Noha N. Atia

Dual separation mode for simultaneous determination of antihypertensive drug combinations by high-performance liquid chromatography.

A simple, reproducible and efficient dual separation mode high performance liquid chromatographic (HPLC) method was developed for simultaneous determination of antihypertensive drug combinations including; hydrochlorothiazide (HCTZ), valsartan (VAL), amiloride (AML) and captopril (CAP). The newly developed Platinum™ column, which provides a dual-mode separation with its polar and non-polar sites, was used for rapid separation of these co-administered drugs. Good resolution was obtained when Platinum™ column was used compared with C(18) column. Additionally, simple isocratic mode with mobile phase containing methanol and 0.02 mole L(-1) phosphate buffer adjusted to pH 3.0 (45:55, v/v) was used for separation. The flow rate was 0.5 mL min(-1) and effluent was monitored at 270 nm. All the investigated drugs were completely separated within less than 6 min. The linearity range obtained for the developed HPLC method was 0.5-100 μg mL(-1) with detection limits of 0.13-1.2 μg mL(-1) for all the studied drugs. The method was validated in accordance with the requirements of ICH guidelines and shown to be suitable for intended applications. The method was successfully used for determination of the studied drugs in pure form and pharmaceutical dosage forms without prior need for separation. The method is valuable for quality control laboratories for simultaneous determination of these co-administered antihypertensive drugs in binary, ternary and quaternary mixtures.

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

&NA; 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 &lgr;ex/&lgr;em 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.

Thin layer chromatography–densitometric determination of some non-sedating antihistamines in combination with pseudoephedrine or acetaminophen in synthetic mixtures and in pharmaceutical formulations

The combination of certain non-sedating antihistamines (NSA) such as fexofenadine (FXD), ketotifen (KET) and loratadine (LOR) with pseudoephedrine (PSE) or acetaminophen (ACE) is widely used in the treatment of allergic rhinitis, conjunctivitis and chronic urticaria. A rapid, simple, selective and precise densitometric method was developed and validated for simultaneous estimation of six synthetic binary mixtures and their pharmaceutical dosage forms. The method employed thin layer chromatography aluminum plates precoated with silica gel G 60 F254 as the stationary phase. The mobile phases chosen for development gave compact bands for the mixtures FXD-PSE (I), KET-PSE (II), LOR-PSE (III), FXD-ACE (IV), KET-ACE (V) and LOR-ACE (VI) [Retardation factor (Rf ) values were (0.20, 0.32), (0.69, 0.34), (0.79, 0.13), (0.36, 0.70), (0.51, 0.30) and (0.76, 0.26), respectively]. Spectrodensitometric scanning integration was performed at 217, 218, 218, 233, 272 and 251 nm for the mixtures I-VI, respectively. The linear regression data for the calibration plots showed an excellent linear relationship. The method was validated for precision, accuracy, robustness and recovery. Limits of detection and quantitation were calculated. Statistical analysis proved that the method is reproducible and selective for the simultaneous estimation of these binary mixtures.

Ultrasound assisted dispersive liquid-liquid microextraction coupled with high performance liquid chromatography designated for bioavailability studies of felodipine combinations in rat plasma

Felodipine (FLD), a calcium channel antagonist, is commonly prescribed for the treatment of hypertension either with Metoprolol (MET) or Ramipril (RAM) in two different drug combinations. FLD has high plasma protein binding ability affecting its extraction recoveries from plasma samples. Hence, a specific ultrasound assisted dispersive liquid-liquid microextraction (UA-DLLME) method coupled with HPLC using photodiode array detector was developed and validated for the simultaneous determination of FLD, MET and RAM in rat plasma after oral administration of these combinations. The factors affecting UA-DLLME were carefully optimized. In this study, UA-DLLME method could provide simple and efficient plasma extraction procedures with superior recovery results. Under optimum condition, all target drugs were separated within 13min. The validation procedures was carried out in agreement with US-FDA guidelines and shown to be suitable for anticipated purposes. Linear calibration ranges were obtained in the range 0.05-2.0μgmL-1 for FLD and MET and 0.1-2.0μgmL-1 for RAM with detection limits of 0.013-0.031μgmL-1 for all the studied drug combinations. The%RSD for inter-day and intra-day precisions was in range of 0.63-3.85% and the accuracy results were in the range of 92.13-100.5%. The validated UA-DLLME-HPLC method was successfully applied for the bioavailability studies of FLD, MET and RAM. The pharmacokinetic parameters were calculated for all the investigated drugs in rats after single-dose administrations of two different drug combinations. Although FLD was bioequivalent in the two formulations, a small increase in plasma levels of MET and RAM was found in the presence of FLD.

Spectrophotometric Determination of Some Non-Sedating Antihistamines Using Erythrosine B

A simple and sensitive spectrophotometric method has been developed for the determination of cetirizine (I), ebastine (II), fexofenadine (III), ketotifen (IV), and loratadine (V) based on ion-pair complex formation with erythrosine B. The pink color of the produced complex was measured at 550 nm without solvent extraction. Appropriate conditions were established by studying the color reaction between erythrosine B and the studied drugs to obtain the maximum sensitivity. Beer-Lamberts law is obeyed in the concentration ranges 1–7, 1–8, and 1–6 g/mL for (I, IV), (II, III), and (V), respectively. The method was validated according to ICH guidelines. The suggested method is applicable for the determination of the five investigated drugs in bulk and pharmaceutical dosage forms with excellent recoveries.

Two Validated Spectrofluorometric Methods for Determination of Gemifloxacin Mesylate in Tablets and Human Plasma

Two new, sensitive, and selective spectrofluorometric methods were developed for the determination of gemifloxacin mesylate (GFX) in tablets and spiked human plasma. Method A was based on measurement of the enhanced fluorescence spectral behaviour of GFX in a sodium dodecyl sulphate (SDS) micellar system. In aqueous solution of acetate buffer pH 5.5, the fluorescence intensity of GFX was greatly enhanced about tenfold in the presence of SDS. The fluorescence intensity was measured at 402 nm after excitation at 274 nm. Method B was based on Hantzsch condensation reaction between the primary amino group of GFX with acetylacetone and formaldehyde in acetate buffer of pH 3.5 yielding a highly yellow fluorescent derivative. The reaction of GFX with acetylacetone-formaldehyde system solution resulted in bathochromic shift of both emission (476 nm) and excitation (420 nm) wavelengths. The fluorescence intensity was directly proportional to the concentration over the range 10–1000 ng/ml and 100–2000 ng/ml for method A and B, respectively. The proposed methods were applied successfully for determination of GFX in its tablets and spiked plasma. Therefore, these methods can be considered of real interest for reliable and practical quality control analysis of GFX.

Lipophilicity estimation of statins as a decisive physicochemical parameter for their hepato-selectivity using reversed-phase thin layer chromatography

HighlightsRP‐TLC behavior of statins was studied using binary mobile phases.Determination of retention paramters (RM0 or C0) of six members of statins.Experimentally‐determined lipophilicity values were correlated with those reported in databases.RM0 or C0 were applied for prediction of physicochemical properties and pharmacokinetics of statins.QSRR and QSPR models using RM0 or C0 values are considered as good predictors for extra‐hepatic distributions of statins. Abstract Lipophilicity plays a crucial role in determining the hepato‐selectivity and hence, the biological activity and the associated side effects of statins. Herein, the employment of RP‐TLC for estimation of lipophilicity of six statins namely; atorvastatin, simvastatin, pravastatin, lovastatin, rosuvastatin and fluvastatin is examined. A very good correlation between the chromatographically‐determined retention parameters (relative lipophilicity (RM0) or lipophilic parameter (C0)) and both experimental and computed log P values were obtained. However, the results indicate that the type of organic modifier in the mobile phase system (methanol, acetonitrile and acetone) has a small influence on RM0 or C0 values. Higher values of RM0 or C0 are ascribed to lipophilic statins and lower values of RM0 or C0 are attributed to hydrophilic ones. Therefore, RM0 or C0 could be effectively used as simple practical predictors of extra‐hepatic distributions of statins and thus their expected side effects. Furthermore, three QSPR (quantitative structure‐property relationship) models were constructed to describe the relationship between RM0 with log P and log D of the statins under investigation. These models can be very useful to predict the lipophilicity of other members of statin drugs and might be expanded to newly synthesized compounds with the same structural features.

HPTLC method for direct determination of gemifloxacin mesylate in human plasma

Novel, simple and sensitive high performance thin-layer chromatography (HPTLC) with fluorescence detection has been successfully developed and validated for determination of gemifloxacin mesylate (GFX) in plasma samples without prior pretreatment. Montelukast (MK) was used as internal standard. GFX and MK in plasma samples were separated using a mobile phase consisting of a mixture of ethyl acetate:methanol:25% ammonia, (8:4.5:3, v/v/v). The emission intensity was measured using optical filter K400 after excitation at 342 nm. The Rf values for GFX and MK were 0.45±0.03 and 0.79±0.02, respectively. Under the optimum conditions, a linear relationship with good correlation coefficient (r=0.9965, n=6) was obtained in concentration range of 3-180 ng/band. The LOD and LOQ of the proposed method were 0.45 and 1.5 ng/band, respectively. The accuracy of the method was proved as the recovery % of GFX from spiked human plasma was 94.21-101.85%. The efficiency of the proposed method was confirmed by in-vivo application on human plasma in real patient samples. Moreover, the stability of GFX in plasma was carefully tested at different conditions and compared to others in aqueous solution.

Dual-wavelength thin-layer chromatographic–densitometric determination of febuxostat in combination with acetaminophen in synthetic mixture and in pharmaceutical formulations

The combination of xanthine oxidase inhibitor febuxostat (FBX) with acetaminophen (ACE) is widely used in the treatment of gout. A rapid, simple, selective, and precise densitometric method was developed and validated for the simultaneous estimation of FBX—ACE mixture in a synthetic binary mixture and in their pharmaceutical dosage forms. The method employed thin-layer chromatography (TLC) aluminum plates precoated with silica gel G 60 F254 as the stationary phase using chloroform—methanol—cyclohexane—acetic acid 96% (7:1:1:0.1, v/v) as the mobile phase. The optimized mobile phase selected for development gave compact bands (RFvalues were 0.65 and 0.46 for FBX and ACE, respectively). Spectrodensitometric scanning integration was performed using dual-wavelength detection at 320 and 250 nm for FBX and ACE, respectively. The linear regression data for the calibration plots showed excellent linear relationship (r = 0.9997 and 0.9992 for FBX and ACE, respectively). The method was validated for precision, accuracy, robustness, and recovery. The limits of detection and quantification were calculated. The statistical analysis proved that the method is reproducible and selective for the estimation of this binary mixture.

Simultaneous determination of triple therapy for Helicobacter pylori in human plasma by reversed phase chromatography with online wavelength switching

The infection of gastric mucosa by Helicobacter pylori (HP) is an essential cofactor in the aetiology of gastroduodenal ulcer and gastric carcinoma. Because of the bacterial resistance, combination therapy containing omeprazole (OME), tinidazole (TNZ) and clarithromycin (CLA) is commonly used for eradication of HP. However, the simultaneous determination of the triple therapy in human plasma was not reported. A simple, reproducible, and selective HPLC method was developed for the simultaneous determination of the triple therapy mixture used for management of HP infections in human plasma. An HPLC procedure based on a liquid-liquid extraction, enrichment of the analytes and subsequent reversed-phase chromatography with UV detection was used. To enable sensitive and selective detection, the method involved the use of online wavelength switching detection, with two different detection wavelengths; 280nm for detection of OME and TNZ and 210nm for detection of CLA. Separations were performed on C18 analytical column with acetonitrile-10mM phosphate buffer of pH=3.0 at flow rate of 1.0mLmin(-1). The linear ranges in human plasma were 0.05-10μgmL(-1) with correlation coefficients >0.9990. The detection limits in human plasma were 0.02-0.07μgmL(-1). Validation parameters were assessed in compliance with US-FDA guidelines. The method proved to be valuable for the therapeutic drug monitoring after oral administration of triple therapy tablets.