Acid Stress Conditions Research Articles

Effects of dietary allspice, Pimenta dioica powder on physiological responses of Oreochromis mossambicus under low pH stress

This study investigated the effects of the supplementation with allspice (0, 5, 10, 15, or 20 g kg−1) on the haemato-immunological and biochemical variables in tilapia, Oreochromis mossambicus under acidic stress condition. In a 60-day feeding trial, 15 aquariums (80-L) were stocked with 18 fish (20.05 ± 0.10 g) each. Then, acidic stress was achieved by exposing the sampled fish to acidic water (pH 5.5) for 3 days. Allspice supplementation influenced the haematological indices, serum glucose, protein, globulin and innate immune parameters such as respiratory burst activity, lysozyme, and myeloperoxidase activities. In general, at acidic pH decreased circulating red blood cell numbers (RBC), increased mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and the innate immune parameters were observed. On the other hand, the inclusion of allspice prevented an increase in blood glucose MCV and MCH, decreases in albumin, RBC, lysozyme activity and respiratory burst avtivity. In conclusion, this study demonstrated that allspice supplementation at 10 g kg−1 for 60 days, has adequate beneficial effects on improvement of haemato-immunological and biochemical status of O. mossambicus after stressful management.

Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid.

Lactobacillus delbrueckii subsp. bulgaricus develops acid tolerance response when subjected to acid stress conditions, such as the induction of enzymes associated with carbohydrate metabolism. In this study, pyk gene encoding pyruvate kinase was over-expressed in heterologous host Lactococcus lactis NZ9000, and SDS-PAGE analysis revealed the successful expression of this gene in NZ9000. The survival rate of Pyk-overproducing strain was 45-fold higher than the control under acid stress condition (pH 4.0). In order to determine the transcription factor (TF) which regulates the expression of pyk by bacterial one-hybrid, we constructed a TF library including 65 TFs of L. bulgaricus. Western blotting indicated that TFs in this library could be successfully expressed in host strains. Subsequently, the promoter of pfk-pyk operon in L. bulgaricus was identified by 5′-RACE PCR. The bait plasmid pH3U3-p01 carrying the deletion fragment of pfk-pyk promoter captured catabolite control protein A (CcpA) which could regulate the expression of pyk by binding to a putative catabolite-responsive element (5′-TGTAAGCCCTAACA-3′) upstream the -35 region. Real-time qPCR analysis revealed the transcription of pyk was positively regulated by CcpA. This is the first report about identifying the TF of pyk in L. bulgaricus, which will provide new insight into the regulatory network.

Growth behavior and temporal enterotoxin D expression of Staphylococcus aureus strains under glucose and lactic acid stress

Ingestion of the staphylococcal enterotoxin D (SED) leads to staphylococcal food poisoning, the most prevalent foodborne intoxication worldwide. Patients suffer from acute signs of gastroenteritis such as violent vomiting, diarrhea, cramps, and fever. As the symptoms result in pronounced electrolyte imbalances and dehydration, the intoxication is particularly dangerous to children and the elderly. SED is formed during growth of Staphylococcus aureus in food. While growth of S. aureus is repressed by competing bacteria in most food matrices, the organism exhibits a crucial competitive growth advantage in foods with low pH or a low aw value (e.g. through high sugar concentrations). To date, the effect of these stress conditions on sed expression is unclear. The objective of this study was to determine sed mRNA expression levels of S. aureus exposed to glucose and lactic acid stress conditions similar to food production and preservation. To this end, temporal sed mRNA expression levels of three S. aureus strains grown at control conditions, glucose stress conditions (30% glucose), and lactic acid stress conditions (pH 6.0) were determined using quantitative Real-Time PCR. Under both glucose and acid stress conditions, the mean lag phase duration was prolonged and maximum cell density in late stationary phase was decreased. In addition, glucose stress slightly increased the growth rate of the tested strains and led to decreased sed expression in late stationary phase. Lactic acid stress had no statistically significant effect on sed expression. Our study provides data on the effect of critical food-related stressors on growth and SE expression of S. aureus, which can be used for risk assessment.

Gibberellic Acid-Stimulated Arabidopsis6 Serves as an Integrator of Gibberellin, Abscisic Acid, and Glucose Signaling during Seed Germination in Arabidopsis.

The DELLA protein REPRESSOR OF ga1-3-LIKE2 (RGL2) plays an important role in seed germination under different conditions through a number of transcription factors. However, the functions of the structural genes associated with RGL2-regulated germination are less defined. Here, we report the role of an Arabidopsis (Arabidopsis thaliana) cell wall-localized protein, Gibberellic Acid-Stimulated Arabidopsis6 (AtGASA6), in functionally linking RGL2 and a cell wall loosening expansin protein (Arabidopsis expansin A1 [AtEXPA1]), resulting in the control of embryonic axis elongation and seed germination. AtGASA6-overexpressing seeds showed precocious germination, whereas transfer DNA and RNA interference mutant seeds displayed delayed seed germination under abscisic acid, paclobutrazol, and glucose (Glc) stress conditions. The differences in germination rates resulted from corresponding variation in cell elongation in the hypocotyl-radicle transition region of the embryonic axis. AtGASA6 was down-regulated by RGL2, GLUCOSE INSENSITIVE2, and ABSCISIC ACID-INSENSITIVE5 genes, and loss of AtGASA6 expression in the gasa6 mutant reversed the insensitivity shown by the rgl2 mutant to paclobutrazol and the gin2 mutant to Glc-induced stress, suggesting that it is involved in regulating both the gibberellin and Glc signaling pathways. Furthermore, it was found that the promotion of seed germination and length of embryonic axis by AtGASA6 resulted from a promotion of cell elongation at the embryonic axis mediated by AtEXPA1. Taken together, the data indicate that AtGASA6 links RGL2 and AtEXPA1 functions and plays a role as an integrator of gibberellin, abscisic acid, and Glc signaling, resulting in the regulation of seed germination through a promotion of cell elongation.

Glutathione is involved in physiological response of Candida utilis to acid stress

Candida utilis often encounters an acid stress environment when hexose and pentose are metabolized to produce acidic bio-based materials. In order to reveal the physiological role of glutathione (GSH) in the response of cells of this industrial yeast to acid stress, an efficient GSH-producing strain of C. utilis CCTCC M 209298 and its mutants deficient in GSH biosynthesis, C. utilis Δgsh1 and Δgsh2, were used in this study. A long-term mild acid challenge (pH3.5 for 6h) and a short-term severe acid challenge (pH1.5 for 2h) were conducted at 18h during batch culture of the yeast to generate acid stress conditions. Differences in the physiological performances among the three strains under acid stress were analyzed in terms of GSH biosynthesis and distribution; intracellular pH; activities of γ-glutamylcysteine synthetase, catalase, and superoxide dismutase; intracellular ATP level; and ATP/ADP ratio. The intracellular GSH content of the yeast was found to be correlated with changes in physiological data, and a higher intracellular GSH content led to greater relief of cells to the acid stress, suggesting that GSH may be involved in protecting C. utilis against acid stress. Results presented in this manuscript not only increase our understanding of the impact of GSH on the physiology of C. utilis but also help us to comprehend the mechanism underlying the response to acid stress of eukaryotic microorganisms.

Stability-Indicating HPTLC Method for Simultaneous Estimation of Flurbiprofen and Chloramphenicol in Ophthalmic Solution.

A specific, accurate and reproducible stability-indicating high performance thin layer chromatography (HPTLC) method was developed for the estimation of flurbiprofen and chloramphenicol in the presence of their degradation products. Degradation studies of both the drugs were carried out in acidic, alkaline, neutral, oxidative, photolytic and thermal stress conditions. Separation was performed on thin layer chromatography plate precoated with silica gel 60 F254 using ethyl acetate : n-hexane : methanol : tri-ethyl amine (5 : 4 : 2 : 0.5, v/v/v/v). Spots at retention factor 0.29 and 0.62 were recognized as flurbiprofen and chloramphenicol, respectively, and were quantified through densitometric measurements at wavelength 267 nm. Method was found to be linear over the concentration range 12-60 ng/spot with correlation coefficient of 0.9997 for flurbiprofen and 200-1,000 ng/spot with correlation coefficient of 0.9977 for chloramphenicol. The proposed method was applied to the estimation of flurbiprofen and chloramphenicol in commercial ophthalmic formulation. The developed HPTLC method can be applied for routine analysis of flurbiprofen and chloramphenicol in the presence of their degradation products in their individual as well as combined pharmaceutical formulations.

Functional profiling in Streptococcus mutans: construction and examination of a genomic collection of gene deletion mutants.

A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene-specific barcoded deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non-essential genes. As S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out-compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared with the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose- or sucrose-containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co-cultures of deletion strains were grown either in vitro in a chemostat to steady-state values of pH 7 and pH 5 or in vivo in an animal model for oral infection. Taken together, these data represent a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.

A transcriptomic analysis of the response of the arctic pteropod Limacina helicina to carbon dioxide-driven seawater acidification

Ocean acidification from the uptake of anthropogenic carbon dioxide (CO2) is regarded as a critical threat particularly to marine calcifying organisms. The arctic pteropod Limacina helicina may be one of the first polar organisms that are expected to display early sensitivity to ocean acidification, but a molecular approach as a foundation for understanding the effect of ocean acidification on this pteropod has rarely been reported. In this study, we examined the sublethal effects of CO2-driven seawater acidification at the transcriptome level in L. helicina. cDNAs, treated under control (pH 8.2), high-CO2 (pH 7.5), and extreme-CO2 (pH 6.5) conditions, generated a total of 31,999,474 reads, comprising a total of 2,271,962,654 bp, using the Illumina platform. De novo assembly yielded 53,121 transcripts comprising 31.79 Mbp. Among the upregulated genes, 346 (0.7 %) and 655 (1.2 %) genes responded to extreme-level CO2 (pH 6.5) and high-level CO2 (pH 7.5), respectively. Also, 76 (0.1 %) transcripts were commonly upregulated in both conditions. Among the downregulated genes, 690 (1.3 %) and 739 (1.4 %) genes were in response to extreme-level CO2 and high-level CO2, respectively. Also, 270 downregulated genes (0.5 %) were affected in both acidic stress conditions. Moreover, 504 transcripts (1 %) of biomineralization-related genes were identified; 16 of these genes showed differential expression in response to acidified seawater. The dataset provides the first comprehensive overview of changes in transcript levels in the arctic pteropod L. helicina in response to increased CO2, emphasizing the potential impact of future environmental change and ocean acidification on Arctic species with external calcified structures.

Identification and characterization of related substances in pomalidomide by hyphenated LC–MS techniques

The current study dealt with the separation, identification and characterization of related substances in pomalidomide by hyphenated techniques. Complete separation was obtained with an Inertsil ODS-SP column (250mm×4.6mm, 5μm) by linear gradient elution using a mobile phase consisting of 0.1% formic acid solution and acetonitrile. They were characterized by hyphenated chromatographic techniques with the accurate mass determination using high resolution LC–TOF–MS methods as well as the product MS spectra determination and elucidation. The degradation behaviors of pomalidomide under ICH prescribed stress conditions were also conducted. Pomalidomide was found to be labile to degrade under acid, alkaline, oxidative and thermal stress conditions, while it was relatively stable to photolytic stress. 13 related substances were detected and identified to be 10 degradation products and three process related substances. The hyphenated LC–MS method with high resolution accurate mass determination facilitated the qualitative analysis of the unknown compounds than that of the conventional HPLC–UV. The related compounds identified are valuable for pomalidomide manufacturing process optimization and quality control.

Sll0751 and Sll1041 are involved in acid stress tolerance in Synechocystis sp. PCC 6803.

The ATP-binding cassette (ABC) transporter is a multi-subunit membrane protein complex involved in lipid transport and acid stress tolerance in the cyanobacterium Synechocystis sp. PCC 6803. This organism has two sets of three ABC transporter subunits: Slr1045 and Slr1344, Sll0751 and Sll1002, and Sll1001 and Sll1041. We previously found that Slr1045 is essential for survival under acid stress condition (Tahara et al. 2012). In the present study, we examined the participation of other ABC transporter subunits in acid stress tolerance using a deletion mutant series of Synechocystis sp. PCC 6803. Although Slr1344 is highly homologous to Slr1045, Δslr1344 cells were not susceptible to acid stress. Δsll0751 and Δsll1041 cells displayed acid stress sensitivity, whereas Δsll1001/sll1002 double mutant cells grew normally. Under high- and low-temperature stress conditions, the growth rate of Δslr1344 and Δsll1001/sll1002 cells did not differ from WT cells, whereas Δsll0751 and Δsll1041 cells showed significant growth retardation, as previously observed in Δslr1045 cells. Moreover, nile red staining showed more lipid accumulation in Δslr1045, Δsll0751, and Δsll1041 cells than in WT cells. These results suggest that Slr1045, Sll0751, and Sll1041 function together as a lipid transport complex in Synechocystis sp. PCC 6803 and are essential for growth under various stresses.

Development and validation of UHPLC-DAD method for the determination of cholesteryl-hexahydrophthaloyl-5-fluorouracil in lipid nanoemulsion

An ultra HPLC-diode array detector (UHPLC-DAD) method was developed and validated for rapid determination of cholesteryl-hexahydrophthaloyl-5-fluorouracil (CH5F) conjugate in standard drug, lipid nanoemulsion and dissolution samples. The chromatographic identification of this conjugate was achieved on Hypersil GOLD 50 × 2.1 mm reversed phase C18 column having a 1.9 μm packing as a stationary phase using methanol-water (80: 20, v/v) as a mobile phase, at a flow rate of 0.4 mL/min with DAD detection at 276 nm. The proposed UHPLC-DAD method is linear in the concentration range of 1–50 μg/mL with correlation coefficient of 0.998. The proposed method is precise, accurate, robust, sensitive and specific for analysis of the conjugate. High assay value (98.7%) of CH5F conjugate in lipid nanoemulsion was obtained by the proposed method. Forced degradation studies indicated that conjugate was sufficiently stable under oxidative stress conditions degraded under acid, base and thermal stress conditions. The proposed UHPLC-DAD method successfully and resolved drug conjugate peak in the presence of its degradation products which established stability and indicating property of the method. Results of the present study indicated that the proposed UHPLC-DAD method can be successfully used for routine determination of CH5F conjugate in standard drug and pharmaceutical formulations.

Robust and Rugged Stability-Indicating HPLC Method for the Determination of Plerixafor and Its Related Impurities in Drug Substances.

A novel, reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of related substances in Plerixafor (PLX) drug substance. PLX is an immunostimulant used to mobilize hematopoietic stem cells in cancer patients. The method is efficient for estimation of all degradation and process-related impurities. The method was developed using the Phenomenex Luna L11 column using the gradient program with mobile phase A and mobile phase B, where mobile phase A consists of pH 2.0 1-heptanesulfonic acid sodium salt buffer and acetonitrile in the ratio of 80:20 (v/v) and mobile phase B consists of pH 2.0 1-heptanesulfonic acid sodium salt buffer and acetonitrile in the ratio of 20:80 (v/v). PLX and its impurities were monitored at 210 nm. The present work is describing the role of ion-pair reagent in the separation of polar compounds. PLX was subjected to various stress conditions of oxidative, acid, base, hydrolytic, thermal, humidity and photolytic degradations. The degradation products were well separated from each other and with the main peak, demonstrating the stability-indicating power of the method. The performance of the method was validated according to the present ICH guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness and robustness.

Selective separation and characterization of the stress degradation products of ondansetron hydrochloride by liquid chromatography with quadrupole time-of-flight mass spectrometry.

Ondansetron hydrochloride was subjected to forced degradation studies under various conditions of hydrolysis (acidic, basic, and neutral), oxidation, photolysis, and thermal as prescribed by International Conference on Harmonisation guideline Q1A (R2). A simple, selective, precise, and accurate high-performance liquid chromatography method was developed on a Waters Xterra C18 (150 × 4.6 mm id, 3.5 μm) column using 10 mM ammonium formate (pH 3.0)/methanol as a mobile phase in gradient elution mode at a flow rate of 0.6 mL/min. The method was extended to liquid chromatography quadrupole time-of-flight tandem mass spectrometry for identification and structural characterization of stress degradation products of ondansetron. The drug showed significant degradation in base hydrolytic and photolytic stress conditions in the liquid state, while it was found to be stable in neutral, acidic, thermal, and oxidative stress conditions. A total of five degradation products were characterized and most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation of the [M + H](+) ions of the drug and its degradation products. Finally, the developed method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonisation guideline Q2 (R1).

The impact of quality filter for RNA-Seq

BackgroundWith the emergence of large-scale sequencing platforms since 2005, there has been a great revolution regarding methods for decoding DNA sequences, which have also affected quantitative and qualitative gene expression analyses through the RNA-Sequencing technique. However, issues related to the amount of data required for the analyses have been considered because they affect the reliability of the experiments. Thus, RNA depletion during sample preparation may influence the results. Moreover, because data produced by these platforms show variations in quality, quality filters are often used to remove sequences likely to contain errors to increase the accuracy of the results. However, when reads of quality filters are removed, the expression profile in RNA-Seq experiments may be influenced. ResultThe present study aimed to analyze the impact of different quality filter values for Corynebacterium pseudotuberculosis (sequenced by SOLiD platform), Microcystis aeruginosa and Kineococcus radiotolerans (sequenced by Illumina platform) RNA-Seq data. Although up to 47.9% of the reads produced by the SOLiD technology were removed after the QV20 quality filter is applied, and 15.85% were removed from K. radiotolerans data set using the QV30 filter, Illumina data showed the largest number of unique differentially expressed genes after applying the most stringent filter (QV30), with 69 genes. In contrast, for SOLiD, the acid stress condition with the QV20 filter yielded only 41 unique differentially expressed genes. Even for the highest quality M. aeruginosa data, the quality filter affected the expression profile. The most stringent quality filter generated a greater number of unique differentially expressed genes: 9 for high molecular weight dissolved organic matter condition and 12 for low P conditions. ConclusionEven high-accuracy sequencing technologies are subject to the influence of quality filters when evaluating RNA-Seq data using the reference approach.

Impact of growth conditions and role of sigB on Listeria monocytogenes fitness in single and mixed biofilms cultured with Lactobacillus plantarum

The role of sigB, a major transcriptional regulator of stress response genes, was assessed in formation of single and mixed species biofilms of Listeria monocytogenes EGD-e and Lactobacillus plantarum WCFS1 as secondary species at 20°C and 30°C using different medium compositions (nutrient-rich medium with and without supplementary manganese, glucose and salt). L. monocytogenes showed significant biofilm development at both temperatures and in all media tested although less biofilm was formed when glucose was supplemented only. The contribution of L. monocytogenes to the mixed species biofilm declined especially at higher temperature in glucose-rich medium in the absence and presence of manganese, due to lactic acid formation with concomitant decrease in culture pH below the pHmin of L. monocytogenes. Using an in-frame sigB deletion mutant and a complementation mutant we showed that sigB contributed to survival under these acid stress conditions. Notably, the additional presence of salt protected L. monocytogenes in the acidic mixed species biofilms resulting in an increase of around 2–3log10cfu/ml and this phenomenon showed to be sigB-dependent.

Genomic analysis of parallel-evolved cyanobacterium Synechocystis sp. PCC 6803 under acid stress.

Experimental evolution is a powerful tool for clarifying phenotypic and genotypic changes responsible for adaptive evolution. In this study, we isolated acid-adapted Synechocystis sp. PCC 6803 (Synechocystis 6803) strains to identify genes involved in acid tolerance. Synechocystis 6803 is rarely found in habitants with pH<5.75. The parent (P) strain was cultured in BG-11 at pH 6.0. We gradually lowered the pH of the medium from pH 6.0 to pH 5.5 over 3months. Our adapted cells could grow in acid stress conditions at pH 5.5, whereas the parent cells could not. We performed whole-genome sequencing and compared the acid-adapted and P strains, thereby identifying 11 SNPs in the acid-adapted strains, including in Fo F1-ATPase. To determine whether the SNP genes responded to acid stress, we examined gene expression in the adapted strains using quantitative reverse-transcription polymerase chain reaction. sll0914, sll1496, sll0528, and sll1144 expressions increased under acid stress in the P strain, whereas sll0162, sll0163, slr0623, and slr0529 expressions decreased. There were no differences in the SNP genes expression levels between the P strain and two adapted strains, except for sll0528. These results suggest that SNPs in certain genes are involved in acid stress tolerance in Synechocystis 6803.

Development and validation of a novel stability-indicating HPLC method for the quantitative determination of eleven related substances in ezetimibe drug substance and drug product

Ezetimibe is a novel lipid-lowering agent that inhibits intestinal absorption of dietary and biliary cholesterol. In the present work, a simple, sensitive and reproducible gradient reverse phase high performance liquid chromatographic (RP-HPLC) method for separation and determination of the related substances of ezetimibe was developed and validated. Eleven potential process-related impurities (starting materials, (3S,4S,3'S)-isomer, degradants and byproducts) were identified in the crude samples. Tentative structures for all the impurities were assigned primarily based on comparison of their retention time and mass spectrometric data with that of available standards and references. This method can be applied to routine analysis in quality control of both bulk drugs and commercial tablets. Separation of all these compounds was performed on a Phenomenex Luna Phenyl-Hexyl (100mm×4.6mm, 5μm) analytical column. The mobile phase-A consists of acetonitrile–water (pH adjusted to 4.0 with phosphoric acid)–methanol at 15:75:10 (v/v/v), and mobile phase-B contains acetonitrile. The eluted compounds were monitored at 210nm. Ezetimibe was subjected to hydrolytic, acid, base, oxidative, photolytic and thermal stress conditions as per ICH serves to generate degradation products that can be used as a worst case to assess the analytical method performance. The drug showed extensive degradation in thermal, acid, oxidative, base and hydrolytic stress conditions, while it was stable to photolytic degradation conditions. The main degradation product formed under thermal, acid, oxidative, base and hydrolytic stress conditions corresponding to (2R,3R,6S)-N, 6-bis(4-fluorophenyl)-2-(4-hydroxyphenyl)-oxane-3-carboxamide (Ezetimibe tetrahydropyran impurity) was characterized by LC-MS/MS analysis. The degradation products were well resolved from the main peak and its impurities, thus proved 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 (LOD), limit of quantification (LOQ), accuracy, precision and robustness.

A new, rapid, stability-indicating UPLC method for separation and determination of impurities in amlodipine besylate, valsartan and hydrochlorothiazide in their combined tablet dosage form

A new rapid stability-indicating UPLC method for separation and determination of impurities in amlodipine besylate, valsartan and hydrochlorothiazide in their combined tablet dosage form was developed. The separation of Ph. Eur. related substances of amlodipine besylate (A, B, D, E, F, G), hydrochlorothiazide (A, B, C), valsartan (B, C), two other valsartan impurities (S)-2-(N-{[2′-cyanobiphenyl-4-yl]methyl}pentanamido)-3-methylbutanoic acid and (S)-3-methyl-2-{[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methylamino}butanoic acid and several unknown impurities was achieved by reversed phase liquid chromatography with UV detection. The detection wavelengths were set as follows: 225nm for valsartan, its impurities and for the impurity D of amlodipine, 271nm for hydrochlorothiazide and its impurities and 360nm for amlodipine and its impurities except for impurity D. Zorbax Eclipse C8 RRHD (100mm×3.0mm, 1.8μm) was used as a separation column and the analytes were eluted within 11min by a programmed gradient mixture of 0.01M phosphate buffer pH 2.5 and acetonitrile. The method was successfully validated in accordance to the International Conference of Harmonization (ICH) guidelines for amlodipine besylate and its impurity D, valsartan and its impurity C and hydrochlorothiazide and its impurities A, B and C. The triple-combined tablets were exposed to thermal, higher humidity, acid, alkaline, oxidative and photolytic stress conditions. Stressed samples were analyzed by the proposed method. All the significant degradation products and impurities were satisfactory separated from each other and from the principal peaks of drug substances. The peak purity test complied for peaks of amlodipine, valsartan and hydrochlorothiazide in all the stressed samples and indicated no co-elution of degradation products. The method was found to be precise, linear, accurate, sensitive, specific, robust and stability-indicating and could be used as a routine purity test method for amlodipine besylate, valsartan, hydrochlorothiazide and their pharmaceutical combinations.

A Novel, Rapid, and Validated Stability-Indicating UPLC Method for the Estimation of Drotaverine Hydrochloride and Ibuprofen Impurities in Oral Solid Dosage Form.

A novel, stability-indicating, reversed-phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the determination of pure drotaverine hydrochloride and ibuprofen in the presence of their impurities and degradation products. The method was developed using a Waters UPLC BEH C18, 100 × 2.1 mm, 1.7 µm column with a flow rate of 0.3 mL/min and detector wavelength at 210 nm. The mobile phase consisted of potassium dihydrogen orthophosphate buffer and acetonitrile. Drotaverine hydrochloride and ibuprofen were subjected to the stress conditions of oxidative, acid, base, photolytic, and thermal degradation. Degradation products resulting from the stress studies were well-resolved, thus confirming the test method as stability-indicating. Validation of the method was carried out as per International Conference on Harmonization guidelines.

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.