Samy Emara

Development and validation of a stability-indicating RP-HPLC method for the determination of paracetamol with dantrolene or/and cetirizine and pseudoephedrine in two pharmaceutical dosage forms

A stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed which can separate and accurately quantitate paracetamol, dantrolene, cetirizine and pseudoephedrine. The method was successfully validated for the purpose of conducting stability studies of the four analytes in quality control (QC) laboratories. The stability-indicating capability of the method was demonstrated by adequate separation of these four analytes from all the degradant peaks. A gradient mobile phase system consisting of (A) 50 mmol L(-1) sodium dihydrogen phosphate, 5 mmol L(-1) heptane sulfonic acid sodium salt, pH 4.2 and (B) acetonitrile was used with Discovery reversed-phase HS C(18) analytical column (250 mm x 4.6 mm i.d., 5 microm particle size). Quantitation was achieved with UV detection at 214 nm, based on peak area. The proposed method was validated and successfully applied for the analysis of pharmaceutical formulations and laboratory-prepared mixtures containing the two multicomponent combinations.

Validated and Optimized High-Performance Liquid Chromatographic Determination of Tizoxanide, the Main Active Metabolite of Nitazoxanide in Human Urine, Plasma and Breast Milk

A high-performance liquid chromatographic method was optimized and validated for the determination of desacetyl nitazoxanide (tizoxanide), the main active metabolite of nitazoxanide in human plasma, urine and breast milk. The proposed method used a CN column with mobile phase consisting of acetonitrile-12mM ammonium acetate-diethylamine in the ratio of 30:70:0.1 (v/v/v) and buffered at pH 4.0 with acetic acid, with a flow rate of 1.5 mL/min. Quantitation was achieved with UV detection at 260 nm using nifuroxazide as internal standard. A simplified direct injection of urine samples without extraction in addition to the urinary excretion pattern were calculated using the proposed method. Also, the effectiveness of protein precipitation and a clean-up procedure were investigated for biological plasma and human breast milk samples. The validation study of the proposed method was successfully carried out in an assay range between 0.2 and 20 µg/mL.

Field-Amplified Sample Stacking β-Cyclodextrin Modified Capillary Electrophoresis for Quantitative Determination of Diastereomeric Saponins

Successful simultaneous diastereomeric separation and sensitive determination of two pairs of triterpenoidal saponins have been achieved by capillary electrophoresis (CE) using β-cyclodextrin (β-CD) as a stereoselective agent to cooperate with borate complexation. A usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to the determination of individual saponins by CE. Soyasaponin I ( S1: ), azukisaponin V ( S2: ), bersimoside I ( S3: ) and bersimoside II ( S4: ) could be well separated within 14 min in a fused-silica capillary (60 cm long to the detector with an additional 10 cm to the cathode; 75 µm i.d.). The background electrolyte was borate buffer (80 mM, pH 10), containing 24 mM β-CD. The separation voltage was 14 kV with a detection wavelength of 195 nm. The sample was electrokinetically injected using a voltage of 16 kV for 12 s. Methanol (70%) was used as the diluent for field-amplified sample stacking after hydrodynamic injection of short water plug (5 cm, 4 s). The method was partially validated for linearity, repeatability, reproducibility, limits of detection and limits of quantification. The correlation coefficients of the calibration curves were all >0.998, and the recoveries were from 98.23 to 96.21%.

Online Pre-Column Derivatization with Chromatographic Separation to Determine Folic Acid

A simple, sensitive, and selective online pre-column derivatization high-performance liquid chromatographic method was developed and validated for the first time to determine trace levels of folic acid (FA). An oxidant cerium (IV) trihydroxyhydroperoxide packed reactor was used for pre-column oxidation and was combined by column switching with a C18 analytical column for sample enrichment and separation. The method was based on oxidative cleavage of FA into highly fluorescence products, 2-amino-4-hydroxypteridine-6-carboxaldehyde and the corresponding 2-amino-4-hydroxypteridine-6-carboxylic acid, during the flow of 0.04 M phosphate buffer (pH 3.5) containing the analyte through packed reactor at a flow rate of 0.2 mL/min and 40°C. The fluorescent products were enriched on the head of the analytical column for the final separation. The separation was performed at room temperature using a mobile phase consisting of phosphate buffer (0.04 M, pH 3.5) and acetonitrile (90:10, v/v). The eluents were monitored at emission and excitation wavelengths of 463 and 367 nm, respectively. The method showed excellent recovery, precision and accuracy with detection limits of 0.067 ng/mL from 500 µL of sample FA. The developed method was successfully applied to the determination of FA in pharmaceutical formulations and showed a recovery of 99.31% and a relative standard deviation of 1.72%.

BACK-FLUSH COLUMN-SWITCHING TECHNIQUE FOR ON-LINE SAMPLE CLEANUP AND ENRICHMENT TO DETERMINE GUAIPHENESIN IN HUMAN SERUM

A direct injection high-performance liquid chromatographic (HPLC) method, with column-switching, for the determination of guaiphenesin (GP) in human serum has been developed. Serum samples were directly injected onto protein-coated RP-8 silica precolumn, where GP was preconcentrated and retained while proteins and very polar components were washed to waste using phosphate buffer saline, pH 7.4. GP was back-flushed from the precolumn by a column-switching technique and separated on a ZORBAX Eclipse XDB-C18 analytical column with a mobile phase consisting of methanol-0.01 M phosphate buffer (containing 1% triethylamine adjusted to pH 3.5 with ortho-phosphoric acid) in the ratio of 45:55 (v/v). The analyte was detected by its UV absorbance at 254 nm. The calibration curve was linear over the concentration range of 25–4000 ng/mL ( r 2 = 0.9994). The method was validated for linearity, accuracy, precision, selectivity, and robustness.

On-line sample cleanup and enrichment chromatographic technique for the determination of ambroxol in human serum.

A sensitive and efficient on-line clean up and pre-concentration method has been developed using column-switching technique and protein-coated µ-Bondapak CN silica pre-column for quantification of ambroxol (AM) in human serum. The method is performed by direct injection of serum sample onto a protein-coated µ-Bondapak CN silica pre-column, where AM is pre-concentrated and retained, while proteins and very polar constituents are washed to waste using a phosphate buffer saline (pH 7.4). The retained analyte on the pre-column is directed onto a C(18) analytical column for separation, with a mobile phase consisting of a mixture of methanol and distilled deionized water (containing 1% triethylamine adjusted to pH 3.5 with ortho-phosphoric acid) in the ratio of 50:50 (v/v). Detection is performed at 254 nm. The calibration curve is linear over the concentration range of 12-120 ng/mL (r(2) = 0.9995). The recovery, selectivity, linearity, precision, and accuracy of the method are convenient for pharmacokinetic studies or routine assays.

Single-Cell Metabolomics

The dynamics of a cell is always changing. Cells move, divide, communicate, adapt, and are always reacting to their surroundings non-synchronously. Currently, single-cell metabolomics has become the leading field in understanding the phenotypical variations between them, but sample volumes, low analyte concentrations, and validating gentle sample techniques have proven great barriers toward achieving accurate and complete metabolomics profiling. Certainly, advanced technologies such as nanodevices and microfluidic arrays are making great progress, and analytical techniques, such as matrix-assisted laser desorption ionization (MALDI), are gaining popularity with high-throughput methodology. Nevertheless, live single-cell mass spectrometry (LCSMS) values the sample quality and precision, turning once theoretical speculation into present-day applications in a variety of fields, including those of medicine, pharmaceutical, and agricultural industries. While there is still room for much improvement, it is clear that the metabolomics field is progressing toward analysis and discoveries at the single-cell level.

Determination of Glucosamine and Carisoprodol in Pharmaceutical Formulations by LC with Pre-Column Derivatization and UV Detection

A simple and reliable precolumn derivatization liquid chromatography method with ultraviolet detection has been developed and validated for the analysis of glucosamine (GS) in various dietary supplement formulations and raw materials. Additionally, the proposed method was used for analysis of carisoprodol (CR) found in ternary mixture with paracetamol (PR) and caffeine (CF). The linearity ranges were 1-100 μg/mL for GS, 1-150 μg/mL for CR, PR and CF. Derivatization was used with 1,2-naphthoquinone-4-sulphonic acid sodium salt in the presence of borate buffer. Chromatographic separation of GS-naphthoquinone derivative was achieved by using a mixture of acetonitrile and water (pH 7.3 adjusted with 0.1 M NaOH) in the ratio 10:90, v/v and flow-rate of 1.0 mL/min. UV detection was carried out at 280 nm. For PR, CF, and CR-naphthoquinone derivative, the chromatographic separation was achieved by using mixture of acetonitrile and 20 mM KH(2)PO(4) (pH 3.0 adjusted with phosphoric acid) in the ratio 20:80, v/v and flow-rate of 1.0 mL/min. UV detection was carried out at 275 nm. The limits of detection were 37.2, 35.9, 30.4 and 40.0 ng/mL for GS, CR, PR and CF, respectively.

On-line solid-phase enrichment coupled to packed reactor flow injection analysis in a green analytical procedure to determine low levels of folic acid using fluorescence detection

BackgroundAnalysis of folic acid (FA) is not an easy task because of its presence in lower concentrations, its lower stability under acidic conditions, and its sensitiveness against light and high temperature. The present study is concerned with the development and validation of an automated environmentally friendly pre-column derivatization combined by solid-phase enrichment (SPEn) to determine low levels of FA.ResultsCerium (IV) trihydroxyhydroperoxide (CTH) as a packed oxidant reactor has been used for oxidative cleavage of FA into highly fluorescent product, 2-amino-4-hydroxypteridine-6-carboxylic acid. FA was injected into a carrier stream of 0.04 M phosphate buffer, pH 3.4 at a flow-rate of 0.25 mL/min. The sample zone containing the analyte was passed through the CTH reactor thermostated at 40°C, and the fluorescent product was trapped and enriched on a head of small ODS column (10 mm x 4.6 mm i.d., 5 μm particle size). The enriched product was then back-flush eluted by column-switching from the small ODS column to the detector with a greener mobile phase consisting of ethanol and phosphate buffer (0.04M, pH 3.4) in the ratio of 5:95 (v/v). The eluent was monitored fluorimetrically at emission and excitation wavelengths of 463 and 367 nm, respectively. The calibration graph was linear over concentrations of FA in the range of 1.25-50 ng/mL, with a detection limit of 0.49 ng/mL.ConclusionA new simple and sensitive green analytical procedure including on-line pre-column derivatization combined by SPEn has been developed for the routine quality control and dosage form assay of FA at very low concentration level. The method was a powerful analytical technique that had excellent sensitivity, sufficient accuracy and required relatively simple and inexpensive instrumentation.

Optimized and validated flow-injection spectrophotometric analysis of topiramate, piracetam and levetiracetam in pharmaceutical formulations

Optimized and validated flow-injection spectrophotometric analysis of topiramate, piracetam and levetiracetam in pharmaceutical formulations Application of a sensitive and rapid flow injection analysis (FIA) method for determination of topiramate, piracetam, and levetiracetam in pharmaceutical formulations has been investigated. The method is based on the reaction with ortho-phtalaldehyde and 2-mercaptoethanol in a basic buffer and measurement of absorbance at 295 nm under flow conditions. Variables affecting the determination such as sample injection volume, pH, ionic strength, reagent concentrations, flow rate of reagent and other FIA parameters were optimized to produce the most sensitive and reproducible results using a quarter-fraction factorial design, for five factors at two levels. Also, the method has been optimized and fully validated in terms of linearity and range, limit of detection and quantitation, precision, selectivity and accuracy. The method was successfully applied to the analysis of pharmaceutical preparations. Optimirana i validirana protočna injekcijska spektrofotometrijska analiza topiramata, piracetama i levetiracetama u farmaceutskim pripravcima Opisana je osjetljiva i brza protočna injekcijska analiza (FIA) za određivanje topira-mata, piracetama i levetiracetama u farmaceutskim pripravcima. Metoda se temelji na reakciji ortho-ftalaldehida i 2-merkaptoetanola u bazičnom puferu i mjerenju apsorbancije na 295 nm u protočnim uvjetima. U svrhu povećanja osjetljivosti i dobivanja reproducibilnih rezultata optimirane su varijable koje utječu na određivanje kao što su volumen injektiranog uzorka, pH, ionska jakost, koncentracija reagensa, brzina protoka reagensa i drugi FIA parametri koristeći četvrt-frakcijski faktorijalni dizajn, za pet faktora na dva nivoa. Metoda je optimirana i potpuno validirana (linearnost, područje određivanja, granica detekcije i kvantifikacije, preciznost, selektivnost i točnost). Metoda je uspješ no primijenjena za analizu farmaceutskih pripravaka.