Hytham M. Ahmed

Validated spectrofluorimetric method for determination of selected aminoglycosides

New, sensitive, and selective spectrofluorimetric method was developed for determination of three aminoglycoside drugs in different dosage forms, namely; neomycin sulfate (NEO), tobramycin (TOB) and kanamycin sulfate (KAN). The method is based on Hantzsch condensation reaction between the primary amino group of aminoglycosides with acetylacetone and formaldehyde in pH 2.7 yielding highly yellow fluorescent derivatives measured emission (471 nm) and excitation (410 nm) wavelengths. The fluorescence intensity was directly proportional to the concentration over the range 10-60, 40-100 and 5-50 ng/mL for NEO, TOB and KAN respectively. The proposed method was applied successfully for determination of these drugs in their pharmaceutical dosage forms.

Determination of metoprolol enantiomers in human plasma and saliva samples utilizing microextraction by packed sorbent and liquid chromatography-tandem mass spectrometry.

A sensitive, accurate and reliable bioanalytical method for the enantioselective determination of metoprolol in plasma and saliva samples utilizing liquid chromatography-electrospray ionization tandem mass spectrometry was developed and validated. Human plasma and saliva samples were pretreated by microextraction by packed sorbent (MEPS) prior to analysis. A new MEPS syringe form with two inputs was used. Metoprolol enantiomers and internal standard pentycaine (IS) were eluted from MEPS sorbent using isopropanol after removal of matrix interferences using aliquots of 5% methanol in water. Complete separation of metoprolol enantiomers was achieved on a Cellulose-SB column (150 × 4.6 mm, 5 μm) using isocratic elution with mobile phase 0.1% ammonium hydroxide in hexane-isopropanol (80:20, v/v) with a flow rate of 0.8 mL/min. A post-column solvent-assisted ionization was applied to enhance metoprolol ionization signal in positive mode monitoring (+ES) using 0.5% formic acid in isopropanol at a flow rate of 0.2 mL/min. The total chromatographic run time was 10 min for each injection. The detection of metoprolol in plasma and saliva samples was performed using triple quadrupole tandem mass spectrometer in +ES under the following mass transitions: m/z 268.08 → 72.09 for metoprolol and m/z 303.3 → 154.3 for IS. The linearity range was 2.5-500 ng/mL for both R- and S-metoprolol in plasma and saliva. The limits of detection and quantitation for both enantiomers were 0.5 and 2.5 ng/mL respectively, in both matrices (plasma and saliva). The intra- and inter-day precisions were presented in terms of RSD values for replicate analysis of quality control samples and were <5%; the accuracy of determinations varied from 96 to 99%. The method was able to determine the therapeutic levels of metoprolol enantiomers in both human plasma and saliva samples successfully, which can aid in therapeutic drug monitoring in clinical laboratories. Copyright

The use of laser-induced fluorescence or ultraviolet detectors for sensitive and selective analysis of tobramycin or erythropoietin in complex samples

Complex samples analysis is a challenge in pharmaceutical and biopharmaceutical analysis. In this work, tobramycin (TOB) analysis in human urine samples and recombinant human erythropoietin (rhEPO) analysis in the presence of similar protein were selected as representative examples of such samples analysis. Assays of TOB in urine samples are difficult because of poor detectability. Therefore laser induced fluorescence detector (LIF) was combined with a separation technique, micellar electrokinetic chromatography (MEKC), to determine TOB through derivatization with fluorescein isothiocyanate (FITC). Borate was used as background electrolyte (BGE) with negative-charged mixed micelles as additive. The method was successively applied to urine samples. The LOD and LOQ for Tobramycin in urine were 90 and 200ng/ml respectively and recovery was >98% (n=5). All urine samples were analyzed by direct injection without sample pre-treatment. Another use of hyphenated analytical technique, capillary zone electrophoresis (CZE) connected to ultraviolet (UV) detector was also used for sensitive analysis of rhEPO at low levels (2000IU) in the presence of large amount of human serum albumin (HSA). Analysis of rhEPO was achieved by the use of the electrokinetic injection (EI) with discontinuous buffers. Phosphate buffer was used as BGE with metal ions as additive. The proposed method can be used for the estimation of large number of quality control rhEPO samples in a short period.

Simple protein precipitation extraction technique followed by validated chromatographic method for linezolid analysis in real human plasma samples to study its pharmacokinetics

Fast and sensitive HPLC method was developed, optimized and validated for quantification of linezolid (LNZ) in human plasma using guaifenesin as an internal standard (IS). Analyte and IS were extracted from plasma by simple protein precipitation extraction technique using methanol as the precipitating solvent. The pretreated samples were injected in a mobile phase formed of acetonitrile:water:methanol (20:70:10v/v/v) in an isocratic mode at a flow rate of 1.5mL/min with UV detection at 251nm. Separation was done using Aglient ODS C18. The method showed linearity in the range of 0.75-50μg/mL with correlation coefficients equals to 0.9991. Precision and accuracy were in conformity with the criteria normally accepted in bio-analytical method validation. The RSDs for intra- and inter-day assays were <3.56 and 4.63%, respectively. The intra- and inter-day accuracies were 94.67-98.28% and 91.25-96.18%, respectively. The mean absolute recoveries ranged from 92.56±1.78 to 95.24±2.84. According to stability results, LNZ was stable in human plasma during the storage and analysis. LNZ a pharmacokinetic behavior was studied by applying the proposed analytical method.

Online post-column solvent assisted and direct solvent-assisted electrospray ionization for chiral analysis of propranolol enantiomers in plasma samples

An Online post-column solvent-assisted ionization (OPSAI) method was developed for enhancing the ionization of the beta-blocker propranolol utilizing normal phase LC-MS/MS. Solvent-assisted electrospray ionization (SAESI) was studied by the introduction of the assistant solvents A: 0.5% Formic acid in Isopropanolol, B: 0.5% Formic acid in Isopropanolol-Water (1:1), and C: 0.5% Formic acid in water into the electrospray ionization chamber using a spray needle. Analyte molecules can be directly ionized by the aid of the assistant solvent spray. Both methods were applied to the chiral separation of propranolol enantiomers using normal phase analysis on cellulose-based chiral column. Interestingly, both methods are easy to handle and offer a wide range of assistant solvents that can be used in order to gain the optimum ionization of the analyte molecules. The both methods considerably improved the analyte signal and the peak area greatly increased. The propranolol average signal-to-noise (S/N) ratio was enhanced from 26±1 and 42±1 to 2341±61 and 1725±29 for R-propranolol and S-propranolol, respectively, when the post-column solvent method (OPSAI) was used with isopropanol-assistant solvent (A). While in case of solvent-assisted electrospray ionization method (SAESI) signal was enhanced from 26±1 and 42±1 to 2223±72 and 2155±58 for R-propranolol and S-propranolol, respectively, with water as an assistant solvent. The limit of detection was 10ng/mL and the method was linear in the range 50-2000ng/mL. The NPLC-MS method was applied for the determination of propranolol enantiomers in human plasma after microextraction by packed C18 sorbent.

New voltammetric analysis of olanzapine in tablets and human urine samples using a modified carbon paste sensor electrode incorporating gold nanoparticles and glutamine in a micellar medium

An effective novel electrochemical sensor for the selective determination of olanzapine (OLA) was introduced. The prepared sensor was based on a carbon paste electrode chemically modified with glutamine (GL) and gold nanoparticles (GNs) in the presence of sodium dodecyl sulphate (SDS) in the medium. The effect of carbon paste composition and scan rate were tested. The working solution pH was 7. The analytical method validation parameters were studied. The linear response was obtained for OLA in the range of 5 × 10−7 to 1.25 × 10−4 M with a correlation coefficient of 0.9986. LOD and LOQ were calculated and found to be 3.58 × 10−9 and 1.19 × 10−8 M, respectively. The utility of this sensor was examined for the determination of OLA in its pharmaceutical dosage form and human urine. Also the proposed method was applied for the simultaneous determination of OLA, fluoxetine (FLX), ascorbic acid (AA) and uric acid (UA).

New valid spectrofluorimetric method for determination of selected cephalosporins in different pharmaceutical formulations using safranin as fluorophore

A new validated spectrofluorimetric method has been developed for the determination of some cephalosporins namely; cefepime, cefaclor, cefadroxil, cefpodoxime and cefexime. The method was based on the reaction of these drugs with safranin in slightly alkaline medium (pH 8.0), to form ion-association complexes. The fluorescent products were extracted into chloroform and their fluorescence intensities were measured at 544-565 nm after excitation at 518-524 nm. The reaction conditions influencing the product formation and stability were investigated and optimized. The relative fluorescence intensity was proportional to the drug concentration in the linear ranges of 0.15-1.35, 0.35-1.25, 0.35-1.25, 0.20-1.44 and 0.20-1.25 μg/mL for cefepime, cefaclor, cefadroxil, cefpodoxime proxetil and cefexime, respectively. The detection limits were 40, 100, 100, 60 and 70 ng/mL, respectively. The performance of the developed method was evaluated in terms of Students t-test and variance ratio F-test to find out the significance of proposed methods over the reference spectrophotometric method. Various pharmaceutical formulations were successfully analyzed using the proposed method and the results were in good agreement with those of the previously reported methods.

Recovery of some valuable components from industrial waste

ABSTRACT The present work studied several ways for wastewater treatment for the waste from the pulp and paper industries including the recovery of some valuable components from this waste and reaching the acceptable environmental limits for the treatment of waste. The acidification technique showed high efficiency for the removal of solids from the black liquor. A proper treatment for the effluent produced from the pulp and paper industry that used rice straw as raw material. The dose of the alum as a coagulant has an effect on the solid process and is due to the high percentage of solids in the black liquor. Hydrochloric acid is used to control the pH of the solution and separating most of the solids from the black liquor. Sodium hydroxide can be used to remove silica from the other solids by forming sodium silicate. The amount of solids precipitated increased by increasing the stirring time, temperature, rpm of the agitator, and decreasing pH. IR spectroscopy was carried out on the solid to assign the functional groups. Thermal analysis and EDX analysis were done to study the thermal properties of the solids. A proper design of the mixer and drier is accomplished to provide high efficiency of the silica removal process.

A validated stability-indicating HPLC-DAD method for simultaneous determination of econazole nitrate, triamcinolone acetonide, benzoic acid and butylated hydroxyanisole in cream dosage form

This study deals with the development and validation of a comprehensive stability-indicating high performance liquid chromatography with diode array detection (HPLC-DAD) method for simultaneous determination of econazole nitrate (EN), triamcinolone acetonide (TA), benzoic acid (BA) and butylated hydroxyanisole (BHA). To the best of our knowledge, no published methods could be found in the scientific literature for analysis of this quaternary mixture. Effective chromatographic separation was achieved using a Thermo Hypersil BDS C8 column (4.6 × 150 mm, 5 μm particle size) with gradient elution of the mobile phase composed of 0.2% w/v phosphoric acid (adjusted to pH 3.0 using ammonia solution) and methanol. The quantification of EN and BA was based on measuring their peak areas at 225 nm, while the quantification of TA and BHA was based on measuring their peak areas at 242 nm and 290 nm, respectively. BA, TA, BHA and EN peaks eluted at retention times of 8.11, 12.45, 14.28 and 18.36 min, respectively. Analytical performance of the proposed HPLC procedure was thoroughly validated with respect to system suitability, linearity ranges, precision, accuracy, specificity, robustness, and detection and quantification limits. The linearity ranges for EN, TA, BA and BHA were 1.5–300, 1–200, 0.6–100 and 1–100 μg mL−1, respectively, with correlation coefficients >0.9999. The analytes were subjected to forced-degradation conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal degradation. The proposed method proved to be stability-indicating by resolution of the analytes from their forced-degradation products. Moreover, specificity of the method was verified by resolution of the four analytes from more than 20 pharmaceutical compounds of various medicinal categories. The validated HPLC method was successfully applied to the analysis of the cited compounds in their cream dosage form. The proposed method made use of DAD as a tool for peak identity and purity confirmation.

Production and analysis of a biosimilar erythropoietin in Egypt

Although management of chronic diseases has been a major challenge for health care systems in developed and developing countries, biopharmaceuticals have been successful in treating many life-threatening conditions. However, the high cost of these agents restricts their availability to countries where patients and/or health care systems are able to afford them. Licensing these biopharmaceuticals as biosimilars after expiration of their patents might increase access to such medicines at an affordable price in developing countries. South Egypt Drug Industries Company (SEDICO) is an Egyptian pharmaceutical company that has had the opportunity to manufacture some of these drugs. SEDICO biotechnology products, such as insulin, erythropoietin, streptokinase, angiokinase, follicle-stimulating hormone, aprotinin, filgrastim, and somatropin, have been available on the Egyptian market for more than 6 years. For this paper, erythropoietin, which has been investigated over a number of years, was chosen as a representative example of SEDICO biotechnology products. Our findings confirm that SEDICO erythropoietin can compete with the originator epoetins on the Egyptian market with high quality and at a lower cost.