Rabbit Monoclonal Antibody-Based Lateral Flow Immunoassay Platform for Sensitive Quantitation of Four Sulfonamide Residues in Milk and Swine Urine

Based on the available rabbit monoclonal antibody (RabMAb), a rapid and sensitive lateral flow immunoassay (LFA) platform has been developed for quantitative detection of four sulfonamide residues(SRs) of sulfadiazine (SD), sulfathiazole (STZ), sulfapyridine (SP), and sulfamethoxazole (SMX).Within the designed LFA competitive format assay, which was based on antigen-antibody properties, the hapten conjugate N¹-[4-(carboxymethyl)-2-thiazolyl] sulfanilamide linked to protein ovalbumin (TS-OVA) and goat anti-rabbit antibody were sprayed as capture and control reagents, respectively, and then the antibody was conjugated to colloidal gold particles as the detection reagent. With quantitative assessment aided by a colorimetric strip reader, the sensitivities of the established LFA method for SD, STZ, SP, and SMX were 0.91 ng mL^?1, 0.10ng mL^?1,0.12ng mL^?1, and 2.13ng mL^?1, and the half-maximum inhibition concentrations (IC₅₀) were 5.19 ng mL^?1, 1.25 ng mL^?1, 0.66 ng mL^?1, and 24.14 ng mL^?1, respectively. The recoveries at three spiked levels (5, 20, 50 ng mL^?1for SD, STZ, and SP; 20, 50, 100 ng mL^?1 for SMX) were in the range of 78.02–135.10% and 76.40–137.16% for milk and swine urine, respectively. More importantly, the detection performance of the established platform was consistent with that of in-parallel LC-MS/MS analysis. In conclusion, the proposed LFA platform has showed the potential for fast, sensitive and relatively accurate quantification of four sulfonamide residues in practical uses.     

Simultaneous Determination of Fluoroquinolones and Sulfonamides in Chicken Muscle by LC with Fluorescence and UV Detection

A simple, rapid and sensitive liquid chromatographic method with programmable fluorescence and ultraviolet detection was developed and validated for simultaneous determination of seven fluoroquinolones (marbofloxacin, ofloxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin and difloxacin) and four sulfonamides (sulfadiazine, sulfapyridine, sulfathiazole and sulfadimidine) in chicken muscle in a single run. The tissue sample was extracted with phosphate buffer (pH 6.0) and cleaned-up with a solid phase extraction cartridge. The mean recoveries for each drug in chicken muscle ranged from 78.0 to 105.2% with a relative standard deviation below 9.3% at 0.2–400 ng g^?1 fortification levels. The limit of quantification was 0.2–4.0 ng g^?1 for fluoroquinolones and 15.0 ng g^?1 for sulfonamides.     

Determination of Danofloxacin and Marbofloxacin in Milk Samples by Micellar Liquid Chromatography with Fluorescence Detection

Abstract

A simple, rapid, and sensitive analytical method has been developed for the determination of two fluoroquinolones, danofloxacin and marbofloxacin, in bovine milk samples. Separation and quantification were performed by micellar liquid chromatography with fluorescence detection (MLC?FD), using sodium dodecyl sulfate (SDS) as a surfactant. The influence of the principal factors, namely, the micelle concentration, the amount of organic modifier, tail‐reducing agents, the pH, and the temperature were studied. The suitable condition was found to be 75 mM SDS?10 mM phosphate buffer–18 mM tetrabutylammonium bromide/3% (v/v) 1‐propanol at pH 3.0 for the separation of marbofloxacin, danofloxacin, and tosufloxacin (internal standard) in about 20 min. The linear concentration range of application was 1.8–30.0 ng · mL^?1 for danofloxacin and 16–120 ng · mL^?1 for marbofloxacin, and the relative standard deviation ranged between 4.9 and 2.7%. The limit of detection found for danofloxacin was 0.5 ng · mL^?1 and 5 ng · mL^?1 for marbofloxacin. These values were lower than the maximum residue limits (MRLs) established by the European Union for these compounds in bovine milk. It was applied to check the eventual existence of these compounds above these limits on commercial milk samples. The validation method was completed with spiked milk samples. Recovery levels obtained were 90.3–108.2%.     

Immunofluorescent Analysis with Magnetic Nanoparticles for Simultaneous Determination of Antibiotic Residues in Milk

An analytical system for simultaneous detection of three antibiotic residues in milk (penicillin, tetracycline, and sulfadimethoxine sodium salt) was developed. The method was based on a fluorescence immunoassay. Magnetic nanoparticles (MNPs) were prepared and then coated with 3-(aminopropyl) triethoxysilane (APTES). Antibodies, against penicillin, tetracycline, and sulfadimethoxine, were immobilized on APTES-MNPs. Two immobilization methods were used: a random and an oriented method by protein A. The fluorochrome-antigen conjugates, containing FITC, ATTO 590, and ATTO 630 were obtained. Three separate immunofluorescent analyses were investigated for penicillin, tetracycline, and sulfadimethoxine, first in buffer and then in milk. Linearity values of standard curves in milk were: for penicillin 4–15 ng mL^?1, for tetracycline 50–500 ng mL^?1 (using random immobilization) and 100–500 ng mL^?1(using oriented immobilization), and for sulfadimethoxine 100–500 ng mL^?1. The linearity and sensitivity of calibration curves of determination of penicillin, tetracycline and sulfadimethoxine in milk were very close to the obtained results for separate determination of three antibiotics. It was shown that the multiple immunofluorescence analysis for antibiotic residues in milk without preliminary treatment is effective and rapid.     

Development of Immunoaffinity Sample-Purification for GC–MS Analysis of Ractopamine in Swine Tissue

A method combining immunoaffinity chromatography with gas chromatography–mass spectrometry (GC–MS) has been established for determination of ractopamine residues in swine liver and urine. After clean-up on an immunoaffinity chromatography column, GC–MS analysis revealed recovery from blank swine liver and urine fortified at 2.5–20 ng g^?1 (ng mL^?1 for urine), respectively, was 68.2–78.6 and 76.2–83.1%. The limits of detection and quantification were 0.5 ng g^?1 (or ng mL^?1) and 2.0 ng g^?1 (or ng mL^?1), respectively. The procedure was used for analysis of ractopamine residues in samples of swine liver and urine in which the levels were unknown. The amounts detected were 9–216 ng g^?1 (ng mL^?1).     

Colorimetric Determination of Lead Using Gold Nanoparticles

A combined homogeneous assay and colorimetric determination method using gold nanoparticles was developed for rapid determination of lead(II) in contaminated natural waters. The presence of lead(II) in the colloidal gold suspension causes a change in the absorbance of the suspension. An increase in the absorption property at 595 nm is accompanied by a change in the size of the gold nanoparticles. High concentrations of lead cause aggregation of the gold colloids. Colloidal gold nanoparticles were synthesized using tannic acid as the reducing agent; this reagent allowed selective determination of lead in 10 µL of water, with a detection limit of 310 ng mL^?1 with an analysis time of 5 min. The coefficient of variation for lead(II) within the working range of the assay (520 ng mL^?1–13 µg mL^?1) varied from 1.3% to 9.2%. The limit of detection using this method with a sample volume of 50 µL was 60 ng mL^?1. The coefficient of variation for lead over the working range of the determined concentrations (80 ng mL^?1–25 µg mL^?1) varied from 0.2% to 9.3%, while the values for the inter-day assay (n = 8) were less than 10%. The method was employed for the analysis of river, lake, marsh, and spring water; the recovery of lead was determined to be 72.5%–130% for 10 µL of water and 93.6%–114.7% for 50 µL.     

Solvent Bar Microextraction with HPLC for Determination and Protein-Binding Characteristics of Oleanolic Acid and Ursolic Acid

An improved solvent bar microextraction (SBME) combined with HPLC was applied to rapidly determine oleanolic acid (OA) and ursolic acid (UA) in Chinese medicinal herbs (CMHs), and to investigate drug–protein binding. Variables influencing SBME were investigated and optimized. Under optimal conditions, the linear ranges of 3.6–1,820 ng mL^?1 for OA and 4.2–2,080 ng mL^?1 for UA were obtained with square correlation coefficients of 0.9996 and 0.9997, respectively. The detection limits were 1.3 ng mL^?1 for OA and 1.5 ng mL^?1 for UA. The relative standard deviations were lower than 9.4 %. The protein-binding rates, the numbers of binding sites, and the binding constants of OA and UA were also obtained via the method. It has been demonstrated that SBME can not only be a simple, rapid and efficient sample preparation method for determination of active components from CMHs but also be a potential research protocol for protein-binding interactions.     

A novel chemiluminescence reaction system for the determination of lincomycin with diperiodatonickelate(IV)

A sensitive and high selective chemiluminescence (CL) method was developed for the determination of lincomycin in acid medium using diperiodatonickelate as a reagent. The mechanism leading to luminescence is discussed by comparing the spectra of fluorescence and CL. Relative CL intensity is linear in the range from 8.0 ng mL^?1 to 1.0 µg mL^?1, the limit of detection is 2.5 ng mL^?1 (3σ), and the relative standard deviation is 4.0% at 0.1 µg mL^?1 of lincomycin (n?=?7). The method was successfully applied to the determination of lincomycin in injections, human urine, and in serum samples.     

Determination of Alkylphenols in Water by Dispersive Liquid–Liquid Microextraction Based on Solid Formation without a Disperser

Dispersive liquid–liquid microextraction based on solid formation without a disperser combined with high-performance liquid chromatography has been developed for the determination of 4-tert-butylphenol, 4-n-nonylphenol, and 4-tert-octylphenol. This method is rapid, easy, and uses only 10 µL of a low toxicity organic solvent (1-hexadecanethiol) for the extraction solvent and no disperser solvent. The extraction time and centrifugation time require less than 10 min. The linear range was 1–500 ng mL^?1 for 4-tert-butylphenol, 2–1000 ng mL^?1 for 4-tert-octylphenol, and 5–500 ng mL^?1 for 4-n-nonylphenol with r² ≥ 0.9986. The detection limits were between 0.2 and 1.5 ng mL^?1. The recoveries of lake and river water samples were in the range of 79% to 108%, and the relative standard deviations were 5% to 10%.     

Biotin-avidin-conjugated metal sulfide nanoclusters for simultaneous electrochemical immunoassay of tetracycline and chloramphenicol

We report on a protocol for a simultaneous competitive immunoassay for tetracycline (TC) and chloramphenicol (CAP) on the same sensing interface. Conjugates of TC and of CAP with bovine serum albumin were first co-immobilized on a glassy carbon electrode modified with gold nanoparticles. In parallel, monoclonal anti-TC and anti-CAP antibodies were conjugated onto CdS and PbS nanoclusters, respectively. In a typical assay, the immobilized haptens and the added target analytes competed for binding to the corresponding antibodies on the nanoclusters. Subsequently, Cd(II) and Pb(II) ions are released from the surface of the corresponding nanoclusters by treatment with acid and then were detected by square wave anodic stripping voltammetry. The currents at the peak potentials for Cd(II) and Pb(II) were used as the sensor signal for TC and CAP, respectively. This multiplex immunoassay enables the simultaneous determination of TC and CAP in a single run with dynamic ranges from 0.01 to 50 ng mL^?1 for both analytes. The detection limits for TC and for CAP are 7.5 pg mL^?1 and 5.4 pg mL^?1, respectively. No obvious nonspecific adsorption and cross-reactivity was observed in a series of analyses. Intra-assay and inter-assay coefficients of variation were less than 10 %. The method was evaluated by analyzing TC and CAP in spiked samples of milk and honey. The recoveries range from 88 % to 107 % for TC, and from 91 % to 119 % for CAP.

Simultaneous Quantitation of Paracetamol, Pseudoephedrine and Chlorpheniramine in Dog Plasma by LC-MS-MS

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry quantitative detection method, using amantadine as internal standard, was developed for the simultaneous analysis of paracetamol, pseudoephedrine and chlorpheniramine concentrations. Analytes were extracted from plasma samples by liquid–liquid extraction with n-hexane–dichloromethane–2-propanol (2:1:0.1, v/v), separated on a C18 reversed-phase column with 0.1% formic acid–methanol (40:60, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves for plasma were linear over the concentration range 10–10,000 ng mL^?1 of paracetamol, 2–2,000 ng mL^?1 of pseudoephedrine and 0.2–200 ng mL^?1 of chlorpheniramine. The method has a lower limit of quantitation of 10 ng mL^?1 for paracetamol, 2.0 ng mL^?1 for pseudoephedrine and 0.2 ng mL^?1 for chlorpheniramine. Recoveries, precision and accuracy results indicate that the method was reliable within the analytical range, and the use of the internal standard was very effective for reproducibility by LC-MS-MS. This method is feasible for the evaluation of pharmacokinetic profiles of a novel multicomponent sustained release formulation containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride and 2 mg of chlorpheniramine maleate. It is the first time the pharmacokinetic evaluation of a novel sustained-action formulation containing paracetamol, pseudoephedrine and chlorpheniramine has been elucidated in vivo using LC-MS-MS.     

LC–MS–MS Method to Simultaneously Determine Six Probe Drugs for CYP450 Isozymes in Human Liver Microsomes

Here, we report a rapid and specific method based on high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) capable of quantifying six CYP450-specific probe substrates in human liver microsomal incubation mixtures simultaneously. These analytes were prepared by single-step extraction and detected in one run by switching polarity of electrospray ionization mode three times. Following optimization of the chromatographic conditions, the peaks were well separated, and retention times ranged between 2.0 and 8.4 min. The total run time for a single injection was within 9 min. This method was fully validated over linear range of 18.8–3,000.0 ng mL^?1 for diclofenac, 0.8–3,000.0 ng mL^?1 for dapson, 1.5–3,000.0 ng mL^?1 for dextromethorphan, 2.0–4,000.0 ng mL^?1 for omeprazole, 75.0–3,000.0 ng mL^?1 for chlorzoxazone and 0.8–3,000.0 ng mL^?1 for phenacetin using diazepam as internal standard. Samples were prepared by protein precipitation and analyzed on the LC–MS–MS equipped with ESI interface. For each analyte, inter- and intra-day precision (RSD%) were <15 % and accuracy was within 85–115 %. The specificity, precision, accuracy, stabilities and matrix effect were evaluated.     

Determination of Protoberberine Alkaloids in Coptis chinensis by Microextraction and High Performance Liquid Chromatography

Sample preparation technique based on an organic filter membrane (pH-resolved filter membrane microextraction) (pH-RFMME) was developed, coupled with high-performance liquid chromatography, and used to determine protoberberine alkaloids (jatrorrhizine, epiberberine, coptisine, palmatine, and berberine) in Coptis chinensis at different pH values through a one-step procedure. This green procedure provides a desirable sample pretreatment technology. The main variables affecting the extraction such as filter membrane area (or volumes of extraction solvents), sample pH, eluent pH, ionic strength, extraction stirring rate, extraction time, and sample volume were optimized. Under the optimized conditions, the enrichment factors of the analytes were 40.4–52.0, the linear ranges were 3.2–6250 ng · mL^?1 for jatrorrhizine and epiberberine, 6.0–12000 ng · mL^?1 for coptisine, 1.8–3600 ng · mL^?1 for palmatine, and 18.8–18800 ng · mL^?1 for berberine, with r ² ≥ 0.9945. The limits of detection were less than 0.3 ng · mL^?1. Satisfactory recoveries (84.8%–115.5%) and precision (1.8%–10.0%) were also achieved. These results confirmed that pH-RFMME is a simple, rapid, practical, and environmentally friendly method to isolate analytes that exhibit significant differences in acidity or alkalinity from complex samples.     

Magnetic Nanoparticle-Based Micro-Solid Phase Extraction and GC�CMS Determination of Oxadiargyl in Aqueous Samples

A new facile, rapid, inexpensive, and sensitive method based on magnetic micro-solid phase extraction (M-??-SPE) coupled to gas chromatography?Cmass spectrometry (GC?CMS) was developed for determination of the herbicide oxadiargyl in environmental water samples. The feasibility of employing non-modified magnetic nanoparticles (MNPs) as sorbent was examined and applied to perform the extraction process. Influential parameters affecting the extraction efficiency along with desorption conditions were investigated and optimized. The limit of detection (LOD, S/N = 3) and limit of quantification (LOQ, S/N = 10) of the method under optimized conditions were 0.005 and 0.030 ng mL^?1, respectively. The relative standard deviations (RSD) (n = 3) at a concentration of 0.10 ng mL^?1 was 11%. The calibration curve of oxadiargyl showed linearity in the range of 0.050?C0.50 ng mL^?1. The developed method was successfully applied to the extraction of oxadiargyl from spiked tap water and Zayande-Rood River water samples and the relative recoveries of 98 and 94% were obtained, respectively.     

Diphenyl diselenide grafted onto a Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>-chitosan composite as a new nanosorbent for separation of metal ions by effervescent salt-assisted dispersive magnetic micro solid-phase extraction

Diphenyl diselenide was immobilized on chitosan loaded with magnetite (Fe₃O₄) nanoparticles to give an efficient and cost-effective nanosorbent for the preconcentration of Pb(II), Cd(II), Ni(II) and Cu(II) ions by using effervescent salt-assisted dispersive magnetic micro solid-phase extraction (EA-DM-μSPE). The metal ions were desorbed from the sorbent with 3M nitric acid and then quantified via microflame AAS. The main parameters affecting the extraction were optimized using a one-at-a-time method. Under optimum condition, the limits of detection, linear dynamic ranges, and relative standard deviations (for n?=?3) are as following: Pb(II): 2.0 ng·mL^?1; 6.3–900 ng·mL^?1; 1.5%. Cd(II): 0.15 ng·mL^?1; 0.7–85 ng·mL^?1, 3.2%; Ni(II): 1.6 ng·mL^?1,.6.0–600. ng·mL^?1, 4.1%; Cu(II): 1.2 ng·mL^?1, 3.0–300 ng·mL^?1, 2.2%. The nanosorbent can be reused at least 4 times.

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Graphical abstract Fe₃O₄-chitosan composite was modified with diphenyl diselenide as a sorbent for separation of metal ions by effervescent salt-assisted dispersive magnetic micro solid-phase extraction.

     

A Novel Electrochemical Immunosensor for Prostate-Specific Antigen Based on Noncovalent Nanocomposite of Ferrocene Monocarboxylic Acid with Graphene Oxide

A novel electrochemical immunosensor was developed for the determination of prostate-specific antigen based on immobilization of appropriate antibodies on gold nanoparticles and a poly-(2,6-pyridinediamine) modified electrode. The nanocomposite of ferrocene monocarboxylic acid hybridized graphene oxide was prepared by a π-π stacking interaction and was used as the electrochemical probe. A sandwich-type complex immunoassay was applied with polyclonal prostate-specific antigen antibodies labeled with the nanocomposite of ferrocene monocarboxylic acid hybridized graphene oxide. In order to improve the sensitivity, a potentiostatic method was used to reduce graphene oxide. Cyclic voltammetry and differential pulse voltammetry were employed to characterize the assembly process and the performance of the immunosensor. Under optimal conditions, the peak current of the immunosensor increased with concentration, showing a linear relationship between the peak current and the logarithm of the prostate-specific antigen concentrations in a wide range of 2.0 pg mL^?1 to 10.0 ng mL^?1 with a low detection limit of 0.5 pg mL^?1. The immunosensor was used for the determination of prostate-specific antigen in serum.     

Simultaneous Determination of Amoxicillin and Clavulanic Acid in Dog Plasma by High-Performance Liquid Chromatography Tandem Mass Spectrometry

A rapid, sensitive, and specific high-performance liquid chromatography tandem mass spectrometric method was developed for the simultaneous determination and confirmation of amoxicillin and clavulanic acid in plasma. Plasma sample was subjected to a simple deproteinization with acetonitrile, and then the supernatant was directly diluted by water. Analysis was performed on a Phenomenex Luna C₈ reversed-phase column by detection with mass spectrometry in negative ions multiple reaction monitoring mode. A gradient elution program with 0.1% formic acid and acetonitrile was performed at a flow of 0.25 mL min^?1. There is good linearity in the range of 0.5–500 ng mL^?1 for both amoxicillin and clavulanic acid. The decision limits of amoxicillin and clavulanic acid were 0.06 ng mL^?1 and 0.08 ng mL^?1 in plasma, respectively, and the detection capabilities of two analytes were below 0.5 ng mL^?1. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The extraction recoveries of amoxicillin and clavulanic acid were between 102% and 115% in plasma at three spiked levels of 0.5, 50, and 500 ng mL^?1, with the relative standard deviations less than 15% for each analyte. The developed method was applied to pharmacokinetic studies of amoxicillin and clavulanic acid tablets in healthy beagles.     

Synthesis and application of a novel magnetic nanosorbent for determination of trace Cd(II), Ni(II), Pb(II), and Zn(II) in environmental samples

In this work for the first time, Fe₃O₄@SiO₂ core–shell nanoparticles functionalized with isatin groups as a magnetic nanosorbent was applied for the simultaneous extraction of trace amounts of cadmium(II), nickel(II), lead(II), and zinc(II). The characterization of this nanosorbent was studied using Fourier transform infrared spectroscopy, scanning electron microscopy, energy-dispersive X-ray spectrometry, X-ray diffraction, vibrating sample magnetometer and thermogravimetric analysis. The effect of several factors such as pH, amount of sorbent, extraction time, type and volume of the eluent, sample volume, sorption capacity, and potentially interfering ions was investigated. In the selected conditions, it was observed that the limits of detection were 0.11 ng mL^?1 for Cd(II), 0.28 ng mL^?1 for Ni(II), 0.47 ng mL^?1 for Pb(II), and 0.21 ng mL^?1 for Zn(II), and the maximum sorption capacity of this suggested magnetic nanosorbent was 120, 112, 100, and 100 mg g^?1 for Cd(II), Ni(II), Pb(II), and Zn(II), respectively. Also, the precision of the method (RSD%) for ten replicate measurements was found 2.5, 2.5, 2.8, and 3.1%, for Cd(II), Ni(II), Pb(II), and Zn(II) ions, respectively. Finally, the suggested procedure was applied for determination of cadmium(II), nickel(II), lead(II), and zinc(II) at trace levels in different water and agricultural products with satisfactory results.     

Quantification of Thiram in Honeybees: Development of a Chemiluminescent ELISA

Abstract

A Chemiluminescence Enzyme‐Linked Immuno‐Sorbent Assay (CL‐ELISA) for determination and quantification of the fungicide thiram in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti‐thiram antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC₅₀ and the limit of detection (LOD) values were 60 ng mL^?1 and 9 ng mL^?1, respectively, similar to those of colorimetric ELISA with a calibration range of 9–15,000 ng mL^?1. Cross reactivity of some related compounds such as some dithiocarbamates, a thiocarbamate, the ethylenethiourea and the tetramethylthiourea were tested. The assay was then applied to honeybees sample extracts obtained by using the liquid‐liquid extraction or the graphitized carbon‐based solid phase extraction.

The calibration curves in honeybee extracts from liquid‐liquid procedure gave an IC₅₀ of 141 ng mL^?1 and a LOD of 17 ng mL^?1. In case of extracts obtained by SPE these values were 139 ng mL^?1 and 15 ng mL^?1, respectively. The average recovery value from honeybee extracts spiked with 75 ng mL^?1 of thiram was 72% for SPE, higher than for liquid‐liquid extraction (60%). On the opposite, when the honeybees were directly spiked with 2 and 10 ppm the average recovery was higher for liquid‐liquid extraction (54%), than for SPE (31%). Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and Russia.     

Determination of Pantoprazole, Rabeprazole, Esomeprazole, Domperidone and Itopride in Pharmaceutical Products by Reversed Phase Liquid Chromatography Using Single Mobile Phase

A simple, sensitive, and precise high performance liquid chromatographic method for the analysis of pantoprazole, rabeprazole, esomeprazole, domperidone and itopride, with ultraviolet detection at 210 nm, has been developed, validated, and used for the determination of compounds in commercial pharmaceutical products. The compounds were well separated on a Hypersil BDS C18 reversed-phase column by use of a mobile phase consisting of 0.05 M, 4.70 pH, potassium dihydrogen phosphate buffer - acetonitrile (720:280 v/v) at a flow rate of 1.0 mL min^?1. The linearity ranges were 400–4,000 ng mL^?1 for pantoprazole, 200–2,000 ng mL^?1 for rabeprazole, 400–4,000 ng mL^?1 for esomeprazole, 300–3,000 ng mL^?1 for domperidone and 500–5,000 ng mL^?1 for itopride. Limits of detection (LOD) obtained were: pantoprazole 147.51 ng mL^?1, rabeprazole 65.65 ng mL^?1, esomeprazole 131.27 ng mL^?1, domperidone 98.33 ng mL^?1 and itopride 162.35 ng mL^?1. The study showed that reversed-phase liquid chromatography is sensitive and selective for the determination of pantoprazole, rabeprazole, esomeprazole, domperidone and itopride using single mobile phase.