with a mobile phase containing a mixture of n-hexane, dichloromethane, 2-propanol, and trifluoroacetic acid in the ratio 82:10:8:0.2 (v/v/v/v). The eluted compounds were monitored at 243 nm and the run time was 18 min. Multivariate analysis and statistical tools were used to develop this highly robust method in a short span of time. The stability-indicating power of the method was established by subjecting Rosuvastatin Calcium to the stress conditions (forced degradation) of acid, base, oxidative, thermal, humidity, and photolytic degradation. Major degradation products were identified and found to be well-resolved from the enantiomer peak, 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 and limit of quantification, precision, linearity, accuracy, and robustness. The method exhibited consistent, high-quality recoveries (100 ± 10%) with a high precision for the enantiomer. Linear regression analysis revealed an excellent correlation between the peak responses and concentrations (r2 value of 0.9977) for the enantiomer. The method is sensitive enough to quantify the enantiomer above 0.04% and detect the enantiomer above 0.015% in Rosuvastatin Calcium. The stability tests were also performed on the drug substances as per ICH norms.

Stability-indicating HPLC method for the determination of nicardipine in capsules and spiked human plasma. Identification of degradation products using HPLC/MS

In this stability-indicating, reversed-phase high-performance liquid chromatographic method for nicardipine (NIC), forced degradation has been employed and the formed degradants were separated on a C18 (150mm×3.9mm, 5μm) analytical column using a mobile phase consisted of 70% methanol: acetic acid containing 0.01M triethylamine with pH 4. The flow rate was 1.0mL/min and the photodiode array detection wavelength was 353nm. Forced degradation of the drug was carried out under acidic, basic, photolytic, and oxidative stress conditions. Chromatographic peak purity data indicated no co-eluting peaks with the main peaks. This method resulted in the detection of seven degradation products. Among these, two major degradation products from basic hydrolysis, one from oxidation by H2O2 and four from photolytic stress were identified by mass spectral data. A good linear response was achieved over the range of 0.5–40μg/mL with a limit of detection (LOD) of 0.011μg/mL and limit of quantification (LOQ) of 0.036μg/mL. The suggested method was successfully applied for the analysis of NIC in its commercial capsules, with mean% recovery value of 100.11±2.26%. The method was extended to the in vitro determination on NIC in spiked human plasma samples with mean% recovery of 99.04±5.67%. The suggested method was utilized to investigate the kinetics of photolytic induced degradation.

A versatile, stability-indicating and high-throughput ultra-fast liquid chromatography method for the determination of isoflavone aglycones in soybeans, topical formulations, and permeation assays

There is a growing interest in the pharmaceutical field concerning isoflavones topical delivery systems, especially with regard to their skin care properties and antiherpetic activity. In this context, the present work describes an ultra-fast liquid chromatography method (UFLC) for determining daidzein, glycitein, and genistein in different matrices during the development of topical systems containing isoflavone aglycones (IA) obtained from soybeans. The method showed to be specific, precise, accurate, and linear (0.1 to 5µgmL−1) for IA determination in soybean acid extract, IA-rich fraction obtained after the purification process, IA loaded-nanoemulsions, and topical hydrogel, as well as for permeation/retention assays in porcine skin and porcine esophageal mucosa. The matrix effect was determined for all complex matrices, demonstrating low effect during the analysis. The stability indicating UFLC method was verified by submitting IA to acidic, alkaline, oxidative, and thermal stress conditions, and no interference of degradation products was detected during analysis. Mass spectrometry was performed to show the main compounds produced after acid hydrolysis of soybeans, as well as suggest the main degradation products formed after stress conditions. Besides the IA, hydroxymethylfurfural and ethoxymethylfurfural were produced and identified after acid hydrolysis of the soybean extract and well separated by the UFLC method. The method’s robustness was confirmed using the Plackett-Burman experimental design. Therefore, the new method affords fast IA analysis during routine processes, extract purification, products development, and bioanalytical assays.

An essential role of a ferritin-like protein in acid stress tolerance of Listeria monocytogenes.

The expression of ten genes of Listeria monocytogenes previously identified as penicillin G-inducible was transcriptionally analyzed in the presence of 0.5 M KCl, pH 5.0 and 42 °C. This study revealed that all the genes are upregulated by osmotic stress, seven by acid stress and four by temperature stress conditions. The contribution of a gene encoding a ferritin-like protein (fri), a two-component phosphate-response regulator (phoP) and an AraC/XylS family transcription regulator (axyR) to temperature, acid and osmotic stress tolerance was further examined by analysis of nonpolar deletion mutants. This revealed that a lack of PhoP or AxyR does not affect the ability to grow under the tested stress conditions. However, the Δ fri strain showed slightly delayed growth under osmotic and clearly impaired growth under acid stress conditions, indicating an important role of the ferritin-like protein in acid stress tolerance.

Stability-Indicating HPLC Method for the Simultaneous Determination of HIV Tablet Containing Emtricitabine, Tenofovir Disoproxil Fumarate, and Rilpivirine Hydrochloride in Pharmaceutical Dosage Forms.

A simple, accurate, rapid, and stability-indicating RP-HPLC method for a combination of tenofovir disoproxil fumarate, emtricitabine, and rilpivirine has been developed and subsequently validated in commercial tablets. The proposed HPLC method utilizes Phenomenex Gemini C18 column (150 mm × 4.6 mm i.d., 5 µm) and mobile phase consisting of MeCN, potassium dihydrogen phosphate buffer (20 mM, pH 3.3), and triethylamine 58.72 : 41.23 : 0.05 (v/v) at a flow rate of 1.7 mL/min. Quantitation was achieved with UV detection at 270 nm. The method was validated in terms of accuracy, precision, linearity, limits of detection, limits of quantitation, and robustness. This optimized method has been successively applied to pharmaceutical formulation and no interference from the tablet excipients was found. TDF, EMT, and RPV and their combination drug product were subjected to acid, base, neutral hydrolysis, oxidation, dry heat, and photolytic stress conditions and the stressed samples were analyzed by the proposed method. As the proposed LC method could effectively separate the drugs from its degradation products, it can be employed as stability-indicating method for the determination of instability of these drugs in bulk and commercial tablets.

The Brucella abortus virulence regulator, LovhK, is a sensor kinase in the general stress response signalling pathway.

In the intracellular pathogen Brucella abortus, the general stress response (GSR) signalling system determines survival under acute stress conditions in vitro, and is required for long-term residence in a mammalian host. To date, the identity of the Brucella sensor kinase(s) that function to perceive stress and directly activate GSR signalling have remained undefined. We demonstrate that the flavin-binding sensor histidine kinase, LovhK (bab2_0652), functions as a primary B. abortus GSR sensor. LovhK rapidly and specifically phosphorylates the central GSR regulator, PhyR, and activates transcription of a set of genes that closely overlaps the known B. abortus GSR regulon. Deletion of lovhK severely compromises cell survival under defined oxidative and acid stress conditions. We further show that lovhK is required for cell survival during the early phase of mammalian cell infection and for establishment of long-term residence in a mouse infection model. Finally, we present evidence that particular regions of primary structure within the two N-terminal PAS domains of LovhK have distinct sensory roles under specific environmental conditions. This study elucidates new molecular components of a conserved signalling pathway that regulates B. abortus stress physiology and infection biology.

Biocompatible click chemistry enabled compartment-specific pH measurement inside E. coli.

Bioorthogonal reactions, especially the Cu(I)-catalyzed azide-alkyne cycloaddition, have revolutionized our ability to label and manipulate biomolecules under living conditions. The cytotoxicity of Cu(I) ions, however, has hindered the application of this reaction in the internal space of living cells. By systematically surveying a panel of Cu(I)-stabilizing ligands in promoting protein labeling within the cytoplasm of E. coli, here we identify a highly efficient and biocompatible catalyst for intracellular modification of proteins by azide-alkyne cycloaddition. This reaction permits us to conjugate an environment-sensitive fluorophore site-specifically onto HdeA, an acid-stress chaperone that adopts pH-dependent conformational changes, in both the periplasm and cytoplasm of E. coli. The resulting protein-fluorophore hybrid pH indicators enable compartment-specific pH measurement to determine the pH gradient across the E. coli cytoplasmic membrane. This construct also allows the measurement of E. coli transmembrane potential, and the determination of the proton motive force across its inner membrane under normal and acid-stress conditions.

AtMyb7, a subgroup 4 R2R3 Myb, negatively regulates ABA-induced inhibition of seed germination by blocking the expression of the bZIP transcription factor ABI5.

Various Myb proteins have been shown to play crucial roles in plants, including primary and secondary metabolism, determination of cell fate and identity, regulation of development and involvement in responses to biotic and abiotic stresses. The 126 R2R3 Myb proteins (with two Myb repeats) have been found in Arabidopsis; however, the functions of most of these proteins remain to be fully elucidated. In the present study, we characterized the function of AtMyb7 using molecular biological and genetic analyses. We used qRT-PCR to determine the levels of stress-response gene transcripts in wild-type and atmyb7 plants. We showed that Arabidopsis AtMyb7 plays a critical role in seed germination. Under abscisic acid (ABA) and high-salt stress conditions, atmyb7 plants showed a lower germination rate than did wild-type plants. Furthermore, AtMyb7 promoter:GUS seeds exhibited different expression patterns in response to variations in the seed imbibition period. AtMyb7 negatively controls the expression of the gene encoding bZIP transcription factor, ABI5, which is a key transcription factor in ABA signalling and serves as a crucial regulator of germination inhibition in Arabidopsis.

Influence of lactic acid and acetic acid on Salmonella spp. growth and expression of acid tolerance-related genes

Salmonella spp. is an important foodborne pathogen, often associated with meat products. This pathogen presents a complex tolerance mechanism in the presence of organic acids, which is regulated by a diversity of genes, including rpoS, nlpD and clpP. The present study aimed to measure the expression of such genes by Salmonella strains subjected to acid stress conditions, and associate these data with microbial growth. A culture collection composed of 79 strains of Salmonella spp. obtained from bovine and swine production chains was subjected to PFGE using XbaI, and 3 strains (serovars Derby, Typhimurium and Meleagridis) were selected for acid tolerance trials. The selected strains were inoculated in meat extract broth (MEB) added to lactic or acetic acids at a final pH of 4.0, 5.0 or 6.0, and incubated at 37°C for 6, 12, 24 and 48h. As controls, Salmonella strains were inoculated in MEB at pH7.0, and incubated in the same conditions. Bacterial populations were monitored by direct plating and gene expression using qPCR. Salmonella presented similar populations to controls and evident expression of rpoS at pH5.0 and 6.0. However, Salmonella populations were not detectable after 6h at pH4.0. The adaptability of Salmonella to pH5.0 and 6.0 emphasizes the importance of adequate monitoring of pH reduction during cleaning procedures in food industries, such as organic acid spraying in bovine carcasses. The data obtained demonstrated the relevance of rpoS in the acid tolerance mechanism of Salmonella strains, prompting further studies to investigate its expression in meat systems.

Separation, Identification, and Characterization of Degradation Products of Erlotinib Hydrochloride Under ICH-Recommended Stress Conditions by LC, LC-MS/TOF

The present research work was carried out to determine the stability of erlotinib hydrochloride (ERLO) drug under different stress conditions recommended by International Conference on Harmonization (ICH) guideline Q1A (R2). The stability of the drug was studied under hydrolytic, oxidative, photolytic, and thermal stress conditions. The drug was found susceptible to degradation under acidic, basic, and photolytic stress conditions, while it was stable under neutral, oxidative, and thermal stress conditions. A total of three degradation products (DPs) were formed. Separation was carried out by using high-performance liquid chromatography system (HPLC). Better separation was achieved on Kromasil®(150 mm × 4.6 mm, 5 µm) C18 column using gradient elution program. A mixture of stressed samples was subjected to LC-MS/TOF studies to obtain mass spectral data. The information obtained from accurate mass study was first utilized to build a complete mass fragmentation pathway of the drug, and later it was used for its degradants. Structures were proposed for each fragment based on the best possible molecular formula. Three novel DPs 6-(2-hydroxyethoxy)-7-(2-methoxyethoxy)quinazolin-4-amine, 6,7-bis(2-methoxyethoxy)quinazolin-4-amine, and N-(3-ethynylphenyl)-6, 7-bis (2-methoxyethoxy)-2-oxy-quinazolin-4-amine were identified.

α-Acetolactate synthase of Lactococcus lactis contributes to pH homeostasis in acid stress conditions

Lactic Acid Bacteria (LAB) are recognized as safe microorganisms with the capacity to improve the quality of dairy products. When the LAB Lactococcus lactis is employed as starter for the production of fermented foods, high quantities of important aroma compounds such as diacetyl are generated by means of the diacetyl/acetoin pathway. Our previous results obtained with L. lactis strains report that this pathway is activated under acidic conditions. In this study, we describe the metabolism of pyruvate, a diacetyl/acetoin precursor, and its contribution to pH homeostasis in this microorganism. L lactis strain IL1403 is able to cometabolize pyruvate and glucose at low pH, producing lactate, acetate as well as diacetyl/acetoin compounds. In contrast, the als defective strain, which is incapable of producing C4 compounds, appeared sensitive to pyruvate under acidic conditions rendering it unable to grow. Accordingly, the als-mutant strain showed a simultaneous inability to alkalinize internal and external media. These results demonstrate that the decarboxylation reactions associated to the diacetyl/acetoin pathway represent a competitive advantage in a condition of intracellular pyruvate accumulation during growth at low pH. Interestingly, a genomic comparative analysis shows that this pathway has been conserved in L. lactis during the domestication of different strains. Also, our analysis shows that the recent acquisition of the cit cluster required for citrate metabolism, which contributes to diacetyl/acetoin production as well, is the specific feature of the biovar. diacetylactis. In this regard, we present for first time genetic evidence supporting the proposal made by Passerini et al. (2013) who postulated that the expression “biovar. citrate” should be more appropriate to define this specific industrial strain.

A Validated Quantitative Estimation and Stability Indicating Reversed-phase-high Performance Liquid Chromatography Method for Balofl oxacin in Bulk and its Tablet Formulation

This paper describes the development of a stability-indicating reversed-phase-high performance liquid chromatography (RP-HPLC) method for balofl oxacin (BFX) in the presence of its degradation products generated from forced decomposition studies. The drug substance was found to be susceptible to stress conditions of acid, base, oxidation, wet heat, and photo degradation. The drug was found to be stable to the dry heat condition attempted. Successful separation of the drug from the degradation products formed under stress conditions was achieved on a Zorbax C 18 column (150 mm × 4.6 mm id, 5 microns particle size) using methanol-water pH adjusted to 2.5 by orthophosphoric acid (40:60, v/v) as the mobile phase at a fl ow rate of 0.5 mL/min at 30°C temperature. Quantifi cation was achieved with photodiode array detection at 293 nm over the concentration range 50-150 μg/ml with mean recovery of 100.0 ± 0.53% for BFX by the RP-HPLC method. Statistical analysis proved the method is repeatable, specifi c, and accurate for estimation of BFX. Because the method could effectively separate the drug from its degradation products, it can be used as a stability-indicating method. Key words: Balofl oxacin, reversed-phase-high performance liquid chromatography, orthophosphoric acid, validation.

Selective determination of diclazuril in the presence of its degradation products.

A simple stability-indicating high-performance liquid chromatographic method was developed and validated for the determination of diclazuril (DIC) in the presence of its induced degradation products. The drug was subjected to stress stability study including acidic, alkaline, photolytic, thermal and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. The developed method utilized a C18 column (250 × 4.6 mm, i.d., 5 μm) in an isocratic separation mode with mobile phase consisting of acetonitrile and 0.2% phosphoric acid at a flow rate of 1.2 mL/min with UV-detection at 275 nm. The proposed method was validated according to the International Conference on Harmonization guidelines. The method was applied in short term and accelerated stability studies for determination of the DIC in bulk powder and in its pharmaceutical formulation.

Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models.

Degradation kinetics study of cabozantinib by a novel stability-indicating LC method and identification of its major degradation products by LC/TOF-MS and LC–MS/MS

The chemical stability of cabozantinib (CBZ) was investigated using a novel stability-indicating LC method. Forced degradation of CBZ was carried out under acidic, basic, thermal, oxidative and photolytic stress conditions. Hydrolysis and oxidation were the primary pathways for this compound and three major unknown degradation products were characterized by LC/TOF-MS and LC–MS/MS. The major oxidative degradation product was isolated by preparative LC and identified by UV, HRMS and NMR techniques to be N-{4-[(N-oxide-6,7-dimethoxyquinolin-4-yl)oxy]phenyl}-N′-(4-fluorophenyl)-cyclopropane-1,1-dicarboxamide. The developed method was validated as per ICH guidelines and then successfully applied to investigate the degradation kinetics of CBZ. Degradation of CBZ followed first-order kinetics under all experimental conditions. A V-shaped pH-rate profile over the pH range 2–10 was observed with maximum stability at pH 6. The effect of temperature on the rate of CBZ degradation was characterized using the Arrhenius equation. The activation energy for hydrolysis was 57.31kJmol−1 in alkaline solution.

Development and validation of a stability-indicating RP-LC method for the estimation of process-related impurities and degradation products of oxcarbazepine in pharmaceutical formulation

Summary A stability-indicating gradient reverse-phase liquid chromatographic method was developed for the quantitative determination of process-related impurities and forced degradation products of oxcarbazepine in pharmaceutical formulation. The method was developed by using Inertsil cyano (250 × 4.6 mm) 5 μm column with mobile phase containing a gradient mixture of solvent A (0.01 M sodium dihydrogen phosphate, pH adjusted to 2.7 with orthophosphoric acid and acetonitrile in the ratio of 80:20 v/v) and B (50:40:10 v/v/v mixture of acetonitrile, water, and methanol). The flow rate of mobile phase was 1.0 mL min−1. Column temperature was maintained at 25°C and detection wavelength at 220 nm. Developed reverse-phase high-performance liquid chromatography (RP-HPLC) method can adequately separate and quantitate five impurities of oxcarbazepine, namely imp-A, imp-B, imp-C, imp-D, and imp-E. Oxcarbazepine was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degra...

Liquid chromatography electrospray ionization tandem mass spectrometry study of nilutamide and its stress degradation products: in silico toxicity prediction of degradation products.

Nilutamide, a nonsteroidal anti-androgen drug, widely used in the treatment of prostate cancer, was subjected to hydrolytic, photolytic, thermal and oxidative stress conditions as per International Conference on Harmonization guidelines Q1A (R2). Nilutamide showed significant degradation under basic hydrolysis and photolytic stress conditions, while it was stable to neutral, acidic and thermal stress conditions. Five degradation products were formed and the chromatographic separation of nilutamide and its degradation products was achieved on a Waters C18 column (4.6 × 250 mm, 5 µm) using a mobile phase consisting of acetonitrile and 0.1% of formic acid in an isocratic elution method. All these degradation products were characterized by LC/MS/MS in negative ion mode, combined with accurate mass measurements. To assign likely structures for the observed degradation products, the fragmentation patterns of the deprotonated drug and its degradation products were compared. The in silico toxicity of the drug and its degradation products was also assessed using TOPKAT software. The carcinogenicity probability of the degradation products, DP-I-IV, was greater than that of nilutamide.