A molecularly imprinted polymer based chemiluminescence array sensor for one-step determination of phenothiazines and benzodiazepines in pig urine.

The residues of phenothiazines and benzodiazepines in foods of animal origin are dangerous to consumers. For inspection of their abuses, this study for the first time reported on the use of a chemiluminescence array sensor for the simultaneous determination of four phenothiazines and five benzodiazepines in pig urine. Two molecularly imprinted polymers were coated in different wells of a conventional 96-well microtiter plate as the recognition reagents. After sample loading, the absorbed analytes were initiated directly by using an imidazole enhanced bis(2,4,6-trichlorophenyl)oxalate-hydrogen peroxide system to emit light. The assay process consisted of only one sample-loading step prior to data acquisition, so one test was finished within 10 min. The limits of detection for the nine drugs in the pig urine were in a range of 0.1 to 0.6 pg/mL, and the recoveries from the fortified blank urine samples were in a range of 80.3 to 95%. Furthermore, the sensor could be reused six times. Therefore, this sensor could be used as a simple, rapid, sensitive and reusable tool for routine screening for residues of phenothiazines and benzodiazepines in pig urine.

Molecularly imprinted polymer based microtiter chemiluminescence array for determination of phenothiazines and benzodiazepines in pork.

In this study, a molecularly imprinted polymer based chemiluminescence array capable of simultaneous determining phenothiazines and benzodiazepines was first reported. Two polymers were coated in different wells of the conventional 96-well microtiter plate as the recognition reagents, and the added analytes competed with a horseradish peroxidase-labeled bi-hapten conjugate to bind the recognition reagents. The light signal was induced by using a highly effective luminol-H2O2-IMP system. The assay procedure consisted of only one sample-loading step prior to data acquisition. Then, the array was used to determine 4 phenothiazines and 5 benzodiazepines in pork simultaneously. The limits of detection for the 9 drugs were in a range of 0.001-0.01 ng/mL, and the recoveries from the fortified blank pork were in a range of 63.5%-94.1%. Furthermore, the array could be reused for 8 times. The detection results for some real pork samples were consistent with an ultra performance liquid chromatography method.

Virtual mutation and directional evolution of anti-amoxicillin ScFv antibody for immunoassay of penicillins in milk.

In this study, an anti-amoxicillin single chain variable fragment (ScFv) antibody was evolved by directional mutagenesis of a contact amino acid residue based on the analysis of virtual mutation. Comparison with its parental ScFv, the mutant showed highly improved affinity for 11 penicillins with up to 6-folds increased sensitivity. Then, its recognition mechanisms for the 11 drugs were studied by using molecular docking. Results showed that the mutant-penicillins intermolecular forces increased and the total binding energies decreased dramatically, which were responsible for the improvement of antibody sensitivity. The ScFv mutant was used to develop an indirect competitive enzyme linked immunosorbent assay for determination of the 11 drugs in milk. The limits of detection were in the range of 0.2-3.0 ng/mL, the crossreactivities were in the range of 31%-132%, and the recoveries from standards fortified blank milk were in the range of 65.7%-92.4%. This is the first study reporting the directional evolution of a ScFv antibody based on virtual mutation and the use of ScFv antibody for determination of penicillins in foods of animal origin.

The intestinal absorption mechanism of chicoric acid and its bioavailability improvement with chitosan.

Chicoric acid (CA), an active phenolic acid of Echinacea purpurea (Linn.) Moench, has been demonstrated to exhibit antioxidative, antiviral and immunological activities. A prior study showed that CA is a water-soluble compound with low bioavailability. The current study was performed to study the intestinal absorption mechanism of CA and improve its bioavailability using natural biodegradable chitosan. A Caco-2 monolayer cell model was established to characterise the mechanisms involved in the intestinal absorption of CA. The bioavailability improvement of CA was studied in Sprague-Dawley rats after oral (20 mg/kg) administration of 0.5% chitosan. In vitro, the results showed that the absorption transport of CA was fairly poor, with Papp values of 8.2 × 10-8 to 2.1 × 10-7 cm/s in the absorption direction and 1.5 × 10-7 to 2.6 × 10-7 cm/s in the secretory direction. The permeability was increased by EDTA and chitosan in both directions. Moreover, the transport through the intestinal monolayer was H+ dependent, and P-glycoprotein and OATP2B1 transporters were involved in the intestinal transport of CA. In vivo, the absorption of CA was increased and accelerated with chitosan in rats because the bioavailability was 1.74-fold that of the prototype drug. The above mentioned results indicated that CA was a poor absorption drug and that paracellular and carrier-mediated trancellular transport both participated in its transport route. Chitosan is an excellent absorption enhancer for CA. The transport characteristics uncovered in this study lay the groundwork for further studies directed toward the development and utilisation of its new formulations.

An LC-MS/MS method for determination of 3,6'-disinapoylsucrose in rat plasma and its application to a pharmacokinetic study.

3,6'-Disinapoylsucrose (DSS), a major active component of traditional Chinese medicine Yuan-Zhi (the roots of Polygala tenuifolia), has significant effects for neuroprotection and improving learning memory. In order to explore the pharmacokinetic properties of DSS so as to further understand its in vivo activities, a sensitive LC-MS/MS method was developed for determination of DSS in rat plasma and applied to a pharmacokinetic study in the present study. After treatment by protein precipitation, the plasma sample was separated on a C(18) HPLC column and analyzed by a mass spectrometry under positive electrospray ionization. Multiple-reaction monitoring was employed to measure the ion transition at m/z 777.4 --> 409.2 for DSS and m/z 557.2 --> 309.1 for forsythin as internal standard. The method was linear over the studied concentration range of 0.5-1000.0 ng/mL. The precision and accuracy ranged from 1.4 to 18.4%, and from -3.7 to -9.5%, respectively, for within-day and between-day assay. Extraction recovery was higher than 86.6%. The limits of detection and quantification were 0.3 and 0.5 ng/mL, respectively. The present method was successfully applied to a pharmacokinetic study. DSS was found to have poor oral absorption with only about 0.5% bioavailability.

Formulation development and bioavailability evaluation of a self-nanoemulsified drug delivery system of oleanolic acid.

This study aims to formulate and evaluate bioavailability of a self-nanoemulsified drug delivery system (SNEDDS) of a poorly water-soluble herbal active component oleanolic acid (OA) for oral delivery. Solubility of OA under different systems was determined for excipient selection purpose. Four formulations, where OA was fixed at the concentration of 20 mg/g, were prepared utilizing Sefsol 218 as oil phase, Cremophor EL and Labrasol as primary surfactants, and Transcutol P as cosurfactant. Pseudo-ternary phase diagrams were constructed to identify self-emulsification regions for the rational design of SNEDDS formulations. Sefsol 218 was found to provide the highest solubility among all medium-chained oils screened. Efficient self-emulsification was observed for the systems composing of Cremophor EL and Labrasol. The surfactant to cosurfactant ratio greatly affected the droplet size of the nanoemulsion. Based on the outcomes in dissolution profiles, stability data, and particle size profiles, three optimized formulations were selected: Sefsol 218/Cremophor EL/Labrasol (50:25:25, w/w), Sefsol 218/Cremophor EL/Labrasol/Transcutol P (50:20:20:10, w/w), and Sefsol 218/Cremophor EL/Labrasol/Transcutol P (50:17.5:17.5:15, w/w). Based on the conventional dissolution method, a remarkable increase in dissolution was observed for the SNEDDS when compared with the commercial tablet. The oral absorption of OA from SNEDDS showed a 2.4-fold increase in relative bioavailability compared with that of the tablet (p < 0.05), and an increased mean retention time of OA in rat plasma was also observed compared with that of the tablet (p < 0.01). These results suggest the potential use of SNEDDS to improve dissolution and oral bioavailability for poorly water-soluble triterpenoids such as OA.

Dummy molecularly imprinted polymer based microplate chemiluminescence sensor for one-step detection of Sudan dyes in egg.

The objective of this study is to report a molecularly imprinted polymer-based chemiluminescence method for determination of Sudan dyes. A dummy-template molecularly imprinted polymer capable of recognizing seven Sudan dyes was first synthesized and its recognition mechanism was studied by using computation simulation method. The polymer was coated in the wells of conventional microplate to prepare a chemiluminescence sensor and the assay process consisted of only one sample-loading step prior to signal acquisition. The optimized sensor was used to determine the seven dyes in egg yolk and the results were confirmed with a high performance liquid chromatography. Results showed that this sensor achieved ultrahigh sensitivity (1.0-5.0 pg/mL), rapid assay process (10 min) and satisfactory recovery (70.5%-92.2%). Furthermore, the sensor could be reused for 5 times. Therefore, this sensor could be used as a useful tool for screening the residues of Sudan dyes in egg.

Magnetic graphene dispersive solid phase extraction combining high performance liquid chromatography for determination of fluoroquinolones in foods.

In this study, a magnetic graphene-based dispersive solid phase extraction method was developed that was combined with high performance liquid chromatography to determine the residues of fluoroquinolone drugs in foods of animal origin. During the experiments, several parameters possible influencing the extraction performance were optimized (amount of magnetic graphene, sample pH, extraction time and elution solution). This extraction method showed high absorption capacities (>6800ng) and high enrichment factors (68-79-fold) for seven fluoroquinolones. Furthermore, this absorbent could be reused for at least 40 times. The limits of detection were in the range of 0.05-0.3ng/g, and the recoveries from the standards fortified blank samples (bovine milk, chicken muscle and egg) were in the range of 82.4-108.5%. Therefore, this method could be used as a simple and sensitive tool to determine the residues of fluoroquinolones in foods of animal origin.

Preparation of dual-template molecularly imprinted polymer coated stir bar based on computational simulation for detection of fluoroquinolones in meat.

In this study, a dual-template molecularly imprinted polymer-coated stir bar capable of recognizing nine fluoroquinolone drugs was prepared. Its recognition abilities for fluoroquinolones and other drugs were studied by using computational simulation method. Results showed that the shape and volume of the imprinted cavities were responsible for the polymer's recognition performance. After optimization of several important parameters, a stir-bar-sorptive-extraction method was developed that was combined with high performance liquid chromatography for determination of the nine drugs in meat. The stir bar showed high enrichment factors (33-47 folds), high capture capacities (4640-4950ng) and high recoveries (>90%) for the nine drugs, and could be reused for thirty times. The limits of detection for the nine drugs were in the range of 0.1-0.3ngg-1, and the recoveries from the fortified blank meat were in the range of 67.4%-99.0%. Therefore, this method could be used as a simple, rapid and specific tool for routine detection of residual fluoroquinolones in meat.

Application of molecularly imprinted polymer based matrix solid phase dispersion for determination of fluoroquinolones, tetracyclines and sulfonamides in meat.

In this study, a type of novel mixed-template molecularly imprinted polymer was synthesized that was able to recognize 8 fluoroquinolones, 8 sulfonamides and 4 tetracyclines simultaneously with recoveries higher than 92%. Then the polymer was used to develop a matrix solid phase dispersion method for simultaneous extraction of the 20 drugs in pork followed by determination with ultra performance liquid chromatography. During the experiments, the MMIP amount, washing solvent and elution solvent were optimized respectively. The limits of detection of this method for the 20 drugs in pork were in the range of 0.5-3.0ngg-1, and the intra-day and inter-day recoveries from the fortified blank samples were in the range of 74.5%-102.7%. Therefore, this method could be used as a rapid, simple, specific and sensitive method for multi-determination of the residues of the three classes of drugs in meat.

Magnetic graphene dispersive solid phase extraction-ultra performance liquid chromatography tandem mass spectrometry for determination of ß-agonists in urine.

In this study, a magnetic graphene-based dispersive solid phase extraction method was first developed for extraction of ß-agonists in urine. During the experiments, the absorbent amount, sample pH, extraction time, elution solution and elution time were optimized respectively. The optimized extraction method was finished within 10min, and showed high enrichment factors for 9 ß-agonists (20-26 folds). Furthermore, this absorbent could be reused for at least 60 times. Then this extraction method was combined with ultra performance liquid chromatography triple quadrupole tandem mass spectrometry to determine the 9 drugs in urine. The limits of detection for the 9 drugs were in a range of 0.015-0.023ngmL-1, and the recoveries from the standards fortified blank urine were in a range of 60.2%-109.4%. Therefore, this method could be used as a simple, rapid, sensitive and accurate tool to determine trace level of ß-agonists in urine.

Dummy template molecularly imprinted polymer for solid phase extraction of phenothiazines in meat based on computational simulation.

The 3D structures of two dummy templates and four phenothiazine drugs were studied by using computational simulation method. Then the two dummy templates were used to synthesize two molecularly imprinted polymers respectively. Results showed that the recognition abilities of the two polymers were consistent with the theoretical calculation. Then a solid phase extraction column was developed for extraction of the four phenothiazines in meat (pork, chicken) followed by determination with high performance liquid chromatography. The column showed high adsorption capacities (850-962ng analyte per milligram of polymer) and high recoveries (93-98%) to the four drugs, and could be recycled for sixty times. The limits of detection were in the range of 1.0-10ng/g, and the recoveries from the fortified blank samples were in the range of 70.3-96.1%. This is the first study reporting the use of molecularly imprinted polymer-based method for determination of phenothiazines residues in foods.

Production and Directional Evolution of Antisarafloxacin ScFv Antibody for Immunoassay of Fluoroquinolones in Milk.

A recombinant antisarafloxacin ScFv antibody was produced by direct transformation of its gene into Rosetta-gami(DE3) for expression, and then its recognition mechanisms for 12 fluoroquinolones were studied using the molecular docking method. On the basis of the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Tyr99 to His. The ScFv mutant showed highly increased affinity for the 12 drugs with up to sevenfold improved sensitivity. Finally, the mutant was used to develop an indirect competitive enzyme linked immunosorbent assay for determination of the 12 drugs in milk. The limits of detection were in the range of 0.3-8.0 ng/mL; the ties were in the range of 5-106%, and the recoveries from the standard fortified blank milk were in the range of 62.0-89.3%. This is the first study reporting the evolution of an ScFv antibody using a directional mutagenesis strategy based on virtual mutation.

Oral absorption and excretion of icaritin, an aglycone and also active metabolite of prenylflavonoids from the Chinese medicine Herba Epimedii in rats.

Icaritin (ICT) is a main aglycone and also active intestinal metabolite of prenylflavonoids from the Chinese medicine Herba Epimedii. In the present study, the oral absorption and excretion of this compound was investigated using rats for exploring its fate in the body, so as to better understanding its in vivo pharmacological activities. The free (parent) and total (parent plus conjugated metabolites) ICT concentrations in rat plasma, urine and bile, after intravenous (i.v.) and oral administration both at 5mg/kg, were determined before and after enzymatic hydrolysis with ß-glucuronidase/sulphatase, respectively, by a HPLC-UV method. The results showed that free ICT plasma concentration after i.v. dose was rapidly decreased with average t(1/2, λ) of 0.43 h, while the total ICT concentration was decreased slowly with t(1/2, λ) of 6.86 h. The area under the curve of ICT conjugated metabolites was about 11-fold higher than that of free ICT. The majority of ICT in the body was excreted from the bile with 68.05% of dose over 8 h after i.v. dosing, in which only 0.15% was in parent form. While very little amount of ICT was excreted from the urine with 3.01% of dose over 24 h, in which the parent form was 0.62%. After oral administration, very little amount of parent ICT was detected only in 0.5, 1 or 2 h plasma samples with the concentration less than LOQ, however, its total plasma concentration after enzymatic hydrolysis treatment was at relative high level with average maximum concentration of 0.49 µg/ml achieved at 1h post dose. The oral bioavailability of ICT was 35% of dose, estimated by its total plasma drug concentrations. It is concluded that ICT can be easily absorbed into the body, and then rapidly conversed to its conjugated metabolites, and finally removed from the body mainly by biliary excretion.

Spray freeze drying with polyvinylpyrrolidone and sodium caprate for improved dissolution and oral bioavailability of oleanolic acid, a BCS Class IV compound.

Spray-freeze-drying (SFD) of oleanolic acid (OA), a BCS Class IV compound, with polyvinylpyrrolidone-40 (PVP-40) as stabilizer and sodium caprate (SC) as wetting agent and penetration enhancer produced kinetically stable, amorphous solid dispersion systems with superior in vitro dissolution performance, and better and more uniform absorption in comparison with commercial OA tablet. Relative to the SC-free formulation, the presence of SC in the formulation resulted in a significant increase in the in vivo absorption rate of OA while exerting no apparent impact on the extent of OA absorption. The SFD-processed OA formulations and commercial OA tablet generally exhibited large inter-animal variability in oral bioavailability, consistent with the absorption characteristics of BCS Class IV compounds. Inclusion of SC coupled with the replacement of OA with its sodium salt (OA-Na) in the formulation was shown to substantially decrease the observed absorption variability. Above results suggested that increases in both dissolution rate and intestinal permeability of BCS Class IV compounds, as exemplified by the SFD-processed dispersion system containing both OA-Na and SC, are critical to reducing the large inter-individual absorption variability commonly observed with this class of drugs.

An LC-MS/MS method for determination of forsythiaside in rat plasma and application to a pharmacokinetic study.

A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of forsythiaside in rat plasma using epicatechin as internal standard. The analytes were extracted by solid-phase extraction and chromatographied on a C(18) column eluted with a gradient mobile phase of acetonitrile and water both containing 0.2% formic acid. The detection was performed by negative ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 623-->161 and m/z 289-->109 for forsythiaside and epicatechin, respectively. The assay was linear over the concentration ranges of 2.0-50.0 and 50.0-5000.0ng/mL with limits of detection and quantification of 0.2 and 1.0ng/mL, respectively. The precision was <10.8% and the accuracy was >91.9%, and extraction recovery ranged from 81.3% to 85.0%. This method was successfully applied to a pharmacokinetic study of forsythiaside in rats after intravenous (20mg/kg) and oral (100mg/kg) administration, and the result showed that the compound was poorly absorbed with an absolute bioavailability being approximately 0.5%.