Combination of vortex assisted binary solvent microextraction and QuEChERS for the determination of prothiofos, oxadiargyl, and gamma-cyhalothrin in water and pineapple samples by gas chromatography mass spectrometry.

An accurate and sensitive dispersive liquid-liquid microextraction method based on binary solvents was used to enrich prothiofos, oxadiargyl, and gamma-cyhalothrin for quantification by GC-MS. The combination of two extraction solvents (binary mixture) resulted in higher extraction efficiencies compared to the single solvent extraction systems. Parameters of the binary extraction method where optimized to enhance the extraction output of the analytes. The limits of detection calculated for the analytes ranged between 0.59 and 1.6 ng/mL. Linear calibration plots of the analytes covered wide concentration ranges with R2 values greater than 0.9996 and percent relative standard deviation lower than 10%. Spiked recovery experiments were performed well and wastewater at two different concentrations and satisfactory results (89-104%) were obtained. The binary solvent microextraction method was combined with QuEChERS to quantify the analytes in pineapple matrix, using matrix matching method to enhance the accuracy of the method to almost 100%.

Lipophilicity evaluation of some thiazolyl-1,3,4-oxadiazole derivatives with antifungal activity.

The chromatographic behavior of a series of thiazolyl-1,3,4-oxadiazoles with antifungal activity was studied by reverse-phase thin-layer chromatography (RP-TLC). The lipophilicity parameters derived from RP-TLC were correlated with the data derived from liquid-chromatography mass-spectrometry. Good linear relationships were observed between the chromatographic lipophilicity parameters and the theoretical lipophilicity descriptors (logP) generated by various computer software and internet modules. Principal component analysis, applied on the experimental chromatographic lipophilicity indices and the theoretically calculated logP, enabled us to obtain a lipophilicity chart for better vizualization of the similarities and differences of the investigated compounds, which were grouped by k-means clustering in two congeneric classes.

Development and validation of a stability-indicating RP-HPLC-FLD method for determination of 5-[(4-chlorophenoxy) methyl]-1, 3, 4-oxadiazole-2-thiol; A novel drug candidate.

The present study describes the development and validation of a simple high performance liquid chromatographic method for the determination of a novel drug candidate, 5-[(4-chlorophenoxy) methyl]-1, 3, 4-oxadiazole-2-thiol. The stability-indicating capacity of the method was evaluated by subjecting the compound's solution to hydrolytic, oxidative, photolytic, transition metal- and thermal- stress. The chromatographic separation was achieved over a C18 column (Promosil, 5 µm, 4.60 × 250 mm), maintained at 25°C, using an isocratic mobile phase comprising a mixture of acetonitrile and acidified water of pH 2.67 (1:1, v/v), at a flow rate of 1.00 mL/min and detection using a fluorescent light detector (excitation at 250 nm and emission at 410 nm). The Beer's law was followed over the concentration range 2.50-50.00 µg/ml. The recovery (98.56-100.19%, SD <5%), intraday accuracy and precision (97.31-100.81%, RSD <5%), inter-day accuracy and precision (97.50-100.75%, RSD <5%) and intermediate accuracy and precision (98.10-99.91%, RSD <5%) indicated that the method was reliable, repeatable, reproducible and rugged. The resolution and selectivity factors of the compound's peak from the nearest resolving peak, particularly in case of dry heat and copper metal stress, were found to be greater than 2 and 1, respectively, which indicated specificity and selectivity. The compound was extensively decomposed in alkaline-hydrolytic, oxidative, metal- and dry heat- stress. However, the compound in acidic and neutral conditions was resistant to photolysis. The results of the present study indicate that the developed method is specific, selective, sensitive and suitable, hence, may be used for quality control, stability testing and preformulation studies.

Synthesis, spectral analysis and antibacterial activity of some novel 5-substituted-2-((6-bromo-3,4-methylenedioxybenzyl)thio)-1,3,4-oxadiazole derivatives.

A number of novel 5-substituted-2-((6-bromo-3,4-methylenedioxybenzyl)thio)-1,3,4-Oxadiazole derivatives (6a-l) have been synthesized to evaluate their antibacterial activity. Using aryl/aralkyl carboxylic acids (1a-l) as precursors, 5-substituted-1,3,4-Oxadiazol-2-thiols (4a-l) were yielded in good amounts. The derivatives, 4a-l, were subjected to electrophilic substitution reaction on stirring with 6-bromo-3,4-methylenedioxybenzyl chloride (5) in DMF to synthesize the required compounds. All the synthesized molecules were well characterized by IR, 1H-NMR, 13C-NMR and EIMS spectral data and evaluated for antibacterial activity against some bacterial strains of Gram-bacteria. The molecule, 6d, demonstrated the best activity among all the synthesized molecules exhibiting weak to moderate inhibition potential.

Development of a liquid chromatography assay for the determination of opicapone and BIA 9-1079 in rat matrices.

Opicapone is a novel potent, reversible and purely peripheral third generation catechol-O-methyltransferase inhibitor, currently under clinical trials as an adjunct to levodopa therapy for Parkinson's disease. To support additional nonclinical pharmacokinetic studies, a novel high-performance liquid chromatographic method coupled to a diode array detector (HPLC-DAD) to quantify opicapone and its active metabolite (BIA 9-1079) in rat plasma and tissues (liver and kidney) is herein reported. The analytes were extracted from rat samples through a deproteinization followed by liquid-liquid extraction. Chromatographic separation was achieved in less than 10 min on a reversed-phase C18 column, applying a gradient elution program with 0.05 M monosodium phosphate solution (pH 2.45 ± 0.05) and acetonitrile. Calibration curves were linear (r(2) ≥ 0.994) within the ranges of 0.04-6.0 µg/mL for both analytes in plasma, 0.04-4.0 µg/mL for opicapone in liver and kidney homogenates, and 0.07-4.0 µg/mL and 0.06-4.0 µg/mL for BIA 9-1079 in liver and kidney homogenates, respectively. The overall intra- and inter-day accuracy ranged from -12.68% to 7.70% and the imprecision values did not exceed 11.95%. This new HPLC-DAD assay was also successfully applied to quantify opicapone and BIA 9-1079 in a preliminary pharmacokinetic study.

Liquid chromatography/tandem mass spectrometry study of forced degradation of azilsartan medoxomil potassium.

RATIONALE: Azilsartan medoxomil potassium (AZM) is a new antihypertensive drug introduced in the year 2011. The presence of degradation products not only affects the quality, but also the safety aspects of the drug. Thus, it is essential to develop an efficient analytical method which could be useful to selectively separate and identify the degradation products of azilsartan medoxomil potassium. METHODS: AZM was subjected to forced degradation under hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress conditions. Separation of the drug and degradation products was achieved by a liquid chromatography (LC) method using an Acquity UPLC(®) C18 CSH column with mobile phase consisting of 0.02% trifluoroacetic acid and acetonitrile using a gradient method. Identification and characterization of the degradation products was carried out using LC/electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOFMS). RESULTS: A total of five degradation products (DP 1 to DP 5) were formed under various stress conditions and their structures were proposed with the help of tandem mass spectrometry (MS/MS) experiments and accurate mass data. A common degradation product (DP 4) was observed under all the degradation conditions. DP 1, DP 2 and DP 5 were observed under acid hydrolytic conditions whereas DP 3 was observed under alkaline conditions. CONCLUSIONS: AZM was found to degrade under hydrolytic, oxidative and photolytic stress conditions. The structures of all the degradation products were proposed. The degradation pathway for the formation of degradation products was also hypothesized. A selective method was developed to quantify the drug in the presence of degradation products which is useful to monitor the quality of AZM.

Chiral separation of D/L-aldoses by micellar electrokinetic chromatography using a chiral derivatization reagent and a phenylboronic acid complex.

A novel method was developed for D/L-isomeric separation of aldopentoses and aldohexoses as their (S)-(+)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole derivatives using phenylboronate buffer containing sodium dodecyl sulfate as a background electrolyte. The combination of derivatization with a chiral labeling reagent and micellar electrokinetic chromatography with phenylboronate made possible the efficient separation of D/L isomers as well as epimeric isomers of aldopentoses and aldohexoses. Laser-induced fluorescence detection permitted the micromolar-level determination of monosaccharide derivatives. The limit of detection was 105 amol (300 nM), and the repeatabilities of the migration times and peak area responses were 0.8 % and 7.9 % (relative standard deviation; n = 6), respectively. The method was applied to the determination of D/L- galactose in red seaweed.

Determination and Method Validation of the New Sulfone Fungicide 2-(4-Fluorophenyl)-5-Methylsulfonyl-1,3,4-Oxadiazole in Tomato and Soil by UPLC in Field Trial Samples from Guizhou Province, China.

A new method was developed and validated for the determination of 2-(4-fluorophenyl)-5-methylsulfonyl-1,3,4-oxadiazole (jiahuangxianjunzuo, JHXJZ) by ultra-performance liquid chromatography equipped with photo-diode array detector. JHXJZ from tomato and soil was extracted with ethyl acetate without further cleanup. The limits of detection and quantification of JHXJZ were 0.0083 and 0.025 mg kg(-1) in tomato, 0.0017 and 0.005 mg kg(-1) in soil, respectively. The average recoveries of tomato and soil were studied at three spiked levels and ranged from 84.51 % to 101.30 % and 85.30 % to 101.53 %, respectively, with relative standard deviations of 2.61 %-4.13 % and 1.21 %-4.80 %, respectively. The results indicated that the reported method could meet the requirement for the analysis of JHXJZ in trace amount in tomato and soil.

Stability-indicating RP-LC method for determination of azilsartan medoxomil and chlorthalidone in pharmaceutical dosage forms: application to degradation kinetics.

A RP-LC method was developed and validated for simultaneous determination of the active components, azilsartan medoxomil (AZL) and chlorthalidone (CLT), in their novel antihypertensive combined recipe. The chromatographic separation was achieved on an Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) column using a mobile phase consisting of methanol/potassium hydrogen phosphate buffer (pH 8, 0.05 M) (40:60, v/v) in isocratic mode. The flow rate was maintained at 0.8 mL min(-1) at ambient temperature. Detection was carried out at 210 nm. The method was validated according to the ICH guidelines. Linearity, accuracy, and precision were satisfactory over the concentration range of 5.0-50.0 and 2.5-25.0 µg mL(-1) for AZL and CLT, respectively (r (2) = 0.9999). LODs for AZL and CLT were 0.90 and 0.32 µg mL(-1), whereas LOQs were 2.72 and 0.98 µg mL(-1), respectively. Both drugs were subjected to forced degradation studies under hydrolysis (neutral, acidic, and alkaline), oxidative, and photolytic extensive stress conditions. The proposed method is stability indicating by the resolution of the investigated drugs from their degradation products. Moreover, the kinetics of the acidic degradation of AZL as well as the kinetics of the alkaline degradation of CLT were investigated. Arrhenius plots were constructed and the apparent first-order rate constants, half-life times, shelf-life times, and the activation energies of the degradation processes were calculated. The method was successfully applied for the determination of the studied drugs simultaneously in their coformulated tablet. The developed method is specific and stability indicating for the quality control and routine analysis of the cited medications in their pharmaceutical preparations.

Structural elucidation of potential impurities in Azilsartan bulk drug by HPLC.

During the synthesis of Azilsartan (AZS), it was speculated that 15 potential impurities would arise. This study investigated the possible mechanism for the formation of 14 of them, and their structures were characterized and confirmed by IR, NMR, and MS techniques. In addition, an efficient chromatographic method was developed to separate and quantify these impurities, using an Inertsil ODS-3 column (250 x 4.6 mm, 5 pm) in gradient mode with a mixture of acetonitrile and the potassium dihydrogen orthophosphate buffer (10 mM, pH adjusted to 3.0 with phosphoric acid). The HPLC method was validated for specificity, precision, accuracy, and sensitivity. LOQ of impurities were in the range of 1.04-2.20 ng. Correlation coefficient values of linearity were >0.9996 for AZS and its impurities. The mean recoveries of all impurities in AZS were between 93.0 and 109.7%. Thus, the validated HPLC method is suitable for the separation and quantification of all potential impurities in AZS.

An HPLC-DAD method for the simultaneous quantification of opicapone (BIA 9-1067) and its active metabolite in human plasma.

Opicapone (BIA 9-1067) is a novel catechol-O-methyltransferase inhibitor presently under clinical development as an adjuvant in the pharmacotherapy of Parkinson's disease. This report describes the development and validation of a bioanalytical assay for the simultaneous quantification of opicapone and its active metabolite (BIA 9-1079) in human plasma. The method herein reported is based on high-performance liquid chromatography coupled with diode-array detection (HPLC-DAD) and the sample preparation consists of a plasma protein precipitation step followed by liquid-liquid extraction. Chromatographic separation of the analytes (opicapone and BIA 9-1079) and the internal standard (tamoxifen) was achieved in less than 10 min on a reversed-phase C18 column at 25 °C by applying a gradient elution program using a mobile phase composed of 0.05 M monosodium phosphate solution adjusted to pH 2.45 (A) and acetonitrile (B) pumped at 0.8 mL min(-1). Opicapone and the internal standard were monitored at 271 nm while BIA 9-1079 was assessed at 257 nm. Calibration curves of both analytes were linear (r(2) ≥ 0.997) in the concentration range of 25-3000 ng mL(-1) and their limits of quantification were established to be 25 ng mL(-1). The overall precision did not exceed 13.2% and the accuracy was within ±11.1%. Several drugs potentially co-administered with opicapone were tested and they did not interfere at the retention times of the analytes (opicapone and BIA 9-1079) and internal standard. The method was then successfully applied for quantifying opicapone and its active metabolite (BIA 9-1079) in plasma samples obtained from a healthy subject enrolled in a clinical trial.

Novel cytochrome P450-mediated ring opening of the 1,3,4-oxadiazole in setileuton, a 5-lipoxygenase inhibitor.

Setileuton [4-(4-fluorophenyl)-7-[({5-[(1S)-1-hydroxy-1-(trifluoromethyl)propyl]-1,3,4-oxadiazol-2-yl}amino)methyl]-2H-1-benzopyran-2-one] is a selective inhibitor of the 5-lipoxygenase enzyme, which is under investigation for the treatment of asthma and atherosclerosis. During the development of setileuton, a metabolite (M5) was identified in incubations with rat, dog, and human liver microsomes that represented the addition of 18 Da to the 1,3,4-oxadiazole portion of the molecule. Based on mass spectral data, a ring opened structure was proposed and confirmed through comparison with a synthetic standard. The metabolic ring opening was examined in vitro in rat liver microsomes and was determined to be mediated by cytochrome P450s (P450s). Upon examination of the specific P450s involved using cDNA-expressed rat P450s, it was shown that CYP1A2 likely was the major isoform contributing to the formation of M5. Studies using stable labeled molecular oxygen and water demonstrated that the oxygen was incorporated from molecular oxygen, rather than water, and confirmed that the metabolic formation was oxidative. An alternative, comparatively slow pathway of chemical hydrolysis also was identified and described. Three potential mechanisms for the two-step metabolic ring opening of the 1,3,4-oxadizole are proposed.

Novel application of low pH-dependent fluorescent dyes to examine colitis.

BACKGROUND: Endoscopy capable of fluorescence observation provides histological information on gastrointestinal lesions. We explored the novel application of low pH-dependent fluorescent dyes for fluorescence observation of crypt structure and inflammatory cell infiltration in the colon. METHODS: Low pH-dependent fluorescent dyes were applied to the colonic mucosa of normal mice for observation under fluorescence stereomicroscopy system. We also examined mouse models of colitis, which were induced by trinitrobenzenesulfonic acid, dextran sulfate sodium or interleukin-10 deficiency. RESULTS: Topical application of low pH-dependent fluorescent dyes revealed crypts as ring-shaped fluorescent stains by visualizing the mucin granules of goblet cells. Because of the minimal fluorescence intensity of the low pH-dependent fluorescent dyes in phosphate-buffered saline, it was not necessary to wash the mucosa before the fluorescence observation. 4-Nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was quicker to achieve complete staining (three minutes) than LysoSensor Green DND-153 and DND-189 (20 minutes). In each type of colitis, NBD-PZ revealed the destruction of the crypts as the disappearance of the ring-shaped fluorescent stains and the infiltration of inflammatory cells as the aggregation of punctate fluorescent stains through visualization of lysosomes. CONCLUSIONS: Low pH-dependent fluorescent dyes, especially NBD-PZ, are suitable for topical application to the colonic mucosa and have characteristics that allow for the histological examination of colitis.

Determination of oxadiargyl residues in environmental samples and rice samples.

Oxadiargyl is a commonly used herbicide in China. We developed a simple, fast, and high-throughput method employing gas chromatography with electron capture detector to determine oxadiargyl residues in food samples (rice, straws) and environmental samples (soil, water). Samples were prepared by a modified QuEChERS procedure. In this method, acetonitrile was used as the extracting solvent. The purifying step was omitted when the chromatographic conditions were optimized. Recoveries ranged from 82.9 to 112.0% for oxadiargyl in all samples, with relative standard deviation values lower than 6.2% at 0.01 mg/kg fortified concentration level.

Qualitative and Quantitative Analysis of the New Sulfone Bactericide 2-(4-Fluorophenyl)-5-(Methylsulfonyl)-1,3,4-Oxadiazole and Identification of Its Degradation Pathways in Paddy Water.

Rapid and simple methods for the determination of Jiahuangxianjunzuo (JHXJZ) in paddy water, brown rice, soil and rice straw was developed and validated. This method involved the use of ultrahigh-performance liquid chromatography equipped with photodiode array detector. The most important factor was chromatographic conditions, as identified through an orthogonal experimental design. This method showed good recoveries and precisions, thereby indicating its suitability for monitoring of JHXJZ residues in paddy water, brown rice, soil and rice straw. Furthermore, hydrolysis experiment was conducted in the laboratory under pH = 7 buffer solutions, and its degradation product was identified as 2-(4-fluorophenyl)-5-methoxy-1,3,4-oxadiazole by high-resolution mass spectrometry. JHXJZ has a major degradation pathway in the water which the OH- nucleophilic attack the C5 of 1,3,4-oxadiazole ring. Then it leaves mesyl to form intermediate 5-(4-fluorophenyl)-1,3,4-oxadiazol-2-ol and the intermediate combined with methanol formed the degradation product 2-(4-fluorophenyl)-5-methoxy-1,3,4-oxadiazole by the loss of one H2O.The degradation pathways of JHXJZ under the present indoor simulation conditions were proposed.

Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy.

We have previously shown that a fluorescent derivative of ceramide, N-(epsilon-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-eryth ro-sphingosin e (C6-NBD-Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6-NBD-Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6-NBD-Cer at 2 degrees C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24 degrees C removed excess C6-NBD-Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland, 1988. J. Histochem. Cytochem. 36:555-559), is described and evidence is presented that C6-NBD-Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6-NBD-Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6-NBD-Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.

Stability-Indicating RP-UPLC Method for Simultaneous Determination of Azilsartan Medoxomil and Chlorthalidone in Tablets in the Presence of Its Degradation Products.

Azilsartan Medoxomil (AZL) angiotensin II receptor blocker and chlorthalidone (CLT) were determined by ultraperformance liquid chromatography (UPLC) method in their combined dosage form, they were both subjected to forced degradation studies under extensive stress conditions. The method is a stability indicating by resolving the investigated drugs from their degradation products. Moreover, the degradation products for both drugs obtained from forced degradation were subjected to LC-MS for structure elucidation. The UPLC technique depends on the measurement of spectra for AZL and CLT at 254 nm. Linearity, accuracy and intermediate precision were acceptable over the concentration range of 67.2-268.8 and 40-160 µg/mL for AZL and CLT, respectively. The method was applied for the determination of the studied drugs in their dosage forms. The UPLC method is inexpensive, simple and considered as green chemistry method for the routine analysis and quality control of both drugs in their combined dosage form.

Diversity oriented high-throughput screening of 1,3,4-oxadiazole modified chlorophenylureas and halogenobenzamides by HPLC with peptidomimetic calixarene-bonded stationary phases.

Retention profiles in series of the neutral and highly hydrophobic 1,3,4-oxadiazoles containing chlorophenylurea and halogenobenzamide moiety and indicating analgesic activity were determined in the isocratic standard- and narrow-bore HPLC systems employing, respectively, various octadecylsilica and different calixarene bonded stationary phases. When acetonitrile - 2.65 mM phosphoric acid (55 : 45, %, v/v), pH* 3.25, mobile phase was applied retention of these compounds increased with decline of their overall hydrophobicity according to the general preference of more polar compounds by calixarene cavity in time of its non-specific host-guest supramolecular interactions with halogenated substances. The size of calixarene nanocavity and its upper-rim substitution did not change the observed retention order, resolution and selectivity of separation for oxadiazoles. Compared to the retention on the non-end-capped and the highly-end-capped octadecylsilica HPLC column a most improved separation of some regioisomers of halogenated 1,3,4-oxadiazoles were observed on both used calixarene-type HPLC supports. In addition, preliminary data on the self-assembled supramolecular crystal structure of exemplary 1,3,4-oxadiazolchlorophenylurea with cis-elongated conformation was reported and formation of the monovalent inclusion host-guest complexes between 1,3,4-oxadiazoles and each calixarene-type stationary phase was studied with molecular modelling MM+ and AM1 methods. The structural, isomeric and energetic factors leading to the hydrogen bond stabilized inclusion complexes between these species were considered and used for explanation of observed retention sequence and selectivity of 1,3,4-oxadiazoles separation in applied calixarene-based HPLC systems. All these data would be useful in future development of optimized procedures enabling encapsulation of 1,3,4-oxadiazolurea-type drugs with calixarenes.

Runoff of pesticides from rice fields in the Ile de Camargue (Rhône river delta, France): field study and modeling.

A field study on the runoff of pesticides was conducted during the cultivation period in 2004 on a hydraulically isolated rice farm of 120 ha surface with one central water outlet. Four pesticides were studied: Alphamethrin, MCPA, Oxadiazon, and Pretilachlor. Alphamethrin concentrations in runoff never exceeded 0.001 microg L(-1). The three other pesticides were found in concentrations between 5.2 and 28.2 microg L(-1) in the runoff water shortly after the application and decreased thereafter. The data for MCPA compared reasonably well with predictions by an analytical runoff model, accounting for volatilization, degradation, leaching to groundwater, and sorption to soil. The runoff model estimated that runoff accounted for as much as 18-42% of mass loss for MCPA. Less runoff is observed and predicted for Oxadiazon and Pretilachlor. It was concluded that runoff from rice paddies carries important loads of dissolved pesticides to the wetlands in the Ile de Camargue, and that the model can be used to predict this runoff.

Reverse-Phase Chromatographic Determination and Intrinsic Stability Behavior of 5-[(4-Chlorophenoxy)Methyl]-1,3,4-Oxadiazole-2-Thiol.

The study describes the development and preliminary validation of a simple reverse-phase chromatographic method for determination of a novel drug candidate, 5-[(4-chlorophenoxy) methyl]-1,3,4-oxadiazole-2-thiol (OXCPM), in bulk and stressed solution, in order to find out the intrinsic stability behavior of the compound. Isocratic elution was carried out at a flow rate of 1.0 mL min-1 through a Promosil C18 column maintained at 25 °C, using the mobile phase comprising acetonitrile and aqueous o-H3PO4 (pH 2.67) (1:1, V/V). Detection was performed at 258 nm. The response of the detector was linear in a concentration range of 1.25-50.00 µg mL-1 with the correlation coefficient of 0.9996 ± 0.0001. Cumulative intra-day, inter-day and inter-instrument accuracy (99.5 ± 1.0, 100.2 ± 1.0 and 100.3 ± 0.4 %, resp.) with RSD less than 5 % indicated that the method was accurate and precise. The resolution and selectivity factor (>2 and >1, resp.), particularly in copper metal- and dry-heat-stress solutions, depicted the selectivity of the method. OXCPM remained stable under hydrolytic (acidic and neutral pH, ≤ 37 °C), photolytic and moist heat stress conditions. Under alkaline conditions (hydrolytic and photolytic), polar products were formed that eluted very fast through the column (tR < 3.75 min). At room temperature, the compound was susceptible to oxidation by hydrogen peroxide and transition metals. The ionogram of most of the stress solutions indicated the presence of a product having m/z 256, which might be a result of N- or Smethylation or -SH oxidation. The results of the study indicate that the method is selective, sensitive and suitable to be used for determination of OXCPM in bulk and under stress conditions.