Assessment of Dentifrices Against Candida Biofilm

The invasion of opportunistic pleiomorphic Candida albicans into oral cavity environment leads to development and progression of its resistance to both naturally occurring antifungal peptides in human saliva as well as commercially available antifungal therapies. As a result of this, the usage and popularity of natural medicine and dentifrices had increased significantly in the last decade. In the present investigation, we have assessed the action of locally available dentifrices against C. albicans biofilm. Disk diffusion test showed maximum zone of inhibition (20?mm) by herbal dentifrice (D-5) as compared to other dentifrices when incubated at 37?°C and 48?h. Assessment of dentifrice D-5 for its effectiveness against C. albicans was further shown in MIC₉₀ (3.12?mg?mL^?1) and SMIC₉₀ (6.2?mg?mL^?1) values for planktonic and sessile cells (biofilm forming), respectively. Our data depicted 80% reduction in the cell surface hydrophobicity when 6.2?mg?mL^?1 of herbal dentifrice D-5 was used against 48-h grown Candida biofilm at 37?°C. Visualization of herbal dentifrice D-5-treated C. albicans biofilm under SEM revealed drastic reduction in the dense network of yeast, hyphae, and pseudohyphae enclosed in its ECM as compared to its control biofilm. The data were further supported by CLSM analysis which depicted C. albicans architecture disruption by herbal dentifrices. From the above data, it is inferred that these studies would provide researchers and medical practitioners with better insight into the antifungal effect of natural herbal dentifrices.     

Control of the Harmful Alga Microcystis aeruginosa and Absorption of Nitrogen and Phosphorus by Candida utilis

This study is aimed at controlling eutrophication through converting the nutrients such as nitrogen and phosphorus into microbial protein and simultaneously inhibiting the growth of Microcystis aeruginosa by Candida utilis. C. utilis and M. aeruginosa (initial cell density was 2.25?×?10⁷ and 4.15?×?10⁷ cells·mL^?1) were cultured together in the absence or presence of a carbon source (glucose) during a 10-day experiment. In the absence of carbon source, the measured removal efficiencies of NH₄ ⁺–N and PO₄ ^3?–P were 41.39?±?2.19 % and 82.93?±?3.95 %, respectively, at the second day, with the removal efficiency of 67.82?±?2.29 % for M. aeruginosa at the fourth day. In contrast, the removal efficiencies of NH₄ ⁺–N and PO₄ ^3?–P were increased to 87.45?±?4.25 % and 83.73?±?3.55 %, respectively, while the removal efficiency of M. aeruginosa decreased to 37.89?±?8.41 % in the presence of the carbon source (C/N?=?2:1). These results showed that the growth of M. aeruginosa was inhibited by C. utilis. Our finding sheds light on a novel potential approach for yeast to consume nutrients and control harmful algal during bloom events.     

A Comparative Study of Anti-Candida Activity and Phenolic Contents of the Calluses from Lythrum salicaria L. in Different Treatments

In the study, anti-Candida activity and phenol contents of Lythrum salicaria L. calli and wild species have been evaluated. The seeds of L. salicaria (Lythraceae), collected from Lahidjan City in the north of Iran, were cultured in Murashige and Skoog medium (MSM) with a supplement, gibberellin, to germinate. Callus inductions were performed from segments of seedling on MSM containing different concentrations of plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The activity of calluses extracts, wild plant, gallic acid, and 3,3′,4′-tri-O-methylellagic acid-4-O-β-d-glucopyranoside (TMEG) as the main phenolic compounds against Candida albicans was assessed using cup plate diffusion method. The total phenols contents of calli and wild plant extracts were analyzed using Folin–Ciocalteu reagent. The callus formation in MSM supplemented with various concentrations of 2,4-D and BAP were 0–100 %. Anti-Candida activity of callus extract which obtained from MSM supplemented with 2,4-D and BAP (1 mg?dm^?3) was similar to the wild plant extract. Minimum inhibitory concentration values of gallic acid and TMEG were obtained as 0.312 and 2.5 mg?cm^?3, respectively. Gallic acid equivalent values in all treatments were from 0 to 288 μg GAE mg^?1. Phenolic contents of plant aerial parts (331?±?3.7 μg GAE mg^?1) and the callus, which developed in MSM including 1 mg?dm^?3 of both 2,4-D and BAP, showed the same phenolic value and exhibited anti-Candida extract activity.     

Adsorption and thermodynamics of biosurfactant,surfactin, monolayers at the air-buffered liquid interface

Adsorption of surfactin, a powerful lipopeptide biosurfactant, at the air-liquid interface has been investigated in this article. The adsorption took place from buffered solutions containing relatively high concentrations of surfactin co- and counterions. Dynamic surface tension measurements were used to follow the self-assembly of surfactin at the interface until equilibrium surface pressure Π ^e is reached at a given surfactin concentration (C _s). Gibbs adsorption equation in conjunction with the Langmuir adsorption isotherm was used to predict surfactin surface excess as a function of the biosurfactant concentration up to the critical micelle concentration (CMC). The predicted surface excess at saturation (Γ _∞ ) is 1.05?±?0.05 μmol m^?2, corresponding to an area per molecule (A _∞ ) of 159?±?8 Å². The adsorption equilibrium constant (K?=?(1.5?±?0.6)?×?10⁶ M^‐?1) was also estimated from the nonlinear regression of Π ^e???C _s data in region B of the Π ^e???ln?C _s plot. The value of K suggests that surfactin has strong affinity for the interface, which is in line with its known high surface activity. Gibbs elasticity (E _G) of the interfacial surfactin monolayers, which is an important thermodynamic property, was also predicted at different surfactin concentrations. The limiting value (at the CMC) of E _G was found to be 183 mN m^?1, which is comparable to those reported in the literature for similar systems. The findings reported in this work reveal that the surface tension measurements coupled with appropriate theoretical analysis could provide useful information comparable to those obtained using highly sophisticated techniques.     

Production of Microbial Surfactants from Oily Sludge-Contaminated Soil by Bacillus subtilis DSVP23

The indigenous microbial community utilizing aliphatic, aromatic, and polar components from the oily sludge as sole source of carbon and energy was selected from the soil samples of Ankleshwar, India for biosurfactant production. Evaluation of biosurfactant production was done using screening assays such as surface tension reduction, hemolytic activity, emulsification activity, drop-collapse assay, and cell surface hydrophobicity studies. Maximum biosurfactant (6.9?g/l) production was achieved after 5?days of growth from Bacillus subtilis DSVP23 which was identified by 16S RNA technique (NCBI GenBank accession no. EU679368). Composition of biosurfactant showed it to be lipopeptide in nature with 15.2% protein content and 18.0% lipid content. Functional group analysis was also done by using Fourier transform infrared spectroscopy which showed it to be a protein-bound lipid thereby imparting them special properties. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric and nuclear magnetic resonance revealed that the major constituents of lipopeptide are leucine and isoleucine. Gas chromatographic analysis data indicated that oily sludge components of chain length C₁₂?CC₃₀ and aromatic hydrocarbons were degraded effectively by B. subtilis DSVP23 after 5?days of incubation. These results collectively points toward the importance of B. subtilis DSVP23 as a potential candidate for bioremediation studies.     

UPLC-PDA Analysis for Simultaneous Quantification of Four Active Compounds in Crude and Processed Rhizome of Polygonum multiflorum Thunb

A novel, rapid and specific ultra performance liquid chromatography-photo diode array detection method was developed for the simultaneous determination of 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), emodin-8-O-β-d-glucoside (EMG), emodin (EM) and physcion (PS). The chromatographic separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm i.d., 1.7 μm). The mobile phase was a mixture of 0.3% acetic acid–water and 0.3% acetic acid–acetonitrile employing gradient elution at the flow rate of 0.4 mL min^?1. The four compounds behaved linearly in the concentration range between 60.80–3040.00 μg mL^?1 (TSG), 0.50–25.00 μg mL^?1 (EMG), 2.16–108.00 μg mL^?1 (EM) and 1.56–78.00 μg mL^?1 (PS), respectively with correlation coefficients >0.999. The precision of the method were below 5% RSD. Recoveries of the four compounds ranged from 95.71 to 102.97%, with RSD values less than 2%.     

Behavior of Wild-type and Transfected S2 Cells Cultured in Two Different Media

An animal protein-free medium composed of IPL-41 containing 6 g L^?1 yeastolate ultrafiltrate, 10 g L^?1 glucose, 2 g L^?1 lactose, 5 g L^?1 glutamine, 1% lipid emulsion, and 0.1% Pluronic F-68 was used for producing recombinant proteins in batch mode employing two cell lines, S2AcRVGP2k expressing the G glycoprotein from rabies virus (RVGP) and S2AcHBsAgHy-9C expressing the surface antigen of hepatitis B virus (HBsAg), both obtained from Drosophila melanogaster S2 cells. Growth of wild-type S2 cells was also evaluated in the same medium. Cell behavior in the tested medium was compared to that verified in Sf900 II®. The results show that in shake flasks, S2AcRVGP2k and S2AcHBsAgHy-9C cells reached around 2?×?10⁷ cells mL^?1 in both media. In supplemented IPL-41 and Sf900 II® media, S2AcRVGP2k cells produced 367 ng RVGP mL^?1 and 638 ng RVGP mL^?1, respectively, while S2AcHBsAgHy-9C cells correspondently produced 573 ng HBsAg mL^?1 and 322 ng HBsAg mL^?1 in the mentioned media. In stirred tanks, S2AcRVGP2k cells reached 3?×?10⁷ cells mL^?1 and produced up to 758 ng RVGP mL^?1. In general, glucose was consumed by cells, while lactate and ammonia were produced.     

Molecular and Kinetic Characterization of Two Extracellular Xylanases Isolated from Leucoagaricus gongylophorus

In this work, the xylanolytic profile of Leucoagaricus gongylophorus was studied, and two extracellular enzymes with xylanolytic activity (XyLg1 and XyLg2) were isolated, purified, and characterized. XyLg1 has a molecular mass of about 38 kDa and pI greater than 4.8. For beechwood xylan substrate, XyLg1 showed an optimum temperature of 40 °C, optimum pH between 8.5 and 10.5, and Km?=?14.7?±?7.6 mg mL^?1. Kinetic studies of the XyLg1 using polygalacturonic acid as substrate were developed, and the enzyme showed optimum pH 5.5, optimum temperature between 50 and 60 °C, and Km?=?2.2?±?0.5 mg mL^?1. XyLg2 has molecular weight of about 24 kDa and pI less than 4.8, and thus is an acid protein. Parameters such as optimum temperature (70 °C) and pH (4.0), as well as the kinetic parameters (Km?=?7.4?±?2.0 mg mL^?1) using beechwood xylan as substrate, were determined for XyLg2. This enzyme has no activity for polygalacturonic acid as substrate. XyLg1 and XyLg2 are the first native xylanases isolated and characterized from L. gongylophorus fungi and, due to their biochemistry and kinetic features, they have potential to be used in biotechnological processes.     

Simple, Sensitive, High-Throughput Method for the Quantification of Mitragynine in Rat Plasma Using UPLC-MS and Its Application to an Intravenous Pharmacokinetic Study

A simple, sensitive and rapid ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method was developed and validated for the quantification of mitragynine in rat plasma using amitriptyline hydrochloride as an internal standard. Sample preparation involved a one-step liquid?Cliquid extraction using methyl t-butyl ether. Mitragynine was separated on an Acquity UPLC^? BEH HILIC column using isocratic elution with a mobile phase of 10 mM ammonium formate buffer containing 0.1% formic acid:acetonitrile (15:85, v/v). At a flow rate of 0.2 mL min^?1, the retention time of mitragynine was found to be 1.3 min. Ionization was performed in the positive ion electrospray mode. The selected mass-to-charge (m/z) ratio transition of mitragynine ion [M + H]⁺ used in the selected ion recording (SIR) was 399.1. The calibration curve was found to be linear over a concentration range of 1?C5,000 ng mL^?1 (r = 0.999) with a lower limit of quantification (LLOQ) of 1 ng mL^?1. Intra- and inter-day assay variations were found to be less than 15%. The extraction recoveries ranged from 85?C93% at the three concentrations (2, 400 and 4,000 ng mL^?1) in rat plasma. This method was successfully used to quantify mitragynine in rat plasma following intravenous administration of the compound.     

Simultaneous Determination of Buprenorphine, Norbuprenorphine and Naloxone in Human Plasma by LC-MS-MS

A novel sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method simultaneously determined buprenorphine (BUP) and its active metabolite, norbuprenorphine (NBUP), and a coformulant, naloxone was developed, validated and applied successfully in humans. Buprenorphine-d ₄ and norbuprenorphine-d ₃ were used as the internal standard. The analysis was performed on a silica column, and the mobile phase was isocratic and composed of acetonitrile:2 mM ammonium formate in H2O (82:18, v/v). Mass spectrometry employed multiple reaction monitoring modes with transitions of m/z 468.1?C55.2 for BUP, 414.2?C101.2 for NBUP, 328.3?C310.3 for naloxone, 472.1?C59.2 for buprenorphine-d ₄ and 417.2?C101.2 for norbuprenorphine-d ₃. Lower limit of quantification (LLOQ) of the analytical method was 0.05 ng mL^?1 for BUP, 0.1 ng mL^?1 for NBUP and 0.025 ng mL^?1 for naloxone, respectively. The standard calibration curves of BUP, NBUP and naloxone were linear over the concentration range of 0.05?C20 ng mL^?1, 0.1?C20 ng mL^?1 and 0.025?C20 ng mL^?1, respectively. The precisions (RSD) and accuracies (RE) of LLOQ and other QC samples were in acceptable range, with RSD < 20% and RE ± 20% for LLOQ and RSD < 15% and RE within ±15% for QC samples. The method was accurate, precise and specific, and was applied to the pharmacokinetic study of buprenorphine in healthy volunteers.     

Ionic liquid/Ammonium Sulfate Aqueous Two-phase System Coupled with HPLC Extraction of Sulfadimidine in Real Environmental Water Samples

Room temperature ionic liquids are novel solvents with a rather specific blend of physical and solution properties that make them of interest for applications in separation science. In this study, a green, simple, and sensitive sample pretreatment procedure coupled with high-performance liquid chromatography (HPLC) was developed for the analysis of sulfadimidine (SM2) that exploits an aqueous two-phase system based on 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF₄) and ammonium sulfate ((NH₄)₂SO₄) using a liquid?Cliquid extraction technique in real environmental water samples. The influences of the concentration of (NH₄)₂SO₄, pH value, temperature, and concentration of SM2 on the extraction efficiency of SM2 were determined. Under optimal conditions, the extraction efficiencies of SM2 were over 93.7%. Calibration curves yielded good linearity (R ² = 0.9998) over the range 3?C240 ng mL^?1, and the limit of detection and limit of quantification for analytes were 0.9 ng mL^?1 and 3 ng mL^?1, respectively. The proposed method was successfully applied to the quantification of SM2 in water samples and recoveries were in the range of 101.2?C107%.     

Application of Lipopeptide Biosurfactant Isolated from a Halophile: Bacillus tequilensis CH for Inhibition of Biofilm

Biosurfactants are amphiphilic molecules having hydrophobic and hydrophilic moieties produced by various microorganisms. These molecules trigger the reduction of surface tension or interfacial tension in liquids. A biosurfactant-producing halophile was isolated from Lake Chilika, a brackish water lake of Odisha, India (19°41′39″N, 85°18′24″E). The halophile was identified as Bacillus tequilensis CH by biochemical tests and 16S rRNA gene sequencing and assigned accession no. KC851857 by GenBank. The biosurfactant produced by B. tequilensis CH was partially characterized as a lipopeptide using thin-layer chromatography, Fourier transform infrared spectroscopy, and nuclear magnetic resonance techniques. The minimum effective concentration of a biosurfactant for inhibition of pathogenic biofilm (Escherichia coli and Streptococcus mutans) on hydrophilic and hydrophobic surfaces was found to be 50 μg ml^?1. This finding has potential for a variety of applications.     

An Effective High-Performance Liquid Chromatographic–Mass Spectrometric Assay for Catecholamines, as the N(O,S)-Ethoxycarbonyl Ethyl Esters, in Human Urine

The purpose of this study was to develop a simple and accurate analytical method for determination of norepinephrine, epinephrine, and dopamine in urine. The method involves liquid–liquid extraction then liquid chromatography–mass spectrometry (LC–MS). Alkyl chloroformate derivatives were prepared, as the N(O,S)-alkoxycarbonyl alkyl esters of the analytes, in the aqueous samples. The optimum derivatizing reagent for preparation of the N(O,S)-alkoxycarbonyl alkyl esters was chosen by comparing the efficiency of LC of the derivatized analytes after liquid–liquid extraction. The optimum conditions for liquid–liquid extraction from the aqueous matrix were pH 3.0, no salt, and diethyl ether as extraction solvent. Limits of detection (LOD) were 0.5 ng mL^?1 for dopamine and epinephrine and 0.1 ng mL^?1 for norepinephrine. Limits of quantification (LOQ) for urine samples were 1.0 ng mL^?1 for all three compounds. The precision of intra- and inter-day assays was 1.65–581 and 7.17–9.73% (relative standard deviation, RSD), respectively. The range of inaccuracy for intra- and inter-day assays was ?6.47 to 11.9% and ?7.5 to 7.76% (bias) at concentrations of 5 and 50 ng mL^?1, respectively.     

Production and Partial Characterization of Cellulases and Xylanases from Trichoderma atroviride 676 Using Lignocellulosic Residual Biomass

Trichoderma atroviride 676 was studied to evaluate its efficiency in the production of some lignocellulolytic enzymes, using lignocellulosic residual biomass. Best results were obtained when 3.0 % (w/v) untreated sugarcane bagasse was used (61.3 U mL^?1 for xylanase, 1.9 U mL^?1 for endoglucanase, 0.25 U mL^?1 for FPase, and 0.17 U mL^?1 for β-glucosidase) after 3–4 days fermentation. The maximal enzymatic activity for endoglucanase, FPase, and xylanase were observed at 50–60 °C and pH?4.0–5.0, whereas thermal stability at 50 °C (CMCase and FPase) or 40 °C (xylanase) was obtained after 8 h. Zymograms have shown two bands of 104 and 200 kDa for endoglucanases and three bands for xylanase (23, 36, and 55.7 kDa). The results obtained with T. atroviride strain 676 were comparable to those obtained with the cellulolytic strain Trichoderma reesei RUT-C30, indicating, in the studied conditions, its great potential for biotechnological application, especially lignocellulose biomass hydrolysis.     

Simultaneous Determination of Pregabalin, Sildenafil and Its Active Metabolite in Rat Plasma Utilising SPE Followed by LC�CMS�CMS

A sensitive and selective analytical method for the quantification of pregabalin, sildenafil and the active desmethyl metabolite of sildenafil (UK-103320) has been developed. The method can simultaneously quantify the three analytes within the expected in vivo concentration ranges using 50 ??L of rat plasma. It utilises solid-phase extraction followed by high performance liquid chromatography coupled with tandem mass spectrometry. Quantitation in rat plasma demonstrated good accuracy and precision over the following dynamic ranges for each analyte: pregabalin (70?C10,000 ng mL^?1), sildenafil (1?C2,000 ng mL^?1) and UK-103320 (1?C2,000 ng mL^?1). For each analyte, the following lower limits of quantitation were obtained: 70 ng mL^?1 for pregabalin and 1 ng mL^?1 for sildenafil and UK-103320, respectively. The method was successfully used to analyse plasma samples from rats when pregabalin and sildenafil were administered in combination.     

Determination of Tangeretin in Rat Plasma by LC-Electrospray-Ion Trap MS

A selective, sensitive, and accurate method has been developed and validated for the quantification of tangeretin in rat plasma. The application of LC-electrospray-ion trap mass spectrometry in full scan and multiple reactions monitoring modes were investigated. Following solid phase extraction using a hydrophilic–lipophilic balance cartridge, the analytes were separated on a C₁₈ column using an isocratic mobile phase composed of acetonitrile/water (50:50, v/v) containing 0.3% formic acid. In full scan mode, the LOQ was 2 ng mL^?1. The standard calibration curve was linear (R ² = 0.9999) over the concentration range 2–200 ng mL^?1. The precision over the concentration range was within 15% (RSD) and the accuracy was ranged from 86 to 115%. In multiple reaction monitoring mode, the LOQ was 1 ng mL^?1 and the standard calibration curve was linear (R ² = 0.9976) over the concentration range 1–100 ng mL^?1 with a precision of 12% and accuracy rangeing from 91 to 113%.     

Determination of Rizatriptan in Human Plasma by Liquid Chromatography Stable Isotope Dilution Electrospray MS�CMS for Application in Bioequivalence Study

A simple, sensitive, selective, rapid, rugged, reproducible and specific liquid chromatography?Ctandem mass spectrometry (LC?CMS/MS) method was used for quantitative estimation of rizatriptan (RZ) in human plasma using rizatriptan-d ₆ (RZD6) as internal standard (IS). Chromatographic separation was performed on Ascentis Express RP Amide C18, 50 × 4.6 mm, 2.7 ??m column with isocratic mobile phase composed of 10 mM ammonium formate:acetonitrile (20:80 v/v) at flow rate of 0.5 mL min^?1. RZ and RZD6 were detected with proton adducts at m/z (amu) 270.2 ?? 201.2 and 276.1 ?? 207.1, respectively, in multiple reaction monitoring (MRM) positive mode. Liquid?Cliquid extraction was used and validated over a linear concentration range of 0.1?C100.0 ng mL^?1 with correlation coefficient r ² ?? 0.9981. The limit of quantification (LOQ) and limit of detection (LOD) were found to be 0.1 ng mL^?1 and 12.5 fg, respectively. Intra- and inter-day precision were within 1.7?C3.1% and 2.8?C3.7%, and accuracy within 96.0?C101.7% and 99.7?C101.4% for RZ. Drug was found to be stable throughout three freeze?Cthaw cycles. The method was successfully employed for analysis of plasma samples following oral administration of RZ (10 mg) in 25 healthy Indian male human volunteers under fasting conditions.     

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.     

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.     

Development and Validation of Atorvastatin by LC–ESI–MS and Application in Bioequivalence Research in Healthy Chinese Volunteers

The aim of this research was to develop a sensitive liquid chromatographic–electrospray ionization–mass spectrometric (LC–MS) method for direct measurement of the concentration of Atorvastatin in human plasma. Plasma samples (1 mL) were extracted with 3 mL ethyl acetate, and by a simple reversed-phase chromatography. Pitavastatin was used as internal standard (IS). The LOQ was 0.25 ng mL^?1 (RSD 4.24%). The assay was linear from 0.25–20 ng mL^?1. And the correlation coefficient for the calibration regression line was 0.9996 or better. Intra-day and inter-day accuracy were better than 15%. The method has been successfully used for a pharmacokinetic study with human subjects. A two-period crossover designed bioequivalence research was also progressed in healthy Chinese volunteers. Among the pharmacokinetic data obtained, T _max was 1.36 ± 0.68 h for reference formulation and 0.81 ± 0.54 h for test formulation. C _max was 8.54 ± 5.06 ng mL^?1 for reference formulation and 9.54 ± 3.68 ng mL^?1 for test formulation. t _1/2 was 8.50 ± 2.74 h for reference formulation and 9.24 ± 3.17 h for test formulation. AUC _0?48h was 54.77 ± 21.82 h ng mL^?1 for reference formulation and 55.66 ± 20.91 h ng mL^?1 for test formulation. The method was successfully applied to the study of pharmacokinetics of Atorvastatin in healthy Chinese volunteers.