Medhat A. Al-Ghobashy

Removal of cationic and anionic dyes from aqueous solution with magnetite/pectin and magnetite/silica/pectin hybrid nanocomposites: kinetic, isotherm and mechanism analysis

Novel adsorbents, magnetite nanoparticles modified with pectin shell and silica/pectin double shell, were fabricated and tested for single dye and dye mixture adsorption from water samples. Cationic dyes methylene blue (MB) and crystal violet (CV) and anionic dyes methyl orange (MO) and Eriochrome black T (EBT) were employed to assess dye removal efficiency. The influence of pH, amount of adsorbent, initial dye concentration and contact time was investigated. Results indicated that the optimum pH for removing cationic dyes was 8.0 and 2.0 for anionic dyes. The kinetic studies showed rapid sorption dynamics following a second-order kinetic model. Dye adsorption equilibrium data were fitted well to the Sips isotherm for cationic and anionic dyes. The maximum monolayer capacity, (qmax) for MB, CV, EBT and MO was calculated from Sips as 197.18, 180.29, 65.35 and 26.75 mg g−1 respectively for magnetite/silica/pectin NPs and 168.72, 140.49, 72.35 and 27.22 mg g−1 respectively for magnetite/pectin nanoparticles. For dye mixture adsorption, a new HPLC assay was proposed for quantitation of dyes in treated samples. The results came in accordance with that of single dye adsorption where the magnetite/pectin NPs showed preferred adsorption to anionic dyes while the magnetite/silica/pectin NPs had more affinity to cationic dyes. Thus, our proposed NPs can be used as cheap and efficient adsorbents for removal of cationic and anionic dyes from aqueous solutions.

Development and validation of a modified QuEChERS protocol coupled to LC–MS/MS for simultaneous determination of multi-class antibiotic residues in honey

LC-MS/MS assay was developed and validated according to EU guidelines for determination of nitrofuran metabolites and nitroimidazole residues in honey. Crude samples were acid-treated to liberate matrix-bound residues and a modified QuEChERS protocol was employed. Nitrofurantoin, furazolidone, furaltadone and nitrofurazone were determined via analysis of their metabolites AHD, AOZ, AMOZ and SEM, respectively while nitroimidazole residues; ronidazole (RNZ) and dimetridazole (DMZ) were determined directly. For all analytes, neat standard calibration curves, after correction for matrix effect were successfully employed. Decision limit (CCα) and detection capability (CCβ) were below the MRPL for nitrofurans (1.00 μg kg(-1)) and the recommended concentration for nitroimidazole (3.00 μg kg(-1)), respectively. The CCα, CCβ, percentage recovery and CV% ranges were 0.12-0.74 μg kg(-1), 0.21-1.27 μg kg(-1), 90.96-104.80% and 2.65-12.58%, respectively. This work is part of the national initiative for establishing a national monitoring program for drug residues in Egyptian honey.

Simultaneous determination of 200 pesticide residues in honey using gas chromatography-tandem mass spectrometry in conjunction with streamlined quantification approach.

A sensitive, accurate and reliable multi-class GC-MS/MS assay protocol for quantification and confirmation of 200 common agricultural pesticides in honey was developed and validated according to EU guidelines. A modified extraction procedure, based on QuEChERS method (quick, easy, cheap, effective, rugged and safe) was employed. Mass spectrophotometric conditions were individually optimized for each analyte to achieve maximum sensitivity and selectivity in MRM mode. The use of at least two reactions for each compound allowed simultaneous identification and quantification in a single run. The pesticides under investigation were separated in less than 31 min using the ultra-inert capillary column (DB-35MS). For all analytes, neat standard calibration curves in conjunction with correction for matrix effect were successfully employed. The detection limits of the assay ranged from 1.00 to 3.00 ng mL(-1) for the studied pesticides. The developed assay was linear over concentration range of 10.00-500.00 ng mL(-1), with correlation coefficient of more than 0.996. At the LOQ, 81% of the studied pesticides were efficiently recovered in the range of 70.00-120.00%, with CV% less than 15.00% while 99.3% compounds had mean percentage recovery of 60.00-140.00%, with CV% less than 21.00% (N=18, over three different days). The proposed assay was successfully applied for the analysis of the studied pesticide residues in one PT sample and 64 commercial honey samples collected over 1 year from different districts around Egypt. Results revealed that only one honey sample out of the 64 analyzed samples was contaminated with tau-Fluvalinate (10.00 μg kg(-1)). This wide scope assay protocol is applicable for monitoring pesticide residues in honey by national regulatory authorities and accredited labs; that should help ensure safety of such widely used product.

Spectrophotometric and TLC-densitometric methods for the simultaneous determination of Ezetimibe and Atorvastatin calcium.

Three sensitive methods were developed for simultaneous determination of Ezetimibe (EZB) and Atorvastatin calcium (ATVC) in binary mixtures. First derivative (D1) spectrophotometry was employed for simultaneous determination of EZB (223.8 nm) and ATVC (233.0 nm) with a mean percentage recovery of 100.23 ± 1.62 and 99.58 ± 0.84, respectively. Linearity ranges were 10.00–30.00 μg mL−1 and 10.00–35.00 μg mL−1, respectively. Isosbestic point (IS) spectrophotometry, in conjunction with second derivative (D2) spectrophotometry was employed for analysis of the same mixture. Total concentration was determined at IS, 224.6 nm and 238.6 nm over a concentration range of 10.00–35.00 μg mL−1 and 5.00–30.00 μg mL−1, respectively. ATVC concentration was determined using D2 at 313.0 nm (10.00–35.00 μg mL−1) with a mean recovery percentage of 99.72 ± 1.36, while EZB was determined mathematically at 224.6 nm (99.75 ± 1.43) and 238.6 nm (99.80 ± 0.95). TLC-densitometry was employed for the determination of the same mixture; 0.10–0.60 μg band−1 for both drugs. Separation was carried out on silica gel plates using diethyl ether–ethyl acetate (7:3 v/v). EZB and ATVC were resolved with Rf values of 0.78 and 0.13. Determination was carried out at 254.0 nm with a mean percentage recovery of 99.77 ± 1.30 and 99.86 ± 0.97, respectively. Methods were validated according to ICH guidelines and successfully applied for analysis of bulk powder and pharmaceutical formulations. Results were statistically compared to a reported method and no significant difference was noticed regarding accuracy and precision.

On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows.

Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni2+ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.

Development and validation of LC–MS/MS assay for the simultaneous determination of methotrexate, 6-mercaptopurine and its active metabolite 6-thioguanine in plasma of children with acute lymphoblastic leukemia: Correlation with genetic polymorphism

Individualized therapy is a recent approach aiming to specify dosage regimen for each patient according to its genetic state. Cancer chemotherapy requires continuous monitoring of the plasma concentration levels of active forms of cytotoxic drugs and subsequent dose adjustment. In order to attain optimum therapeutic efficacy, correlation to pharmacogenetics data is crucial. In this study, a specific, accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed for determination of methotrexate (MTX), 6-mercaptopurine (MP) and its metabolite 6-thioguanine nucleotide (TG) in human plasma. Based on the basic character of the studied compounds, solid phase extraction using a strong cation exchanger was found the optimum approach to achieve good extraction recovery. Chromatographic separation was carried out using RP-HPLC and isocratic elution by acetonitrile: 0.1% aqueous formic acid (85:15v/v) with a flow rate of 0.8mL/min at 40°C. The detection was performed by tandem mass spectrometry in MRM mode via electrospray ionization source in positive ionization mode. Analysis was carried out within 1.0min over a concentration range of 6.25-200.00ng/mL for the studied analytes. Validation was carried out according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained. The applicability of the assay for the monitoring of the MTX, MP and TG and subsequent application to personalized therapy was demonstrated in a clinical study on children with acute lymphoblastic leukemia (ALL). Results confirmed the need for implementation of reliable analysis tools for therapeutic dose adjustment.

Development and validation of LC–MSMS assay for the determination of the prodrug dabigatran etexilate and its active metabolites in human plasma

Dabigatran etexilate (DABE) is a low-molecular-weight prodrug that is converted after oral administration to dabigatran (DAB)-a directly acting oral anticoagulant. In this study, an LC-MSMS assay was developed and validated for the determination of DABE, free DAB and its equipotent O-glucuronide conjugates in plasma. Owing to the susceptibility of DABE and DAB to chemical hydrolysis, cleavage of the O-glucuronide moiety was carried out using β-glucuronidase enzyme. Free and total plasma concentrations of DAB were determined in incurred plasma samples before and after enzymatic cleavage (50°C and 3 h), respectively. RP-HPLC separation was carried out using acetonitrile: water (30:70, v/v), adjusted to pH 3.0 using formic acid. Tandem mass spectrometric detection at positive electrospray ionization in the MRM mode was then employed for the determination of DABE and DAB. The analysis was carried out within 5.0 min over a linear concentration range of 1.00-600.00 ng/mL for the prodrug and its active metabolite. Validation was carried out according to FDA guidelines for bioanalytical method. The recoveries were higher than 89.48%, the accuracy was within 98.33-110.12% and the RSD was below 10% for the studied compounds in both incurred plasma and quality control samples. Results of incurred sample re-analysis and incurred sample stability revealed less than 10% variability. This indicated good assay precision and sufficient stability of target analytes in their real matrix at the employed experimental conditions. The applicability of the assay for therapeutic drug monitoring and the determination of the pharmacokinetic parameters were demonstrated.

Probing the interaction between recombinant human myelin basic protein and caseins using surface plasmon resonance and diffusing wave spectroscopy.

An intrinsically unstructured human myelin basic protein (hMBP) was expressed in the milk of transgenic cows (TGmilk) and found exclusively associated with the casein micellar phase. The interaction between the recombinant protein and milk caseins was investigated using surface plasmon resonance (SPR). An anti‐human myelin basic protein antibody was covalently immobilized to the surface of the sensor chip. Subsequently the interaction between the recombinant protein (captured by this antibody) and caseins was studied in comparison to that noted with its human counterpart. Results showed a calcium‐mediated interaction between the recombinant protein and caseins. The order of magnitude of this interaction was in agreement with the number of phosphorylated residues carried by each type of casein (αs‐ > β‐ > κ‐casein). This selective interaction was not noted between the human protein and milk caseins indicating that the recombinant protein was phosphorylated to a higher extent than the human protein. The obtained results indicated that the co‐expression of the recombinant protein and caseins by the mammary gland along with the recombinant proteins ability to form calcium bridges played a key role in the association of the recombinant human myelin basic protein (rhMBP) with the casein micelles of milk. Despite this association between the recombinant protein and milk caseins, light scattering investigations using diffusing wave spectroscopy (DWS) showed no significant differences between the milks of the transgenic and the non‐transgenic control cows, with respect to both the average micelle size and surface charges. This was attributed to the low expression levels of the recombinant protein in milk. Copyright

Chromatographic and electrophoretic assessment of Filgrastim biosimilars in pharmaceutical formulations

An orthogonal testing protocol was developed and validated to assess the quality of Filgrastim biosimilars. Results were compared to those obtained from the innovator product. Initial screening was carried out using reducing and non-reducing gel electrophoresis. RP-LC was employed for the determination of Filgrastim in the presence of its oxidative degradation products. SEC and CIEF were used under non-denaturing conditions to reveal high molecular weight and charged impurities, respectively. RP-LC assay was found accurate (99.78±0.89) and precise over a linear concentration range of 9.38-300.00μg/ml with a LOD of 8.26μg/ml (0.44mM). SEC was carried out over a molecular weight range of 5.0-150.0kDa. CIEF was optimized using neutrally coated capillaries over a wide-range pH gradient (pH 3.0-10.0). Differences between the studied products were revealed using all these techniques. Impurities above the acceptable limits were detected in both biosimilar products. CIEF revealed heterogeneity in the active ingredient that has not been investigated by the manufacturers. Correlation of the obtained results indicated the presence of not only product-related impurities, but also process-related impurities. Results confirmed the need for in-house validated orthogonal testing protocols to be developed by local regulatory authorities. This should prevent access of substandard biosimilars to price-sensitive markets.

Stability of catechins in green tea nutraceutical products: Application of solid phase extraction–thin layer chromatography densitometry

Epigallocatechin gallate (EGCG) is a powerful antioxidant and commonly used nutraceutical. Accelerated stability of EGCG in tablet formulations was investigated. LLE and SPE were employed for sample clean-up and enrichment of EGCG over caffeine. Samples were analysed after spiking with fixed concentration of gallic acid (GA), in order to verify reproducibility of analysis. A TLC-densitometric assay was developed and validated for determination of % loss EGCG. EGCG, GA and caffeine were resolved with Rf values 0.54, 0.69 and 0.80, respectively. LC-MS/MS was used to verify identity and purity of the EGCG band. Determination was carried out over a concentration range of 0.50-5.00μg/band and 0.20-2.40μg/band for GA and caffeine, respectively. Results showed significant reduction in EGCG content after one, three and six months: 24.00%, 28.00% and 52.00% respectively. Results continue to demonstrate that stability of nutraceutical products should be investigated in-depth using industry-oriented protocols before granting marketing authorisation.