Mamdouh R. Rezk

Novel spectrophotometric methods for simultaneous determination of timolol and dorzolamide in their binary mixture

Two smart and novel spectrophotometric methods namely; absorbance subtraction (AS) and amplitude modulation (AM) were developed and validated for the determination of a binary mixture of timolol maleate (TIM) and dorzolamide hydrochloride (DOR) in presence of benzalkonium chloride without prior separation, using unified regression equation. Additionally, simple, specific, accurate and precise spectrophotometric methods manipulating ratio spectra were developed and validated for simultaneous determination of the binary mixture namely; simultaneous ratio subtraction (SRS), ratio difference (RD), ratio subtraction (RS) coupled with extended ratio subtraction (EXRS), constant multiplication method (CM) and mean centering of ratio spectra (MCR). The proposed spectrophotometric procedures do not require any separation steps. Accuracy, precision and linearity ranges of the proposed methods were determined and the specificity was assessed by analyzing synthetic mixtures of both drugs. They were applied to their pharmaceutical formulation and the results obtained were statistically compared to that of a reported spectrophotometric method. The statistical comparison showed that there is no significant difference between the proposed methods and the reported one regarding both accuracy and precision.

Development of a sensitive UPLC-ESI-MS/MS method for quantification of sofosbuvir and its metabolite, GS-331007, in human plasma: Application to a bioequivalence study

A rapid and simple LC-MS/MS method was developed and validated for the simultaneous estimation of sofosbuvir (SF) and its metabolite GS-331007 (GS) using famotidine as an internal standard (IS). The Xevo TQD LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Extraction with ethyl acetate was used in sample preparation. The prepared samples were chromatographed on Acquity UPLC HSS C₁₈ (50 mm × 2.1 mm, 1.8 μm) column by pumping 0.1% formic acid and acetonitrile (50:50, v/v) in an isocratic mode at a flow rate of 0.3 ml/min. Method validation was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 10-2500 ng/ml for both SF and its metabolite. The intra-day and inter-day precision and accuracy results were within the acceptable limits. A very short run time of 1.2 min made it possible to analyze more than 300 human plasma samples per day. The developed assay method was successfully applied to a bioequivalence study in human volunteers.

Development and validation of sensitive and rapid UPLC-MS/MS method for quantitative determination of daclatasvir in human plasma: Application to a bioequivalence study

A rapid and sensitive UPLC-MS/MS method was developed and validated for determination of daclatasvir (DAC) in human plasma using sofosbuvir (SOF) as an internal standard (IS). The Xevo TQD LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Precipitation with acetonitrile was used in sample preparation. The prepared samples were chromatographed on Acquity UPLC HSS C18 (50×2.1mm, 1.8μm) column by pumping 10mM ammonium formate (pH 3.5) and acetonitrile in an isocratic mode at a flow rate of 0.30ml/min. Method validation was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 5-4000ng/ml for DAC. The intra-day and inter-day precision and accuracy results were within the acceptable limits. A very short run time of 1.2min made it possible to analyze more than 500 human plasma samples per day. The wider range of quantification of DAC allowed the applicability of the developed method for its determination in a bioequivalence study in human volunteers.

Stability-indicating determination of meropenem in presence of its degradation product

Stability-indicative determination of meropenem (MERM) in the presence of its open-ring degradation product, the metabolite, is investigated. The degradation product has been isolated, via acid-degradation, characterized and confirmed. Selective quantification of MERM, singly in bulk form, pharmaceutical formulations and/or in the presence of its major degradate is demonstrated. The indication of stability has been undertaken under conditions likely to be expected at normal storage. Among the analytical techniques adopted for quantification are spectrophotometry [first-derivative ((1)D), first-derivative of ratio spectra ((1)DD) and bivariate analysis], as well as chromatography [coupled TLC-densitometry and HPLC].

Selective determination of ertapenem in the presence of its degradation product

Stability-indicative determination of ertapenem (ERTM) in the presence of its beta-lactam open-ring degradation product, which is also the metabolite, is investigated. The degradation product has been isolated, via acid-degradation, characterized and elucidated. Selective quantification of ERTM, singly in bulk form, pharmaceutical formulations and/or in the presence of its major degradant is demonstrated. The indication of stability has been undertaken under conditions likely to be expected at normal storage conditions. Among the spectrophotometric methods adopted for quantification are first derivative ((1)D), first derivative of ratio spectra ((1)DD) and bivariate analysis.

Novel and sensitive UPLC-MS/MS method for quantification of sofosbuvir in human plasma: application to a bioequivalence study

A novel and sensitive LC-MS/MS method was developed and validated for determination of sofosbuvir (SF) using eplerenone as an internal standard. The Xevo TQD LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Extraction with tert-butyl methyl ether was used in sample preparation. The prepared samples were chromatographed on Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 μm) column by pumping 0.1% formic acid and acetonitrile in an isocratic mode at a flow rate of 0.35 mL/min. Method validation was performed as per the US Food and Drug Administration guidelines and the standard curves were found to be linear in the range of 0.25-3500 ng/mL for SF. The intra- and inter-day precision and accuracy results were within the acceptable limits. A very short run time of 1 min made it possible to analyze more than 500 human plasma samples per day. A very low quantification limit of SF allowed the applicability of the developed method for determination of SF in a bioequivalence study in human volunteers. Copyright

Multi-residues determination of antimicrobials in fish tissues by HPLC–ESI-MS/MS method

A rapid, simple, sensitive and specific LC-MS/MS method was developed and validated for the simultaneous quantification of four antimicrobials commonly used in aquaculture, namely ciprofloxacin (CPX), trimethoprim (TMP), sulphadimethoxine (SDM) and florphenicol (FLOR) in fish tissues. The LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Sample preparation involves simple liquid extraction step followed by post-extraction clean-up step with n-hexane. The purified extracts were chromatographed on Agilent Poroshell 120 EC, C18 (50 mm × 3 mm, 2.7 μm) column by pumping an isocratic mobile phase consisting of 0.1% formic acid in water:0.1% formic acid in methanol (20:80, by volume) at a flow rate of 0.4 mL/min. A detailed validation of the method was performed as per FDA guidelines and the standard curves were found to be linear in the range of 1-100 ng/g for both CPX and TMP, 0.5-100 ng/g for SDM and 1-50 ng/g for FLOR. The intra-day and inter-day precision and accuracy of the results were within the acceptable limits. A run time of 1.5 min for each sample made it possible to analyze multiple fish tissue samples per day. The developed assay method was successfully applied for the detection of antimicrobials in real fish tissue samples obtained from different fish farms.

Development, optimization and validation of a highly sensitive UPLC-ESI-MS/MS method for simultaneous quantification of amlodipine, benazeprile and benazeprilat in human plasma: application to a bioequivalence study.

A rapid, simple, sensitive and specific LC-MS/MS method has been developed and validated for the simultaneous estimation of amlodipine (AML), benazepril (BEN) and benazeprilat (BNT) using eplerenone and torsemide as internal standards (IS). The Xevo TQD LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Sample preparation involves both extraction and precipitation techniques. The reconstituted samples were chromatographed on Acquity UPLC BEH C18 (50mm×2.1mm, 1.7μm) column by pumping 0.1% formic acid and acetonitrile in a gradient mode at a flow rate of 0.45ml/min. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 0.1-5ng/ml for AML; 5-1200ng/ml for both BEN and BNT. The intra-day and inter-day precision and accuracy results were within the acceptable limits. A run time of 2.5min for each sample made it possible to analyze more than 300 human plasma samples per day. The developed assay method was successfully applied to a bioequivalence study in human volunteers.

Comparative study of novel versus conventional two-wavelength spectrophotometric methods for analysis of spectrally overlapping binary mixture

Smart spectrophotometric methods have been applied and validated for the simultaneous determination of a binary mixture of chloramphenicol (CPL) and prednisolone acetate (PA) without preliminary separation. Two novel methods have been developed; the first method depends upon advanced absorbance subtraction (AAS), while the other method relies on advanced amplitude modulation (AAM); in addition to the well established dual wavelength (DW), ratio difference (RD) and constant center coupled with spectrum subtraction (CC-SS) methods. Accuracy, precision and linearity ranges of these methods were determined. Moreover, selectivity was assessed by analyzing synthetic mixtures of both drugs. The proposed methods were successfully applied to the assay of drugs in their pharmaceutical formulations. No interference was observed from common additives and the validity of the methods was tested. The obtained results have been statistically compared to that of official spectrophotometric methods to give a conclusion that there is no significant difference between the proposed methods and the official ones with respect to accuracy and precision.

Novel spectrophotometric determination of chloramphenicol and dexamethasone in the presence of non labeled interfering substances using univariate methods and multivariate regression model updating

Smart and novel spectrophotometric and chemometric methods have been developed and validated for the simultaneous determination of a binary mixture of chloramphenicol (CPL) and dexamethasone sodium phosphate (DSP) in presence of interfering substances without prior separation. The first method depends upon derivative subtraction coupled with constant multiplication. The second one is ratio difference method at optimum wavelengths which were selected after applying derivative transformation method via multiplying by a decoding spectrum in order to cancel the contribution of non labeled interfering substances. The third method relies on partial least squares with regression model updating. They are so simple that they do not require any preliminary separation steps. Accuracy, precision and linearity ranges of these methods were determined. Moreover, specificity was assessed by analyzing synthetic mixtures of both drugs. The proposed methods were successfully applied for analysis of both drugs in their pharmaceutical formulation. The obtained results have been statistically compared to that of an official spectrophotometric method to give a conclusion that there is no significant difference between the proposed methods and the official ones with respect to accuracy and precision.