Orientational order of dyes in a lyotropic chromonic liquid crystal.

Absorption measurements allow the orientational order parameter of four dyes in the lyotropic chromonic liquid crystal di-sodium cromoglycate (DSCG) to be determined. The dye order parameters are small, except for dyes that intercalate between the DSCG molecules of the rod-like assemblies. The dye order parameters decrease with increasing temperature faster than the nematic order parameter of the DSCG assemblies. For intercalating dyes, the measured dye order parameter varies with the wavelength of the measurement because both intercalated and non-intercalated dye molecules contribute. On the contrary, measurements of the dye order parameter using fluorescence are sensitive only to intercalated dye molecules and produce values that reflect the order parameter of the DSCG assemblies. Therefore, the temperature and concentration dependence of the DSCG order parameter is also explored, since data of this kind on this often-studied system are lacking. Finally, the association constant of one of the intercalating dyes with the DSCG assemblies is determined, yielding a value considerably less than what is found for the same dye with DNA.

Structures and topological defects in pressure-driven lyotropic chromonic liquid crystals.

Lyotropic chromonic liquid crystals are water-based materials composed of self-assembled cylindrical aggregates. Their behavior under flow is poorly understood, and quantitatively resolving the optical retardance of the flowing liquid crystal has so far been limited by the imaging speed of current polarization-resolved imaging techniques. Here, we employ a single-shot quantitative polarization imaging method, termed polarized shearing interference microscopy, to quantify the spatial distribution and the dynamics of the structures emerging in nematic disodium cromoglycate solutions in a microfluidic channel. We show that pure-twist disclination loops nucleate in the bulk flow over a range of shear rates. These loops are elongated in the flow direction and exhibit a constant aspect ratio that is governed by the nonnegligible splay-bend anisotropy at the loop boundary. The size of the loops is set by the balance between nucleation forces and annihilation forces acting on the disclination. The fluctuations of the pure-twist disclination loops reflect the tumbling character of nematic disodium cromoglycate. Our study, including experiment, simulation, and scaling analysis, provides a comprehensive understanding of the structure and dynamics of pressure-driven lyotropic chromonic liquid crystals and might open new routes for using these materials to control assembly and flow of biological systems or particles in microfluidic devices.

From Combinations to Single-Molecule Polypharmacology-Cromolyn-Ibuprofen Conjugates for Alzheimer's Disease.

Despite Alzheimer's disease (AD) incidence being projected to increase worldwide, the drugs currently on the market can only mitigate symptoms. Considering the failures of the classical paradigm "one target-one drug-one disease" in delivering effective medications for AD, polypharmacology appears to be a most viable therapeutic strategy. Polypharmacology can involve combinations of multiple drugs and/or single chemical entities modulating multiple targets. Taking inspiration from an ongoing clinical trial, this work aims to convert a promising cromolyn-ibuprofen drug combination into single-molecule "codrugs." Such codrugs should be able to similarly modulate neuroinflammatory and amyloid pathways, while showing peculiar pros and cons. By exploiting a linking strategy, we designed and synthesized a small set of cromolyn-ibuprofen conjugates (4-6). Preliminary plasma stability and neurotoxicity assays allowed us to select diamide 5 and ethanolamide 6 as promising compounds for further studies. We investigated their immunomodulatory profile in immortalized microglia cells, in vitro anti-aggregating activity towards Aß42-amyloid self-aggregation, and their cellular neuroprotective effect against Aß42-induced neurotoxicity. The fact that 6 effectively reduced Aß-induced neuronal death, prompted its investigation into an in vivo model. Notably, 6 was demonstrated to significantly increase the longevity of Aß42-expressing Drosophila and to improve fly locomotor performance.

Duodenal acidification induces gastric relaxation and alters epithelial barrier function by a mast cell independent mechanism.

Duodenal hyperpermeability and low-grade inflammation in functional dyspepsia is potentially related to duodenal acid exposure. We aimed to evaluate in healthy volunteers the involvement of mast cell activation on the duodenogastric reflex and epithelial integrity during duodenal acidification. This study consisted of 2 parts: (1) Duodenal infusion of acid or saline during thirty minutes in a randomized, double-blind cross-over manner with measurement of intragastric pressure (IGP) using high resolution manometry and collection of duodenal biopsies to measure epithelial barrier function and the expression of cell-to-cell adhesion proteins. Mast cells and eosinophils were counted and activation and degranulation status were assessed. (2) Oral treatment with placebo or mast cell stabilizer disodiumcromoglycate (DSCG) prior to duodenal perfusion with acid, followed by the procedures described above. Compared with saline, acidification resulted in lower IGP (P < 0.01), increased duodenal permeability (P < 0.01) and lower protein expression of claudin-3 (P < 0.001). Protein expression of tryptase (P < 0.001) was increased after acid perfusion. Nevertheless, an ultrastructural examination did not reveal degranulation of mast cells. DSCG did not modify the drop in IGP and barrier dysfunction induced by acid. Duodenal acidification activates an inhibitory duodenogastric motor reflex and, impairs epithelial integrity in healthy volunteers. However, these acid mediated effects occur independently from mast cell activation.

In vitro assessment of an idealized nose for nasal spray testing: Comparison with regional deposition in realistic nasal replicas.

An idealized nasal replica that mimics average regional deposition of nasal spray pump droplets in human nasal airways would potentially be useful in expediting the development of nasal spray products. The aim of this study was to validate an idealized nose, previously developed using in silico simulations, by comparing with regional deposition in realistic, sectioned nasal replicas obtained from in vitro deposition experiments. The realistic nasal airway replicas of five subjects obtained from computerized tomography were manufactured in plastic using rapid prototyping. The idealized nose was made using the same build procedure. A commercial nasal spray pump (NasalCrom, 5.2 mg cromolyn sodium per spray) was then actuated repeatably into each replica under a steady inspiratory flow of 7.5 L/min at two different orientations (45° and 60° from the horizontal). Sectioned replicas were disassembled, and the mass fraction of drug deposited on the surface of each anatomical region was determined. It was found that regional deposition of spray droplets in the idealized replica agreed well with average regional deposition in the realistic replicas. Regional deposition also agreed with previously published in vivo regional deposition using the same spray pump.

Bulk Alignment of Chromonic Aggregates During Swelling of Hydrogels.

This study demonstrates that the bulk alignment of chromonic aggregates can be achieved during the swelling of hydrogels. Swelling of an ionic hydrogel immersed in an aqueous solution of disodium cromoglycate reorients the chromonic aggregates, and millimeter-thick optically anisotropic hydrogels are obtained. These anisotropic hydrogels contain the chromonic aggregates at a condensed concentration as high as in the columnar phase of a normal chromonic aqueous solution, although the X-ray diffraction results show much less stacking order and orientational order of the aggregates. Furthermore, anisotropic mechanical properties of the hydrogels are observed due to the anisotropic alignment of the chromonic aggregates.

Characterization of interactions between cromolyn sodium and bovine serum albumin by spectroscopic, calorimetric and computational methods.

Cromolyn sodium (CS), an anti-inflammatory drug is used in the treatment of allergic disorders. Bovine serum albumin (BSA) a blood plasma protein is used as a model protein for studying protein folding and ligand binding mechanism as it is the main transporter protein which decides the disposition and pharmacodynamics of numerous drugs. In this study, interaction of CS with BSA was investigated using isothermal titration calorimetry, UV-vis, fluorescence, circular dichroism (CD) spectroscopy and molecular docking techniques. Steady state fluorescence data revealed that BSA-CS complex formation occurred through static mode of quenching. Negative values of Gibbs free energy change and enthalpy change showed that BSA-CS complexation was spontaneously favorable and enthalpy driven. CS preferentially interacted at Sudlow's site I (sub-domain IIA) of BSA and the finding was further substantiated by molecular docking study. The binding of CS induced changes in secondary motif of BSA resulting decrease of α-helical content as evident from CD. We explored detailed thermodynamic and structural parameters of interaction of CS to BSA that will be helpful for understanding the more precise binding mechanism of the drug at molecular level.Communicated by Ramaswamy H. Sarma.

Cromolyn-crosslinked chitosan nanoparticles for the treatment of allergic rhinitis.

The aim of this work was to prepare new mucoadhesive nasal decongestant nanoparticles obtained by direct crosslinking between the cationic polymer chitosan and the anionic drug cromolyn. Different chitosan/cromolyn molar ratios were used in order to obtain nanoparticles of suitable size, encapsulation efficiency/drug loading and mucoadhesion. Moreover, the ability of the nanoparticles to deliver cromolyn into and through the nasal mucosa was evaluated. The obtained positively charged nanoparticles, sized 180-400 nm, showed interesting properties in terms of yield, mucoadhesion, encapsulation efficiency and drug loading. Release and permeation/penetration data indicated the ability of the nanoparticles to retain a high amount of cromolyn inside the mucosa, which is rich in mast cells. These findings suggest developing decongestant nanoparticles for potential treatment of allergic rhinitis.

Protective effect of sodium stearate on the moisture-induced deterioration of hygroscopic spray-dried powders.

Amorphous powders are thermodynamically unstable, significantly impacting the processing, storage and performance of a product. Therefore, stabilization of the amorphous contents is in demand. In this study, disodium cromoglycate (DSCG) powder was chosen as a model drug because it is amorphous and highly hygroscopic after spray drying. Sodium stearate (NaSt) was co-spray dried with DSCG at various concentrations (10, 50 and 90% w/w) to investigate its effect against moisture-induced deterioration on the in vitro aerosolization performance of DSCG. Particle size distribution and morphology were measured by laser diffraction and scanning electron microscopy (SEM). Physicochemical properties of the powders were analysed by X-ray powder diffraction (XRPD) and dynamic vapour sorption (DVS). Particle surface chemistry was analysed by the time-of-flight secondary ion mass spectrometry (ToF-SIMS). In vitro dissolution behaviours of the spray-dried (SD) powders were tested by the Franz cell apparatus. In vitro aerosolization performance of SD formulations stored at different relative humidity (RH) was evaluated by a multi-stage liquid impinger (MSLI), using an Osmohaler® at 100 L/min. Results showed that adding NaSt in the formulation not only increased the aerosolization performance of DSCG significantly, but also effectively reduced the deleterious impact of moisture. No significant difference was found in the fine particle fraction (FPF) of formulations containing NaSt before and after storage at both 60% and 75% RH for one week. However, after one month storage at 75% RH, SD formulation containing 10% NaSt showed a reduction in FPF, while formulations containing 50% or 90% NaSt showed no change. The underlying mechanism was that NaSt increased the crystallinity of the powders and its presence on the particle surface reduced particle aggregations and cohesiveness. However, NaSt at high concentration could reduce dissolution rate, which needs to be taken into consideration.

Excitation of triplet states of hypericin in water mediated by hydrotropic cromolyn sodium salt.

Hypericin (Hyp) is a hydrophobic pigment found in plants of the genus Hypericum which exhibits low levels of solubility in water. This work shows that the solubility of Hyp can be significantly increased through the addition of cromolyn disodium salt (DSCG). Performed studies using UV-VIS absorption and fluorescence spectroscopies demonstrate that Hyp remains in a predominantly biologically photodynamic active monomeric form in the presence of DSCG at concentrations ranging from 4.6×10-3 to 1.2×10-1mol·L-1. The low association constant between Hyp and DSCG (Ka=71.7±2M-1), and the polarity value of 0.3 determined for Hyp in a DSCG-water solution, lead to a suggestion that the monomerization of Hyp in aqueous solution can be explained as a result of the hydrotropic effect of DSCG. This hydrotropic effect is most likely a result of interactions between two relative rigid aromatic rings of DSCG and a delocalized charge on the surface of the Hyp molecule. The triplet-triplet (T-T) electronic transition observed in is Hyp in the presence of DSCG suggests a possible production of reactive oxygen species once Hyp is irradiated with visible light in a DSCG aqueous solution.

Cromolyn chitosan nanoparticles as a novel protective approach for colorectal cancer.

Colorectal cancer is the third most common cancer in the world. Cromolyn is a mast cell stabilizer and was proposed as an anticancer agent; however its high polarity limits its bioavailability by rapid washing from the body. We formulated 10 cromolyn chitosan nanoparticles (CCSNPs)1 following ionic gelation technique to improve its bioavailability and investigated the protective anticancer effect of the optimum formula against colorectal cancer in dimethylhydrazine-induced model in rats. Rats were divided into seven groups, group-1: normal control, group-2: cromolyn control, group-3: CCSNPs control, groups-4 to 7 received dimethylhydrazine for 16 weeks to induce colorectal cancer. Groups-5 to 7 received cromolyn solution, non-medicated chitosan nanoparticles and CCSNPs, respectively as protective treatments. Optimum CCSNPs (size 112.4 nm, charge +39.9 mV, enclosed 93.6% cromolyn and showed a sustained drug release pattern over 48 h) significantly reduced tumor-signaling molecules and the number of aberrant crypt foci compared to dimethylhydrazine. Histopathological examination of colon samples revealed that CCSNPs exerted an augmented protective anticancer effect by ameliorating tumor pathology compared to cromolyn solution. In conclusion, CCSNPs ameliorated tumor pathology and malignant oncogenic signaling molecules in colorectal cancer tissue. Thus, CCSNPs may provide a novel protective approach in colorectal cancer treatment. Moreover, encapsulating cromolyn in chitosan nanoparticles augmented the protective anticancer effect of the drug.

Unraveling the thermodynamics, binding mechanism and conformational changes of HSA with chromolyn sodium: Multispecroscopy, isothermal titration calorimetry and molecular docking studies.

Cromolyn sodium is an anti-allergic drug effective for treatment in asthma and allergic rhinitis. In this project, interaction of chromolyn sodium (CS) with human serum albumin (HSA) has been investigated by various techniques such as UV-vis, fluorescence, circular dichorism (CD), fourier transform infrared (FT-IR) spectroscopy, isothermal titration calorimetric (ITC) and molecular docking. The fluorescence quenching results revealed that there was static quenching mechanism in the interactions of CS with HSA. The binding constant (Kb), enthalpy change (ΔH°), entropy change (ΔS°) and Gibbs free energy change (ΔG°) were calculated. The negative values of TΔS° and ΔH° obtained from fluorescence spectroscopy and isothermal titration calorimetry, indicate that hydrogen bonding and van der Waal's forces played major role in the binding process and the reaction is exothermic in nature. The binding constant (Kb) was found to be in the order of 104M-1 which depicts a good binding affinity of CS towards HSA. The conformational changes in the HSA due to interaction of CS were investigated from CD and FT-IR spectroscopy. The binding site of CS in HSA was sub-domain IIA as evident from site probing experiment and molecular docking studies.

Mast cells mediate early neutrophil recruitment and exhibit anti-inflammatory properties via the formyl peptide receptor 2/lipoxin A4 receptor.

BACKGROUND AND PURPOSE: In recent years, studies have focused on the resolution of inflammation, which can be achieved by endogenous anti-inflammatory agonists such as Annexin A1 (AnxA1). Here, we investigated the effects of mast cells (MCs) on early LPS-induced neutrophil recruitment and the involvement of the AnxA1-formyl peptide receptor 2/ALX (FPR2/ALX or lipoxin A4 receptor) pathway. EXPERIMENTAL APPROACH: Intravital microscopy (IVM) was used to visualize and quantify the effects of LPS (10 µg per mouse i.p.) on murine mesenteric cellular interactions. Furthermore, the role that MCs play in these inflammatory responses was determined in vivo and in vitro, and effects of AnxA1 mimetic peptide Ac2-26 were assessed. KEY RESULTS: LPS increased both neutrophil endothelial cell interactions within the mesenteric microcirculation and MC activation (determined by IVM and ruthenium red dye uptake), which in turn lead to the early stages of neutrophil recruitment. MC recruitment of neutrophils could be blocked by preventing the pro-inflammatory activation (using cromolyn sodium) or enhancing an anti-inflammatory phenotype (using Ac2-26) in MCs. Furthermore, MCs induced neutrophil migration in vitro, and MC stabilization enhanced the release of AnxA1 from neutrophils. Pharmacological approaches (such as the administration of FPR pan-antagonist Boc2, or the FPR2/ALX antagonist WRW4) revealed neutrophil FPR2/ALX to be important in this process. CONCLUSIONS AND IMPLICATIONS: Data presented here provide evidence for a role of MCs, which are ideally positioned in close proximity to the vasculature, to act as sentinel cells in neutrophil extravasation and resolution of inflammation via the AnxA1-FPR2/ALX pathway.

Simultaneous Determination of Cromolyn Sodium Combined Dosage Forms Using Isocratic HPLC Method.

A simple and selective reversed-phase high-performance liquid chromatography method was developed for the estimation of cromolyn sodium (CRM) with either oxymetazoline hydrochloride (OMZ) or xylometazoline hydrochloride (XMZ) in their binary mixtures. The method is based on the simultaneous separation of each drug in a reversed-phase Waters symmetry® C18 column (250 mm × 4.6 mm intradermally, 5-µm particle size) at 25°C. Elution was performed with a mobile phase consisting of methanol : 0.1 M phosphate buffer (60:40, v/v, pH 4.0). Quantitation was achieved with ultraviolet detection at 220 nm. The method could determine the three drugs, with linearity, in the range of 2.0-100.0 µg/mL for CRM and 0.8-8.0 µg/mL for OMZ and for XMZ. Aspirin was used as internal standard. Optimization of the separation in terms of mobile phase composition is critical to the method development, which is discussed in detail. The suggested procedure was successfully applied to the analysis of the studied drugs in their nasal preparations. Statistical evaluation of the data obtained by the proposed and comparison methods revealed good accuracy of the proposed method.

Formulation and Characterization of Polysaccharide Microparticles for Pulmonary Delivery of Sodium Cromoglycate.

Sodium cromoglycate (SC) is an antiasthmatic and antiallergenic drug commonly used for chronic inhalation therapy; however, many daily intakes are required due to the fast drug clearance from airways. For these reasons, SC polymeric particles for inhalatory administration with adequate aerosolization and mucoadhesive properties were designed to prolong the drug residence time in the site of action. Sodium carboxymethylcellulose (CMCNa), sodium hyaluronate, and sodium alginate were selected to co-process SC by spray drying. The influence of these polysaccharides on the spray drying process and powder quality was evaluated (among others, morphology, size, moisture content, hygroscopicity, flowability, densities, liquid sorption, and stability). In vitro aerosolization, drug release, and mucoadhesion performance were also studied. Particularly, a novel method to comparatively evaluate the interaction between formulations and mucin solution (mucoadhesion test) was proposed as a rapid methodology to measure adhesion properties of inhalable particles, being the results as indicative of clearance probability. Among all the studied formulations, the powder based on SC and CMCNa exhibited the best mucoadhesion and aerosolization performance, the highest process yield and adequate moisture content, hygroscopicity, and stability. SC-CMCNa formulation arose as a promising inhalatory system to reduce the daily intakes and to increase the patient compliance.

The Hidden Side of Disodium Cromolyn: from Mast Cell Stabilizer to an Angiogenic Factor and Antitumor Agent.

Scattered data suggested that disodium cromolyn, well known as a mast cell stabilizer shows some effects on tumor cells and tumor-associated newly formed vascular networks. Most of these studies used tumor cell lines assessed by in vitro studies. Nor disodium cromolyn effects on melanoma cell lines were studied yet, neither its influence on recruited tumor blood vessels or angiogenic growth factors expression. We designed here a study regarding disodium cromolyn effects on A375 melanoma tumor cells implanted on chick embryo chorioallantoic membrane (CAM) and on blood vessels recruited by the experimental melanoma in the absence of mast cells, knowing that within CAM, the existence of mast cells are not certified yet. We also assessed the role of disodium cromolyn on the expression of several angiogenic growth factors. Disodium cromoglycate differentially acts on tumor cells and blood vessels. Extensive necrotic areas of experimental melanoma together with an increased number of peritumor blood vessels were observed in treated specimens as compared with untreated tumors. Disodium cromolyn inhibited VEGF and PDGF-BB expression, and had no effects on EG VEGF expression between treated and non treated specimens in a mast cells free microenvironment. Our results sustain the direct antitumor effects of sodium cromolyn and suggest the involvement of several growth factors in the recruitment of tumor vessels by A375 melanoma tumor cells. The expression of growth factors is differentially influenced by sodium cromolyn treatment.

Interpreting the behavior of concentration-response curves of hyaluronidase inhibitors under DMSO-perturbed assay conditions.

Hyaluronan-degrading enzyme (hyaluronidase) is involved in tumor growth and inflammation, and as such, hyaluronidase inhibitors have received recent attention as potential therapeutics. The previous studies have successfully discovered a wide range of inhibitors, but unfortunately most of them are dissimilar to original ligand hyaluronan and the mode of action is poorly understood. The present study mechanistically characterized these structurally unrelated inhibitors by interpreting the behavior of concentration-response curves under several in vitro assay conditions. Detergent-addition conditions definitely identified aggregation-based inhibitors. Subsequently, DMSO-perturbed conditions, though preliminary, highlighted the inhibitors that might bind to enzyme non-specifically. Here, an intriguing implication of the latter description is that DMSO-perturbed conditions would generate non-productive but not-denatured enzyme that is an assembly of effective species to capture non-specific binding molecules, and thereby would attenuate their inhibitory activities.

Identification of benzopyrone as a common structural feature in compounds with anti-inflammatory activity in a zebrafish phenotypic screen.

Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well-regulated, persistent neutrophils can cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real time. Finally, our studies reveal that clinically available 'cromones' are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo These findings could have implications for the therapeutic use of benzopyrones in inflammatory disease.

Influence of Proton and Salt Concentration on the Chromonic Liquid Crystal Phase Diagram of Disodium Cromoglycate Solutions: Prospects and Limitations of a Host for DNA Nanostructures.

Lyotropic chromonic liquid crystals have recently been suggested for use as a self-organized host for dispersing and aligning self-organized DNA origami nanostructures. However, an appropriate pH value and a suitable cation concentration are necessary to stabilize such nanostructures and to avoid unfolding of the DNA. The present study shows that the nematic and columnar liquid crystal phases appearing in aqueous solutions of disodium cromoglycate are robust against the replacement of deionized water by a neutral or alkaline buffer solution. However, disodium cromoglycate precipitates when an acidic buffer is used or when the concentration of magnesium cations exceeds a critical concentration of about 0.6-0.7 mmol/L.

Stability-Indicating RP-HPLC Methods for the Determination of Fluorometholone in Its Mixtures with Sodium Cromoglycate and Tetrahydrozoline Hydrochloride.

Two stability-indicating reversed-phase liquid chromatographic methods were developed and validated for the determination of fluorometholone (FLU) in its mixtures with sodium cromoglycate (SCG) and tetrahydrozoline hydrochloride (THZ). The first HPLC method (Method 1) was based on isocratic elution of FLU and SCG along with their alkaline degradation products on a reversed phase C18 column (250 × 4.6 mm id)-ACE Generix 5, using a mobile phase consisting of methanol-water (70 : 30, v/v), pH adjusted to 2.5 using orthophosphoric acid at a flow rate of 1.2 mL min(-1) Quantitation was achieved with UV detection at 240 nm. The second HPLC method (Method 2) was based on isocratic elution of FLU, its alkaline degradation product and THZ on a reversed phase C8 column (250 × 4.6 mm)-ACE Generix 5, using a mobile phase consisting of acetonitrile-50 mM potassium dihydrogen orthophosphate (40 : 60, v/v) at a flow rate of 2 mL min(-1) Quantitation was achieved by applying dual-wavelength detection, where FLU and its alkaline degradation product were detected at 240 nm and THZ was detected at 215 nm at ambient temperatures. Linearity, accuracy and precision were found to be acceptable over the concentration range of 5-50 and 10-500 µg mL(-1) for FLU and SCG (Method 1) and over the concentration range of 5-80 and 5-60 µg mL(-1) for FLU and THZ (Method 2), respectively. Besides, the FLU alkaline degradation product was verified using IR, NMR and LC-MS spectroscopy. The two proposed methods could be successfully applied for the routine analysis of the studied drugs either in their pure bulk powders or in their pharmaceutical preparations without any preliminary separation step.