Microbial-based metabolites associated with degradation of imidacloprid and its impact on stress-responsive proteins.

Imidacloprid (IMD), a neonicotinoid insecticide, is intensively used in agricultural fields for effective protection against aphids, cane beetles, thrips, stink bugs, locusts, etc., is causing serious environmental concerns. In recent years, seed treatment with Imidacloprid is being practiced mainly to prevent sucking insect pests. In India, due to the increase in application of this insecticide residue has been proven to have an impact on the quality of soil and water. In view of this, the current investigation is focussed on sustainable approach to minimize the residual effect of IMD in agricultural fields. The present study reveals a most promising imidacloprid resistant bacterium Lysinibacillus fusiformis IMD-Bio5 strain isolated from insecticide-contaminated soil. The isolated bacterial strain upon tested for its biodegradation potential on mineral salt medium (MSM) showed a significant survival growth at 150 g/L of IMD achieved after 3 days, whereas immobilized cells on MSM amended with 200 g/L of IMD as the sole carbon source provided degradation of 188 and 180 g/L of IMD in silica beads and sponge matrices, respectively. The liquid chromatography mass spectrometry was performed to test the metabolite responsive for IMD biodegradation potential of L. fusiformis IMD-Bio5 which showed the induced activity of the metabolite 6-Chloronicotinic acid. Furthermore, as compared to the untreated control, the Lysinibacillus fusiformis IMD-Bio5 protein profile revealed a range of patterns showing the expression of stress enzymes. Thus, results provided a most effective bacterium enabling the removal of IMD-like hazardous contaminants from the environment, which contributes to better agricultural production and soil quality, while long-term environmental advantages are restored.

Dissipation and residues of imidacloprid in amaranth under greenhouse and open field cultivations.

Despite the extensive exposure to imidacloprid residues in food plants, there has been little research on imidacloprid residues in amaranth. The dissipation trend and residue behavior of imidacloprid were evaluated to provide guidelines for imidacloprid application on amaranth under open field and greenhouse. The dissipation rate of imidacloprid in amaranth conformed to the first-order kinetic equation, and the half-lives of imidacloprid in amaranth ranged from 0.29 days in open field to 1.29 days in the greenhouse. After 7 and 14 days from the application of imidacloprid (pesticide dosage, 45 or 67.5 g a.i./ha), the amaranth under the open field and greenhouse growth could be consumed safely with average residues of 0.19 and 0.38 mg/kg, respectively. This result demonstrated that the cultivation has the dominant influence on imidacloprid residue, and the residue of imidacloprid in amaranth planting on open field was much lower than that in the greenhouse, indicating a significant difference in the pesticide residues between the two cultivations with a p-value less than 0.05.

Low doses of the neonicotinoid insecticide imidacloprid induce ROS triggering neurological and metabolic impairments in Drosophila.

Declining insect population sizes are provoking grave concern around the world as insects play essential roles in food production and ecosystems. Environmental contamination by intense insecticide usage is consistently proposed as a significant contributor, among other threats. Many studies have demonstrated impacts of low doses of insecticides on insect behavior, but have not elucidated links to insecticidal activity at the molecular and cellular levels. Here, the histological, physiological, and behavioral impacts of imidacloprid are investigated in Drosophila melanogaster, an experimental organism exposed to insecticides in the field. We show that oxidative stress is a key factor in the mode of action of this insecticide at low doses. Imidacloprid produces an enduring flux of Ca2+ into neurons and a rapid increase in levels of reactive oxygen species (ROS) in the larval brain. It affects mitochondrial function, energy levels, the lipid environment, and transcriptomic profiles. Use of RNAi to induce ROS production in the brain recapitulates insecticide-induced phenotypes in the metabolic tissues, indicating that a signal from neurons is responsible. Chronic low level exposures in adults lead to mitochondrial dysfunction, severe damage to glial cells, and impaired vision. The potent antioxidant, N-acetylcysteine amide (NACA), reduces the severity of a number of the imidacloprid-induced phenotypes, indicating a causal role for oxidative stress. Given that other insecticides are known to generate oxidative stress, this research has wider implications. The systemic impairment of several key biological functions, including vision, reported here would reduce the resilience of insects facing other environmental challenges.

Trace determination of imidacloprid and its major metabolites in wheat-soil system.

Trace analysis method is a reliable basis for studying the translocation and metabolism of imidacloprid used as an insecticide in wheat, and it clarifies whether biologically active metabolites including residual imidacloprid, have long-lasting insecticidal potency against wheat aphids under seed treatment during the entire growth period. In this study, a highly sensitive analytical method was established to determine the residues of imidacloprid and its six metabolites (5-hydroxy imidacloprid, imidacloprid olefin, imidacloprid guanidine, imidacloprid urea, 6-chloronicotinic acid, and imidacloprid nitrosimine) in wheat-soil systems, such as in wheat leaves, wheat ears, wheat grains, roots, and soil. All the compounds were extracted using an ACN:water (8:2, v/v) mixture and purified by dispersive solid-phase extraction. The average recoveries ranged from 74.4% to 109.5% for all matrices, with intra- and inter-day variations of less than 14.9%. The limit of quantitation was in the range of 0.001-0.005 mg/kg. The method is demonstrated to be sensitive and accurate for monitoring imidacloprid and its metabolites at trace levels during the entire growth period under field conditions.

A novel stability-indicating HPLC method for the determination of enantiomeric purity of eluxadoline drug: Amylose tris(3,5-dichlorophenyl carbamate) stationary phase.

A simple and sensitive stability-indicating chiral HPLC method has been developed and validated per International Conference on Harmonization guidelines for the determination of enantiomeric purity of eluxadoline (Exdl). The impact of different mobile phase compositions and chiral stationary phases on the separation of Exdl enantiomer along with process- and degradation-related impurities has been studied. Homogeneity of Exdl and stable results of Exdl enantiomer in all degraded samples reveal the fact that the proposed method was specific (stability indicating). Amylose tris(3,5-dichlorophenyl carbamate) stationary phase column Chiralpak IE-3 (150 × 4.6 mm, 3 µm) provided better resolution with polar organic solvents than cellulose derivative, crown ether, and zwitterion stationary phases and nonpolar solvents. The mobile phase consisted of acetonitrile, tetrahydrofuran, methanol, butylamine, and acetic acid in the ratio of 500:500:20:2:1.5 (v/v/v/v/v). Isocratic elution was performed at a flow rate of 1.0 mL/min, column temperature of 35°C, injection volume of 10 µL, and UV detection of 240 nm. The United States Pharmacopeia (USP) resolution of the Exdl enantiomer was found to be more than 4.0 within a 65-min run time. Exdl enantiomer detector response linearity over the concentration range of 0.859-4.524 µg/mL was found to be R2  = 0.9985. The limit of detection, limit of quantification, and average percentage recovery values were established as 0.283 µg/mL, 0.859 µg/mL, and 96.0, respectively.

HPLC Fluorescence Method for Eugenols in Basil Products Derivatized with DIBI.

Eugenols (Eugs) such as eugenol (Eug), methyleugenol (MeEug), and linalool (Lin) in basil product are the main bioactive components of basil products and have a terminal double-bond. A sensitive HPLC-fluorescence method for Eugs derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl)iodobenzene (DIBI) was developed. Good separation of DIB-Eugs was achieved within 20 min on an Atlantis T3 column (50 × 2.1 mm i.d., 3 µm) with a mobile phase of methanol-water. The calibration curves obtained with Eug standards showed good linearities in the range of 0.1-50 µM (r ≥ 0.999). The limits of detection at a signal-to-noise ratio (S/N) = 3 for Eug, MeEug, and Lin were 1.0, 6.0, and 4.8 nM, respectively. The limits of quantitation (S/N = 10) of the Eugs were lower than 19.9 nM. The accuracies for the Eugs were within 96.8-104.6%. The intra- and inter-day precisions as relative standard deviations for the Eugs were less than 1.2 and 9.6% (n = 3). The recoveries of Eug, MeEug, and Lin were 99.0 ± 0.1, 98.0 ± 0.2, and 96.0 ± 0.4% (n = 3), respectively. The DIB-Eugs were confirmed to be stable for 2 h (>90%) at room temperature and 24 h (>95%) at 4 °C. These parameters of the proposed method were useful for the simultaneous determination of Eugs in basil products. Therefore, the developed method may be a powerful tool for the quality evaluation of dried commercially available basil products.

Mechanical stimulus-evoked signal transduction between keratinocytes and sensory neurons via extracellular ATP.

The skin is exposed to various external stimuli. Keratinocytes, which are the main cell type in the epidermis, interact with peripheral sensory neurons and modulate neuronal activity. Recent studies have revealed that keratinocytes play crucial roles in nociception, and that ATP is one of the main mediators of signal transduction from keratinocytes to sensory neurons. However, no quantitative cellular level analyses of ATP-mediated information flow from keratinocytes to sensory dorsal root ganglion (DRG) neurons have been conducted. In this study, we performed simultaneous imaging of cell surface ATP and intracellular Ca2+ signals using both iATPSnFR, a genetically encoded ATP probe localized to the outside of the cell membrane, and the Ca2+ probe, Fura-red. Upon mechanical stimulation of the keratinocyte with a glass needle, an increase in Ca2+ and ATP release were observed around the stimulated area, and these phenomena were positively correlated. In cultured DRG neurons and keratinocytes neighboring the stimulated keratinocyte, increased intracellular Ca2+ concentration and levels of cell surface ATP on the side closer to the stimulated cell were detected. The ratio of Ca2+ response to input ATP signal was significantly larger in DRG neurons than in keratinocytes. We found that DRG neurons were more sensitive to ATP than keratinocytes, and therefore, only DRG neurons responded to ATP at 1 µM or lower concentrations when in co-culture with keratinocytes. Moreover, signals caused by moderate mechanical stimulation of keratinocytes were transmitted predominantly to DRG neurons. These findings would be important in the further determination of the detailed mechanism of nociception in the epidermis.

Electrochemical detection of alkaline phosphatase activity through enzyme-catalyzed reaction using aminoferrocene as an electroactive probe.

As a nonspecific phosphomonoesterase, alkaline phosphatase (ALP) plays a pivotal role in tissue mineralization and osteogenesis which is an important biomarker for the clinical diagnosis of bone and hepatobiliary diseases. Herein, we described a novel electrochemical method that used aminoferrocene (AFC) as an electroactive probe for the ALP activity detection. In the condition with imidazole and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the AFC probe could be directly labeled on single-stranded DNA (ssDNA) by one-step conjugation. Specifically, thiolated ssDNA at 3'-terminals was modified to the electrode surface through Au-S bond. In the condition without ALP, AFC could be labeled on ssDNA by conjugating with phosphate groups. In the presence of ALP, phosphate groups were catalyzed to be removed from the 5'-terminal of ssDNA. The AFC probe cannot be labeled on ssDNA. Thus, the electrochemical detection of ALP activity was achieved. Under optimal conditions, the strategy presented a good linear relationship between current intensity and ALP concentration in the range of 20 to 100 mU/mL with the limit of detection (LOD) of 1.48 mU/mL. More importantly, the approach rendered high selectivity and satisfactory applicability for ALP activity detection. In addition, this method has merits of ease of operation, low cost, and environmental friendliness. Thus, this strategy presents great potential for ALP activity detection in practical applications. An easy, sensitive and reliable strategy was developed for the detection of alkaline phosphatase activity via electrochemical "Signal off".

Bees prefer foods containing neonicotinoid pesticides.

The impact of neonicotinoid insecticides on insect pollinators is highly controversial. Sublethal concentrations alter the behaviour of social bees and reduce survival of entire colonies. However, critics argue that the reported negative effects only arise from neonicotinoid concentrations that are greater than those found in the nectar and pollen of pesticide-treated plants. Furthermore, it has been suggested that bees could choose to forage on other available flowers and hence avoid or dilute exposure. Here, using a two-choice feeding assay, we show that the honeybee, Apis mellifera, and the buff-tailed bumblebee, Bombus terrestris, do not avoid nectar-relevant concentrations of three of the most commonly used neonicotinoids, imidacloprid (IMD), thiamethoxam (TMX), and clothianidin (CLO), in food. Moreover, bees of both species prefer to eat more of sucrose solutions laced with IMD or TMX than sucrose alone. Stimulation with IMD, TMX and CLO neither elicited spiking responses from gustatory neurons in the bees' mouthparts, nor inhibited the responses of sucrose-sensitive neurons. Our data indicate that bees cannot taste neonicotinoids and are not repelled by them. Instead, bees preferred solutions containing IMD or TMX, even though the consumption of these pesticides caused them to eat less food overall. This work shows that bees cannot control their exposure to neonicotinoids in food and implies that treating flowering crops with IMD and TMX presents a sizeable hazard to foraging bees.

Analysis of pesticide residues by a support vector machine combined with fluorescence spectroscopy.

Pesticide residues enter a lake through the water cycle, causing harm to the water environment and human health. It is necessary to select highly sensitive fluorescence spectroscopy to detect pesticides (bifenthrin, prochloraz, and cyromazine), and a support vector machine (SVM) is used to analyze the concentration of pesticides. In addition, this paper adopts K-fold cross validation and a grid search to optimize the SVM algorithm. The performance evaluation index and running time prove the reliability of the results of this experiment. They show that fluorescence spectroscopy combined with SVM is efficient in predicting pesticide residue content.

In situ controllable synthesis of Schiff base networks porous polymer coatings for open-tubular capillary electrochromatography.

A uniform Schiff base network (SNW) film was synthesized in situ in a controllable way through continuous flow of reactants inside the capillary. The properties and application of the as-prepared capillary was investigated in capillary electrochromatography. The effects of reaction monomer concentration and reaction time on coating thickness were studied by SEM. The results show that the reaction condition has a significant influence on the morphology and thickness of the SNW films. The thickness of the film can be controlled by changing the concentration of reaction solution and reaction time. Capillaries coated under different conditions were employed to separate four nucleotides by capillary electrochromatography, which demonstrated significant variation of migration time, peak order, and separation efficiency. Analytes containing nitrogen heterocycle structures, such as nucleotides, methylimidazole isomers, and ß-lactam antibiotics, were successfully separated with the prepared open-tubular columns. Under the selected separation conditions, theoretical plate number of four nucleotides is in a range 45,237-104,505 plates·m-1, and the resolutions are 1.98-8.07. A resolution of 1.75 is obtained for methylimidazole isomers. The nucleotides in a real sample, chicken essence seasoning, were determined using the prepared capillary column with satisfactory recoveries in the range 95 to 105%.

L-Histidine-DNA interaction: a strategy for the improvement of the fluorescence signal of poly(adenine) DNA-templated gold nanoclusters.

An interesting phenomenon is described that the fluorescence signal of poly(adenine) (A) DNA-templated gold nanoclusters (AuNCs) is greatly improved in the presence of L-histidine by means of L-histidine-DNA interaction. The modified nanoclusters display strong fluorescence emission with excitation/emission maxima at 290/475 nm. The fluorescence quantum yield (QY) is improved from 1.9 to 6.5%. Fluorescence enhancement is mainly ascribed to the L-histidine-DNA interaction leading to conformational changes of the poly(A) DNA template, which offer a better microenvironment to protect AuNCs. The assay enables L-histidine to be determined with good sensitivity and a linear response that covers the 1 to 50 nM L-histidine concentration range with a 0.3 nM limit of detection. The proposed method has been applied to the determination of imidazole-containing drugs in pharmaceutical samples. A turn-on fluorescent method has been designed for the sensitive detection of L-histidine as well as imidazole-containing drugs on the basis of the L-histidine-DNA interaction.

LC and NMR Studies for Identification and Characterization of Degradation Byproducts of Olmesartan Acid, Elucidation of Their Degradation Pathway and Ecotoxicity Assessment.

The discovery of various sartans, which are among the most used antihypertensive drugs in the world, is increasingly frequent not only in wastewater but also in surface water and, in some cases, even in drinking or groundwater. In this paper, the degradation pathway of olmesartan acid, one of the most used sartans, was investigated by simulating the chlorination process normally used in a wastewater treatment plant to reduce similar emerging pollutants. The structures of nine isolated degradation byproducts (DPs), eight of which were isolated for the first time, were separated via chromatography column and HPLC methods, identified by combining nuclear magnetic resonance and mass spectrometry, and justified by a proposed mechanism of formation beginning from the parent drug. Ecotoxicity tests on olmesartan acid and its nine DPs showed that 50% of the investigated byproducts inhibited the target species Aliivibrio fischeri and Raphidocelis subcapitata, causing functional decreases of 18% and 53%, respectively.

RP-HPLC method development and validation for quantification of daclatasvir dihydrochloride and its application to pharmaceutical dosage form.

Daclatasvir dihydrochloride is an antiviral drug used in the treatment of Hepatitis C and for its estimation in drug product, no Pharmacopeial method is available. Therefore, a simple, rapid, precise and accurate isocratic RP-HPLC method was developed and validated for quantification of daclatasvir dihydrochloride in pharmaceutical dosage form. The quantification was carried out using Hypersil ODS - C18 Column (250mm, 4.6mm, 5µm), Shimadzu LC-2030 Prominence-I Series. The mobile phase composed of phosphate buffer (pH 3.5, adjusted with ortho phosphoric acid) and acetonitrile (60:40 v/v). The flow rate was 1.0ml/min with UV detection at 308 nm. The validation of developed method was conducted for specificity, linearity, accuracy, precision, LOD and LOQ. A linearity was established in the concentration range of 0.5-150% with coefficient of correlation 0.9993. The limit of detection (LOD) was 0.005µg/ml and the limit of quantification (LOQ) was 0.01µg/ml. The method was successfully applied to the assay and in-vitro dissolution studies of daclatasvir dihydrochloride in tablet dosage form. It can be concluded that this method can be very helpful in the quality control estimation of daclatasvir dihydrochloride in different pharmaceutical products intended for hepatitis C infections.

Electrochemical Quantitation of Supramolecular Excipient@Drug Complexation: A General Assay Strategy Based on Competitive Host Binding with Surface-Immobilized Redox Guest.

The macrocyclic cucurbit[7]uril (CB[7]) host has exhibited great application potential as a pharmaceutical excipient due to its versatile abilities to modulate the chemical/physical properties of drug molecules (guests) and to control their in vivo delivery and release (upon complexation). The formation of stable CB[7]@drug complexes is the prerequisite for these promising applications; we report herein a general assay strategy to quantitate the complexation based on competitive binding with surface-immobilized redox guests in conjunction with conventional electrochemical techniques (e.g., cyclic voltammetry). Particularly, by incubating a mixture of CB[7] and a drug molecule with ferrocene (Fc)-terminated self-assembled monolayers (SAMs) on gold, the competitive host@guest binding between the CB[7]@drug complex formed in solution and the CB[7]@Fc complex formed on surface can be quantified with direct cyclic voltammetry measurements. On the basis of the known concentrations of CB[7]/drug and electrochemically determined surface densities of free/complexed Fc groups, the formation constant of CB[7]@drug complex can be determined. With several drug molecules as examples, we have demonstrated the capability of this method for quantitative studies of the formation of supramolecular excipient@drug complexes that are of interest in pharmaceutical and biomedical sciences. More importantly, this work promises a general assay strategy that allows electrochemical quantitation of a wide range of electro-inactive analytes based on the competitive supramolecular host@guest binding at redox-tagged molecular interfaces.

HPLC method transfer study for simultaneous determination of seven angiotensin II receptor blockers.

In this study, a sensitive high-performance liquid chromatography method was developed and validated for the simultaneous determination of seven angiotensin II receptor blockers, namely, hydrochlorothiazide, chlorthalidone, eprosartan mesylate, valsartan, losartan potassium, irbesartan, and candesartan cilexetil. Different chromatographic parameters were tested and fully optimized. Best chromatographic separation was accomplished on a reversed-phase octadecylsilyl column (250 × 4.6 mm id; 5 µm) under gradient elution using methanol/sodium phosphate monobasic buffer (0.01 M, pH 6.5) as mobile phase. The detection of target analytes was obtained at 254 nm. The pH of the buffer has been selected according to Marvin® sketch software. The proposed method was validated according to ICH guidelines and showed good precision (relative standard deviation < 1), good linearity (square of correlation coefficient ≥ 0.999), and high accuracy (between 98 and 102%) with detection limit and quantitation limit (40 and 160 ng/mL, respectively) for all the detected analytes.

In vitro metabolites characterization of ponatinib in human liver microsomes using ultra-high performance liquid chromatography combined with Q-Exactive-Orbitrap tandem mass spectrometry.

Ponatinib is an oral drug for the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia, which has been reported to increase the risk of hepatotoxicity. The aim of this study was to characterize the metabolites of ponatinib in human liver microsomes as well as its reactive metabolites. Ponatinib was incubated with human liver microsomes in the presence of NADPH and trapping agents (glutathione or potassium cyanide). The metabolites were characterized by liquid chromatography in combination with Q-Exactive-Orbitrap-MS. Under the current conditions, six metabolites were detected and structurally identified on the basis of their accurate masses, fragmentation patterns, and retention times. M3 (N-demethylation) was unambiguously identified by matching its retention time and fragment ions with those of its reference standard. N-demethylation and oxygenation were proved to be the predominant metabolic pathways of ponatinib. In addition, two reactive metabolites (cyano adducts) were detected in human liver microsomes in the presence of potassium cyanide and NADPH, suggesting that ponatinib underwent CYP450-mediated metabolic activation, which could be one of the causative mechanisms for its hepatotoxicity. The current study provides new information regarding the metabolic profiles of ponatinib and would be helpful in understanding the effectiveness and toxicity of ponatinib, especially the mechanism of hepatotoxicity.

The Dissipation of Cyazofamid and Its Main Metabolite CCIM During Wine-Making Process.

Few studies have focused on the residues of cyazofamid and its main metabolite CCIM (4-chloro-5-p-tolylimidazole-2-carbonitrile) in the wine making process, which is crucial to evaluate the potential food risk of cyazofamid and CCIM. In this work, detailed study has been conducted on the evaluation of the fate of cyazofamid and its main metabolite CCIM during the wine-making process. The targeted compounds cyazofamid and CCIM were separated and determined by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) and processing procedure including washing, peeling, fermentation, and clarification. Results showed that residues of cyazofamid and CCIM decreased significantly in wine processing. The dissipation of cyazofamid in the fermentation process followed the first-order of kinetics, and the half-life of cyazofamid was 46.2-63.0 h, whereas, the residues of CCIM, in the three treatments, decreased with time elapse. The processing factors (PFs) were all less than one in different processing processes, and the PFs ranges of cyazofamid and CCIM were 0.003-0.025 and 0.039-0.067 in three treatments in the overall process. The outcome indicated that the whole process could significantly reduce the residues of cyazofamid and CCIM in red and white wines. The results might provide more precise risk assessments of cyazofamid in the wine-making process.

Optimization of a Simplified and Effective Analytical Method of Pesticide Residues in Mealworms (Tenebrio molitor Larvae) Combined with GC-MS/MS and LC-MS/MS.

An effective analytical method was optimized for residues including chlorpyrifos-methyl, deltamethrin, fenoxanil, thiobencarb and fludioxonil in mealworms, the larval form of Tenebrio molitor. They are listed for pest control during wheat cultivation and can be found in wheat-bran feed for growing mealworms in South Korea. Analytes were extracted using acetonitrile and salt packet. Four clean-up methods ((1) MgSO4 + 25 mg PSA + 25 mg C18; (2) MgSO4 + 50 mg PSA + 50 mg C18; (3) EMR-lipidTM tube; and (4) 10 mL n-hexane) were investigated and the method (1) was selected due to its robustness. Low-temperature precipitation of fat and proteins improved the recoveries. Recoveries from the Method (1) were satisfying with 70-120% with <20% relative SD at a spiking level of 0.01 mg/kg. With the simultaneous sample preparation, fenoxanil, thiobencarb and fludioxonil were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and chlorpyrifos-methyl and deltamethrin by gas chromatography tandem mass spectrometry (GC-MS/MS). Quantification limits for LC-MS/MS and GC-MS/MS were 0.5 and 2.5 µg/L, respectively. No pesticides of interest were detected in 30 real samples collected across the nation. However, the data can be provided for establishing maximum residue limits for the pesticides in mealworms in response to the positive list system.

HPLC Determination of Imidazoles with Variant Anti-Infective Activity in Their Dosage Forms and Human Plasma.

A suitable HPLC method has been selected and validated for rapid simultaneous separation and determination of four imidazole anti-infective drugs, secnidazole, omeprazole, albendazole, and fenbendazole, in their final dosage forms, in addition to human plasma within 5 min. The method suitability was derived from the superiority of using the environmentally benign solvent, methanol over acetonitrile as a mobile phase component in respect of safety issues and migration times. Separation of the four anti-infective drugs was performed on a Thermo Scientific® BDS Hypersil C8 column (5 µm, 2.50 × 4.60 mm) using a mobile phase consist of MeOH: 0.025 M KH2PO4 (70:30, v/v) adjusted to pH 3.20 with ortho-phosphoric acid at room temperature. The flow rate was 1.00 mL/min and maximum absorption was measured with UV detector set at 300 nm. Limits of detection were reported to be 0.41, 0.13, 0.18, and 0.15 µg/mL for secnidazole, omeprazole, albendazole, and fenbendazole, respectively, showing a high degree of the method sensitivity. The method of analysis was validated according to Food and Drug Administration (FDA)guidelines for the determination of the drugs, either in their dosage forms with highly precise recoveries, or clinically in human plasma, especially regarding pharmacokinetic and bioequivalence studies.