Medhat Al-Ghobashy

n/a

Abstract 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 one 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.

Abstract 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.

Environmentally friendly gallic acid coated magnetic nanoparticles (GA-MNP) have been synthesized and evaluated as a novel photocatalyst for degradation of Meloxicam; a commonly prescribed nonsteroidal anti-inflammatory drug. The synthesized GA-MNP were characterized using transmission electron microscopy (TEM){,} Fourier transform infrared (FTIR) spectroscopy and dynamic light scattering. Results showed the formation of core-shell MNP with a mean hydrodynamic diameter of 160.55 +/-5.02 nm and zeta potential of -42.4 +/-1.6 mV. A validated RP-HPLC stability-indicating assay was developed for monitoring of Meloxicam concentration in the presence of its degradation products and for determination of the kinetics of degradation. Full factorial design (24) was employed in order to investigate the effects of pH{,} irradiation time{,} GA-MNP loading and initial Meloxicam concentration on the efficiency of the process. The irradiation time was found the most significant parameter followed by initial Meloxicam concentration and GA-MNP loading{,} respectively. At the optimized conditions{,} increasing GA-MNP loading to 5.00 mg/mL demonstrated superior photocatalytic activity when compared to bare MNP and TiO2NP. Meloxicam degradation was found to follow pseudo first order rate kinetics with Kobs and t0.5 of -0.0029 min-1 and 239 min respectively. The protocol was successfully applied for treatment of incurred water samples collected during various cleaning validation cycles. A percentage degradation of 89.10 +/- 0.13% was achieved upon irradiation of samples containing 64.57 +/- 0.09 [small micro]g/mL with UV light (1012 [small micro]W/cm2{,} 8 h) in the presence of 5 mg/mL GA-MNP at pH 9.0 +/- 0.05. It could be suggested that treatment of waste waters collected during the cleaning validation of each pharmaceutical product{,} before pooling into the general waste pool{,} should improve the efficiency and economics of pharmaceutical waste water treatment.

Four simple, accurate, sensitive and precise spectrophotometric methods were developed and validated for simultaneous determination of Troxerutin (TXN) and Carbazochrome (CZM) in their bulk powders, laboratory prepared mixtures and pharmaceutical dosage forms. Method A is first derivative spectrophotometry (D1) where TXN and CZM were determined at 294 and 483.5 nm, respectively. Method B is first derivative of ratio spectra (DD1) where the peak amplitude at 248 for TXN and 439 nm for CZM were used for their determination. Method C is ratio subtraction (RS); in which TXN was determined at its λmax (352 nm) in the presence of CZM which was determined by D1 at 483.5 nm. While, method D is mean centering of the ratio spectra (MCR) in which the mean centered values at 300 nm and 340.0 nm were used for the two drugs in a respective order. The two compounds were simultaneously determined in the concentration ranges of 5.00–50.00 μg mL−1 and 0.5–10.0 μg mL−1 for TXN and CZM, respectively. The methods were validated according to the ICH guidelines and the results were statistically compared to the manufacturer’s method.

Abstract Midodrine (MD) is a prodrug that is converted after oral administration to Desglymidodrine (DMD). In this study, an LC–MS/MS assay was developed and validated for investigation of the pharmacokinetics of \{MD\} and \{DMD\} in non azotemic patients with liver cirrhosis and tense ascites. Results were compared to those noted with healthy volunteers following the adminstration of a single oral dose of MD. Sample preparation was performed by liquid–liquid extraction using t-butyl methyl ether. \{HPLC\} separation was carried out using \{RP\} \{C18\} column (4.6 mm × 50 mm, 5 μm). Isocratic elution was performed using methanol:0.2% formic acid (70:30, v/v) as the mobile phase, at a flow rate of 0.7 mL/min. Tandem mass spectrometric detection was employed at positive electrospray ionization in \{MRM\} mode for the determination of \{MD\} and DMD. Analysis was carried out within 1.0 min over a concentration range of 0.50–40.00 ng/mL for the prodrug and its active metabolite. The assay was validated according to \{FDA\} guidelines for bioanalytical method validation and satisfactory results were obtained. The applicability of the assay for the determination of the pharmacokinetic parameters of \{MD\} and \{DMD\} and personalized therapy was demonstrated in healthy volunteers and ascitic patients. Results revealed significant differences in pharmacokinetic parameters among the studied groups. Such differences were explained on the basis of the medical condition and co-adminstered medications exerting possible drug–drug interaction. Results confirmed the need for implementation of reliable analysis tools for therapeutic dose adjustment.

Abstract 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.

Abstract Rivaroxaban (RIVA), an amide group-containing oral anticoagulant was subjected to stress conditions commonly required for the registration of pharmaceuticals: base and acid-catalyzed hydrolysis (0.1 M, 60 °C, 3–6 h), oxidation (10% H2O2, 24 h), photodegradation (300–800 nm, 24 h) and thermal decomposition (50 °C, 6 h). Two major degradation products were separated and identified using \{TLC\} and LC/MS/MS, respectively. An orthogonal stability-indicating testing protocol (RP-HPLC and NP-TLC-densitometry) was developed and validated according to İCH\} guidelines. Both assays enabled the determination of \{RIVA\} in the presence of its degradation products as well as the kinetics of degradation. Determination was carried out over a concentration range of (5.00–50.00 μg/mL) and (0.40–12.00 μg/band) with an accuracy of (100.81% ± 1.03) and (100.29% ± 1.08) for \{HPLC\} and TLC-densitometry, respectively. Results indicated that \{RIVA\} was stable towards oxidation, photodegradation and thermal decomposition but extremely sensitive to hydrolysis. Two major degradation products were detected in the case of base-catalyzed hydrolysis while only one degradation product was detected upon acid-catalyzed hydrolysis. This could be attributed to the presence of amide groups in \{RIVA\} structure of different stability profiles. The kinetics of hydrolysis was investigated in more detail and the reaction was found to follow the pseudo first order kinetics, as confirmed by the results of both \{HPLC\} and TLC-densitometric assays. The applicability of the assay for the determination of \{RIVA\} content and dissolution pattern of the innovator product as well as three generic formulations was demonstrated.

Covalently attaching polyethylene glycol (PEGylation) to therapeutic proteins is an increasingly important tool for improving stability, pharmacokinetic and pharmacodynamic properties. In this work, degradation of pegylated interferon alpha-2b (Mono-PEG-IFN) was induced using various physicochemical stress conditions (mechanical agitation, pH, temperature, and repeated freeze-thaw). Stability-indicating SE-HPLC assay was validated and employed for monitoring Mono-PEG-IFN in the presence of all degradation products. Results were expressed in terms of percentage decrease in Mono-PEG-IFN concentration (%Degradation) and peak area normalization method (%Purity). Separation was carried out using a mobile phase of phosphate buffer (100 mM, pH 6.8): 1-propanol (80:20 v/v) at 1.0 mL/min and 214 nm. Incubation at pH 4.0-10.0, 37°C for up to four weeks resulted in formation of aggregates, small molecular weight peptide fragments and mostly depegylated interferon. Similar degradation pattern but to lower extent was noted under short term storage conditions (24 h at 2-8°C and 37°C). No degradation was noted when the lyophilized powder was stored for 30 months at 2–8°C, under real-time stability conditions. It should be noted that expression of the results using %Purity, currently employed for batch release was not a reliable approach. Alternatively, the stability of Mono-PEG-IFN should be expressed as %Degradation that was shown to reveal minor changes in product stability. Results raised a concern about the efficacy and safety of reconstituted multi-dose vials of pegylated therapeutics that are stored refrigerated. The need for in-house validated testing protocols developed by local regulatory authorities to prevent access of substandard biotherapeutics to local markets is discussed.

An orthogonal testing protocol was developed and validated to assess the quality of Filgrastim biosimilars in Egyptian market. 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 - 300.00 µg/mL with a LOD of 8.26 µg/mL (0.44 mM). SEC was carried out over a molecular weight range of 5.0 – 150.0 kDa. 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 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.

In this work, the electrophoretic behavior of zwitterionic buffers is investigated in the
absence of electroosmotic flow (EOF). Electro mobilization of capillary contents is noted when
zwitterionic buffers are employed as the background electrolyte at a pH where the buffering moiety
carries a net charge. The bulk flow of capillary contents was demonstrated via monitoring the
migration of a neutral marker as well as a free and micellar negatively charged marker and
SDS–protein complexes. This electrolyte-driven mobilization (EDM) was investigated in detail
using 4-(2-hydroxyethyl)piprazine-1-ethanesulfonic acid (HEPES) buffer over a wide pH range
(pH 4.0–8.0). Results confirmed that at a pH where HEPES molecules carry a net negative charge,
a bulk flow toward the anode is observed. This was attributed to the migration of HEPES ions
toward the anode along with their hydration shells. The relatively large difference in size and solvation
number between the ionic buffering moiety and its counter-migrating ions (Na+ or H+)
resulted in such a net movement. Results indicated that at constant voltage, plotting the measured
current versus buffer pH can be used for determination of the isoelectric point of the zwitterionic
buffering moiety. Furthermore, this novel mobilization modality was demonstrated using five different
HEPES analogs over pH range 5.0–8.0. More in depth investigations are required in order to
explore the applicability of EDM in coated capillaries of different wall chemistries and dimensions.

Combination drug products containing ezetimibe (EZB) and atorvastatin calcium (ATVC) are widely used for treatment of hyperlipidemia. A rapid, simple and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for determination of EZB and ATVC in spiked human plasma has been developed and validated. Plasma samples were subjected to solid phase extraction before analysis with an extraction recovery of 90.59 - 98.02%. Chromatographic separation was performed on a C18 column using isocratic elution within 2 min run time. The mobile phase consisted of acetonitrile: 1% of formic acid in water (8: 2, v/v) and was pumped at a flow rate of 0.8 mL/min. Detection of analytes was achieved by with electrospray ionization (ESI) in negative ion mode for EZB and positive ion mode for ATVC. The calibration curves were linear over the range of 1.67 – 83.33 ng/mL for both drugs. The precision at the lower limit of quantitation for EZB (12.97%) and ATVC (8.99%) was determined. The intra- and inter-day precisions were within 9.16%, while the accuracy ranged from 94.18 to 106.94%. The validated LC-MS/MS method was successfully employed for the determination of EZB and ATVC in spiked human plasma for application in therapeutic drug monitoring.

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 analyzed 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 EGCG band. Determination was carried out over a concentration range of (0.50 – 5.00 μg/band) and (0.20 – 2.40 μg/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 authorization.

A simple, sensitive, and specific method was developed for simultaneous determination of ezetimibe (EZB) and atorvastatin calcium (ATVC) by high performance liquid chromatography without previous separation. Satisfactory resolution was achieved using a RP-C18 chromatographic column, Kromasil RP-18 column (250 mm x 4.6 mm i.d) and a mobile phase consisting of acetonitrile: water (6: 4, v/v) at a flow rate 0.7 mL/min and the wavelength detection was 260.0 nm. The retention time for EZB and ATVC was 7.47 ± 0.02and 4.67 ± 0.02 min; respectively. The described method was linear over a range of 10-100 μg /ml for EZB and 5 -100μg/ml for ATVC. The mean percent recoveries were 99.87 ± 0.769 and 100.04 ± 0.480 for EZB and ATVC, respectively. Method was validated according to ICH guidelinesand successfully applied for analysis of bulk powder, pharmaceutical formulations and spiked human plasma.

A method for the simultaneous determination of ezetimibe (EZB) and atorvastatin calcium (ATVC) was developed, based on the measurement of their native fluorescence signals, by using second-derivative spectrofluorimetry to resolve the mixture. EZB was measured at λem = 455.0 nm, and ATVC was measured at λem = 394.0 nm. Instrumental parameters were optimized, and the emission spectra were recorded between 380.0 and 470.0 nm, at λex = 246.0 nm and excitation and emission slit widths of 10.0 nm. The calibration graphs were linear over the ranges of 200 – 900 ng/mL for both EZB and ATVC, with a mean percentage recovery of 99.64 ± 0.88 and 99.88 ± 0.61, respectively. Methods were validated according to ICH guidelines and successfully applied for analysis of bulk powder and pharmaceutical formulations. The results were statistically compared to a reported method and no significant difference was noticed regarding accuracy and precision.

Two rapid, simple and sensitive spectrophotometric methods for the quantitative analysis of ezetimibe (EZB) and atorvastatin calcium (ATVC) in bulk powder and combined tablet form have been developed and validated. The first method is bivariate calibration and the second method is ratio subtraction in conjunction with second derivative (D2) spectrophotometry. These methods are tested by analyzing synthetic binary mixtures of the above drugs and they are applied to commercial pharmaceutical preparation of the subjected drugs. Standard deviation is < 2 in the assay of raw materials and tablets. Methods are validated as per ICH guidelines and statistical comparison of the suggested methods with the reported spectrophotometric one using F and t tests showed no significant difference regarding both accuracy and precision.