Development of sensitive spectrofluorometric approach based on formation of pyrrolidone derivative for determination of linagliptin through condensation reaction with ninhydrin and phenylacetaldehyde: Application to content uniformity and real plasma.

The new drug linagliptin belongs to the class of dipeptidyl peptidase-4 enzyme inhibitors. Linagliptin is used to treat type 2 diabetes and is taken orally either alone or in combination with other drugs. In this instance, a new, simple, and economical technique for analyzing linagliptin was developed by the effective use of a pyrrolidone derivative. The primary amine group of linagliptin permits its condensation with ninhydrin (0.14% w/v) to produce a fluorescent product in the presence of phenylacetaldehyde (0.02% v/v). All experimental parameters were carefully examined and adjusted in order to monitor the generation of the pyrrolidone derivative at excitation and emission wavelengths of 385 and 475 nm, respectively. The calibration graph was made by plotting the intensity of the fluorescence in relation to linagliptin concentration. A significant linearity was found for values ranging from 20 to 460 ng/mL. The process's validity has been verified by a thorough assessment of the instructions provided by the International Conference on Harmonization (ICH). The results indicate excellent uniformity with a reference method, showing that there is no substantial difference in precision and accuracy. The proposed approach was utilized for determining linagliptin in real rat plasma successfully owing to its high sensitivity. Additionally, the proposed approach was evaluated using the Eco-Scale evaluation tool and showed a high degree of eco-friendliness (86/100).

Innovative HPTLC method with fluorescence detection for assessment of febuxostat-montelukast combination and study of their protective effects against gouty arthritis.

The present study was designed to evaluate the potential protective effects of the cysteinyl leukotriene receptor blocker montelukast (MNK) and the xanthine oxidase inhibitor febuxostat (FBX) and their combination on a model of acute gouty arthritis induced by intraarticular injection of monourate sodium crystal (MUC) injection in rats. Additionally, we established an HPTLC method for the quantitative determination of both drugs simultaneously and applied this method to detect this combination in rat plasma. In this study, the treated male Wistar rats were grouped as follows: a negative control group injected with phosphate buffer saline (PBS) and a positive control group injected with MUC in their tibiotarsal joint. Four groups were treated orally with diclofenac (4 mg kg-1), MNK (10 mg kg-1), FBX (5 mg kg-1) and MNK plus FBX before MUC injection in their tibiotarsal joints. MUC injection in joints without pretreatment led to a progressive significant increase in joint diameter and heavy leucocytic infiltration compared to the PBS-injected joints. Treatment with diclofenac or a combination of MNK and FBX significantly decreased both joint diameters and leucocytic infiltration compared to the MUC group. MNK or FBX treatment attenuated leucocytic infiltration and significantly decreased joint diameters compared to the MUC group. Thus, MNK and FBX can prevent the development of MUC-induced acute gouty arthritis in rats. Also, MNK can be an alternative preventive treatment, particularly in elderly patients who cannot tolerate NSAIDs or corticosteroid. Furthermore, a new thin-layer chromatographic method combined with fluorescence detection was developed and validated for the simultaneous estimation of a mixture of FBX and MNK in spiked human plasma. In this method, we employed TLC aluminium plates precoated with silica gel G 60F254 as the stationary phase and chloroform : methanol (9 : 1, v/v) as the mobile phase. The optimized mobile phase selected for development gives compact bands (Rf values are 0.28 and 0.63 for FBX and MNK, respectively). The emission intensities were measured using a K400 optical filter after excitation at 322 nm. The linear regression data for the calibration plots showed excellent linear relationship (r = 0.9990 and 0.9996 for FBX and MNK, respectively), and the linearity range was 10.0-800.0 ng per band for both drugs. According to ICH-guidelines, this method was validated for precision, accuracy, robustness and selectivity. Also, the limits of detection and quantitation were calculated. In addition, stability studies on the studied mixture were performed. Statistical analysis proved that this method is reproducible and selective for the estimation of a mixture of FBX and MNK.

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.

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.

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.

Fluorimetric determination of febuxostat in dosage forms and in real human plasma via Förster resonance energy transfer.

A rapid, simple, selective and precise fluorimetric method was developed and validated for determination of a selective xanthine oxidase inhibitor; febuxostat (FBX) in pharmaceutical formulations and in human plasma. The proposed method is based on quenching effect of FBX on the fluorescence intensity of terbium (Tb3+ ) through fluorescence resonance energy transfer (FRET) from Tb3+ to FBX. The formed complex was measured at λex. 320 nm/λem. 490 nm against a reagent blank. Fluorescence intensity of Tb3+ was diminished when FBX was added. A linear relationship between the fluorescence quenching value of the formed complex ΔF=FTb3+-FFBX-Tb3+ and the concentration of FBX was investigated. The reaction conditions and the fluorescence spectral properties of the complex have been studied. The linearity range of the developed method was 1.0-16.0 µg/ml. The suggested method was applied successfully for the estimation of FBX in bulk powder, dosage forms and spiked plasma samples with excellent recoveries (96.79-98.89%). In addition, the developed method has been successfully applied for determination of FBX in real plasma samples collected from healthy volunteers with good recoveries (82.06-85.65%). All obtained results of the developed method were statistically analyzed and validated according to ICH (International Conference on Harmonization) guidelines.

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.

Determination of Some Non-sedating Antihistamines via Their Native Fluorescence and Derivation of Some Quantitative Fluorescence Intensity - Structure Relationships.

A validated simple, novel, and rapid spectrofluorimetric method was developed for the determination of some non-sedating antihistamines (NSAs); namely cetirizine (CTZ), ebastine (EBS), fexofenadine (FXD), and loratadine (LOR). The method is based on measuring the native fluorescence of the cited drugs after protonation in acidic media and studying their quantitative fluorescence intensity - structure relationships. There was a linear relationship between the relative fluorescence intensity and the concentration of the investigated drug. Under the optimal conditions, the linear ranges of calibration curves for the determination of the studied NSAs were 0.10-2.0, 0.20-6.0, and 0.02-1.0 [Formula: see text] for (CTZ, FXD), (EBS), and (LOR); respectively. The factors affecting the protonation of the studied drugs were carefully studied and optimized. The method was validated according to ICH guidelines. The suggested method is applicable for the determination of the four investigated drugs in bulk and pharmaceutical dosage forms with excellent recoveries (97.67-103.80%). Quantitative relationships were found between the relative fluorescence intensities of the protonated drugs and their physicochemical parameters namely: the pKa, log P, connectivity indexes (χ(v)) and their squares. Regression equations (76) were obtained and not previously reported. Six of these equations were highly significant and used for the prediction of RFI of the studied NSAs.

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.

Frontal analysis of drug-plasma lipoprotein binding using capillary electrophoresis.

High performance frontal analysis coupled with capillary electrophoresis (HPFA/CE) was applied to the ultramicroanalysis of enantioselective binding of drug to plasma lipoproteins. A small volume (ca. 80 nl) of (R)- or (S)-propranolol (PRO, 25-150 microM) and human high-density lipoprotein (HDL, 2.63 g/l) or human low-density lipoprotein (LDL, 4.37 g/l) mixed solution, which was in the state of binding equilibrium, was introduced hydrodynamically into a non-coated fused silica capillary. Positively charged unbound PRO enantiomers migrated toward cathodic end much faster than negatively charged lipoproteins and the bound form. Once unbound PRO migrated apart from lipoprotein, the bound PRO was quickly released from the lipoprotein to maintain the binding equilibrium. Thus, PRO migrated as a zone in the capillary, giving a peak with a plateau region, where the concentration is the same as the unbound PRO concentration in the original sample solution. The unbound PRO concentration calculated form the plateau height agreed with that determined by a conventional ultrafiltration method used as a reference method. It was found that the bindings of PRO to HDL and PRO to LDL were not enantioselective, while the total binding affinity of PRO to LDL (4.01 x 10(5) per M) was 17 times higher than that of PRO-HDL binding (2.38 x 10(4) per M).