Simultaneous quantitation of two direct acting hepatitis C antivirals (sofosbuvir and daclatasvir) by an HPLC-UV method designated for their pharmacokinetic study in rabbits.

Sofosbuvir (SOF) and daclatasvir (DCS) are novel, recently developed direct acting antiviral agents characterized by potent anti-hepatitis C virus action. A fast and efficient HPLC-UV method was developed, validated and applied for simultaneous determination of SOF and DCS in pharmaceutical formulations and biological fluids based on coupling liquid-liquid extraction with ultrasound and dual wavelength detection at λmax; 260 and 313 nm for SOF and DCS, respectively. This approach provided simple, sensitive, specific and cost-effective determination of the SOF-DCS mixture with good recoveries of the analytes from plasma. Analytes were separated within 7 min on C18 analytical column with acetonitrile-10 mM acetate buffer of pH 5.0 at a flow rate of 1.0 mL min-1. The linear ranges were 1-20 µg mL-1 for SOF and 0.6-6 µg mL-1 for DCS with correlation coefficients ≥0.9995. The detection limits in spiked rabbit plasma were 0.20 and 0.19 µg mL-1 for SOF and DCS, respectively. The method was validated according to ICH and US-FDA guidelines. Finally, the method was successfully applied for simultaneous pharmacokinetic studies of SOF and DCS in rabbits using rofecoxib as internal standard.

New, simple and sensitive HPTLC method for simultaneous determination of anti-hepatitis C sofosbuvir and ledipasvir in rabbit plasma.

Sofosbuvir (SOF) and ledipasvir (LDS) represent anti-hepatitis C binary mixture. Herein, a fast high-performance thin-layer chromatography (HPTLC) method was developed, validated and applied for simultaneous determination of SOF and LDS in biological matrix. An innovative strategy was designed which based on coupling dual wavelength detection with HPTLC. This strategy enabled sensitive, specific, high sample throughput and cost-effective determination of the SOF-LDS binary mixture. The developed HPTLC procedure is based on a simple liquid-liquid extraction, enrichment of the analytes and subsequent separation with UV detection. Separations were performed on HPTLC silica gel 60 F254 aluminum plates with a mobile phase consisting of ethyl acetate-glacial acetic acid (100:5, v/v). The Rf values for SOF and LDS were 0.62 and 0.30, respectively. Dual wavelength scanning was carried out in the absorbance mode at 265 and 327 nm for SOF and LDS, respectively. The linear ranges were 40-640 and 9-144 ng/band for SOF and LDS, respectively with correlation coefficients of 0.9998. The detection limits were 10.61 and 2.54 ng/band and the quantitation limits were 32.14 and 7.70 ng/band for SOF and LDS, respectively indicating high sensitivity of the proposed method. Consequently, this permits in vitro and in vivo application of the proposed method in rabbit plasma with good percentage recovery (95.68-103.26%). Validation parameters were assessed according to ICH guidelines. The proposed method represents a simple, high sample throughput and economic alternative to the already existing more complicated reported LC-MS/MS techniques. The method would afford an efficient tool for therapeutic drug monitoring and bioavailability studies of SOF and LDS.

Efficient HPTLC-dual wavelength spectrodensitometric method for simultaneous determination of sofosbuvir and daclatasvir: Biological and pharmaceutical analysis.

Sofosbuvir (SOF) and daclatasvir (DCS) are newly discovered anti-hepatitis C drugs that have direct antiviral activity. A novel and simple high-performance thin-layer chromatography (HPTLC) method was designed for simultaneous determination of SOF and DCS in miscellaneous matrices. The method adopts coupling HPTLC with dual wavelength spectrodensitometry. Consequently, this enabled sensitive, specific and cost-effective determination of the SOF-DCS mixture. The developed HPTLC procedure is based on a simple liquid-liquid extraction, enrichment of the analytes and subsequent chromatographic separation with UV detection. Separations were performed on HPTLC silica gel 60 F254 aluminum plates with a mobile phase consisting of ethyl acetate-isopropanol (85:15, v/v). Dual wavelength scanning was carried out in the absorbance mode at 265 and 311 nm for SOF and DCS, respectively. The linear ranges were 40-640 and 20-320 ng band-1 for SOF and DCS, respectively with correlation coefficients of ≥0.9997. The detection limits were 11.3 and 6.5 ng band-1 for SOF and DCS, respectively indicating high sensitivity of the proposed method. Consequently, this permits in vitro and in vivo application of the proposed method in human plasma with good percentage recovery (94.1-103.5%). Validation parameters were assessed according to ICH guidelines and US-FDA guidelines. Furthermore, the application was extended to analysis of SOF and DCS in their pharmaceutical formulations.

Ultrasensitive spectrofluorimetric method for rapid determination of daclatasvir and ledipasvir in human plasma and pharmaceutical formulations.

Direct-acting antivirals (DAAs) represent a revolution in the treatment of chronic hepatitis C which have emerged at an extremely rapid pace over the past few years. DAAs act directly on the hepatitis C virus at various points in the viral life cycle to inhibit viral production. Among these novel DAAs, are daclatasvir (DCS) and ledipasvir (LDS). Herein, a novel, fast, simple, ultrasensitive and cost-effective spectrofluorimetric method was designed for determination of DCS and LDS in miscellaneous matrices. The method is based on investigation of the native fluorescence of the cited drugs. The relative fluorescence intensity (RFI) was measured at λex/λem equal to 315/381 nm for DCS and 332/387 nm for LDS. Under the optimum conditions, the linear ranges of calibration curves were 0.2-30 and 6-120 ng mL-1 for DCS and LDS, respectively with correlation coefficients ≥0.9998. The detection limits were 0.047 and 1.939 ng mL-1 for DCS and LDS, respectively indicating ultrasensitivity of the proposed method. Consequently, this permits in vitro and in vivo application of the proposed method in spiked and real human plasma with good percentage recovery (96.6-103.6%). The method was validated in compliance with ICH guidelines and US-FDA guidelines. Furthermore, the application was extended to analysis of DCS and LDS in its pharmaceutical formulations (either alone or in presence of other co-formulated drugs) and in synthetic mixture with sofosbuvir or ribavirin.

Spectrofluorimetric method for determination of some angiotensin II receptor antagonists.

A simple, rapid, accurate and highly sensitive spectrofluorimetric method has been developed for determination of some angiotensin II receptor antagonists (AIIRA's), namely Losartan potassium (Los-K), Irbesartan (Irb), Valsartan (Val) and Candesartan cilexetil (Cand) in pure forms as well as in their pharmaceutical dosage forms. All the variables affecting the relative fluorescence intensity (RFI) were studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.9982-0.9991) were obtained over the concentration range from 0.006 µg/mL to 1.7 µg/mL. Good accuracy and precision were successfully obtained for the analysis of tablets containing each drug alone or combined with hydrochlorothiazide (HCTZ) without interferences from the co-formulated HCTZ or the additives commonly present in tablets.

Selective densitometric determination of four alpha-aminocephalosporins using ninhydrin reagent.

A simple, selective, and precise densitometric method for analysis of four alpha-aminocephalosporins, namely cefaclor monohydrate, cefadroxil monohydrate, cefalexin anhydrous, and cefradine anhydrous, both in bulk drugs and in formulations was developed and validated. The method employed thin-layer chromatography (TLC) aluminium sheets precoated with silica gel G 60 F(254) as the stationary phase. The solvent system consists of ethyl acetate-methanol-water with different ratios for all studied drugs (R(f) values of 0.40-0.60). The separated spots were visualized as blue to violet color after spraying with ninhydrin reagent. The linear regression analysis data for the calibration plots of all studied drugs produced a good linear relationship with correlation coefficients ranging from 0.9990 to 0.9996 and coefficients of determination ranging from 0.9986 to 0.9992 over the concentration range 2-10 microg/spot. The limits of detection and quantitation for all studied drugs ranged from 0.09 to 0.23 and from 0.27 to 0.84 microg/spot, respectively. The developed method was applied successfully for the determination of the studied drugs in their pharmaceutical dosage forms with good precision and accuracy. Also, the method can be employed as a promising stability-indicating assay.

Kinetic spectrophotometric determination of certain cephalosporins using oxidized quercetin reagent.

A simple, precise and accurate kinetic spectrophotometric method for determination of cefoperazone sodium, cefazolin sodium and ceftriaxone sodium in bulk and in pharmaceutical formulations has been developed. The method is based upon a kinetic investigation of the reaction of the drug with oxidized quercetin reagent at room temperature for a fixed time of 30 min. The decrease in absorbance after the addition of the drug was measured at 510 nm. The absorbance concentration plot was rectilinear over the range 80-400 microg mL(-1) for all studied drugs. The concentration of the studied drugs was calculated using the corresponding calibration equation for the fixed time method. The determination of the studied drugs by initial rate, variable time and rate-constant methods was feasible with the calibration equations obtained but the fixed time method has been found to be more applicable. The analytical performance of the method, in terms of accuracy and precision, was statistically validated; the results were satisfactory. The method has been successfully applied to the determination of the studied drugs in commercial pharmaceutical formulations. Statistical comparison of the results with a well established reported method showed excellent agreement and proved that there is no significant difference in the accuracy and precision.

Analysis of cephalosporin antibiotics.

A comprehensive review with 276 references for the analysis of members of an important class of drugs, cephalosporin antibiotics, is presented. The review covers most of the methods described for the analysis of these drugs in pure forms, in different pharmaceutical dosage forms and in biological fluids.