Validated Microwell-Based Spectrofluorimetric Method for Quantification of Ravidasvir (New Anti-Chronic Hepatitis C Virus-GT4) in Rat Plasma and Its Application to Pharmacokinetic Study.

BACKGROUND: Ravidasvir (RAV) has been regarded as a potent new NS5A inhibitor with a magnificent safety and tolerability in the management of genotype 4 hepatitis C virus (HCV) patients. Suitable analytical techniques are needed for the measurement of RAV in different biological matrices. METHODS: We have developed a fast, sensitive and economical 96-microwell-based spectrofluorimetric technique combined with one-step protein precipitation extraction strategy for the measurement of RAV in rat plasma. RESULTS: Under the optimum conditions, the direct relationship in rat plasma was accomplished between the RAV concentrations and the fluorescence (FL) intensity in a scope of 2.5-200 ng/mL with 0.9998 and 0.9999 for the quantification and correlation coefficients, respectively. The lower limit of detection (LLOD) was 0.840 ng/mL and this demonstrates the high sensitivity of the proposed assay. The accuracy (RE%) ranged from 95.34% to 102.29%, and the precision (RSD%) was less than 3.59%. The recovery was ranged from 93.12% to 96.26%. The stability of RAV in rat plasma was carried out and established its good stability in the range of room conventional temperature and at long-term stability (-80°C, 30 days). The developed technique was validated as stated by the United States Food and Drug Administration (US-FDA) guidelines for bioanalytical technique verification. CONCLUSION: The approved technique was effectively applied for a pharmacokinetic (PK) study after single oral gavage administration of RAV at a dose of 35 mg/kg and it could be presumed that the proposed assay can be applied to clinical trials.

Synthesis of hapten, generation of specific polyclonal antibody and development of ELISA with high sensitivity for therapeutic monitoring of crizotinib.

Crizotinib (CZT) is a potent drug used for treatment of non-small cell lung cancer (NSCLC); however, its circulating concentration variability has been associated with acquired resistance and toxicity, restricting the success of cancer treatment. As such, the development of an assay that monitors CZT plasma concentrations in patients is a valuable tool in cancer treatment. In this study, a hapten of CZT was synthesized by introducing the acetohydrazide moiety as a spacer into the chemical structure of CZT. The chemical structure of the CZT acetohydrazide (hapten) was confirmed by mass, 1H-, and 13C-NMR spectrometric techniques. The hapten was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. CZT-KLH conjugate was used for immunization and generation of a polyclonal antibody recognizing CZT with high affinity (IC50 = 0.5 ng/mL). The polyclonal antibody was used in the development of an ELISA for determination of CZT. The ELISA involved a competitive binding reaction between CZT, in its samples, and immobilized CZT-BSA conjugate for the binding sites on a limited amount of the anti-CZT antibody. The assay limit of detection was 0.03 ng/mL and the working range was 0.05 - 24 ng/mL. Analytical recovery of CZT from spiked plasma was 101.98 ± 2.99%. The precisions of the assay were satisfactory; RSD was 3.2 - 6.5% and 4.8 - 8.2%, for the intra- and inter-assay precision, respectively. The assay is superior to all the existing chromatographic methods for CZT in terms of its procedure simplicity, convenience, and does not require treatment of plasma samples prior to the analysis. The proposed ELISA is anticipated to effectively contribute to the therapeutic monitoring of CZT in clinical settings.

Development and validation of an ELISA with high sensitivity for therapeutic monitoring of afatinib.

AIM: To support the therapeutic drug monitoring of afatinib (AFT), an ELISA was required. RESULTS: A hapten for AFT was prepared and linked to each of BSA and KLH proteins by diazotization/coupling reaction. A polyclonal antibody recognizing AFT with high affinity (IC50 = 40 ng ml-1) was generated and used in the development of a competitive ELISA for quantitation of AFT in plasma samples. The assay limit of detection was 2 ng ml-1. The assay accuracy and precision were proved. CONCLUSION: The assay is an appropriate alternative to the existing LC-MS/MS assays for AFT and it is anticipated to effectively contribute to the therapeutic drug monitoring of AFT in clinical settings.

Isolation of flavonoids from Delonix elata and determination of its rutin content using capillary electrophoresis.

Delonix elata (L.) Gamble (Fabaceae) is an important, traditionally used plant in Saudi Arabia. It is used to relieve rheumatic pain, flatulence and the seeds are employed as purgatives. The aim of the present study was to isolate chemical constituents of the n-butanol fraction (BF) of D. elata and to find out, by capillary electrophoresis (CE), percentage of rutin present in this BF. Three quercetin glycosides and one kaempferol rutinoside were isolated from the BF of aerial parts of D. elata; namely, Quercetin 3-O-rutinoside-7-O-glucoside (1), Quercetin 3,7-diglucoside (2), Quercetin 3-O-rutinoside (RUT) (3) and Kaempferol 3-O-rutinoside (4). Rutin, an active constituent has been reported to possess good pharmacological as well as therapeutic potentials. A sensitive and rapid procedure for quantitative determination of RUT by capillary electrophoresis was developed and its content was found to be 7.349 mg/gm, relative to n-butanol fraction and 18.373 mg%, relative to the dry powder of D. elata. The method could be recommended for approval and use in the pharmaceutical and food industries.

Enantioselective quantification of atenolol in mouse plasma by high performance liquid chromatography using a chiral Stationary phase: application to a pharmacokinetic study.

Enantiomeric resolution of atenolol was achieved on the HPLC vancomycin macrocyclic antibiotic chiral stationary phase Chirobiotic V. The polar ionic mobile phase consisted of methanol-glacial acetic acidtriethylamine (100 + 0.025 + 0.75, v/v/v) at a flow rate of 0.8 mL/min. Fluorescence detection at 2751305 nm for excitation and emission, respectively, was used. Plasma samples were purified using SPE on Oasis HLB cartridges. The calibration curves in plasma were linear over the range of 5-400 ng/mL (r = 0.999) for each enantiomer with an LOD of 1.0 ng/mL. The proposed method was validated in compliance with International Conference of Harmonization guidelines in terms of linearity, accuracy, precision, LOD, LOQ, and selectivity. The overall recoveries for S-(-)- and R-(+)-atenolol enantiomers from plasma were 95.0-99.5%; RSD ranged from 2.5 to 3.3%. The developed method was applied for the trace analysis of atenolol enantiomers in plasma and for the pharmacokinetic investigation of atenolol enantiomers in mouse plasma.

Validated stability-indicating capillary electrophoresis method for the separation and determination of a fixed-dose combination of carvedilol and hydrochlorothiazide in tablets.

A novel, fast, sensitive, and specific capillary electrophoresis (CE) technique coupled to a diode array detector has been developed for the separation and simultaneous determination of carvedilol (CRV) and hydrochlorothiazide (HCT) in two combination formulations. The proposed method utilized a fused silica capillary (55 cm x75 microm id) and the background electrolyte solution phosphate buffer (12.5 mM, pH 7.4)-methanol (95+5, v/v). The separation was achieved at 30 kV applied voltage and 24 degree C. Atorvastatin (80 microg/mL) was chosen as the internal standard. The described method was linear over the range of 1-200 and 0.2-150 microg/mL for CRV and HCT, respectively. Intraday and interday RSD (n = 6) was < or =1.4%. The LOD values of CRV and HCT were 0.26 and 0.07 microg/mL, respectively. The validated CE method was successfully applied to the analysis of two commercial tablet dosage forms. Forced degradation studies were performed on bulk samples of the two drugs using thermal, photolytic, hydrolytic, and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. Degradation products produced as a result of stress studies did not interfere with the determination of CRV and HCT; the assay could, therefore, be considered stability-indicating.

4-Benzyl-3-(thio-phen-2-yl)-4,5-di-hydro-1H-1,2,4-triazole-5-thione.

In the title compound, C13H11N3S2, the triazole and thio-phene rings are coplanar [dihedral angle = 6.22 (13)°]. By contrast, the phenyl ring is perpendicular to the triazole ring [dihedral angle = 85.58 (13)°], so that the mol-ecule has an L-shape. The thio-phene S atom is syn with the ring imine N atom. In the crystal, eight-membered {⋯HNCS}2 synthons form between centrosymmetrically related mol-ecules, leading to dimeric aggregates that are connected into a supra-molecular layer parallel to (101) by π-π inter-actions between centrosymmetrically related triazole rings [centroid-centroid distance = 3.6091 (15) Å] and C-H⋯π inter-actions.

1-(Adamantan-1-yl)-3-(4-fluoro-phen-yl)thio-urea.

In the title mol-ecule, C(17)H(21)FN(2)S, the mean planes of the benzene ring and the thio-urea fragment form a dihedral angle of 61.93 (9)°. In the crystal, pairs of weak N-H⋯S inter-actions link the mol-ecules, forming inversion dimers.

3-(Adamantan-1-yl)-4-(prop-2-en-1-yl)-1H-1,2,4-triazole-5(4H)-thione.

The title mol-ecule, C(15)H(21)N(3)S, exists as the thione tautomer in the solid state. The 1,2,4-triazole ring is almost planar (r.m.s. deviation = 0.004 Å) and the prop-2-en-1-yl chain is close to being perpendicular to this plane [C-N-C-C torsion angle = 77.1 (5)°]. In the crystal, centrosymmetric dimeric aggregates are formed by pairs of N-H⋯S hydrogen bonds as parts of eight-membered (⋯HNCS)(2) synthons. These are connected into layers parallel to (101) via C-H⋯π inter-actions, where the π-system is the triazole ring. The investigated sample was a nonmerohedral twin; the refined domain ratio was 0.655 (4):0.345 (4).

Novel spectrophotometric method for determination of cinacalcet hydrochloride in its tablets via derivatization with 1,2-naphthoquinone-4-sulphonate.

This study represents the first report on the development of a novel spectrophotometric method for determination of cinacalcet hydrochloride (CIN) in its tablet dosage forms. Studies were carried out to investigate the reaction between CIN and 1,2-naphthoquinone-4-sulphonate (NQS) reagent. In alkaline medium (pH 8.5), an orange red-colored product exhibiting maximum absorption peak (λmax) at 490 nm was produced. The stoichiometry and kinetic of the reaction were investigated and the reaction mechanism was postulated. This color-developing reaction was employed in the development of a simple and rapid visible-spectrophotometric method for determination of CIN in its tablets. Under the optimized reaction conditions, Beer's law correlating the absorbance with CIN concentration was obeyed in the range of 3 - 100 µg/ml with good correlation coefficient (0.9993). The molar absorptivity (ε) was 4.2 × 105 l/mol/cm. The limits of detection and quantification were 1.9 and 5.7 µg/ml, respectively. The precision of the method was satisfactory; the values of relative standard deviations (RSD) did not exceed 2%. No interference was observed from the excipients that are present in the tablets. The proposed method was applied successfully for the determination of CIN in its pharmaceutical tablets with good accuracy and precisions; the label claim percentage was 100.80 - 102.23 ± 1.27 - 1.62%. The results were compared favorably with those of a reference pre-validated method. The method is practical and valuable in terms of its routine application in quality control laboratories.

HPLC separation technique for analysis of bufuralol enantiomers in plasma and pharmaceutical formulations using a vancomycin chiral stationary phase and UV detection.

A sensitive and selective high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of bufuralol enantiomers in plasma and pharmaceutical formulations. Enantiomeric resolution was achieved on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with UV detection set at 254 nm. The polar ionic mobile phase (PIM) consisting of methanol-glacial acetic acid-triethylamine (100:0.015:0.010, v/v/v) has been used at a flow rate of 0.5 ml/min. The method is highly specific where other coformulated compounds did not interfere. The stability of bufuralol enantiomers under different degrees of temperature was also studied. The results showed that the drug is stable for at least 7 days at 70 degrees C. The method was validated for its linearity, accuracy, precision and robustness. An experimental design was used during validation to evaluate method robustness. The calibration curves in plasma were linear over the range of 5-500 ng/ml for each enantiomer with detection limit of 2 ng/ml. The mean relative standard deviation (RSD) of the results of within-day precision and accuracy of the drug were 0.05) between inter- and intra-day studies for each enantiomer which confirmed the reproducibility of the assay method. The mean extraction efficiency for S-(-)- and R-(+)-bufuralol from plasma was in the range 97-102% at 15-400 ng/ml level for each enantiomer. The overall recoveries of bufuralol enantiomers from pharmaceutical formulations was in the range 99.6-102.2% with %RSD ranging from 1.06 to 1.16%. The assay method proved to be suitable as chiral quality control for bufuralol formulations by HPLC and for therapeutic drug monitoring.

Enantioanalysis of bisoprolol in human plasma with a macrocyclic antibiotic HPLC chiral column using fluorescence detection and solid phase extraction.

A sensitive, enantioselective, high-performance liquid chromatographic (HPLC) method was developed and validated to determine S-(-)- and R-(+)-bisoprolol in human plasma. Baseline resolution was achieved using the 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 : 0.02 : 0.025, v/v/v) at a flow rate of 1.5 ml/min and fluorescence detection set at 275 nm for excitation and 305 nm for emission. All analyses with S-(-)-atenolol as the internal standard were conducted at ambient temperature. The assay involved the use of a solid-phase extraction procedure for human plasma samples prior to HPLC analysis. The C18 cartridge gave good recovery rates for both enantiomers without any interference. The method was validated over the range of 20-200 ng/ml for each enantiomer concentration. Recovery rates for S-(-)- and R-(+)-bisoprolol enantiomers were in the range of 95-102%. The method proved to be precise (within-run precision expressed as % RSD ranged from 1.0-6.2% and between-run precision ranged from 0.9-6.7%) and accurate (within-run accuracies expressed as percentage error ranged from 0.2-4.8% and between-run accuracies ranged from 0.3-1.7%). The limit of quantitation and limit of detection for each enantiomer in human plasma were 20 and 5 ng/ml, respectively.

Direct enantiomeric resolution of betaxolol with application to analysis of pharmaceutical products.

A high-performance liquid chromatographic (HPLC) method has been developed for the separation and determination of S- and R-enantiomers of betaxolol in tablets and ophthalmic preparations. Baseline resolution was achieved by using teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with fluorescence detection at excitation/emission wavelengths 275/305 nm. The polar ionic mobile phase (PIM) consists of methanol-glacial acetic acid-triethylamine, (100:0.020:0.025, v/v/v) has been used at a flow rate of 1.5 ml/min. All analytes with S-(-)-atenolol as internal standard were conducted at ambient temperature. The method is highly specific where another coformulated compounds did not interfere. The stability of betaxolol enantiomers under different degree of temperature also studied. The results showed that it is stable for at least 7 days at 70 degrees C. The method validated for its linearity, accuracy, precision and robustness. Experimental design was used during validation to evaluate method robustness. Using the chromatographic conditions described, S- and R-betaxolol were well resolved with mean retention times of 11.3 and 12.6 min, respectively. Linear response (r > 0.997) was observed over the range of 10-500 ng/ml of betaxolol enantiomers, with detection limit of 5 ng/ml. The recoveries of S- and R-betaxolol from tablets and ophthalmic preparation ranged from 97.4 to 101.4% and 98.0 to 102.0%, respectively. The mean relative standard deviation (R.S.D.%) for both enantiomers were 1.1-1.4% and 1.3-1.7% in tablets and ophthalmic solution, respectively.