Mohamed M. Hefnawy

Spectrofluorometric determination of alpha-aminocephalosporins in biological fluids and pharmaceutical preparations.

A selective and highly sensitive fluorometric method was developed for the determination of four alpha-aminocephalosporins, namely cefaclor, cefadroxil, cephalexin and cephradine. The method involves the reaction of the target compounds with fluorescamine at a specific pH, ranging from 7.8 to 8.4. The produced derivatives exhibit maximum fluorescence intensities at 472-478 nm after excitation at 370-372 nm. The method is highly specific because other alpha-aminocephalosporins whose alpha amino group was blocked do not react similarly and hence do not interfere. At the specific pH range of the reaction where no degradation may occur with that medium the proposed method can be utilised as a stability-indicating assay. The different experimental parameters affecting the derivatisation reaction were carefully studied and incorporated into the procedure. Under the described conditions, the proposed method is linear over the concentration range of 0.05(-1) microg/ml(-1) for both cefaclor and cephalexin, and 0.05-0.65 and 0.025-0.5 microg/ml(-1) for cefadroxil and cepharadine, respectively and the coefficients of determination were greater than 0.999 (n = 3). The recoveries of the title compounds from spiked serum ranged from 88.6 to 89.7% and from spiked urine from 92.2 to 93.3% with a limit of quantitation (LOQ) of 25-50 ng/ml(-1) and limit of detection (LOD) of 5 ng/ml(-1) (S/N = 2) for all drugs. The mechanism of the fluorometric reaction is proposed and the advantages of the proposed method are discussed.

Rapid and sensitive simultaneous determination of ezetimibe and simvastatin from their combination drug products by monolithic silica high-performance liquid chromatographic column

A simple, precise and rapid high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of ezetimibe (EZE) and simvastatin (SIM) from their combination drug products. The applicability of monolithic LC phases in the field of quantitative analysis has been evaluated. The existing method with UV detection set at 240 nm was successfully transferred from a conventional silica column to a 10 cm x 4.6 mm i.d. monolithic silica column. By simply increasing the mobile phase flow rate, run time was about five-fold reduced and the consumption of mobile phase was about two-fold decreased, while the chromatographic resolution of the analytes remain unaffected. Ranitidine (RAN) was used as internal standard to guarantee a high level of quantitative performance. The method used a mobile phase consisted of acetonitrile-ammonium acetate (50 mM pH 5.0) (65:35, v/v). It was validated with respect to system suitability, specificity, limit of quantitation (LOQ) and detection (LOD), linearity, precision, accuracy, and recovery, respectively. The described method was linear over the range of 40-1200 ng ml(-1) (r=0.999) for both drugs. The LOD for EZE and SIM were 13.2+/-0.4029 and 13.3+/-0.4772 ng ml(-1), respectively. The LOQ were found to be 39.9+/-1.221 and 39.5+/-1.446 ng ml(-1) for EZE and SIM, respectively. The method is fast (less than 2.0 min) and is suitable for high-throughput analysis of the drug and ones can analyze 700 samples per working day, facilitating the processing of large-number batch samples.

A Selective Spectrofluorimetric Method for the Determination of Some α-Aminocephalosporins in Formulations and Biological Fluids.

Abstract A selective, highly sensitive fluorimetric method is described for the determination of three α-aminocephalosporins, namely cephalexin, cefaclor and cephradine. Other cephalosporins free from the amino group did not interfere with the assay. The proposed method involves acid-hydrolysis of the drugs and subsequent alkalinization before measurement. The different experimental parameters affecting the fluorescence intensity were carefully studied and incorporated into the procedure. The method permits the determination of 0.02–0.40, 0.04–0.60 and 0.08–0.80 ug.ml−1 for cephalexin, cefaclor and cephradine respectively with minimum detectability of 0.2 ng.ml−1 for all cephalosporins. The proposed method was successfully applied to the determination of these drugs in formulations and biological fluids. The advantages of the described method over other existing methods were discussed. A route for the reaction pathway proposed.

Rapid Determination of Sildenafil Citrate in Pharmaceutical Preparations Using Monolithic Silica HPLC Column

Abstract A simple, rapid, and sensitive high performance liquid chromatography (HPLC) method has been developed and validated for the determination of sildenafil citrate in pharmaceutical preparations. The method was developed utilizing a 100 × 4.6 mm I.D. monolithic silica column and an isocratic elution of 60:40, v/v acetonitrile/water. The elution of the analyte was monitored at 292 nm with a flow rate of 2.0 mL/min. All analyses were conducted at ambient temperature. Linearity was observed in the concentration range from 50–3000 ng/mL, with a correlation coefficient (R 2) greater than 0.999. The limit of detection was 25 ng/mL. Parameters of validation prove the precision of the method and its applicability for the determination of sildenafil citrate in pharmaceutical tablet formulations. The method is fast (less than 1 min) and is suitable for high throughput analysis of the drug.

Enantioselective high-performance liquid chromatographic method for the determination of baclofen in human plasma.

A new analytical method for the separation and determination of R-(-)- and S-(+)- baclofen enantiomers in human plasma by high-performance liquid chromatography (HPLC) with UV detection was developed. Enantioselective resolution of the baclofen enantiomers was achieved by using teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar ionic mobile phase (PIM) consisting of methanol: glacial acetic acid: triethylamine, 100:0.1:0.1, (v/v/v) at a flow rate of 0.5 ml min(-1) and UV detection set at 220 nm. The analytes of interest with S-(+)-sulpiride as the internal standard were extracted from human plasma using liquid-liquid extraction procedure with ethyl ether under alkaline condition prior to HPLC analysis. Recoveries for R-(-)- and S-(+)-baclofen enantiomers were in the ranges of 96-103% at 60-2500 ng ml(-1) level. Intra-day and inter-day precision calculated as %RSD was in the ranges of 1.2-5.2 and 1.3-4.3% for both enantiomers, respectively. Intra-day and inter-day accuracy calculated as percentage error were in the ranges of 1.2-3.9 and 1.1-3.9% for both enantiomers, respectively. Linear calibration curves in the concentration ranges of 20-3000 ng ml(-1) for each enantiomer showed correlation coefficient (r) of 0.9997. The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 20 and 10 ng ml(-1) (S/N=3) respectively.

Development of square-wave adsorptive stripping voltammetric method for determination of acebutolol in pharmaceutical formulations and biological fluids

A validated simple, rapid, sensitive and specific square-wave voltammetric technique is described for the determination of acebutolol (AC) following its accumulation onto a hanging mercury drop electrode in a Britton-Robinson universal buffer of pH 7.5. The optimal procedural conditions were: accumulation potential Eacc = - 0.8 V versus Ag/AgCl/KCl, accumulation duration tacc = 30 s, pulse-amplitude = 70 mV, scan rate = 100 mV/s, frequency = 30 Hz, surface area of the working electrode = 0.6 mm2 and the convection rate = 2000 rpm. Under these optimized conditions, the adsorptive stripping voltammetry (AdSV) peak current was proportional over the concentration range 5 × 10-7 - 6 × 10-6 M (r = 0.999). Recoveries for acebutolol from human plasma and urine were in the range 97-103% and 96-104% respectively. The method proved to be precise (intra-day precision expressed as %RSD in human plasma ranged from 2.9 - 3.2% and inter-day precision expressed as %RSD ranged from 3.4 - 3.8%) and accurate (intra-day accuracies expressed as % error in human urine ranged from -3.3 - 2.8% and inter-day accuracies ranged from -3.3 - 1.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for acebutolol were 1.7 × 10-7 and 5 × 10-7 M, respectively. Possible interferences by substances usually present in the pharmaceutical formulations were investigated with a mean recovery of 101.6 ± 0.64%. Results of the developed square-wave adsorptive stripping voltammetry (SW-AdSV) method were comparable with those obtained by reference analytical method.

Development of Capillary Electrophoresis Technique for Simultaneous Measurement of Amlodipine and Atorvastatin from Their Combination Drug Formulations

Abstract A simple, accurate, precise, and sensitive capillary electrophoresis (CE) technique coupled to a diode array detector (DAD) has been developed for the separation and simultaneous determination of amlodipine (AM) and atorvastatin (AT) from their combination formulations. The proposed method utilized fused silica capillary (50 cm × 75 µm ID) and background electrolyte (BGE) composed of phosphate buffer (pH 6.5, 25 mM)-methanol, (80:20, v/v). The separation was achieved at 15 KV applied voltage and 25°C. Losartan was chosen as the internal standard to guarantee a high level of quantitative performance. The two drugs were subjected to thermal, photolytic, hydrolytic, and oxidative stress conditions and the stressed samples were analyzed by the proposed method. The method has shown adequate separation for AM and AT from its main degradation products (UK-55-410) & (PD 0162910-00), respectively, which demonstrated the specificity of the assay. The described method was linear over the range of 1 – 50 µg/mL (r = 0.9998) for both drugs (2.4 × 10−6 − 1.2 × 10−4 M for AM and 1.8 × 10−6 −8.6 × 10−5 M for AT). Intra- and inter-day RSD (n = 6) was ≤2.2%. The limits of detection for AM and AT were 0.5 µg/mL. The percentage recoveries (n = 6) of the two drugs from their tablet formulations were 99.97 ± 1.84 and 100.96 ± 1.12, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of AM and AT and the assay can thus be considered stability indicating.

Liquid chromatographic high-throughput analysis of ketamine and its metabolites in human plasma using a monolithic silica column and solid phase extraction

A rapid and sensitive liquid chromatographic (LC) assay was developed for the simultaneous determination of ketamine (KE) and its two main metabolites, namely, norketamine (NK) and dehydronorketamine (DHNK) in human plasma. Each compound together with an internal standard (Labetalol) was extracted from the plasma matrix using solid phase extraction (SPE). The applicability of monolithic LC phases in the field of quantitative bioanalysis has been evaluated. The existing method with UV detection set at 220nm was successfully transferred from a conventional reversed phase column to a 10cm x 4.6mm i.d. monolithic silica column. By simply increasing the mobile phase flow-rate, run times were about six-fold reduced and consumption of mobile phase were about two-fold decreased, while the chromatographic resolution of the analytes remain unaffected. The method was validated over the range 25-2000ng/mL for KE, 25-1500ng/mL for NK, and 15-750ng/mL for DHNK. The method proved to be precise (within-run precision ranges from 2.2 to 7.2% and between-run precision ranges from 3.7 to 8.2%) and accurate (within-run accuracies ranged from 1.3 to 7.2% and between-run accuracies ranged from 1.5 to 8.7%). The mean absolute recoveries were 95.3, 96.9, and 103.9% for KE, NK and DHNK, respectively. The limit of quantitation (LOQ) and limit of detection (LOD) for KE and NK in human plasma were 25 and 12.5ng/mL, respectively, and for DHNK were 15 and 7.5ng/mL (S/N = 3). The assay should be suitable for use in routine determination of KE and its metabolites in human plasma.

High Throughput Analysis of Lamivudine in Pharmaceutical Preparations Using Monolithic Silica HPLC Column

Abstract A new, rapid and sensitive high performance liquid chromatography (HPLC) assay was developed and validated, which utilized a monolithic column (100 × 4.6 mm I.D.) for the determination of lamivudine in pharmaceutical preparations. The method used a mobile phase consisting of acetonitrile/water (65:35, v/v) and flow rate of 2.0 mL/min. The elution of the analyte was monitored at 285 nm and conducted at ambient temperature. The method was validated with respect to linearity range, limit of detection and quantitation, precision, accuracy, selectivity, and robustness. The method exhibited low limit of detection (LOD) of 12.5 ng/mL, wide linearity range of 25–800 ng/mL and correlation coefficient (R 2) greater than 0.999. Parameters of validation prove the precision of the method and its applicability for the determination of lamivudine in pharmaceutical tablet formulations. The method is fast (less than one minute) and is suitable for high throughput analysis of the drug.

Analysis of certain tranquilizers in biological fluids.

A review with 282 references is presented that deals with the reported methods of analysis of phenothiazines, thioxanthenes, and benzodiazepine derivatives of pharmaceutical interest. The review includes the methods adapted in biological fluids.