Ehab F. Elkady

Liquid chromatographic determination of sitagliptin either alone or in ternary mixture with metformin and sitagliptin degradation product

Two reversed-phase liquid chromatographic (RP-LC) methods have been developed for the determination of sitagliptin phosphate monohydrate (STG). The first method comprised the determination of STG alone in bulk and plasma; and in its pharmaceutical preparation. This method was based on isocratic elution of STG using a mobile phase consisting of potassium dihydrogen phosphate buffer pH (7.8)-acetonitrile (70:30, v/v) at a flow rate of 1 mL min(-1) with flourometric detection. The flourometric detector was operated at 267 nm for excitation and 575 nm for emission. In the second method, the simultaneous determination of STG and metformin (MET) in the presence of sitagliptin alkaline degradation product (SDP) has been developed. In this method, the ternary mixture of STG, MET and SDP was separated using a mobile phase consisting of potassium dihydrogen phosphate buffer pH (4.6)-acetonitrile-methanol (30:50:20, v/v/v) at a flow rate of 1 mL min(-1) with UV detection at 220 nm. Chromatographic separation in the two methods was achieved on a Symmetry(®) Waters C18 column (150 mm×4.6 mm, 5 μm). Linearity, accuracy and precision were found to be acceptable over the concentration ranges of 0.25-200 μg mL(-1) for STG with the first method and 5-160 μg mL(-1), 25-800 μg mL(-1) for STG and MET, respectively with the second method. The optimized methods were validated and proved to be specific, robust and accurate for the quality control of the cited drugs in pharmaceutical preparations.

Simultaneous determination of diclofenac potassium and methocarbamol in ternary mixture with guaifenesin by reversed phase liquid chromatography

New, simple, rapid and precise reversed phase liquid chromatographic (RP-LC) method has been developed for the simultaneous determination of diclofenac potassium (DP) and methocarbamol (MT) in ternary mixture with guaifenesin (GF), degradation product of methocarbamol. Chromatographic separation was achieved on a Symmetry Waters C18 column (150 mm x 4. 6mm, 5 microm). Gradient elution based on phosphate buffer pH (8)-acetonitrile at a flow rate of 1 mL min(-1) was applied. The UV detector was operated at 282 nm for DP and 274 nm for MT and GF. Linearity, accuracy and precision were found to be acceptable over the concentration ranges of 0.05-16, 0.5-160 and 0.5-160 microg mL(-1) for DP, MT and GF, respectively. The optimized method proved to be specific, robust and accurate for the quality control of the cited drugs in pharmaceutical preparation.

A Rapid and Optimized LC-MS/MS Method for the Simultaneous Extraction and Determination of Sofosbuvir and Ledipasvir in Human Plasma.

A new validated bioanalytical method based on LC tandem MS has been developed for the simultaneous extraction and determination of sofosbuvir and ledipasvir in human plasma using antiviral daclatasvir as an internal standard (IS). Liquid-liquid extraction of samples was used for the purification and preconcentration of the analytes from a human plasma matrix. Good and consistent recoveries were obtained, with average extraction recoveries of 91.61 and 88.93% for sofosbuvir and ledipasvir, respectively. The chromatographic separation of the three analytes was achieved within only 2.8 min by an isocratic mobile phase consisting of 10 mM ammonium acetate, which was then adjusted to pH 4.0 by acetic acid-acetonitrile-0.1% methanolic formic acid (12 + 25 + 63, v/v/v) flowing through a C18 Zorbax eclipse plus column (5 μm, 100 × 4.6 mm; Agilent). Multiple reaction monitoring transitions were measured in positive ion mode for sofosbuvir, ledipasvir, and daclatasvir (IS). A detailed validation of the method was performed and the standard curves were found to be linear in the range of 0.5 to 2500 and 5 to 2100 ng/mL for sofosbuvir and ledipasvir, respectively, applying weighted (1/X(2)) linear regression. The developed method was applied to the analysis of the two drugs after a single oral administration of Harvoni 400/90 mg film-coated tablets containing 400 mg sofosbuvir and 90 mg ledipasvir to four healthy volunteers.

LC–MS–MS Simultaneous Determination of Atorvastatin and Ezetimibe in Human Plasma

Atorvastatin and ezetimibe are lipid-lowering drugs prescribed for the treatment of hypercholesterolemia. An LC-MS-MS method has been developed and validated for the simultaneous estimation of atorvastatin and ezetimibe in human plasma using pitavastatin as an internal standard. Liquid-liquid extraction was used for the purification and preconcentration of analytes from human plasma matrix. The chromatographic separation was achieved within 3.0 min by an isocratic mobile phase consisting of 0.2% formic acid in water-acetonitrile (30:70, v/v), flowing through Agilent Eclipse-plus C18, 100 × 4.6 mm, 3.5 µm analytical column, at a flow rate of 0.6 mL min(-1). Multiple reaction monitoring transitions were measured in the positive ion mode for atorvastatin and internal standard, while ezetimibe was measured in negative ion mode. A detailed validation of the method was performed as per US-FDA guidelines and the standard curves were found to be linear in the range of 0.2-30.0 ng mL(-1) with a mean correlation coefficient >0.999 for both drugs. In human plasma, atorvastatin and ezetimibe were stable for at least 36 days at -70 ± 5 °C and 6 h at ambient temperature. After extraction from plasma, the reconstituted samples of atorvastatin and ezetimibe were stable in an autosampler at ambient temperature for 6 h. Also, the cited drugs were stable in plasma samples upon subjecting to three freeze thaw cycles. The method is simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies of this combination.

Improved reversed phase liquid chromatographic method with pulsed electrochemical detection for tobramycin in bulk and pharmaceutical formulation

Tobramycin is one of the aminoglycoside antibiotics that lack a UV absorbing chromophore. However, the application of pulsed electrochemical detection (PED) has been used successfully for the analysis of this and similar antibiotics. This work describes an improved liquid chromatographic (LC) method combined with PED, which is able to separate much more impurities than before. Using a Discovery C-18 RP column (250 mm×4.6 mm i.d., 5 μm), isocratic elution was carried out with a mobile phase, containing sodium sulfate (35 g/L), sodium octanesulphonic acid (1 g/L), tetrahydrofuran (14 mL/L) and 0.2 M phosphate buffer pH 3.0 (50 mL/L). Using these experimental conditions, the limit of quantification (LOQ, S/N=10) was 5 ng. The linearity was examined in the range LOQ-60 μg/mL and the coefficient of determination was 0.998. The method also proved to be repeatable and the recovery was close to 100%. The influence of the different chromatographic parameters on the separation was investigated by means of an experimental design. The proposed method is useful in quality control of tobramycin drug substances and drug products.

Spectrophotometric and Spectrofluorimetric Studies on Azilsartan Medoxomil and Chlorthalidone to Be Utilized in Their Determination in Pharmaceuticals

The recently approved angiotensin II receptor blocker, azilsartan medoxomil (AZL), was determined spectrophotometrically and spectrofluorimetrically in its combination with chlorthalidone (CLT) in their combined dosage form. The UV-spectrophotometric technique depends on simultaneous measurement of the first derivative spectra for AZL and CLT at 286 and 257 nm, respectively, in methanol. The spectrofluorimetric technique depends on measurement of the fourth derivative of the synchronous spectra intensities of AZL in presence of CLT at 298 nm in methanol. The effects of different solvents on spectrophotometric and spectrofluorimetric responses were studied. For, the spectrofluorimetric study, the effect of pH and micelle-assisted fluorescence enhancement were also studied. Linearity, accuracy, and precision were found to be satisfactory over the concentration ranges of 8–50 μg mL−1 and 2–20 μg mL−1 for AZL and CLT, respectively, in the spectrophotometric method as well as 0.01–0.08 μg mL−1 for AZL in the spectrofluorimetric method. The methods were successfully applied for the determination of the studied drugs in their co-formulated tablets. The developed methods are inexpensive and simple for the quality control and routine analysis of the cited drugs in bulk and in pharmaceuticals.

Spectrofluorometric Determination of Certain Antihyperlipidemic Agents in Bulk and Pharmaceutical Preparations

A simple, rapid, and sensitive spectrofluorometric method was developed for the determination of three antihyperlipidemic drugs, namely, rosuvastatin calcium (RSV), ezetimibe (EZE), and pitavastatin calcium (PIT). The method is based on measuring the native fluorescence of the cited drugs at their optimum excitation and emission wavelengths. The fluorescence intensity was measured at λem 362 nm, 309 nm, and 373 nm upon excitation at λex 315 nm, 260 nm, and 245 nm for RSV, EZE, and PIT, respectively. The calibration graphs were linear over the concentration ranges 0.50–10.0, 0.25–4.0, and 0.10–3.00 μg mL−1 for RSV, EZE, and PIT, respectively. Besides, a spectrofluorometric method for the simultaneous determination of RSV and EZE was developed. The fluorescence was measured at λem 309 nm for EZE and 432 nm for RSV upon excitation at λex 260 nm for both. The proposed methods were applied to the determination of the cited drugs either in bulk and pharmaceutical preparations.

TWO LIQUID CHROMATOGRAPHIC METHODS FOR THE SIMULTANEOUS DETERMINATION OF IBUPROFEN AND METHOCARBAMOL OR CHLORZOXAZONE IN THE PRESENCE OF THEIR DEGRADATION PRODUCTS

Two RP-LC methods have been developed for the simultaneous determination of ibuprofen (IBU) and two skeletal muscle relaxants, namely methocarbamol (MET) or chlorzoxazone (CHZ), in the presence of their degradation products. In the first method, the simultaneous determination of IBU and MET in the presence of guaifenesin (GUF), a degradation product of MET, was developed. Chromatographic separation was achieved on an Inertsil CN-3 column (250 mm × 4.6 mm, 5 µm). Gradient elution based on 0.5% aqueous phosphoric acid (pH 2.8) - acetonitrile at a flow rate of 1 mL min−1 was applied with UV detection at 229 nm for IBU and 274 nm for MET and GUF. Linearity, accuracy, and precision were found to be acceptable over the concentration ranges of 5–160 µg mL−1 for IBU and 2.5–160 µg mL−1 for MET and GUF. In the second method, the simultaneous determination of IBU and CHZ in the presence of CHZ degradation product, 2-amino-4-chlorophenol (CDE), was developed. In this method, chromatographic separation was achieved on Reprosil-Pur C8 (250 mm × 4.6 mm, 5 µm). A gradient mobile phase system consisting of acetonitrile and 0.2% aqueous triethylamine (pH 5.1) at a flow rate of 1 mL min−1 was applied with UV detection at 229 nm for the three compounds. Linearity, accuracy, and precision were found to be acceptable over the concentration ranges of 2.5–250 µg mL−1 IBU and CHZ, and 2.5–150 µg mL−1 CDE. The optimized methods were validated and proved to be specific, robust, and accurate for the quality control of the cited drugs in synthetic mixtures and pharmaceutical preparations.

Development and validation of a sensitive UHPLC-MS/MS method for the simultaneous analysis of tramadol, dextromethorphan chlorpheniramine and their major metabolites in human plasma in forensic context: application to pharmacokinetics

The prerequisites for forensic confirmatory analysis by LC/MS/MS with respect to European Union guidelines are chromatographic separation, a minimum number of two MS/MS transitions to obtain the required identification points and predefined thresholds for the variability of the relative intensities of the MS/MS transitions (MRM transitions) in samples and reference standards. In the present study, a fast, sensitive and robust method to quantify tramadol, chlorpheniramine, dextromethorphan and their major metabolites, O-desmethyltramadol, dsmethyl-chlorpheniramine and dextrophan, respectively, in human plasma using ibuprofen as internal standard (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction method using ethyl acetate-diethyl-ether (1:1). Extracted samples were analyzed by ultra-high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase containing acetonitrile, water and formic acid (89.2:11.7:0.1) for 2.0 min at a flow rate of 0.25 μL/min into a Hypersil-Gold C18 column, 20 × 2.0 mm (1.9 µm) from Thermoscientific, New York, USA. The calibration curve was linear for the six analytes. The intraday precision (RSD) and accuracy (RE) of the method were 3-9.8 and -1.7-4.5%, respectively. The analytical procedure herein described was used to assess the pharmacokinetics of the analytes in 24 healthy volunteers after a single oral dose containing 50 mg of tramadol hydrochloride, 3 mg chlorpheniramine maleate and 15 mg of dextromethorphan hydrobromide.

Simultaneous spectrophotometric determination of diclofenac potassium and methocarbamol in binary mixture using chemometric techniques and artificial neural networks

In this study, the simultaneous determination of diclofenac potassium (DP) and methocarbamol (MT) by chemometric approaches and artificial neural networks using UV spectrophotometry has been reported as a simple alternative to using separate models for each component. Three chemometric techniques-classical least-squares (CLS), principal component regression (PCR), and partial least-squares (PLS)-along with radial basis function-artificial neural network (RBF-ANN) were prepared by using the synthetic mixtures containing the two drugs in methanol. A set of synthetic mixtures of DP and MT was evaluated and the results obtained by the application of these methods were discussed and compared. In CLS, PCR, and PLS, the absorbance data matrix corresponding to the concentration data matrix was obtained by the measurements of absorbances in the range 260-310 nm in the intervals with Δλ = 0.2 nm in their zero-order spectra. Then, calibration or regression was obtained by using the absorbance data matrix and concentration data matrix for the prediction of the unknown concentrations of DP and MT in their mixtures. In RBF-ANN, the input layer consisting of 251 neurons, 9 neurons in the hidden layer, and 2 output neurons were found appropriate for the simultaneous determination of DP and MT. The accuracy and the precision of the four methods have been determined and they have been validated by analyzing synthetic mixtures containing the two drugs. The proposed methods were successfully applied to a pharmaceutical formulation containing the examined drugs.