Development of an innovative turn-on fluorescent probe for targeted in-vivo detection of nitric oxide in rat brain extracts as a biomarker for migraine disease.

Nitric oxide (NO) is one of the reactive nitrogen species (RNS) that has been proposed to be a key signaling molecule in migraine. Migraine is a neurological disorder that is linked to irregular NO levels, which necessitates precise NO quantification for effective diagnosis and treatment. This work introduces a novel fluorescent probe, 2,3-diaminonaphthelene-1,4-dione (DAND), which was designed and synthesized to selectively detect NO in-vitro and in-vivo as a migraine biomarker. DAND boasts high aqueous solubility, biocompatibility, and facile synthesis, which enable highly selective and sensitive detection of NO under physiological conditions. NO reacts with diamine moieties (recognition sites) of DAND, results in the formation of a highly fluorescent product (DAND-NO) known as 1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione at λem 450 nm. The fluorescence turn-on sensing mechanism operates through an intramolecular charge transfer (ICT) mechanism. To maximize fluorescence signal intensity, parameters including DAND concentration, reaction temperature, reaction time and pH were systematically optimized for sensitive and precise NO determination. The enhanced detection capability (LOD = 0.08 µmol L-1) and high selectivity of the probe make it a promising tool for NO detection in brain tissue homogenates. This demonstrates the potential diagnostic value of the probe for individuals suffering from migraine. Furthermore, this study sheds light on the potential role of zolmitriptan (ZOLM), an antimigraine medication, in modulating NO levels in the brain of rats with nitroglycerin-induced migraine, emphasizing its significant impact on reducing NO levels. The obtained results could have significant implications for understanding how ZOLM affects NO levels and may aid in the development of more targeted and effective migraine treatment strategies.

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).

QuEChERS-assisted ion pair chromatography/fluorescence detection method for determination of antimigraine combination therapy in rabbit plasma samples: Application to a pharmacokinetic study.

Antimigraine combination therapy has shown significant effectiveness in relieving pain, as well as reducing the frequency, duration, and severity of migraine attacks if compared to a single migraine medication. This work represents the first analytical investigation for emphasizing the synergistic effect of combining ophthalmic beta blockers with triptans in migraine treatment. The presented study was conducted to investigate the pharmacokinetic profile of almotriptan (ALM), a serotonin (5-HT1B/1D) receptor agonist used to treat migraine, when coadministered with timolol (TIM) or verapamil (VER) which are considered as an adjuvant therapy in migraine prevention. Ion pair chromatography (IPC) with online fluorescence detection was applied to simultaneously detect and quantify the binary mixtures of ALM/TIM and ALM/VER in rabbit plasma samples. The separation was achieved using a Platinum C18 analytical column with a mobile phase composed of methanol: 35 mmol L-1 phosphate buffer solution containing 10 mmol L-1 SDS at pH = 6.8 (60:40 v/v). Several parameters were evaluated during the optimization of separation conditions including mobile phase composition, buffer concentration, buffer pH and concentration of ion pair reagent. A thorough investigation of the retention mechanism was performed, and the results showed that Coulomb forces were the main contributors to the overall retention mechanism, which may be hydrophobically assisted. QuEChERS extraction technique was utilized to extract the investigated drugs from plasma samples and a detailed study was carried out to optimize partition/extraction solvents, pH, extraction salts, sample volume and clean-up step. The method had a limit of detection and quantitation of 5.6 and 16.9 ng mL-1 for ALM in ALM/TIM mixture and 2.5 and 7.6 ng mL-1 for ALM in ALM/VER mixture, with an overall recovery not less than 95.22%. This newly proposed method offers a faster alternative to existing chromatographic methods for extraction and determination of ALM in binary mixtures with TIM or VER in rabbit plasma and provides a platform for studying pharmacokinetic parameters. The coadministration of either TIM or VER with ALM resulted in a notable rise in Cmax (maximum plasma concentration) and AUC (area under the plasma concentration-time curve) of ALM, implying possible alterations in the absorption and overall exposure of ALM.

Vortex-assisted dispersive solid phase microextraction using Fe3O4/FeOOH magnetic nanocomposites for high-performance thin-layer chromatographic determination of zolmitriptan in rabbit plasma samples.

In this work, a fast, versatile, and convenient dispersive solid-phase micro-extraction (DSPME) method is combined with a spectro-densitometric technique for the analysis of zolmitriptan (ZOLM) in biological fluids. Fe3O4/FeOOH magnetic nanocomposites (MNCs) were prepared by a co-precipitation method in aqueous solutions and utilized subsequently as a sorbent in DSPME. By coupling DSPME with high-performance thin-layer chromatography (HPTLC) with fluorescence detection, the preconcentration and determination of (ZOLM) in presence of metoclopramide (MET) and paracetamol (PARA), which are prescribed as an adjuvant therapy with ZOLM, was accomplished. Adsorption capability was assessed using both Langmuir and Freundlich adsorption isotherm models. The adsorption data was fitted to Langmuir adsorption isotherm model as reflected by high determination coefficient (R2 = 0.9944). Moreover, adsorption kinetics was assessed by pseudo-first and pseudo-second order kinetic models. The data was fitted to pseudo-second order kinetics, which proves that ZOLM interaction with the adsorbent is a chemisorption process. Surface complexation with MNCs was suggested to explain the pH dependence of ZOLM sorption. The key parameters of extraction and desorption steps (including pH, extraction time, sample volume, magnetic adsorbent amount, and desorption circumstances) were evaluated. Optimized conditions for solid phase microextraction of ZOLM were pH 2.9, 5.0 mg Fe3O4/FeOOH MNCs, 15 min vortex-assisted extraction time and 3 × 200 µL of methanol: 33% ammonia; 4:1 as eluent. The analysis was achieved using ACN: dichloromethane: 33% ammonia (22.5: 6.0: 1.5, v/v/v) as a mobile phase and the fluorescence detection was carried out at 223 nm. The proposed DSPME method was successfully applied for trace quantification of ZOLM in rabbits' plasma (n = 6) after oral administration with a linearity range of 50.0 - 400.0 ng mL-1 (R2 = 0.9931), a detection limit of 12.0 ng mL-1 and extraction recovery of 97.27-99.89% with an RSD < 2% (n = 9). Moreover, the selectivity of the proposed approach for analysis of ZOLM in the presence of MET and PARA is demonstrated.

A rapid FTIR spectroscopic assay for quantitative determination of Memantine hydrochloride and Amisulpride in human plasma and pharmaceutical formulations.

A selective, new, rapid and nondestructive Fourier transform Infrared spectroscopic assay has been developed for simultaneous determination of Memantine hydrochloride and Amisulpride in human plasma and their pharmaceutical formulations without interference from common dugs excipients. A binary mixture of ME and nonselective ß-blocker namely; carvidalol has been determined the solid-state by FTIR spectroscopy for the first time. The linear range had been extent from 1.0 to 8.0 and 1.0 to 10.0 µg/mg, for ME and AMS respectively. The detection limits were 0.29 and 0.23 µg/mg while quantitation limits were 0.90 and 0.71 µg/mg for ME and AMS respectively. The developed assay has been validated according to ICH & USP recommendations and successfully applied for quantitative determination of selected drugs in biological fluid.

A new spectrofluorimetric assay for quantification of Amisulpride and Memantine hydrochloride in real human plasma sample and pharmaceutical formulations via Hantzsch reaction.

A new, selective and accurate spectrofluorimetric assay has been described for detection of Amisulpride and Memantine hydrochloride in pharmaceutical formulations and real plasma samples. The described assay depends on the reaction between the primary amino group of the selected drugs with acetyl acetone & formaldehyde in an acetate buffer of pH4.8. The derivatized product showed yellow fluorescence at λex=418nm and λem=484.5nm. The calibration graph was linear in the range of 0.05-0.5 and 0.2-1µgmL-1 for AMS and ME, orderly. The limits of detection were 0.0085 and 0.0153µgmL-1, and the limits of quantitation were 0.026 and 0.0464µgmL-1 for AMS and ME respectively. Validation of the described assay was in consonance with ICH guideline. Due to the sensitivity of the prescribed assay, it permits the determination of selected medications in biological sample quantitatively.

Utility of chromogenic property of 2, 2-dihydroxyindane-1, 3-dione for quantification of Memantine hydrochloride in pure, pharmaceutical preparation and application to uniformity testing.

Accurate, simple, sensitive, and fast spectrophotometric assay was applied for the quantification of certain anti-Alzheimer drug namely; Memantine hydrochloride, in its bulk and pharmaceutical preparation. The described assay has been established on the reaction between the primary amino moiety of the cited drug and 2,2-dihydroxyindane-1,3-dione reagent in N,N'-dimethylformamide medium in boiling water bath. Which give Ruhemann's purple color that can be determined at λmax = 595 nm. Beer's law has been obeyed within drug concentration range from 10-120 µg per milliliter. Detection limit and quantitation limit have been 1.6 & 4.9 µg per milliliter respectively. Developed procedure has been validated in agreement with International Conference of Horizon recommendations and applied successfully for detection of cited drug in bulk, pharmaceutical dosage form and content uniformity testing.

Innovative HPTLC method for simultaneous determination of ternary mixture of certain DMARDs in real samples of rheumatoid arthritis patients: an application of quality by design approach.

A uniquely developed high performance thin-layer chromatographic (HPTLC) method coupled with UV detection was applied using quality by design approach (QbD) for simultaneous determination of methotrexate (MTX), sulfasalazine (SSZ) and hydroxychloroquine (HCQ) in serum and urine samples of rheumatoid arthritis patients. MTX, SSZ with HCQ are the most common disease-modifying antirheumatic drugs (DMARDs) combination used for the treatment of rheumatoid arthritis. This ternary mixture with montelukast (MK) added as internal standard, were separated by using a mixture of ethyl acetate: methanol: 25% ammonia, (8: 2: 3, v/v/v) as a mobile phase system. The separation was achieved on HPTLC precoated silica gel plate 60 F254 and the detection was carried out at 306 nm for MTX and at 340 nm for both SSZ and HCQ. The design was planned to obtain the most optimum retardation factors (Rf) with best resolution. The Rf values for MTX, SSZ, MK and HCQ were of 0.31 ±â€¯0.03, 0.62 ±â€¯0.02, 0.71 ±â€¯0.02 and 0.83 ±â€¯0.03; respectively. The interactive response optimizer achieved the most favorable conditions with acceptable composite desirability of 0.9703. Linear relationship with good correlation coefficients (r = 0.9990-0.9994) were also obtained with detection and quantification limits of 13.94-260.64 and 46.84-1810.01(ng/ml); respectively. The suggested method was established in accordance with the guidelines of Food and Drug Administration (FDA). The established QbD-HPTLC method achieved simple, sensitive and selective quantification of the studied drugs in serum and urine samples in the presence of their metabolites with no interferences. This method can be extended effectively for therapeutic drug monitoring and pharmacokinetics studies of these drugs.

Novel sublingual tablets of Atorvastatin calcium/Trimetazidine hydrochloride combination; HPTLC quantification, in vitro formulation and characterization.

BACKGROUND: Ischemic heart disorders and accumulation of lipids in blood vessels could contribute to angina pectoris. Therefore, the aim of this study was to formulate sublingual tablets containing a novel combination of Atorvastatin calcium (ATOR) and Trimetazidine HCl (TMZ) for efficient treatment of coronary heart disorders. METHODS: The dissolution rate of water-insoluble ATOR was enhanced via complexation with sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD) and addition of soluplus as a polymeric solubilizer excipient. The solubilized ATOR and TMZ were compressed into a sublingual tablets by direct compression technique and evaluated for their tableting characteristics. In addition, a new validated method based on High Performance Thin Layer Chromatography (HPTLC) was developed for simultaneous determination of both drugs in pure forms and sublingual tablets. RESULTS: The developed HPTLC method showed LODs of 0.056 and 0.013 µg/band and LOQs of 0.17, 0.040 µg/band for TMZ and ATOR, respectively and proved to be linear, accurate, precise and robust. The optimum formulation containing mixture of superdisintegrants; Ac-Di-Sol and crospovidone (4.8% w/w, each) showed the shortest disintegration time (65 s) and enhanced release profiles of both drugs. CONCLUSIONS: The prepared sublingual tablets combining ATOR and TMZ will be a promising dosage form for coronary heart disease patients with an instant action and improved patient compliance.

Selectivity enhanced cation exchange chromatography for simultaneous determination of peptide variants.

If strong cation exchange chromatography (SCX) is combined with ion-pair chromatography, then the solute could be retained selectively with the power of mixed separation modes. This combination is termed selectivity enhanced strong cation exchange chromatography (SE-SCX). Macroporous polystyrene-divinylbenzene (PS/DVB) resin with sulfonate coating that conveys ion exchange and reversed phase characteristics was employed. Sodium dodecyl sulfate (SDS) was utilized as a selectivity modifier and an ion-pair reagent. This separation strategy is exploited for a challenging simultaneous separation of peptide variants having similar isoelectric points (pIs) and comparable retention behaviour. Insulin variants were used as a model in this study. The selective separation of insulin and five structurally-related analogues namely; ASPART, LISPRO, GLULISIN, GLARGIN, and DETEMIR was conducted using gradient elution mode. Three eluents were used for the separation of the target compounds. Eluent A was a mixture of acetonitrile and 10 mmol L-1 SDSat ratio (1:1) and was kept at 20% through the run. Eluent B was 20 mmol L-1 KH2PO4 adjusted at pH = 4.0 and eluent C was eluent B plus 1 mol L-1 NaCl that was increased linearly till 80% at 20 min. It was found that the retention of the tested variants can be modeled mainly by electrostatic interaction that might be hydrophobically-assisted. The developed method was validated in accordance with ICH guidelines and was appropriate for the intended purposes. Finally, this study introduces SE-SCX as a new selective separation strategy for peptides and proteins that may open the door for novel mixed mode perspectives in protein analysis.

A new sensitive approach for spectrofluorimetric assay of Milnacipran and Amisulpride in real plasma and pharmaceutical preparations via complexation with Eosin Y dye.

An accurate, economic, rapid, reliable spectrofluorimetric assay was developing for the assay of definite anti-depressant drugs namely Amisulpride and Milnacipran hydrochloride, in those pharmaceutical preparation and biological fluid. The suggested method was established on the detection of quenching process resulting from the action of AMS and MCN to the native fluorescent EosinY. A binary complex between selected medications and Eosin Y was established in acetate buffer (0.2 M) at pH 3.3 & 4.0 for AMS and MCN respectively. The relative fluorescence capacity was determined at λex = 301.8 nm and λem = 542.7 nm. The calibration graphs had been linear through extent from 0.02 to 0.3 and 0.1-1 µg mL-1, to both dugs respectively. Detection limits have been 0.0047 & 0.0188 µg mL-1 while quantitation limits have been 0.0141 & 0.0569 µg mL-1 to AMS & MCN respectively. Developed assay has been validated in agreement with ICH recommendations. Due to high sensitivity of the described assay, it allows the quantitation of anti-depressant drugs through biological fluid.

Controlled microwave derivatization reaction for reproducible trace analysis of budesonide in human plasma.

Microwave reactors with on-line controlled reaction conditions have been recently innovated to improve reaction kinetics and reproducibility. Herein, a modern microwave reactor was dedicated to develop a new, sensitive and reproducible approach for trace analysis of budesonide (BUD) in human plasma. The method was based on fast microwave reaction of BUD with dansyl hydrazine (DNS-HZ) reagent under controlled conditions coupled with high-performance liquid chromatography (HPLC)-fluorescence detection. The microwave irradiation and dansylation conditions were optimized for the best sensitivity and selectivity. The controlled microwave derivatization reaction (CMDR) decreased the reaction time, amplified the reaction yield and enhanced product purities by reducing the unwanted side reactions. The chromatographic separation was attained by isocratic elution on reversed phase column via a mobile phase consisted of methanol and phosphate buffer (10 mM, pH 7.0) at ratio 80:20 (v/v). The fluorescence detector was set at 500 nm after excitation at 330 nm. Betamethasone dipropionate (BDP) was used as an internal standard. CMDR-HPLC method validation was performed in agreement with bioanalytical method validation guidelines by the US food and drug administration (US-FDA). The obtained linearity range was 0.2-100 ng mL-1 with correlation coefficient 0.9991 and the lower limit of quantitation (LLOQ) in human plasma was 0.21 ng mL-1. The developed CMDR -HPLC method was applied successfully for assessment of plasma levels of BUD in allergic rhinitis patients after intranasal administration of the micronized BUD.

A single run, rapid polarity switching method for determination of 30 pharmaceuticals and personal care products in waste water using Q-Exactive Orbitrap high resolution accurate mass spectrometry.

The analytical capability of the UPLC-Q Exactive™ Orbitrap MS was exploited for simultaneous determination of 30 acidic and basic PPCPs in a single run, using rapid polarity switching of the electrospray ionisation source. Full scan MS mode at resolution of 35,000 FWHM, Automatic gain control (AGC) target of 1E6 ions at injection time of 50 ms provided the optimum parameters for high sensitivity, together with sufficient data points per peak (≥15) for improved reproducibility. In addition to chromatographic retention times, method selectivity was achieved via applying high resolution accurate mass with low mass tolerance filter (<5 ppm) for identification of each target compound. Six-point linear calibration curves (R2 > 0.95) were established for all target analytes over a concentration range of 1-1500 ng/ml. Good results were obtained for method accuracy (% recovery = 76-104%), inter- and intra-day precision (relative standard deviation <15%) at 3 concentration levels. Instrumental detection and quantification limits ranged from (0.02-1.21 ng/ml) and (0.07-4.05 ng/ml), respectively. While optimised MS/MS analysis through parallel reaction monitoring (PRM) mode provided slightly higher sensitivity, Full scan MS mode allowed for higher mass resolution (selectivity), more data points per peak (reproducibility) and more importantly, the potential for post-acquisition screening of non-target compounds. Following solid phase extraction (SPE) of target analytes, the method was successfully applied to provide first data on PPCPs occurrence in effluent and surface water samples (n = 10) from Egypt. Moreover, screening for non-target compounds revealed the presence of bisphenol A, which was further confirmed via matching with an authentic standard. Overall, this study provides first insight into the high analytical capabilities of the Q-Exactive™ Orbitrap platform for both targeted/non-targeted analysis of PPCPs in environmental matrices.

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.

Application of salting-out thin layer chromatography in computational prediction of minimum inhibitory concentration and blood-brain barrier penetration of some selected fluoroquinolones.

The 2017 FDA safety review regarding the CNS (central nervous system) side effects associated with the systemic use of fluoroquinolones antibacterials (FQs) was the key motivation to carry out this work. The main objective of this study is to investigate lipophilicity and retention parameters of some selected fluoroquinolones antibacterials (FQs) namely; levofloxacin (LEV), ofloxacin (OFL), gatifloxacin (GAT), norfloxacin (NOR), sparfloxacin (SPA), ciprofloxacin (CIP) and lomefloxacin (LOM) using salting-out thin layer chromatography (SOTLC). Statistically significant correlations between the chromatographically-obtained retention parameters and experimental log P values were found and expressed as quantitative structure retention relationship (QSRR) equations. Principal component analysis was carried out to explain the variation between chromatographic and both experimental and computed lipophilicity parameters. In another aspect of this study, a comparison between the chromatographically-determined retention parameters (for five of the drugs under study) obtained using SOTLC (current study) and relative lipophilicity (RM0) determined using a previously reported RP (reversed-phase)-TLC method was carried out. Statistically significant correlation between the two methods was found, although RM0 values obtained using SOTLC was lower than those reported using RP-TLC. Multiple linear regression analysis was performed to predict MIC (minimum inhibitory concentration) and blood brain barrier (BBB) penetration of the examined drugs in which efficient QSAR (quantitative structure-activity relationship) and QSPR (quantitative structure-property relationship) models were generated using the calculated chromatographic parameters (RM0 and C0). The described models can provide a useful approach to predict MIC and BBB penetration of newly synthesized FQs targeting to increase their activity against Gram-positive organisms and to minimize the associated CNS side effects.

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.

3-Amino-5-pyridin-3-yl-1,2,4-triazole, a novel fluorescence probe for trace analysis of silymarin in bulk material, pharmaceutical dosage forms and human plasma: Further insights on reaction mechanism using computational molecular modeling and NMR spectroscopy.

A rapid, highly sensitive and roubst spectrofluorimetric method was developed for trace analysis of silymarin (SLM) in active pharmaceutical ingredient (API), pharmaceutical preparations and human plasma. The proposed method is based on reaction of SLM with a novel reagent; 3-amino-5-pyridin-3-yl-1,2,4-triazole (3-APT); in the presence of 0.04 M sodium hydroxide. The formed fluorescent product was formed within 5 min and was measured at 504 nm after excitation at 390 nm. All reaction parameters were optimized and the proposed method was validated according to ICH guidelines. The developed method was linearly correlated at the concentration range of 0.05-8 µg mL-1 with good correlation coefficient 0.9993, limit of detection 10.79 ng mL-1 and limit of quantitation 32.71 ng mL-1. The relative standard deviations %RSD values were 1.59-2.69% and 1.47-2.62% in case of intra- and inter-day precision, respectively. Computational molecular modeling and NMR spectroscopy were used to identify the reaction mechanism between SLM and 3-APT. The proposed method was employed for determination of SLM in API or bulk material, pharmaceutical capsules and sachets. Further, the method was sensitive enough to be applied for analysis of the free (unconjugated) SLM flavonolignans in human plasma samples.

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.

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.