Acid Stress Conditions Research Articles

Development and Validation of LC–MS Method for the Estimation of N-Acetyl-Tryptophan and its Impurities Under Stress Conditions

A highly sensitive and simple high-performance liquid chromatographic–tandem mass spectrometric (LC–MS–MS) assay was developed and validated for the quantification of N-acetyl tryptophan (NAT) in formulations and to identify impurities under different stress conditions. N-acetyl tryptophan was analyzed using a reversed-phase gradient elution after treatment under acidic, basic, oxidative, hydrolytic and thermal stress conditions. Linearity in the calibration curve was obtained at a concentration range of 10–100 µgmL−1 (R2 = 0.9916). The lower limits of detection and quantification were 3.53 and 10.69 µgmL−1. The degradation of NAT was observed maximum under oxidation stress (52.84%) and minimum under thermal stress (10.22%). Three major degradation products were formed under acidic and basic stress conditions, of which tryptophan was the major one. Thermal stress yielded a single major impurity at m/z 230. Water hydrolysis could form dihydroxy-N acetyl tryptophan at m/z 279. Oxidation stress led to the formation of seven major degradation products. The most effective stress condition was found to be oxidative which leads to 52.84% degradation of the drug followed by acidic stress (34.64%) and basic stress (15.66%). The present study showed an accurate, precise and sensitive LC–MS–MS method for the systematic investigation of NAT and its impurities in formulations.

Isolation, identification and characterization of new degradation products of Carfilzomib using high resolution mass spectrometry and nuclear magnetic resonance

Carfilzomib is a newly approved tetra peptide epoxy ketone proteasome inhibiting agent for relapsed or refractory multiple myeloma. Very few stability studies are available on this drug. Carfilzomib (CFZ) is subjected to forced degradation under neutral, acid, base, oxidative, thermal and photolytic stress conditions as per ICH guidelines which results in formation of six degradation products in base hydrolysis. Out of six, four degradation products are reported in literature. UPLC method has been developed for separation of degradation products from CFZ drug. DP-1 and DP-2 which are novel degradants structurally elucidated by high resolution mass spectrometry and nuclear magnetic resonance.

Comparative Analysis of Listeria monocytogenes Plasmids and Expression Levels of Plasmid-Encoded Genes during Growth under Salt and Acid Stress Conditions.

Listeria monocytogenes strains are known to harbour plasmids that confer resistance to sanitizers, heavy metals, and antibiotics; however, very little research has been conducted into how plasmids may influence L. monocytogenes’ ability to tolerate food-related stresses. To investigate this, a library (n = 93) of L. monocytogenes plasmid sequences were compared. Plasmid sequences were divided into two groups (G1 and G2) based on a repA phylogeny. Twenty-six unique plasmid types were observed, with 13 belonging to each of the two repA-based groups. G1 plasmids were significantly (p < 0.05) smaller than G2 plasmids but contained a larger diversity of genes. The most prevalent G1 plasmid (57,083 bp) was observed in 26 strains from both Switzerland and Canada and a variety of serotypes. Quantitative PCR (qPCR) revealed a >2-fold induction of plasmid-contained genes encoding an NADH peroxidase, cadmium ATPase, multicopper oxidase, and a ClpL chaperone protein during growth under salt (6% NaCl) and acid conditions (pH 5) and ProW, an osmolyte transporter, under salt stress conditions. No differences in salt and acid tolerance were observed between plasmid-cured and wildtype strains. This work highlights the abundance of specific plasmid types among food-related L. monocytogenes strains, the unique characteristics of G1 and G2 plasmids, and the possible contributions of plasmids to L. monocytogenes tolerance to food-related stresses.

Chemical Stability and Spectroscopic Characterization of Balofloxacin and its Stress Degradation Products by LC-PDA, LC-MS/MS, NMR and FT-IR

The present manuscript describes the preparative isolation and characterization of three degradation products of Balofloxacin (BFX). The drug is subjected to different stress conditions under acidic, basic, hydrolysis, UV, thermal and oxidative stress conditions, three degradation products (DP-1 to DP-3) were formed under acidic, basic, oxidative stress conditions and no considerable degradation was observed in hydrolytic, thermal, UV stress conditions. The degradants were adequately separated by using stationary phase of LiChrospher® RP-18 HPLC C18 (250 mm × 4.6 mm i.d., 5 μm) column with mobile phase consisting of 20 mM aqueous ammonium acetate: acetonitrile: methanol (55:25:20, v/v/v) subsequently the degradation products (DP-1; m/z 194, DP-2; m/z 359, DP-3; m/z 365) were isolated by semi preparative chromatography and further characterized by LC-ESI-MS/MS, HRMS, NMR and FT-IR methods. On the evidence of spectral data three major DP’s were confirmed and the developed assay method was validated as per ICH Q2 (R1) guideline. Finally, the proposed degradation pathways were extenuated by LC-ESI-MS studies. The proposed analytical stability indicating assay method was successfully applied to bulk drugs and formulations.

Hypocholesterolaemic activity of a novel autochthonous potential probiotic Lactobacillus plantarum YS5 isolated from yogurt

Lactobacillus plantarum YS5, which was isolated from home-made yogurt, demonstrated high percent cholesterol-removal capacity by growing cells (84%) and moderate cholesterol-removal capacity by resting (41.14%) and dead (32.71%) cells. Lactobacillus plantarum ATCC 14917 as a control strain showed moderate cholesterol-removal capability by growing cells (29%), whereas it showed less cholesterol-removal capacity by resting (11.2%) and dead (7.23%) cells. Furthermore, the effect of this strain on serum cholesterol profile of rats is evaluated. Male rats are fed with a high-fat diet supplemented with or without L. plantarum YS5 and normal diet for eight weeks. L. plantarum YS5 supplementation significantly decreased serum total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels, but increased high-density lipoprotein cholesterol. This strain displayed considerable tolerance to acid and bile stress conditions, desirable antagonistic activity against Gram-positive and Gram-negative pathogens, remarkable hydrophobicity, and auto-aggregation. Moreover, L. plantarum YS5 did not represent any side effects, such as hemolytic activity, BSH activity, and antibiotic resistance. The findings of this work indicated that L. plantarum YS5 may be a promising probiotic candidate for application in food industry for production of low-cholesterol products which have hypocholesterolaemic activity in the host.

RELATED SUBSTANCE METHOD DEVELOPMENT AND VALIDATION OF LOXAPINE SUCCINATE IN CAPSULE DOSAGE FORM BY REVERSE PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The present study gives a simple, rapid, and accurate stability indicating reverse phase high-performance liquid chromatography method for the determination of loxapine succinate and its related substance (related compound A) in capsule dosage form.&#x0D; Methods: Loxapine succinate and its related substance were attained on a C18 Purospher star (250 mm × 4.6 mm, 5 μm particle size) column at 254 nm detection wavelength, 1.0 ml/min as a flow rate, and 10 μl injection volume. Water:methanol: Triethylamine: Tetrahydrofuran (50:40:1:10) was used a mobile phase, and column oven temperature was 30°C.&#x0D; Results: The resolution between loxapine succinate and known unknown impurities was &gt;2.0. The correlation coefficient (0.999) value indicates the linear relationship between the concentration and peak areas. The accuracy study was performed by spiking method. Loxapine succinate was exposed to the stress condition of hydrolysis (acid and base), oxidative, thermal, and photolytic degradation. Loxapine succinate was found to degrade unquestionably in acid and base stress condition and almost stable in oxidative, thermal, and photolytic conditions.&#x0D; Conclusion: The degradant products were well resolved from leading peak and its related compound A peak and any other unknown peak justifying the stability indicating capability of the method. The developed method was validated as per the ICH guidelines. This method is used for periodic analysis in laboratory.

Novel tetrahydrofuran (THF) degradation-associated genes and cooperation patterns of a THF-degrading microbial community as revealed by metagenomic

Our understanding of the tetrahydrofuran (THF) degradation in complex environment is limited. The majority of THF degrading genes reported are group V soluble diiron monooxygenases and share greater than 95% homology with one another. In this study, we used sole-carbon-source incubation combined with high-throughput metagenomic sequencing to investigate this contaminant's degradation in environmental samples. We identified as-yet-uncultivated microbe from the genera Pseudonocardia and fungi Scedosporium sp. (Scedosporium sp. was successfully isolated) as THF degraders as containing THF degradation genes, while microbes from the genera Bordetella, Pandoraea and Rhodanobacter functioned as main cooperators by utilizing acidic intermediates and providing anti-acid mechanisms. Furthermore, a 9387-bp THF degradation cluster designated thmX from the as-yet-uncultivated Pseudonocardia (with 6 main ORFs and with 79–93% amino acid sequence identity with previously reported clusters) was discovered. We also found a THF-degrading related cytochrome P450 monooxygenase from the genus Scedosporium and predicted its cognate reductase for the first time. All the genes and clusters mentioned above were successfully amplified from samples and cloned into the suitable expression vectors. This study will provide novel insights for understanding of THF degradation mechanisms under acid stress conditions and mining new THF degradation genes.

Acid stress response ofStaphylococcus xylosuselicits changes in the proteome and cellular membrane

Coagulase-negative Staphylococcus xylosus strains are used as starter organisms for sausage fermentation. As those strains have to cope with low pH-values during fermentation, the aim of this study was to identify the acid adaptation mechanisms of S. xylosus TMW 2.1523 previously isolated from salami. A comparative proteomic study between two different acid tolerant mutants was performed. Therefore, both S. xylosus mutants were grown pH-static under acid stress (pH 5·1) and reference conditions (pH 7·0). Proteomic data were supported by metabolite and cell membrane lipid analysis. Staphylococcus xylosus acid stress adaptation is mainly characterized by a metabolic change towards neutral metabolites, enhanced urease activity, reduced ATP consumption, an increase in membrane fluidity and changes in the membrane thickness. This study corroborates mechanisms as previously described for other Gram-positive bacteria. Additionally, the adjustment of membrane structure and composition in S. xylosus TMW 2.1523 play a prominent role in its acid adaptation. This study demonstrates for the first time changes in the membrane lipid composition due to acid stress adaptation in staphylococci.

Comparative stress response to food preservation conditions of ST19 and ST213 genotypes of Salmonella enterica serotype Typhimurium

The replacement of the most prevalent Salmonella enterica genotypes has been documented worldwide. Here we tested the hypothesis that the current prevalent sequence type ST213 of serotype Typhimurium in Mexico has a higher resistance to stressful food preservation conditions than the displaced sequence ST19. ST19 showed higher cell viability percentages than ST213 in osmotic (685 mM NaCl) and acidic (pH 3.5) stress conditions and in combination with refrigeration (4 °C) and ambient (≈22 °C) temperatures. Both genotypes showed the same poststress recovery growth. ST213 formed biofilm and filamentous cells (FCs) under stress, whereas ST19 did not. ST213 cells also showed higher motility. The capacity of ST213 to form FCs may explain its lower viability percentages when compared with ST19, i.e., ST213 cells divided less under stress conditions, but FCs had the same recovery capacity of ST19 cells. ST213 presented a higher unsaturated/saturated fatty acids ratio (0.5–0.6) than ST19 (0.2–0.5), which indicates higher membrane fluidity. The transcript levels of the rpoS gene were similar between genotypes under the experimental conditions employed. Biofilm formation, the generation of FCs, cell motility and membrane modification seem to make ST213 more resistant than ST19 to food preservation environments.

Survival of Lactobacillus paracasei subsp. paracasei LBC 81 in Fermented Milk Enriched with Green Banana Pulp Under Acid Stress and in the Presence of Bile Salts.

This study evaluated the in vitro effect of 3.0, 6.0, and 9.0% of green banana pulp (GBP) incorporation in fermented milk on the survival of Lactobacillus paracasei subsp. paracasei LBC 81 subjected to acid stress conditions and in the presence of bile salts. Tolerance to acid stress in pH2.0 and in the presence of 0.30% of bile salts was evaluated right after the incorporation of the fermented milk in each of these conditions, and also during 3 and 4h of exposure, respectively. The addition of GBP (3.0%) gives a protective effect on L. paracasei LBC 81 when exposed to stress conditions evaluated, while of 9.0% there is a marked decrease of L. paracasei LBC 81. In the absence of GBP, a decrease of L. paracasei LBC 81 is observed, but lower in the presence of GBP (9.0%).

Ribosomal maturation factor (RimP) is essential for survival of nontuberculous mycobacteria Mycobacterium fortuitum under in vitro acidic stress conditions.

Mycobacterium fortuitum is an important human pathogenic NTM, which resists stress conditions inside macrophages by exploitation of specific genes. TnphoA-based transposon mutagenesis was employed to identify membrane genes responsible for survival of M. fortuitum under such stress conditions. A library of about 450 mutants was constructed after electroporation of vector pRT291 into wild-type M. fortuitum. On the basis of blue color development and alkaline phosphatase assay, 20 mutants were shortlisted to screen for growth and survival under acidic stress at pH 6.5, 5.5, 4.5, and 3.5. Mutant MT727 showed reduced growth and survival under acidic stress. The acid susceptible mutant MT727 was subjected to other in vitro stress conditions prevalent inside macrophages including oxidative, nutrient starvation and nitrosative stress. However, the mutant showed no appreciable difference in growth behavior under oxidative, nutrient starvation and nitrosative stress conditions in comparison to the wild type. Genomic and bioinformatics analysis of MT727 led to identification of putative ribosomal maturation factor RimP of M. fortuitum to be affected by mutagenesis, showing closest homology to M. abscessus RimP. In silico functional interaction of RimP protein using STRING database showed its interaction with proteins of ribosomal assembly and maturation. Results indicate role of rimP gene in survival of M. fortuitum under acidic stress conditions which may be further explored for use as a potential drug target against M. fortuitum and other mycobacterial infections.

Impaired oxidative stress and sulfur assimilation contribute to acid tolerance of Corynebacterium glutamicum.

The industrial organism Corynebacterium glutamicum is often subjected to acid stress during large-scale fermentation for the production of bio-based chemicals. The capacity of the cells to thrive in acidic environments is a prerequisite for achieving high product yields. In this study, we obtained an acid-adapted strain using an adaptive laboratory evolution strategy. Physiological characterizations revealed that the adapted strain achieved improved cell viability after acid-stress challenge, with a higher cytoplasmic pHin level, a lower intracellular reactive oxygen species (ROS), and an enhanced morphological integrity of the cells, when compared to those of the original control strain. Transcriptome analysis indicated that several important cellular processes were altered in the adapted strain, including sulfur metabolism, iron transport, and central metabolic pathways. Further research displayed that KatA and Dps cooperatively mediated intracellular ROS scavenging, which was required for resistance to low-pH stress in C. glutamicum. Furthermore, the repression of sulfur assimilation by the McbR regulator also contributed to the improvement of acid-stress tolerance. Moreover, two copper chaperone genes cg1328 and cg3292 were found to be involved in promoting cell survival under acid-stress conditions. Finally, a new recombinant C. glutamicum strain with enhanced acid tolerance was generated by the combined overexpression of katA, dps, mcbR, and cg1328, showing 18.4 ± 2.5% higher biomass yields than the wild-type strain under acid-stress conditions. These findings will provide new insights into the understanding and genetic improvement of acid tolerance in C. glutamicum.

Overexpression of RCK1 improves acetic acid tolerance in Saccharomyces cerevisiae

Mixed sugars derived from lignocellulosic biomass can be converted into biofuels and chemicals by engineered microorganisms, but toxic fermentation inhibitors produced from harsh depolymerization processes of lignocellulosic biomass pose a critical challenge for economic production of biofuels and chemicals. Unlike other fermentation inhibitors generated from sugar degradation, acetic acid is inevitably produced from acetylated hemicellulose, and its concentrations in cellulosic hydrolysates are substantially higher than other fermentation inhibitors. The aim of this study was to identify novel genetic perturbations for improved acetic acid tolerance in Saccharomyces cerevisiae. Through a genomic library-based approach, we identified an overexpression gene target RCK1 coding for a protein kinase involved in oxidative stress. Overexpression of RCK1 significantly improved glucose and xylose fermentation under acetic acid stress conditions. Specifically, the RCK1-overexpressing strain exhibited a two-fold higher specific ethanol productivity than the control strain in glucose fermentation under the presence of acetic acid. Interestingly, the engineered S. cerevisiae overexpressing RCK1 showed 40% lower intracellular reactive oxygen species (ROS) levels as compared to the parental strain when the strains were exposed to acetic acid, suggesting that RCK1 overexpression might play a role in reducing the oxidative stress caused by acetic acid.

Large scale microbial cell disruption using hydrodynamic cavitation: Energy saving options

Cell disruption is a crucial step in the recovery of intracellular products and has a huge impact on further downstream processing steps and associated cost. The non-selective recovery of intracellular product by conventional method is the major cause for the higher cost of bioproducts. Hydrodynamic cavitation is an emerging technology for the selective recovery of intracellular product at the cell disruption stage. In the present study, the hybrid strategy for the large-scale cell disruption has been developed using hydrodynamic cavitation with different pretreatment strategies such as exposure to the acidic stress, hypoosmotic acidic stress condition and reducing agent. The maximum enzyme recovery of 55.78U/ml and selectivity of 495U/mg were obtained at an optimized pretreatment of hypoosmotic acidic stress (3.6 pH, 0.05 M, 12 h) and reducing agent (2 mM Dithiothreitol, 2 h) followed by the cell disruption using hydrodynamic cavitation. The pretreatment prior to cell disruption by hydrodynamic cavitation increases the selectivity up to 4.79 times and energy efficiency up to 63.19 times as compared to high pressure homogenizer as a conventional method for the cell disruption.

Identification and Characterisation of Rucaparib Degradation Products and Their Comparison with Known Impurities

A reversed-phase HPLC (high-performance liquid chromatographic) method is employed to identify and quantify the degradation impurities and related substances of Rucaparib (RCB), API bulk drug. There is no literature available that elucidates degradation phenomenon of RCB. Thus, the present study focuses on understanding the degradation phenomenon of RCB. The chromatographic separation is achieved within 25 min run time, where Zorbax Bonus RP column is used with a gradient elution of trifluoroacetic acid–ACN–water as the mobile phase. In this study, various forced degradation conditions such as photolysis, hydrolysis, oxidation, and thermal degradation were employed on RCB. Oxidative, basic, and acidic stress conditions showed a significant impact on degradation compared to thermal and photolysis. LC–MS technique was used for the characterization of degradation products and the plausible pathways of fragmentation were proposed. Qualified reference standards were used to quantify the stressed samples and the mass balance was found close to 99.5% (w/w) considering the response of the degradants to be equivalent to RCB. Results of this investigation confirmed the efficacy of the proposed novel method to determine drug stability of RCB, and ICH guidelines were also considered to authenticate our results.

Improved acid-stress tolerance of Lactococcus lactis NZ9000 and Escherichia coli BL21 by overexpression of the anti-acid component recT.

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.

Study of forced degradation behavior of fluorometholone by reversed-phase high-performance liquid chromatography

Introduction: The present proposed work describes the development and validation of a new liquid chromatographic method for the determination of fluorometholone (FM) in pharmaceutical products. Materials and Methods: Chromatography was performed on Shimadzu Model CBM-20A/20 Alite with C8 Phenomenex column (250 mm × 4.6 mm i.d., 5 μm particle size) using 0.1 M ammonium formate and methanol as mobile phase (flow rate 0.8 ml/min) (ultraviolet detection at 241 nm). Results and Discussion: The method was validated. The linear regression equation was found to be y = 35,207x + 29,529 (R² = 0.9995). FM was exposed to forced degradation conditions - acidic, alkaline, and thermal and oxidation stress conditions - and found that the drug is highly resistant. Conclusion: The proposed method was found to be robust and specific and can be used for the assay of FM.

Bacillus megaterium adapts to acid stress condition through a network of genes: Insight from a genome-wide transcriptome analysis

RNA-seq analysis of B. megaterium exposed to pH 7.0 and pH 4.5 showed differential expression of 207 genes related to several processes. Among the 207 genes, 11 genes displayed increased transcription exclusively in pH 4.5. Exposure to pH 4.5 induced the expression of genes related to maintenance of cell integrity, pH homeostasis, alternative energy generation and modification of metabolic processes. Metabolic processes like pentose phosphate pathway, fatty acid biosynthesis, cysteine and methionine metabolism and synthesis of arginine and proline were remodeled during acid stress. Genes associated with oxidative stress and osmotic stress were up-regulated at pH 4.5 indicating a link between acid stress and other stresses. Acid stress also induced expression of genes that encoded general stress-responsive proteins as well as several hypothetical proteins. Our study indicates that a network of genes aid B. megaterium G18 to adapt and survive in acid stress condition.

METHOD DEVELOPMENT AND VALIDATION OF A REVERSED-PHASE LIQUID CHROMATOGRAPHIC METHOD FOR THE SIMULTANEOUS ESTIMATION OF SELECTED ANTIDIABETIC DRUGS IN THE PRESENCE OF THEIR DEGRADATION PRODUCTS

Objective: This study was designed to conduct forced degradation and validation studies for the simultaneous estimation of metformin, sitagliptin, pioglitazone, and glimepiride. Methods: Analytes were separated on an Agilent XDB-C18, 150 × 4.6 mm, 5 μm column using an isocratic elution mode having mobile phase composition of 20 mM potassium dihydrogen phosphate buffer (pH 4.0):acetonitrile (65:35% v/v). Analytes were detected at a wavelength of 225 nm. The optimized method was validated as per the ICH Q2 guidelines. Results: The retention times of metformin, sitagliptin, pioglitazone, and glimepiride were 3.47, 4.83, 5.83, and 9.44 min, respectively. The linearity was 25–100 μg/ml for metformin, 2.5–10 μg/ml for sitagliptin, 1–4 μg/ml for pioglitazone, and 0.75–3 μg/ml for glimepiride. The correlation coefficient for calibration curves was >0.99, and accuracy was between 98 and 102% for each analyte. Inter- and intra-day precisions were calculated <2% relative standard deviation for each analyte. Conclusion: A significant degradation was observed in the presence of acidic, basic, neutral, oxidative, and photolytic stress conditions. The method is simple, precise, accurate, robust, and reproducible and was able to successfully separate and quantify metformin, sitagliptin, pioglitazone, and glimepiride in the presence of their degradation products.

Micelle and inclusion complex enhanced spectrofluorimetric methods for determination of Retigabine: Application in pharmaceutical and biological analysis.

Two new, simple, selective, and highly sensitive spectrofluorimetric methods were developed and validated for the determination of the antiepileptic drug; retigabine (RTG). The first method (Method-I) depends on enhancement of the weak native fluorescence of RTG via the use of an organized medium; sodium dodecyl sulphate (SDS) in acetate buffer (pH 3.74). The second method (Method-II) depends on the enhancement of RTG weak native fluorescence through complexation with a macromolecule; beta cyclodextrin (β-CD) in phosphate buffer (pH 3.20). A full study of different experimental parameters influencing the fluorescence intensity was carried out. In addition, a thorough investigation of the fluorescence quantum yield, fluorophore brightness and mechanism of fluorescence enhancement was performed. A seven-fold improvement in the fluorescence intensity was brought by the first method, whereas a six and half-fold enhancement of the fluorescence intensity was obtained by the second one. Linearity was achieved over wide ranges (0.05-12.5 μg mL-1) and (0.05-15 μg mL-1) with low limits of detection (LOD) of 10.6 and 14.3 ng mL-1, and limits of quantification (LOQ) of 32.0 and 43.2 ng mL-1 for (Method-I) and (Method-II), respectively. The proposed methods were validated according to ICH and US-FDA guidelines. The applicability of the proposed methods was tested for determination of RTG in its pharmaceutical dosage forms, and to study the stability of RTG under different stress conditions according to ICH guidelines including alkaline, acidic, oxidative, thermal, and photolytic stress conditions. Moreover, the high sensitivity achieved by the proposed methods permitted the determination and detection of RTG in both spiked and real rabbit plasma samples utilizing a simple protein precipitation step followed by liquid-liquid extraction method. Percentage recoveries from rabbit plasma samples were within the acceptable limits; (93.47-104.74%) and (91.33-105.70%) for (Method-I) and (Method-II), respectively.