Acid Stress Conditions Research Articles

Characterization and quantitative analysis of related substances in Coenzyme A by HPLC and LC–MS/MS

Ten related substances in coenzyme A (CoA) were detected using a newly developed gradient reverse phase high performance liquid chromatographic (HPLC) method. A highly specific and efficient LC–MS/MS method was developed to characterize process-related substances and the major degradation products, and three unknown related substances were further synthesized and characterized by H-NMR and C-NMR spectroscopy. Synthesized samples of the related substances were used for quantitative analysis by HPLC. The method was validated according to ICH guidelines with respect to specificity, precision, accuracy and linearity. The forced degradation studies included acidic, alkaline, oxidative, photolytic and thermal stress conditions. Furthermore, we depicted and speculated the probable mechanism of formation of related substances and the plausible fragmentation mechanism showed that all the related substances came from the manufacturing process. Characterization, synthesis and quantitative analysis of related substances were discussed in detail, and were critical for quality control, manufacturing process optimization and CoA monitoring, all of which were important to ensure the security of CoA.

Liming improves soil microbial growth, but trash blanket placement increases labile carbon and nitrogen availability in a sugarcane soil of subtropical Australia

Liming has been widely used to decrease soil acidity, but its effects on soil nitrogen (N) availability and microbial processes in sugarcane fields are largely unknown. Adjacent sugarcane soils at 26 months after liming (26ML), 14 months after liming (14ML) and with no lime amendment (CK) in Bundaberg, Australia, were selected to investigate the effect of liming on soil N bioavailability and microbial activity in a long-term subtropical sugarcane cropping system. Liming in both 14ML and 26ML treatments significantly increased soil pH (by 1.2–1.4 units) and exchangeable Ca2+ (>2-fold) compared with the CK treatment. The lower concentrations of hot water extractable organic carbon (C) and total N and ammonium-N in the 14ML, compared with the CK and 26ML treatments, can be attributed to the absence of trash blanket placement in the former. Enhanced microbial immobilisation due to improved soil pH by liming (14ML and 26ML treatments) led to increased soil microbial biomass C and N, particularly in the presence of a trash blanket (26 ML treatment), but decreased soil respiration and metabolic quotient indicated that acidic stress conditions were alleviated in the liming treatments. Soil pH was the main factor governing soil enzyme activities, with an overall decrease in all enzyme activities in response to liming. Overall, liming and trash blanket practices improved sugarcane soil fertility. Further study is warranted to investigate the shifts in soil microbial community composition and the diversity and abundance of N-associated functional genes in response to liming in sugarcane fields.

STABILITY INDICATING RP-LC ASSAY METHOD FOR CARISOPRODOL

Objective: A reverse phase stability-indicating HPLC method was developed for the determination of Carisoprodol in pharmaceutical dosage forms. The chromatographic elution was achieved on C18, 250 mm × 4.6 mm, 5-μm particle size column.Methods: The mobile phase contains a mixture of water and acetonitrile in ratio of 60:40 v/v. The flow rate was 1.0 ml min-1 and was detected by Refractive index detector.Results: The method was proven to be linear over a range of 1 to 4 mg/ml with a mean correlation coefficient of 0.99998. The %mean recovery is in the range of 100.55% to 101.11% and %RSD was less than 1.0% between preparations. The % RSD for Assay results of initial sample preparation in different intervals of 0hr, 24 h, 30 h and 48 h was less than 1.0%. To establish stability-indicating capability of the method, drug product was subjected to the stress conditions of acid, base, oxidative, hydrolytic, thermal and photolytic degradation. The degradation products were well resolved from Carisoprodol.Conclusion: The developed method was validated as per international ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness.

Alkaline degradation of lyophilized DMSA prior to labeling with 99mTc: Identification and development of the degradation pathway by HPLC and MS

IntroductionComplexes of technetium-99m (99mTc) with meso-dimercaptosuccinic acid (DMSA) have been widely used as diagnostic agents in nuclear medicine. The degradation products (DP) of DMSA formed under different forced conditions have been identified through HPLC-DAD and LC-MSn studies. In this study, the DMSA kit was subjected to forced degradation under hydrolysis conditions as prescribed by the International Conference on Harmonization (ICH) guideline Q1A. MethodsChromatographic separation was accomplished on a reverse phase Shim-Pack VP-ODS (150 mm × 4.6 mm; 5 μm) analytical column using the gradient elution method. LC-MSn analysis was performed using an Esquire 3000 Plus ion trap mass spectrometer, operating under electrospray ionization (ESI). ResultsNo products were found under acidic or neutral stress conditions. All the products found were identified through LC-MSn analyses and their fragmentation pathways were proposed. The DMSA standard degraded into an adduct DMSA dimer (2DMSA[-2H+Na]+) and adduct DMSA bound to fumaric acid and dithioglucolic acid (DTGA). In the DMSA kit, the degradation products were dimers and trimers of DMSA with tin. A possible degradation pathway is presented. ConclusionsThis method proved to be convenient and effective since it provided fast and efficient separation of DMSA from its degradation products. The degradation studies carried out were able to delineate the stability of the DMSA standard and the DMSA kit.

Vibrational spectroscopy combined with transcriptomic analysis for investigation of bacterial responses towards acid stress.

The ability of bacteria to tolerate acid stress plays an important role in their growth and survival. In particular, aciduric bacteria have several survival systems that prevent cell damage from acid stress. In this study, the effect of the bacterial stress induced by pre-adaptation at different pH values on the cellular macromolecules of Lactobacillus plantarum was investigated using Raman spectroscopy and Fourier transform infrared spectroscopy. The expression of key genes was also quantified to provide understanding of the transcriptional response of the cells to lethal acid stress conditions. Principal component analysis of the spectra exhibited marked differences in the spectral regions associated with carbohydrates, lipids, proteins, and nucleic acids for all acid-stressed cells compared to those of untreated control cells. The changes in spectroscopic and transcriptomic profiles that were observed revealed alterations in bacterial cell wall composition after acid treatment. The results suggest the existence of a complex bacterial stress response in which modifications of cellular compounds from pre-adaption at low pH are involved. This study demonstrates the potential application of vibrational spectroscopy techniques to discriminate between intact and injured bacterial cells as well as to study their stress responses after exposure to acid environments during food processing.

Development of a validated HPLC method for the quantitative determination of trelagliptin succinate and its related substances in pharmaceutical dosage forms

Trelagliptin succinate is a dipeptidyl peptidase IV (DPP-4) inhibitor which is used as a new long-acting drug for once-weekly treatment of type 2 diabetes mellitus (DM). In the present study, a rapid, sensitive and accurate high-performance liquid chromatography (HPLC) method was developed and validated for separation and determination of trelagliptin succinate and its eight potential process-related impurities. The chromatographic separation was achieved on a Waters Xselect CSH™ C18 (250mm×4.6mm, 5.0μm) column. The mobile phases comprised of 0.05% trifluoroacetic acid in water as well as acetonitrile containing 0.05% trifluoroacetic acid. The compounds of interest were monitored at 224nm and 275nm. The stability-indicating capability of this method was evaluated by performing stress test studies. Trelagliptin succinate was found to degrade significantly in acid, base, oxidative and thermal stress conditions and only stable in photolytic degradation condition. The degradation products were well resolved from the main peak and its impurities. In addition, the major degradation impurities formed under acid, base, oxidative and thermal stress conditions were characterized by ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap). The method was validated to fulfill International Conference on Harmonisation (ICH) requirements and this validation included specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and robustness. The developed method in this study could be applied for routine quality control analysis of trelagliptin succinate tablets, since there is no official monograph.

Identification, Separation and Characterization of Acidic and Alkali Degradation of Rivaroxaban Under ICH Recommended tress Condition by HPTLC with MS/TOF

The stability of Rivaroxaban (RIVA) drug was determined under different stress conditions in accordance with International Conference on Harmonization (ICH) guidelines Q1A (R2). The chromatogram was developed on precoated silica gel 60F254 TLC plates using ethyl acetate: acetonitrile (5:5 v/v) as a mobile phase. There was no chromatographic interference from the tablet excipients and compact spots were observed for RIVA (Rf = 0.28 ± 0.05). When studied under varied stress conditions, the drug was found susceptible for degradation under acidic and oxide stress conditions. However, it was stable under neutral, thermal and photolytic stress conditions. In total, two degradation products (DPs) were formed. IR and MS/TOF with NMR studies were conducted on a mixture of stressed samples to obtain functional group and mass spectral data. The study data from precise mass study was initially used to pattern a complete mass fragmentation pathway of the drug and later for its degradation products. Considering the best possible molecular formula, structures were suggested for each fragment. Two novel degradation products namely, (R-4-(4-(5-(Aminomethyl)-2-Oxooxazolidin-3-yl) phenyl-Morpholin-3-One HCl and (R-4-(4-(5-(dioxymethyl)-2-Oxooxazolidin-3-yl) phenyl-Morpholin-3-One were identified.

Use of the hyphenated LC-MS/MS technique and NMR/IR spectroscopy for the identification of exemestane stress degradation products during the drug development

Exemestane (6-Methyleneandrosta-1,4-diene-3,17-dione) active pharmaceutical ingredient (EE-3) was subjected to thermal, photolytic, oxidative, acidic and base stress conditions prescribed by the ICH (International Conference on Harmonization) guideline Q1A(R2). EE-3 was found to degrade in base, acidic and oxidative conditions. Eleven new degradation products of EE-3 were characterized by the LC-MS/MS technique. One of these impurities was isolated and identified by the LC-MS/MS, NMR and IR techniques. The LC-MS/MS studies were carried out to establish fragmentation pathways of EE-3 and its new impurity. Based on the results obtained from different spectroscopic studies, this impurity was characterized as 3-hydroxy-1,6-dimethyl-oestratetraen-(1, 3, 5(10), 6)-17-one (EE-3Z). The degradation pathway of EE-3 leading to the generation of eleven products was proposed and this has not been reported so far. The separation of EE-3 from its impurities (process-related and degradants) was achieved using a Gemini C18 column (150mm×4.6mm×3μm) with gradient elution. The degradation products were well resolved from the main peak and its impurities, thus proving the method's stability and indicating power of the method. The method was validated according to the ICH guidelines for parameters such as specificity, limit of detection, limit of quantitation, precision, linearity, accuracy, robustness and system suitability.

Identification of degradation impurities in aripiprazole oral solution using LC–MS and development of validated stability indicating method for assay and content of two preservatives by RP-HPLC

ABSTRACTA simple and simultaneous reverse phase high-performance liquid chromatographic method was developed for the quantification of aripiprazole (ARI) and two preservatives, namely, methyl paraben and propyl paraben in ARI oral solution. The method was developed on ACE C18 (4.6 × 250 mm, 5 µm) column using gradient elution of 0.1% v/v trifluoroacetic acid in water and acetonitrile as mobile phase components. Flow rate of 1.0 mL/min and 30°C column temperature were used for the method at quantification wavelength of 254 nm. The developed method was validated in accordance with International Conference on Harmonization guideline for various parameters. Forced degradation study was conducted in acid, base, peroxide, heat, and light stress conditions. ARI was found to degrade in oxidation, acid hydrolysis, and heat while it was stable under the remaining conditions. Specificity of the method was verified using Photo Diode Array (PDA) detector by evaluating purity of peaks from degradation samples. Major degradation impurities formed during stress study were identified using liquid chromatography–mass spectrometry method. The present method was useful for determining the content of all the three main analytes present in the oral solution without interference from degradation impurities. The method was robust under the deliberately modified conditions.

Development and Validation of a Novel Stability Indicating UPLC Method for Analysis of Related Compounds and Assay of Bexarotene, an Anti-Cancer Agent

A novel liquid chromatographic method for the analysis of four potential impurities in the anti-cancer agent Bexarotene has been developed and validated using efficient chromatographic separation, achieved on a C18 column (50 mm × 2.1 mm, 1.7-μm particles) with a simple isocratic mobile phase at a flow rate of 0.5 mL min−1. The mobile phase consisted of a 70:30:0.1 (v/v/v) mixture of acetonitrile, water and trifluoroacetic acid, and quantification was achieved by use of ultraviolet detection at 260 nm. Resolution between Bexarotene and its four potential impurities was greater than 2.0. Regression analysis showed the r value (correlation coefficient) was >0.999 for Bexarotene and its four impurities. The method was capable of detecting all four impurities of Bexarotene at levels below 0.10 μg in a Bexarotene test concentration of 0.5 mg mL−1 using an injection volume of 5 μL. The recovery for Bexarotene in assay method at a 100% level was observed to be 99.1 ± 0.32% with % RSD value of 0.5% for drug substance and 98.9 ± 0.46% for Bexarotene capsules with % RSD value of 0.4%. Recovery of imp-1, imp-2, imp-3, and imp-4 from bulk drug samples ranged from 96.3 to 102.0%. Recovery of imp-1, imp-2, imp-3, and imp-4 from Bexarotene Capsule samples ranged from 97.2 to 101.4%. A solution of Bexarotene in ethanol was stable for 48 h. The drug was also subjected to stress conditions as prescribed by ICH Guidelines. Degradation was found to occur under acidic and basic hydrolysis stress conditions; however, the drug was stable to oxidative, photolytic, and thermal stress. Assay of the stressed samples was calculated relative to a qualified reference standard and the mass balance was found to be close to 99.8%. The method was validated for linearity, accuracy, precision, and robustness.

An eco-friendly and low-cost method for the quantification of cefazolin sodium in powder for injectable solution using thin-layer chromatography assisted by digital images

Cefazolin sodium (CFZ) is a semi-synthetic first-generation cephalosporin used for the treatment of infections caused by Gram‑positive and Gram‑negative microorganisms. Although it is a drug used worldwide, there are only a few green analytical methods described in the literature for its quantification. This work proposes an eco-friendly, low-cost, and rapid analytical method to analyze CFZ in lyophilized powder for injectable solution based on digital images, using thin-layer chromatography (TLC). For the TLC method, the mobile phase was comprised of ethanol and water, and the spots revelation was performed using iodine vapors. The TLC spots were analyzed through digital images, obtained by flatbed scanner, and evaluated using the free software “The Gimp” (GNU Image Manipulation Program), by quantification of pixels in pictures. This method was validated according to the International Conference on Harmonization (ICH) guidelines and presented adequate linearity, accuracy, precision, and robustness. It also showed its selectivity and stability-indicating capability (under acid, alkaline, aqueous, oxidative, and photolytic stress conditions). This is an innovative, easy, and low-cost method that can be used for the analysis of cefazolin sodium in lyophilized powder. Considering that this method does not use toxic organic solvents for the environment and operators, it contributes to the development of green chemistry.

VALIDATED GRADIENT STABILITY INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS QUANTIFICATION OF 11 RELATED SUBSTANCES IN THE COMBINED DOSAGE FORMS OF LAMIVUDINE AND TENOFOVIR DISOPEOXIL FUMARATE

Objective: Development of a stability-indicating reverse phase liquid chromatographic (RP-HPLC) method for the simultaneous quantification of 11 impurities in the combined dosage forms of lamivudine and tenofovir disoproxil fumarate drug substances.Methods: Efficient chromatographic separation of all analytes was achieved on a Waters X-terra RP18 column (150 x 4.6 mm, 3.5 mm) using mobile phase A (ammonium acetate buffer, pH adjusted to 5.0±0.05 with dilute orthophosphoric acid) and mobile phase B (mixture of methanol and ammonium acetate buffer in the ratio of 20:80) with the flow rate of 1.0 ml/min in gradient elution mode at 260 nm.Results: The method was validated in terms of the limit of detection, limit of quantification, linearity, accuracy, precision and robustness according to the international conference on harmonisation (ICH Q2R1). Regression analysis showed that the correlation coefficient (r2) is greater than 0.997 for individual active drug substances as well as their related substances. The method has proven very accurate (94.6 % to 108.2 % with % RSD not more than 4.9), highly precise (% RSD of the Intra-day and the inter-day study was not more than 8.9) and robust enough to deliver accurate results, when the chromatographic conditions were altered intentionally. Forced degradation studies were conducted in acidic, basic, thermal, photolytic, humid and peroxide stress conditions, where all the degradation peaks were monitored. Highest degradation of lamivudine was observed under oxidative stress condition and tenofovir was more susceptible to degradation under acidic and alkaline conditions.Conclusion: The present method is able to separate all the related compounds with each other and with the main drug substances with the resolution more than 2.0. The test solution was found to be stable in diluent up to 24 h. The mass balance of forced degradation of formulations, close to 99 %, made this method as a stability indicating method.

Identification of stress degradation products of iloperidone using liquid chromatography coupled with an Orbitrap mass spectrometer.

Iloperidone (ILOP) is an atypical antipsychotic drug used for the treatment of schizophrenia and related psychotic disorders. Comprehensive stress testing of the ILOP drug was carried out as per ICH guidelines to understand its degradation profile. The presence of degradation products in a drug affects not only the quality, but also the safety and efficacy of drug formulation. Thus, it is essential to develop an efficient analytical method which can be useful for the separation, identification and characterization of all possible degradation products of ILOP. ILOP was subjected to various stress conditions such as acidic, basic, neutral hydrolysis, oxidation, photolysis and thermal conditions; and the resulting degradation products were investigated using LC-PDA-HRMS and MS/MS. An efficient and simple ultra-high-performance liquid chromatography (UHPLC) method has been developed on Acquity UPLC® BEH C18 column (2.1×100mm, 1.7 μm) using a gradient elution of heptafluorobutyric acid (0.1% HFBA) and acetonitrile as mobile phase. ILOP was found to degrade under acidic and basic hydrolysis and oxidative stress conditions, whereas it was stable under neutral hydrolysis, thermal and photolytic conditions. A total of seven degradation products (DP1 to DP7) were identified and characterized by LC/MS/MS in positive ion mode with accurate mass measurements. The hydrolytic degradation under acidic and basic conditions produced two DPs (DP1 and DP2) and four DPs (DP4 to DP7), respectively, whereas DP3 was formed under oxidative conditions. In silico toxicity predictions showed higher probability values for DP4, DP6 and DP7, which indicates these DPs have the potential to mutate DNA. ILOP was found to be labile under hydrolytic and oxidative conditions. The structures of the degradation products were rationalized by appropriate mechanisms. The proposed method can be effectively used for the determination and detection of ILOP and its degradation products.

Forced degradation studies of lansoprazole using LC-ESI HRMS and 1 H-NMR experiments: in vitro toxicity evaluation of major degradation products.

Regulatory agencies from all over the world have set up stringent guidelines with regard to drug degradation products due to their toxic effects or carcinogenicity. Lansoprazole, a proton-pump inhibitor, was subjected to forced degradation studies as per ICH guidelines Q1A (R2). The drug was found to degrade under acidic, basic, neutral hydrolysis and oxidative stress conditions, whereas it was found to be stable under thermal and photolytic conditions. The chromatographic separation of the drug and its degradation products were achieved on a Hiber Purospher, C18 (250×4.6mm, 5μ) column using 10mM ammonium acetate and acetonitrile as a mobile phase in a gradient elution mode at a flow rate of 1.0ml/min. The eight degradation products (DP1-8) were identified and characterized by UPLC/ESI/HRMS with in-source CID experiments combined with accurate mass measurements. DP-1, DP-2 and DP-3 were formed in acidic, DP-4 in basic, DP-5 in neutral and DP-1, DP-6, DP-7 and DP-8 were in oxidation stress condition Among eight degradation products, five were hitherto unknown degradation products. In addition, one of the major degradation products, DP-2, was isolated by using semi preparative HPLC and other two, DP-6 and DP-7 were synthesized. The cytotoxic effect of these degradation products (DP-2, DP-6 and DP-7) were tested on normal human cells such as HEK 293 (embryonic kidney cells) and RWPE-1(normal prostate epithelial cells) by MTT assay. From the results of cytotoxicity, it was found that lansoprazole as well as its degradation products (DP-2, DP-6 and DP-7) were nontoxic up to 50-μM concentrations, and the latter showed slightly higher cytotoxicity when compared with that of lansoprazole. DNA binding studies using spectroscopic techniques indicate that DP-2, DP-6 and DP-7 molecules interact with ctDNA and may bind to its surface. Copyright © 2017 John Wiley & Sons, Ltd.

Analytical Method Development and Validation of Metformin, Losartan and Glimepiride in Bulk and Combined Tablet Dosage form by Gradient RP-HPLC

Purpose : A simple, sensitive, linear, precise, and accurate method by gradient reversed-phase-high performance liquid chromatography for the simultaneous estimation of metformin (MET), losartan (LOS) and glimepiride (GLI) in bulk and in their combined tablet dosage form was developed and validated. Methodology : The separation of the three drugs was based on the use of Luna c18 (250 ~ 4.6 mm, i.e. 5 ƒEm) column in a gradient mode. Mobile phase consisted of Methanol (solvent A) and 0.1% Orthophosphoric acid [OPA] (solvent B) was set with gradient programming for 18 min and was delivered at 1 ml/min flow rate and effluents are achieved with variable wavelength: Photodiode array detector at 284 nm. The retention times of MET, LOS and GLI were found to be 3.11, 7.12 and 13.52mins respectively. The percentage assay of MET, LOS and GLI was found to be 100.5%, 100.5 and 100.4%, respectively. Calibration curves were linear for MET, LOS and GLI at concentration ranges of 30- 450 ng/ml, and 15-225ng/ml and 1-18ng/ml with the regression coefficient of 0.999 for all the three drugs and precise with (% RSD <2). The drug was subjected to various stress conditions of acid and base hydrolysis, oxidation, photolysis, thermal degradation and condition. Findings : Considerable degradation was found under all stress conditions and the degradation products were well resolved from Metformin (MET),Llosartan (LOS) and Glimepiride (GLI) in the proposed gradient RP-HPLC method. Conclusion : The method was validated by determining its linearity, accuracy, precision, system suitability and can be employed for routine quality control analysis.

Heterologous Expression of theCarrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

Stability behavior of antiretroviral drugs and their combinations. 7: Comparative degradation pathways of lamivudine and emtricitabine and explanation to their differential degradation behavior by density functional theory

The interest in this study was to establish comparative degradation behavior of lamivudine (3TC) and emtricitabine (FTC) under solution and solid state stress conditions. Structurally, the two drugs differ only in terms of additional fluorine at 5 position in FTC. Along with the known degradation products of both the drugs, one additional degradation product was observed in each case, which was characterized by mass spectrometry. Both the drugs degraded via the same route, but at a differential rate in acid, base and oxidative stress conditions. The variable rate of degradation in acid and base conditions was justified by the application of density functional theory (DFT).

Changes in thermo-tolerance and survival under simulated gastrointestinal conditions of Salmonella Enteritidis PT4 and Salmonella Typhimurium PT4 in chicken breast meat after exposure to sequential stresses

This study assessed changes in thermo-tolerance and capability to survive to simulated gastrointestinal conditions of Salmonella Enteritidis PT4 and Salmonella Typhimurium PT4 inoculated in chicken breast meat following exposure to stresses (cold, acid and osmotic) commonly imposed during food processing. The effects of the stress imposed by exposure to oregano (Origanum vulgare L.) essential oil (OVEO) on thermo-tolerance were also assessed. After exposure to cold stress (5°C for 5h) in chicken breast meat the test strains were sequentially exposed to the different stressing substances (lactic acid, NaCl or OVEO) at sub-lethal amounts, which were defined considering previously determined minimum inhibitory concentrations, and finally to thermal treatment (55°C for 30min). Resistant cells from distinct sequential treatments were exposed to simulated gastrointestinal conditions. The exposure to cold stress did not result in increased tolerance to acid stress (lactic acid: 5 and 2.5μL/g) for both strains. Cells of S. Typhimurium PT4 and S. Enteritidis PT4 previously exposed to acid stress showed higher (p<0.05) tolerance to osmotic stress (NaCl: 75 or 37.5mg/g) compared to non-acid-exposed cells. Exposure to osmotic stress without previous exposure to acid stress caused a salt-concentration dependent decrease in counts for both strains. Exposure to OVEO (1.25 and 0.62μL/g) decreased the acid and osmotic tolerance of both S. Enteritidis PT4 and S. Typhimurium PT4. Sequential exposure to acid and osmotic stress conditions after cold exposure increased (p<0.05) the thermo-tolerance in both strains. The cells that survived the sequential stress exposure (resistant) showed higher tolerance (p<0.05) to acidic conditions during continuous exposure (182min) to simulated gastrointestinal conditions. Resistant cells of S. Enteritidis PT4 and S. Typhimurium PT4 showed higher survival rates (p<0.05) than control cells at the end of the in vitro digestion. These results show that sequential exposure to multiple sub-lethal stresses may increase the thermo-tolerance and enhance the survival under gastrointestinal conditions of S. Enteritidis PT4 and S. Typhimurium PT4.

Genotypes Associated with Listeria monocytogenes Isolates Displaying Impaired or Enhanced Tolerances to Cold, Salt, Acid, or Desiccation Stress.

The human pathogen Listeria monocytogenes is a large concern in the food industry where its continuous detection in food products has caused a string of recalls in North America and Europe. Most recognized for its ability to grow in foods during refrigerated storage, L. monocytogenes can also tolerate several other food-related stresses with some strains possessing higher levels of tolerances than others. The objective of this study was to use a combination of phenotypic analyses and whole genome sequencing to elucidate potential relationships between L. monocytogenes genotypes and food-related stress tolerance phenotypes. To accomplish this, 166 L. monocytogenes isolates were sequenced and evaluated for their ability to grow in cold (4°C), salt (6% NaCl, 25°C), and acid (pH 5, 25°C) stress conditions as well as survive desiccation (33% RH, 20°C). The results revealed that the stress tolerance of L. monocytogenes is associated with serotype, clonal complex (CC), full length inlA profiles, and the presence of a plasmid which was identified in 55% of isolates. Isolates with full length inlA exhibited significantly (p < 0.001) enhanced cold tolerance relative to those harboring a premature stop codon (PMSC) in this gene. Similarly, isolates possessing a plasmid demonstrated significantly (p = 0.013) enhanced acid tolerance. We also identified nine new L. monocytogenes sequence types, a new inlA PMSC, and several connections between CCs and the presence/absence or variations of specific genetic elements. A whole genome single-nucleotide-variants phylogeny revealed sporadic distribution of tolerant isolates and closely related sensitive and tolerant isolates, highlighting that minor genetic differences can influence the stress tolerance of L. monocytogenes. Specifically, a number of cold and desiccation sensitive isolates contained PMSCs in σB regulator genes (rsbS, rsbU, rsbV). Collectively, the results suggest that knowing the sequence type of an isolate in addition to screening for the presence of full-length inlA and a plasmid, could help food processors and food agency investigators determine why certain isolates might be persisting in a food processing environment. Additionally, increased sequencing of L. monocytogenes isolates in combination with stress tolerance profiling, will enhance the ability to identify genetic elements associated with higher risk strains.

Stability-Indicating HILIC Method for the Determination of Citicoline and Characterization of its Degradation Products by LC–MS/TOF, 1H and 13C NMR

A stability-indicating HILIC method was developed for analysis of Citicoline in the presence of its degradation products. It was performed using Atlantis HILIC Si column (50 × 4.6 mm, 3 µm particle size) and a mobile phase constituted by 70% of acetonitrile in formate buffer 0.02 M (v/v) adjusted to pH 3. The column’s thermostat compartment was adjusted at 30 °C. Citicoline was subjected to oxidative, acidic, basic, hydrolytic, thermal and UV light stress conditions. The column effluents were monitored at 270 nm by photodiode array and mass detectors. Depending on the stress, three degradation products appeared; one after acidic or photolytic action, the second by NaOH reaction and the third with oxidative stress. LC–ESI–MS/TOF, 1H and 13C NMR were used to characterize them. The most probable formation mechanisms of the identified impurities were proposed. The developed method was validated in terms of specificity, limit of detection and quantification, linearity, accuracy and precision as per ICH guidelines. The proposed method was successfully applied for the determination of Citicoline in dosage forms.