Nasr Y. Khalil

Development of a novel 96-microwell assay with high throughput for determination of olmesartan medoxomil in its tablets

A novel 96-microwell-based spectrophotometric assay has been developed and validated for determination of olmesartan medoxomil (OLM) in tablets. The formation of a colored charge-transfer (CT) complex between OLM as a n-electron donor and 2, 5-dichloro-3, 6-dihydroxy-1, 4-benzoquinone (p-chloranilic acid, pCA) as a π-electron acceptor was investigated, for the first time, and employed as a basis in the development of the proposed assay. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 490 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient was found between the absorbance and the concentration of OLM in the range of 1-200 μg ml-1. The limits of detection and quantitation were 0.3 and 1 μg ml-1, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from hydrochlorothiazide and amlodipine that are co-formulated with OLM in some formulations. The assay was successfully applied to the analysis of OLM in tablets with good accuracy and precision. The assay described herein has great practical value in the routine analysis of OLM in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for OLM, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed.

Spectrofluorimetric determination of vigabatrin and gabapentin in urine and dosage forms through derivatization with fluorescamine

A stability-indicating, sensitive, simple and selective spectrofluorimetric method was developed for the determination of vigabatrin (VG) and gabapentin (GB). The method is based on the reaction between the two drugs and fluorescamine in borate buffer of pH 8.2 to give highly fluorescent derivatives that are measured at 472 nm using an excitation wavelength of 390 nm for both drugs. The optimum conditions were ascertained and the method was applied for the determination of VG and GB over the concentration range of 0.20-4.00 and 0.1-1.0 microg/ml, respectively with detection limits of 0.05 microg/ml (2.9 x 10(-7) M) and 0.06 microg/ml (2.3 x 10(-7) M) for VG and GB, respectively. The suggested method was applied, without any interference from the excipients, to the determination of the two drugs in their pharmaceutical formulations. Furthermore, the method was extended to the in-vitro determination of both drugs in spiked human urine. Interference from endogenous amino acids could be eliminated through selective complexation with copper acetate, the % recovery (n=4) is 98.0 +/- 7.05. Co-administered drugs such as lamotrigine, phenobarbitone, valproic acid, clopazam, carbamazepine, clonazepam and cimitidine did not interfere with the assay. The method is also stability-indicating; as the degradation product of vigabatrin: 5-vinylpyrrolidin-2-one, produced no interference with its analysis.

Novel automated flow-based immunosensor for real-time measurement of the breast cancer biomarker CA15-3 in serum

A novel automated immunosensor assay has been developed for real-time measurement of the breast cancer biomarker CA15-3 in serum. The assay employed the kinetic-exclusion analytical technology of the KinExA™ 3200 instrument. Polymethylmethacrylate (PMMA) beads coated with CA15-3 were used as capturing reagent, mouse anti-CA15-3 monoclonal antibody was used as primary antibody, and the fluorescence was monitored and recorded during the flow of the fluorescent-labeled antibody through the beads. The fluorescence signal retained on the beads was plotted versus CA15-3 concentration to generate a calibration curve. The concentrations of CA15-3 in the samples were then obtained by interpolation on the curve. The assay limit of detection was 0.2 IU mL(-1). This highly sensitive automated system allowed rapid and reliable quantification of CA15-3 without any matrix effect; analytical recovery of serum-spiked CA15-3 was 90.7%-108.6%±2.05%-7.45%. The precision of the sensor was satisfactory; relative standard deviation (RSD) was 3.8%-5.1% and 5.2%-7.4% for the intra- and inter-assay precision, respectively. The analytical performance of the proposed sensor was superior to the non-competitive sandwich immunoassays for CA15-3. The automated analysis by the sensor facilitated the processing of a large number of specimens, and the new sensor-based assay is anticipated to have a great value in measurement of CA15-3.

Selective Spectrophotometric and Spectrofluorometric Methods for the Determination of Amantadine Hydrochloride in Capsules and Plasma via Derivatization with 1,2-Naphthoquinone-4-sulphonate

New selective and sensitive spectrophotometric and spectrofluorometric methods have been developed and validated for the determination of amantadine hydrochloride (AMD) in capsules and plasma. The methods were based on the condensation of AMD with 1,2-naphthoquinone-4-sulphonate (NQS) in an alkaline medium to form an orange-colored product. The spectrophotometric method involved the measurement of the colored product at 460  nm. The spectrofluorometric method involved the reduction of the product with potassium borohydride, and the subsequent measurement of the formed fluorescent reduced AMD-NQS product at 382  nm after excitation at 293  nm. The variables that affected the reaction were carefully studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.9972–0.9974) and low LOD (1.39 and 0.013 μg mL−1) were obtained in the ranges of 5–80 and 0.05–10  μg mL−1 for the spectrophotometric and spectrofluorometric methods, respectively. The precisions of the methods were satisfactory; RSD ≤2.04%. Both methods were successfully applied to the determination of AMD in capsules. As its higher sensitivity, the spectrofluorometric method was applied to the determination of AMD in plasma; the recovery was 96.3–101.2 ± 0.57–4.2%. The results obtained by the proposed methods were comparable with those obtained by the official method

A highly sensitive HPLC method with automated on-line sample pre-treatment and fluorescence detection for determination of reboxetine in human plasma

A fully automated, rapid and highly sensitive HPLC method with automated sample pre-treatment by column-switching system and fluorescence detection has been developed for the trace quantitative determination of the new antidepressant reboxetine (RBX) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. Paroxetine (PXT) was used as an internal standard. Plasma samples containing both RBX and PXT, after filtration, were derivatized by heating with NBD-F in borate buffer of pH 8 at 70 degrees C for 30min. The derivatized plasma samples were injected into the HPLC system where an on-line sample clean up was achieved on the pre-treatment column (Co-sense Shim-pack MAYI-ODS) with a washing mobile phase (acetonitrile:2% acetic acid; 40:60, v/v) at a flow rate of 5mLmin(-1) for 1min. After an automated on-line column switching to the analytical Hypersil phenyl 120A column (250mmx4.6mm, 5microm), the separation of the derivatized RBX and PXT was performed using a mobile phase consisting of sodium acetate buffer (pH 3.5):tetrahydrofuran:acetonitrile (55:35:10, v/v/v) at a flow rate of 2.0mLmin(-1). The eluted derivatives were monitored by a fluorescence detector set at an excitation wavelength of 470nm and an emission wavelength of 530nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r=0.9995, n=5) was found between the peak area ratio of RBX to PXT and RBX concentration in the range of 2-500ngmL(-1), with limits of detection and quantification of 0.5 and 1.7ngmL(-1), respectively. The intra- and inter-day precisions were satisfactory; the relative standard deviations were 2.25 and 3.01% for the intra- and inter-day precisions, respectively. The accuracy of the method proved as the mean recovery values were 100.11+/-2.24% and 100.99+/-2.98% for the intra- and inter-day assay runs, respectively. The proposed method involved simple and minimum sample preparation procedure and short run-time (<12min) and therefore it can be applied to the routine therapeutic monitoring and pharmacokinetic studies of RBX.

SENSITIVE HPLC METHOD WITH FLUORESCENCE DETECTION AND ON-LINE WAVELENGTH SWITCHING FOR SIMULTANEOUS DETERMINATION OF VALSARTAN AND AMLODIPINE IN HUMAN PLASMA

This study describes the development and validation of a highly sensitive high-performance liquid chromatographic method with fluorescence detection and on-line emission wavelength switching for the simultaneous determination of valsartan (VAL) and amlodipine (AML) in human plasma. Irbesartan (IRB) was used as internal standard (IS). VAL, AML, and IRB were isolated from plasma by nonextractive procedure; simple protein precipitation with methanol. Separations were performed in low pressure gradient mode on Hypersil phenyl 120A analytical column using a mobile phase consisting of phosphate buffer (pH 4.0 ± 0.1):acetonitrile:methanol (60:30:10, v/v/v) at a flow rate of 0.8 mL/min. The detection of VAL and IRB (IS) was carried out at 253 nm (for excitation) and 374 nm (for emission). After elution of VAL and IRB, the detection wavelengths were switched on-line to 393 nm (excitation) and 446 nm (emission) for detection of AML. The linear ranges of the assay were 1–100 and 10–1000 ng/mL for VAL and AML, respectively. The limits of detection (LOD) were 0.3 and 1.6 ng/mL for VAL and AML, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 5.1%. The accuracy of the method was proved; the recovery of VAL and AML from spiked human plasma were 96.6–100.9 and 92.0–104.4% for VAL and AML, respectively. The method had higher throughput as it involved simple sample preparation procedure and short run-time (15 min). The results demonstrated that the proposed method would have great value in pharmacokinetic studies for VAL and AML.

Novel microwell-based spectrophotometric assay for determination of atorvastatin calcium in its pharmaceutical formulations

The formation of a colored charge-transfer (CT) complex between atorvastatin calcium (ATR-Ca) as a n-electron donor and 2, 3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as a π-electron acceptor was investigated, for the first time. The spectral characteristics of the CT complex have been described, and the reaction mechanism has been proved by computational molecular modeling. The reaction was employed in the development of a novel microwell-based spectrophotometric assay for determination of ATR-Ca in its pharmaceutical formulations. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 460 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient (0.9995) was found between the absorbance and the concentration of ATR-Ca in the range of 10-150 μg/well. The limits of detection and quantitation were 5.3 and 15.8 μg/well, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from the drugs that are co-formulated with ATR-Ca in its combined formulations. The assay was successfully applied to the analysis of ATR-Ca in its pharmaceutical dosage forms with good accuracy and precision. The assay described herein has great practical value in the routine analysis of ATR-Ca in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for ATR-Ca, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed.

Voltammetric determination of josamycin (a macrolide antibiotic) in dosage forms and spiked human urine.

The voltammetric behaviour of josamycin (a macrolide antibiotic) has been studied using direct current (DC(t)) alternating current (AC(t)) and differential pulse polarography (DPP). In Britton-Robinson buffers, josamycin developed cathodic waves over the pH range 7-12. At pH 10, a well-defined cathodic wave with diffusion current constant of 1.06 +/- 0.19 (n = 5) was obtained. The wave was characterized as being diffusion-controlled; and partially affected by adsorption phenomenon. The current-concentrations plots are rectilinear over the range 10-60 and 6-50 microg/ml using DC(t) mode and DPP mode, respectively. The minimum detectability limit was 1.2 microg/ml (1.9 x 10(-6) M) adopting the DPP mode. A method was proposed for the determination of josamycin in its tablets adopting both DC(t) and DPP modes. The results obtained were in good agreement with those given by the manufacturer. The method was extended to the in-vitro determination of the drug in spiked human urine; the % recovery was 98.06 +/- 1.76% (n = 5). The number of electrons involved in the reduction process was accomplished and a proposal of the electrode reaction was presented.

A simple and sensitive high performance liquid chromatography assay with a fluorescence detector for determination of canagliflozin in human plasma

Canagliflozin is the first sodium–glucose co-transporter-2 inhibitor approved for the treatment of type 2 diabetes mellitus. In this study, a simple and sensitive HPLC assay with a florescence detector was developed for accurate quantification of canagliflozin in human plasma using telmisartan as the internal standard (IS). Plasma samples were extracted by a liquid–liquid extraction method using diethyl ether as an extracting solvent. Chromatographic separation of canagliflozin and IS was performed on a Nucleodur Isis C18 column with an isocratic mobile phase of 20 mM potassium dihydrogen orthophosphate : acetonitrile (45 : 55, v/v) at a flow rate of 1 mL min−1. Canagliflozin and IS were eluted at 2.8 and 5.8 min, respectively, and detected at 280 and 325 nm for excitation and emission, respectively. The plasma calibration curve displayed excellent linearity over the concentration range of 16.13–6000 ng mL−1. The assay was fully validated in terms of selectivity & specificity, linearity of the calibration curve, accuracy & precision, recovery and stability under various storage conditions. To the best of our knowledge, this is the first validated HPLC-florescence detector assay for the quantification of canagliflozin in human plasma.

HPLC method with monolithic column for simultaneous determination of irbesartan and hydrochlorothiazide in tablets

Abstract A simple, sensitive and accurate HPLC method with high throughput has been developed and validated for the simultaneous determination of irbesartan (IRB) and hydrochlorothiazide (HCT) in combined pharmaceutical dosage forms. The proposed method employed, for the first time, a monolithic column in the analysis. Optimal chromatographic separation of the analytes was achieved on Chromolith® Performance RP-18e column using a mobile phase consisting of phosphate buffer (pH 4)/acetonitrile (50:50, V/V) pumped isocratically at a flow rate of 1.0 mL min-1. The eluted analytes were monitored with a UV detector set at 270 nm. Under the optimum chromatographic conditions, linear relationship with a good correlation coefficient (R ≥ 0.9997) was found between the peak area and the corresponding concentrations of both IRB and HCT in the ranges of 10-200 and 1-20 ng mL-1. The limits of detection were 2.34 and 0.03 ng mL-1 for IRB and HCT, respectively. The intra- and inter-assay precisions were satisfactory as the RSD values did not exceed 3 %. The accuracy of the proposed method was > 97 %. The proposed method had high throughput as the analysis involved a simple procedure and a very short run- -time of < 3 min. The results demonstrated that the method is applicable in the quality control of combined pharmaceutical tablets containing IRB and HCT