Acid Stress Conditions Research Articles

Development and validation of a stability indicating LC-PDA-MS/MS method for separation, identification and characterization of process related and stress degradation products of rivaroxaban

An isocratic reversed phase high-performance liquid chromatography coupled with a photo diode array and electrospray ionization tandem mass spectrometry (LC-PDA-ESI-MS/MS) method was developed for the identification and characterization of stress degradation products (DPs) and an unknown process related impurity (PR) of rivaroxaban (RVR) in bulk drugs. RVR, a selective and direct factor Xa inhibitor, was subjected to hydrolysis (acidic, alkaline and neutral), photolysis, thermolysis, and oxidation as per ICH guidelines and found susceptible to acid and base hydrolytic stress conditions. In total, three DPs (DP-1, DP-2, and DP-3) were formed. All the DPs, PR and RVR were well separated on a Kinetex C18 (150 × 4.6 mm, 5 μm) column using a mobile phase consisting of 20 mM ammonium acetate and acetonitrile (65 : 35 v/v) at a flow rate of 1.0 ml min−1. To elucidate the structures of PR and DPs, MS/MS experiments were carried out to study the molecular ion fragmentation of RVR, PR, and DPs. The product ions of PR and DPs were compared with those of the RVR molecular ion to assign most possible structures. Potential impurities were isolated by semi-preparative HPLC and subjected to NMR spectroscopy. The LC-PDA method was validated with respect to specificity, linearity, accuracy, precision and robustness as per ICH guidelines.

Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex

Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications.

DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING RP-HPLC METHOD FOR THE ESTIMATION OF DIACEREIN IMPURITIES IN API AND PHARMACEUTICAL FORMULATION

A sensitive, stability-indicating gradient RP-HPLC method has been developed for the quantitative determination of impurities of Diacerein in API and pharmaceutical formulation. Efficient chromatographic separation was achieved on a Hypersil BDS C18, 250 × 4.6 mm, 5 µm column with mobile phase containing a gradient mixture of solvents A and B. The flow rate of the mobile phase was 1.0 mL min−1 with column temperature of 40°C and detection wavelength at 254 nm. The relative response factor values of Rhein, Aloe Emodin, Monoacetyl Rhein I, and Monoacetyl Rhein II were 0.86, 0.80, 0.81, and 0.89, respectively. Diacerein formulation sample was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Diacerein was found to degrade significantly in acid, base, and peroxide stress conditions. The degradation products were well-resolved from Diacerein and its impurities. The peak purity test results confirmed that the Diacerein 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 ICH guidelines with respect to specificity, linearity, limits of detection and quantification, accuracy, precision, and robustness.

Roles of rpoS‐activating small RNAs in pathways leading to acid resistance of Escherichia coli

Escherichia coli and related enteric bacteria can survive under extreme acid stress condition at least for several hours. RpoS is a key factor for acid stress management in many enterobacteria. Although three rpoS-activating sRNAs, DsrA, RprA, and ArcZ, have been identified in E. coli, it remains unclear how these small RNA molecules participate in pathways leading to acid resistance (AR). Here, we showed that overexpression of ArcZ, DsrA, or RprA enhances AR in a RpoS-dependent manner. Mutant strains with deletion of any of three sRNA genes showed lowered AR, and deleting all three sRNA genes led to more severe defects in protecting against acid stress. Overexpression of any of the three sRNAs fully rescued the acid tolerance defects of the mutant strain lacking all three genes, suggesting that all three sRNAs perform the same function in activating RpoS required for AR. Notably, acid stress led to the induction of DsrA and RprA but not ArcZ.

Enhanced inactivation of Escherichia coli and Listeria monocytogenes by exposure to 405 nm light under sub-lethal temperature, salt and acid stress conditions

The antimicrobial effects of 405nm light have generated interest in its use as an emerging disinfection technology with potential food-related applications. The aim of this study was to assess the bactericidal efficacy of 405nm light for inactivation of Escherichia coli and Listeria monocytogenes under sub-lethally stressed environmental conditions. Bacteria were exposed to 405nm light from a light emitting diode (LED) array under various temperature, salt (NaCl) and acid conditions to determine if bacterial susceptibility to 405nm light inactivation is affected when exposed under these conditions. Non-stressed bacterial populations (105CFU/mL) were exposed to increasing doses of 405nm light (~70mW/cm2) and the inactivation results were compared with those generated under stress conditions. Bacteria were held at various temperatures (4°C, 22°C and 45°C), acid concentrations (pH3, 3.5 and 7) and salt concentrations (0%, 0.8%, 10% and 15% NaCl), and simultaneously exposed to 405nm light. Enhanced inactivation of both E. coli and L. monocytogenes was achieved when light exposure was combined with each of the sub-lethal stresses, with significantly increased inactivation rates compared to non-stressed populations (P≤0.05). One exception was with L. monocytogenes when light-exposed in the presence of 15% salt, as this combination reduced bacterial inactivation. The greatest enhancement of 405nm light inactivation for both bacterial species was achieved when light exposure was combined with sub-lethal acid stress conditions at pH3. This was demonstrated by a 5-log10 reduction of E. coli following a 405nm light dose of 84J/cm2 compared to 378J/cm2 for non-stressed populations (77% reduction in dose) and by a 5-log10 reduction of L. monocytogenes achieved with a dose of 42J/cm2 which corresponded to 50% of the dose required for the equivalent reduction of non-stressed populations. This acid-enhanced 405nm light inactivation effect was demonstrated with E. coli and L. monocytogenes when dispersed in liquid suspension and when deposited on a test surface. Overall, results from this study have shown that sub-lethally stressed bacteria have increased susceptibility to 405nm light inactivation, thereby providing a synergistic inactivation effect, findings which increase the potential of this new light-based decontamination technology for food related applications.

LC–MS/MS characterization of forced degradation products of zofenopril

A rapid, specific and reliable isocratic LC–MS/MS method has been developed and validated for the identification and characterization of stressed degradation products of Zofenopril. Zofenopril, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and base hydrolysis stress conditions. However, it was stable to thermal, acid, neutral and photolysis stress conditions. A total of 6 degradation products were observed and the chromatographic separation of the drug and its degradation products were achieved on Phenomenex (Luna) C18 (250mm×4.6mm, i.d., 5μm) column using 20mM ammonium acetate: acetonitrile (50:50, v/v) as a mobile phase. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. The LC–MS method was validated with respect to specificity, linearity, accuracy and precision. No previous reports were found in the literature regarding the degradation behavior of zofenopril.

Development and Validation of a Stability-Indicating RP-HPLC Method for the Estimation of Drotaverine Impurities in API and Pharmaceutical Formulation.

A sensitive, stability-indicating gradient RP-HPLC method with PDA detection has been developed for the simultaneous analysis of drotaverine impurities in active pharmaceutical ingredient (API) and pharmaceutical formulations. Efficient chromatographic separation was achieved on an XTerra RP18, 150 × 4.6 mm, 5 μm column using gradient elution at 230 nm detection wavelength. The optimized mobile phase consisted of a 0.02 M potassium dihydrogen orthophosphate buffer of pH 3.0 as solvent A and acetonitrile as solvent B. The flow rate of the mobile phase was 1.0 mL min−1 with a column temperature of 25°C. The method showed linearity over the range of 0.251–10.033 μg/mL, 0.231–9.995 μg/mL, 0.230–10.089 μg/mL, 0.334–10.011 μg/mL, and 0.324–10.050 μg/mL for impurities 1, 2, 3, 4, and drotaverine, respectively, with a correlation coefficient greater than 0.999. The relative retention times and relative response factors of impurities 1, 2, 3, 4 were 0.36, 0.90, 1.42, 1.55 and 1.04, 0.84, 1.10, 1.30, respectively. The drotaverine formulation sample was subjected to the stress conditions of acid, base, oxidative, thermal, humidity, and photolytic degradation. Drotaverine was found to degrade significantly in peroxide, base, and heat stress conditions. The degradation products were well-resolved from drotaverine and its impurities. The peak purity test results confirmed that the drotaverine 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 ICH guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, and robustness.

Development and Validation of a Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Guaifenesin and Dextromethorphan Impurities in Pharmaceutical Formulations

A sensitive, stability-indicating gradient RP-HPLC method has been developed for the simultaneous estimation of impurities of Guaifenesin and Dextromethorphan in pharmaceutical formulations. Efficient chromatographic separation was achieved on a Sunfire C18, 250 × 4.6 mm, 5 µm column with mobile phase containing a gradient mixture of solvents A and B. The flow rate of the mobile phase was 0.8 mL min−1 with column temperature of 50°C and detection wavelength at 224 nm. Regression analysis showed an r value (correlation coefficient) greater than 0.999 for Guaifenesin, Dextromethorphan, and their impurities. Guaifenesin and Dextromethorphan formulation sample was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Guaifenesin was found stable and Dextromethorphan was found to degrade significantly in peroxide stress condition. The degradation products were well resolved from Guaifenesin, Dextromethorphan, and their impurities. The peak purity test results confirmed that the Guaifenesin and Dextromethorphan 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 ICH guidelines with respect to specificity, linearity, limits of detection and quantification, accuracy, precision, and robustness.

Channel-mediated lactic acid transport: a novel function for aquaglyceroporins in bacteria

MIPs (major intrinsic proteins), also known as aquaporins, are membrane proteins that channel water and/or uncharged solutes across membranes in all kingdoms of life. Considering the enormous number of different bacteria on earth, functional information on bacterial MIPs is scarce. In the present study, six MIPs [glpF1 (glycerol facilitator 1)-glpF6] were identified in the genome of the Gram-positive lactic acid bacterium Lactobacillus plantarum. Heterologous expression in Xenopus laevis oocytes revealed that GlpF2, GlpF3 and GlpF4 each facilitated the transmembrane diffusion of water, dihydroxyacetone and glycerol. As several lactic acid bacteria have GlpFs in their lactate racemization operon (GlpF1/F4 phylogenetic group), their ability to transport this organic acid was tested. Both GlpF1 and GlpF4 facilitated the diffusion of D/L-lactic acid. Deletion of glpF1 and/or glpF4 in Lb. plantarum showed that both genes were involved in the racemization of lactic acid and, in addition, the double glpF1 glpF4 mutant showed a growth delay under conditions of mild lactic acid stress. This provides further evidence that GlpFs contribute to lactic acid metabolism in this species. This lactic acid transport capacity was shown to be conserved in the GlpF1/F4 group of Lactobacillales. In conclusion, we have functionally analysed the largest set of bacterial MIPs and demonstrated that the lactic acid membrane permeability of bacteria can be regulated by aquaglyceroporins.

Development and Validation of a Stability Indicating RP-UPLC Method for Analysis of Imipramine Hydrochloride in Pharmaceuticals

The objective of the current study was the development of a simple, rapid, and accurate isocratic reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method for the routine control analysis of imipramine hydrochloride (IMH) in bulk drug and in pharmaceutical formulations. This work was carried out in order to reduce analysis time and maintaining good efficiency which in turn is focused on high-speed chromatographic separations. The method was developed using Waters Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with mobile phase consisting of a mixture of acetonitrile and ammonium acetate buffer of pH-5 (80 : 20, v/v/v). UV detection was performed at 220 nm for eluted compound. An excellent linearity was observed in the concentration range 0.2–3 µg/mL IMH with a regression coefficient () value of 0.9999. The method developed was validated and forced degradation was performed as per ICH guidelines. The limit of detection () was 0.2532 ng/mL and the limit of quantitation () was found to be 0.7672 ng/mL. The drug IMH was subjected to hydrolytic, acidic, basic, thermal, photolytic, and oxidative stress conditions according to ICH regulations. IMH was found to be stable in basic, thermal, and photolytic conditions and degrades in acidic, hydrolytic, and oxidative stress conditions.

Stability-Indicating HPLC Method for Determination of Fosamprenavir Calcium

A novel stability-indicating reverse-phase high-performance liquid chromatographic (HPLC) method has been developed for quantitative determination of Fosamprenavir Calcium, HIV-1 protease inhibitor. Chromatographic separation was achieved using an YMC Pack ODS AQ (150 mm × 4.6 mm × 3.0 μm) HPLC column in isocratic mode employing 0.05 M Potassium dihydrogen orthophosphate monohydrate (pH 6.8) buffer and Acetonitrile in the ratio 60:40 (v/v) with a flow rate of 0.8 mL min(-1). Detector wavelength was monitored at 265 nm and column temperature was maintained at 40 °C. Fosamprenavir calcium was exposed to thermal, photolytic, humidity, water, acid, base and oxidative stress conditions. Considerable degradation of the drug substance was found to occur under acid, base and oxidative stress conditions. Peak homogeneity data of Fosamprenavir Calcium obtained by photodiode array detection demonstrated the specificity of the method in the presence of degradants. The degradation products were well resolved from the main peak of Fosamprenavir, indicating that the method is specific and stability-indicating. The HPLC method was validated as per International Conference on Harmonization guidelines with respect to specificity, precision, linearity, accuracy and robustness. Regression analysis showed a correlation coefficient value greater than 0.999. The accuracy of the method was established based on the recovery obtained for Fosamprenavir Calcium.

Development and validation of a stability‐indicating assay including the isolation and characterization of degradation products of metaxalone by LC‐MS

A stability-indicating reverse-phase high-performance liquid chromatography-mass spectrometric method was developed and validated for the assay of metaxalone through forced degradation under acidic, alkaline, photo, oxidative and peroxide stress conditions. Separation of degradation products was accomplished on a reverse-phase Phenomenex C18 (250 × 4.6 mm, 5 µm) column thermostated at 25 °C using 10 mM aqueous ammonium acetate: methanol (35:65 v/v) as mobile phase in an isocratic mode of elution. The eluents were detected at 275 nm by photo diode array detector and mass detectors connected in series. Two unknown base hydrolysis products of metaxalone were identified and characterized as (a) methyl 3-(3,5-dimethylphenoxy)-2-hydroxypropylcarbamate and (b) 1-(3,5-dimethylphenoxy)-3-aminopropan-2-ol by MS, (1)H NMR and FTIR spectroscopy. The method was validated as per International Conference on Harmonization guidelines and metaxalone was selectively determined in presence of its degradation impurities, demonstrating its stability-indicating nature.

Pullulan production and physiological characteristics of Aureobasidium pullulans under acid stress

In this study, batch processes of pullulan production by Aureobasidium pullulans CCTCC M 2012259 under different pH environments were evaluated. The pH of the medium decreased quickly to an acid stress condition under batch fermentation without pH control. A higher pullulan production was always obtained with a lower biomass under a given glucose concentration with constant pH control, and vice versa. Based on the nonlinear regression analysis of the results obtained from diverse pH control modes, a constant controlled pH of 3.8 was predicted as an optimum pH for efficient pullulan production using a one-element cubic equation. A maximum pullulan concentration of 26.8g/L and a minimum biomass of 8.1g/L were achieved under the optimal pH of 3.8, which were in good agreement with the results predicted by the mathematical model. Further information on the physiological characteristics of A. pullulans CCTCC M 2012259 such as intracellular pH, NADH/NAD(+), ATP/ADP, and glutathione generation under moderate or severe acidic conditions were investigated, and the results presented more evidence on why pullulan biosynthesized with high efficiency under moderate acid stress (e.g., pH3.8), which would also help us to better understand the response of the cells to acid stress.

Stability-Indicating UPLC Method for Determining Related Substances and Degradants in Dronedarone

A simple, sensitive and reproducible method was developed on ultra-performance liquid chromatography coupled with photodiode array detection for the quantitative determination of dronedarone hydrochloride (DRO) in drug substance and pharmaceutical dosage forms. The method is applicable for the quantification of related substances and assays of drug substances. Chromatographic separation was achieved on Acquity UPLC BEH C8 100 mm, 2.1 mm and 1.7 µm columns, using gradient elution within a short run time of 10.0 min. The eluted compounds were monitored at 288 nm, the flow rate was 0.5 mL/min and the column oven temperature was maintained at 40°C. The resolution of DRO and 11 impurities (potentials and by-products) was greater than 2.0 for all pairs of components. The high correlation coefficient value (>0.9995) indicates the clear correlations between the concentrations of investigated compound and their peak areas within the test ranges. The repeatability and intermediate precision, expressed by the relative standard deviation, were less than 2.5%. The accuracy and validity of the method were further ascertained by performing recovery studies via a spike method. The accuracy of the method, expressed as relative error, was satisfactory. No interference was observed from concomitant substances normally added to the tablets. DRO was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. DRO was found to degrade significantly in acid and base stress conditions and to remain stable in thermal, photolytic degradation, oxidative and hydrolytic conditions. The degradation products were well resolved from primary peak and its impurities, proving that the method is stability indicating. The developed method was validated as per International Conference on Harmonization guidelines with respect to specificity, limit of detection, limit of quantification, linearity, accuracy, precision, solution stability and robustness. This method is also suitable for the determination of DRO drug substance and pharmaceutical dosage forms.

Novel validated stability-indicating UPLC method for the determination of Metoclopramide and its degradation impurities in API and pharmaceutical dosage form

AimTo develop a stability-indicating reversed phase ultra performance liquid chromatographic (RP-UPLC) method for the determination of related substances in Metoclopramide bulk drugs and pharmaceutical dosage form. MethodThe chromatographic separation was achieved using a Waters X-terra RP18 (150 × 4.6 mm), 3.5 μm particle size column using the gradient program with mobile phase consisting of solvent A: 30 mM monobasic sodium phosphate and 2.3 mM of pentane-1-sulphonic acid sodium salt (pH 3.0 buffer) and solvent-B (Acetonitrile). A flow rate of 1.2 mL/min and UV detector at 273 nm was used. The runtime was 18 min within which Metoclopramide and its four impurities, ACETYLMETO, ACMA, CLEE and ACME were well separated. Results and discussionThe drug was subjected to stress conditions such as oxidative, acid & base hydrolysis, thermal and photolytic degradation. Metoclopramide was found to degrade significantly in photolytic, oxidative & thermal stress conditions and stable in acid, base, hydrolytic & humidity stress conditions. The major degradation impurities in oxidation and photolytic degradation were identified by LCMS. The degradation products were well resolved from the main peak and its impurities, thus proved the stability-indicating power of the method. ConclusionThe developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. The calibration curves obtained for the four impurities were linear over the range 0.062–3.040 μg/mL.

Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Ceftaroline Fosamil in Bulk and Its Parenteral Dosage Forms

The present method describes the development of a validated RP-HPLC method for determination of ceftaroline fosamil in presence of its degradation products or other pharmaceutical excipients. The drug substance was subjected to stress conditions of acid, alkali, and oxidative and thermal degradation studies. Separation was carried out on a C-18 X-terra column (Waters Corporation, 250 mm × 4.6 mm I.D.; particle size 5 μm) using 40 : 30 : 30 [buffer : acetonitrile : methanol] as mobile phase at a flow rate of 1.0 ml/min. UV detection was performed at 242 nm. The method was validated with respect to specificity, selectivity, linearity, accuracy, precision, and robustness. The assay method was found to be linear in the range of 40 to 120 μg/mL with a correlation coefficient of 0.9999. The percentage recovery of active pharmaceutical ingredient from parenteral dosage form ranged from 99.5 to 100.2%. The method precision for determination of ceftaroline was below 0.85%. The results showed that the developed RP-HPLC method is suitable for determination of ceftaroline fosamil in bulk as well as stability samples of pharmaceutical dosage forms containing various excipients.

Identification and analysis of MKK and MPK gene families in canola (Brassica napusL.)

BackgroundEukaryotic mitogen-activated protein kinase (MAPK/MPK) signaling cascades transduce and amplify environmental signals via three types of reversibly phosphorylated kinases to activate defense gene expression. Canola (oilseed rape, Brassica napus) is a major crop in temperate regions. Identification and characterization of MAPK and MAPK kinases (MAPKK/MKK) of canola will help to elucidate their role in responses to abiotic and biotic stresses.ResultsWe describe the identification and analysis of seven MKK (BnaMKK) and 12 MPK (BnaMPK) members from canola. Sequence alignments and phylogenetic analyses of the predicted amino acid sequences of BnaMKKs and BnaMPKs classified them into four different groups. We also examined the subcellular localization of four and two members of BnaMKK and BnaMPK gene families, respectively, using green fluorescent protein (GFP) and, found GFP signals in both nuclei and cytoplasm. Furthermore, we identified several interesting interaction pairs through yeast two-hybrid (Y2H) analysis of interactions between BnaMKKs and BnaMPKs, as well as BnaMPK and BnaWRKYs. We defined contiguous signaling modules including BnaMKK9-BnaMPK1/2-BnaWRKY53, BnaMKK2/4/5-BnaMPK3/6-BnaWRKY20/26 and BnaMKK9-BnaMPK5/9/19/20. Of these, several interactions had not been previously described in any species. Selected interactions were validated in vivo by a bimolecular fluorescence complementation (BiFC) assay. Transcriptional responses of a subset of canola MKK and MPK genes to stimuli including fungal pathogens, hormones and abiotic stress treatments were analyzed through real-time RT-PCR and we identified a few of BnaMKKs and BnaMPKs responding to salicylic acid (SA), oxalic acid (OA), Sclerotinia sclerotiorum or other stress conditions. Comparisons of expression patterns of putative orthologs in canola and Arabidopsis showed that transcript expression patterns were generally conserved, with some differences suggestive of sub-functionalization.ConclusionsWe identified seven MKK and 12 MPK genes from canola and examined their phylogenetic relationships, transcript expression patterns, subcellular localization, and protein-protein interactions. Not all expression patterns and interactions were conserved between canola and Arabidopsis, highlighting the limitations of drawing inferences about crops from model species. The data presented here provide the first systematic description of MKK-MPK-WRKY signaling modules in canola and will further improve our understanding of defense responses in general and provide a basis for future crop improvement.

The Streptococcus mutans Aminotransferase Encoded by ilvE Is Regulated by CodY and CcpA

The aminotransferase IlvE was implicated in the acid tolerance response of Streptococcus mutans when a mutation in its gene resulted in an acid-sensitive phenotype (B. Santiago, M. MacGilvray, R. C. Faustoferri, and R. G. Quivey, Jr., J. Bacteriol. 194:2010-2019, 2012). The phenotype suggested that amino acid metabolism is important for acid adaptation, as turnover of branched-chain amino acids (bcAAs) could provide important signals to modulate expression of genes involved in the adaptive process. Previous studies have demonstrated that ilvE is regulated in response to the external pH, though the mechanism is not yet established. CodY and CcpA have been shown to regulate expression of branched-chain amino acid biosynthetic genes, suggesting that the ability to sense carbon flow and the nutritional state of the cell also plays a role in the regulation of ilvE. Electrophoretic mobility shift assays using the ilvE promoter and a purified recombinant CodY protein provided evidence of the physical interaction between CodY and ilvE. In order to elucidate the signals that contribute to ilvE regulation, cat reporter fusions were utilized. Transcriptional assays demonstrated that bcAAs are signaling molecules involved in the repression of ilvE through regulation of CodY. In a codY deletion background, ilvE transcription was elevated, indicating that CodY acts a repressor of ilvE transcription. Conversely, in a ccpA deletion background, ilvE transcription was reduced, showing that CcpA activated ilvE transcription. The effects of both regulators were directly relevant for transcription of ilvE under conditions of acid stress, demonstrating that both regulators play a role in acid adaptation.

A validated stability-indicating RP-LC method for the estimation of process-related impurities and degradation products of quetiapine fumarate in solid oral dosage form

Summary A simple, selective, and stability-indicating reverse phase liquid chromatographic method has been developed and validated for the simultaneous determination of impurities and forced degradation products of quetiapine fumarate. The chromatographic separation was achieved on Inertsil-3 C8, 150 mm × 4.6 mm, 5 μm column at 35°C with UV detection at 217 nm using gradient mobile phase at a flow rate of 1.0 mL/min. Mobile phase A contains a mixture of 0.01 M di-potassium hydrogen orthophosphate (pH 6.8) and acetonitrile in the ratio of 80:20 (v/v), respectively, and mobile phase B contains a mixture of 0.01 M di-potassium hydrogen orthophosphate (pH 6.8) and acetonitrile in the ratio of 20:80 (v/v), respectively. The drug product was subjected to the stress conditions of oxidative, hydrolysis (acid and base), hydrolytic, thermal, and photolytic degradation. Quetiapine fumarate was found to degrade significantly in acid, base, and oxidative stress conditions. The degradation products were well resolved f...

Selective separation, detection of zotepine and mass spectral characterization of degradants by LC–MS/MS/QTOF

A simple, precise, accurate stability-indicating gradient reversed-phase high-performance liquid chromatographic (RP–HPLC) method was developed for the quantitative determination of zotepine (ZTP) in bulk and pharmaceutical dosage forms in the presence of its degradation products (DPs). The method was developed using Phenomenex C18 column (250mm×4.6mm i.d., 5µm) with a mobile phase containing a gradient mixture of solvents, A (0.05% trifluoroacetic acid (TFA), pH=3.0) and B (acetonitrile). The eluted compounds were monitored at 254nm; the run time was within 20.0min, in which ZTP and its DPs were well separated, with a resolution of >1.5. The stress testing of ZTP was carried out under acidic, alkaline, neutral hydrolysis, oxidative, photolytic and thermal stress conditions. ZTP was found to degrade significantly in acidic, photolytic, thermal and oxidative stress conditions and remain stable in basic and neutral conditions. The developed method was validated with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness as per ICH guidelines. This method was also suitable for the assay determination of ZTP in pharmaceutical dosage forms. The DPs were characterized by LC–MS/MS and their fragmentation pathways were proposed.