Combined eutexia and amorphization for simultaneous enhancement of dissolution rate of triamterene and hydrochlorothiazide: preparation of orodispersible tablets.

BACKGROUND: Triamterene is an oral antihypertensive drug with dissolution-limited poor bioavailability. It can be used as monotherapy or in fixed dose combination with hydrochlorothiazide which also suffers from poor dissolution. Moreover, co-processing of drugs in fixed dose combination can alter their properties. Accordingly, pre-formulation studies should investigate the effect of co-processing and optimize the dissolution of drugs before and after fixed dose combination. This is expected to avoid deleterious interaction (if any) and to hasten the biopharmaceutical properties. OBJECTIVE: Accordingly, the aim of this work was to optimize the dissolution rate of triamterene alone and after fixed dose combination with hydrochlorothiazide. METHODOLOGY: Triamterene was subjected to dry co-grinding with xylitol, HPMC-E5 or their combination. The effect of co-grinding with hydrochlorothiazide was also tested in absence and presence of xylitol and HPMC-E5. The products were assessed using Fourier-transform infrared (FTIR), differential scanning calorimetry, X-ray powder diffraction (XRPD), in addition to dissolution studies. Optimum formulations were fabricated as oral disintegrating tablets (ODT).Results: Co-processing of triamterene with xylitol formed eutectic system which hastened dissolution rate. HPMC-E5 resulted in partial amorphization and improved triamterene dissolution. Co-grinding with both materials combined their effects. Co-processing of triamterene with hydrochlorothiazide resulted in eutexia but the product was slowly dissolving due to aggregation. This problem was vanished in presence of HPMC-E5 and xylitol. Compression of the optimum formulation into ODT underwent fast disintegration and liberated acceptable amounts of both drugs. CONCLUSION: The study introduced simple co-processing with traditional excipients for development of ODT of triamterene and hydrochlorothiazide.

Development and validation of an analytical method for trace-level quantification of genotoxic nitrosamine impurities in losartan and hydrochlorothiazide fixed-dose combination tablets using ultra performance liquid chromatography triple quadrupole mass spectrometry.

RATIONALE: Since June 2018, globally large numbers of pharmaceuticals have been recalled due to the unexpected presence of nitrosamines. Beginning with the class of pharmaceuticals known as sartans, subsequent lines of inquiry included antidiabetic medicines, antihistamines, and antibiotics. A critical review of the U.S. Food and Drug Administration database reveals that the highest number of products recall due to the presence of unacceptable levels of nitrosamines were losartan potassium drug products and their coformulations with other drug substances. The problem can be mainly attributed to naively adopted approval revisions and the lack of sufficient current analytical technologies to detect those contaminants in time. In this work, we developed a specific, selective, accurate, precise, and robust ultra-performance liquid chromatography-triple quadrupole-mass spectrometry (UPLC-TQ-MS/MS) method for the estimation of eight genotoxic nitrosamine impurities in losartan and hydrochlorothiazide (HCTZ) tablets, which is the only fixed-dosage combination approved by the USFDA to treat hypertension. METHODS: All the nitrosamine impurities along with the drug substances were separated using an Agilent Pursuit XRs Ultra diphenyl column (150 × 2.0 mm, 2.8 µm) with mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in methanol) at a flow rate of 0.4 ml/min using the gradient elution program. The proposed method was validated per ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) Q2 (R1) guidelines to ensure the method is suitable for its intended purpose. RESULTS: Limit of detection and limit of quantification were obtained in the range of 0.25-0.5 ng/mL, which was very low compared to levels specified by the USFDA, European Medicines Agency (EMA), and other regulatory authorities that ensure the sensitivity of the method in its entire life cycle. CONCLUSIONS: The developed method can be incorporated into an official monograph and applied for routine quality control analysis of losartan and HCTZ fixed-dose combination tablets.

Facile immunochromatographic assay based on metal-organic framework-decorated polydopamine for the determination of hydrochlorothiazide adulteration in functional foods.

Herein, a novel immunochromatographic assay (ICA) based on metal-organic framework-decorated polydopamine (MOF@PDA) was firstly developed for the determination of hydrochlorothiazide (HCTZ) adulteration in functional foods. The coupling rate of MOF@PDA carrier to HCTZ antibody was as high as 91.7 %. The detection limits of the developed MOF@PDA-ICA in functional tablets and capsules were 5.93 and 4.72 µg/kg, the linear ranges were 11.2-91.91 µg/kg and 9.11-86.78 µg/kg, respectively. The sensitivity was 27-fold higher than that of the reported ICA. The recovery was 82.5-116.6 %, and coefficient of variation was 6.9-14.2 %. The results can be achieved and analyzed in 8 min with the smartphone-based detection device. The parallel tests of 23 commercial functional tablets and capsules showed that the results of the MOF@PDA-ICA were consistent with that of the LC-MS/MS (R2 > 0.99). Therefore, our method is facile, sensitive, portable, and can provide a reliable technical mean for the detection of HCTZ adulteration in functional foods.

Synthesis of 1,2,3,triazole modified analogues of hydrochlorothiazide via click chemistry approach and in-vitro α-glucosidase enzyme inhibition studies.

The current study was aimed to discover potent inhibitors of α-glucosidase enzyme. A 25 membered library of new 1,2,3-triazole derivatives of hydrochlorothiazide (1) (HCTZ, a diuretic drug also being used for the treatment of high blood pressure) was synthesized through click chemistry approach. The structures of all derivatives 2-26 were deduced by MS, IR, 1H-NMR, and 13C-NMR spectroscopic techniques. All the compounds were found to be new. Compounds 1-26 were evaluated for α-glucosidase enzyme inhibition activity. Among them, 18 compounds showed potent inhibitory activity against α-glucosidase with IC50 values between 24 and 379 µM. α-Glucosidase inhibitor drug acarbose (IC50 = 875.75 ± 2.08 µM) was used as the standard. Kinetics studies of compounds 6, 9, 11, 12, 15, 20, 23, and 24 revealed that only compound 15 as a mixed-type of inhibitor, while others were non-competitive inhibitors of α-glucosidase enzyme. All the compounds were found to be non-cytotoxic when checked against mouse fibroblast 3T3 cell line.

Development and validation of a stability-indicating UPLC method for the determination of olmesartan medoxomil, amlodipine and hydrochlorothiazide degradation impurities in their triple-combination dosage form using factorial design of experiments.

The current work describes the development and validation of a stability-indicating UPLC method for the determination of olmesaratan medoxomil (OLM), amlodipine besylate (AMB), hydrochlorothiazide (HCT) and their degradation products in the triple-combination tablet dosage form. The separation was achieved using a Zorbax Eclipse plus C8 RRHD (100 mm × 3.0 mm), 1.8 µm column with gradient elution of mobile phase A containing 0.02 m of sodium phosphate buffer (pH 3.35) and mobile phase B as acetonitrile and water (90:10, v/v). The detector signal was monitored at UV 250 nm. Analytical performance of the optimized UPLC method was validated as per International Conference on Harmonization guidelines. The linearity ranges for OLM, AMB and HCT were 0.59-240, 0.30-60 and 0.37-150 µg/ml, respectively, with correlation coefficients >0.999. The dosage form was subjected to forced-degradation conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal and photodegradation. The method was proved to be stability indicating by demonstrating the specificity of the drugs from degradation products. The robustness of the method was evaluated through a two-level, three-factorial design with a multivariate approach. Statistical data analysis with best model fit P-value < 0.05 from an ANOVA test indicated that the influence of individual factors is relatively higher than the interaction effects. The method is useful for the analysis of drug products.

Egg White Protein Carrier-Assisted Development of Solid Dispersion for Improved Aqueous Solubility and Permeability of Poorly Water Soluble Hydrochlorothiazide.

Hydrochlorothiazide (HTZ) is a first-line drug used in the treatment of hypertension suffered from low oral bioavailability due to poor aqueous solubility and permeability. Hence, lyophilized egg white protein-based solid dispersion (HTZ-EWP SD) was developed to explore its feasibility as a solid dispersion carrier for enhanced aqueous solubility and permeability of HTZ. The HTZ-EWP SD was prepared using the kneading method. HTZ-EWP SD was characterized using scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier transforms infrared spectroscopy (FT-IR), powder X-ray diffractometer (PXRD), solubility, in vitro dissolution, and ex vivo permeation studies. The physico-chemical evaluation suggested the formation of the solid dispersion. Optimized HTZ-EWP SD4 drastically enhanced (~32-fold) aqueous solubility (~16.12 ± 0.08 mg/mL) over to pure HTZ (~ 0.51 ± 0.03 mg/mL). The dissolution study in phosphate buffer media (pH 6.8) revealed that HTZ-EWP SD4 significantly enhanced the release rate of HTZ (~ 87 %) over to HTZ (~ 25 %). The permeation rate of HTZ from optimized HTZ-EWP SD4 was enhanced significantly (~ 84 %) compared to pure HTZ (~ 24 %). Optimized HTZ-EWP-SD4 enhanced the rate of HTZ dissolution (~ 86 %) in FeSSIF (fed state simulated intestinal fluid), compared to a low dissolution rate (~ 72 %) in FaSSIF (fasted state simulated intestinal fluid) state after 2-h study. Obtained results conclude that lyophilized egg white protein can be utilized as an alternative solid dispersion carrier for enhancing the solubility and permeability of HTZ.

Determination of co-crystal phase purity by mid infrared spectroscopy and multiple curve resolution.

Multivariate Curve Resolution (MCR) was used to determine the phase purity of pharmaceutical co-crystals from mid infrared spectra. An in-silico coformer screening was used to choose one of ten potential coformers. This analysis used quantum chemistry simulation to predict which coformers are thermodynamically inclined to form cocrystals with the model drug, hydrochlorothiazide. The coformer chosen was nicotinamide. An experimental solvent screening by ultrasound assisted slurry co-crystallization was performed to evaluate the capacity of the method to determine phase purity. Afterwards, slurry and slow evaporation co-crystallizations were performed at 10, 25, and 40 °C using 7 solvent systems, and two levels of agitation for the evaporation co-crystallization (on and off). Mid infrared spectroscopy (MIRS) analysis of the products of these co-crystallizations was used to develop an MCR model to determine co-crystal phase purity. The MCR results were compared with a reference co-crystal. Experimental design (DoE) was used to investigate the effect of solvents, temperature, and agitation on the purity of co-crystals produced by slurry and evaporation co-crystallization. DoE revealed that evaporation co-crystallization with agitating at 65 rpm formed co-crystals with greater phase purity. The optimal temperature varied with the solvent used.

Employment of Quality by Design Approach via Response Surface Methodology to Optimize and Develop Modified-release Formulation of Hydrochlorothiazide.

BACKGROUND: Heart attack predominantly occurs during the last phase of sleep and early morning hours, causing millions of death worldwide. Hydrochlorothiazide (HCTZ) is a recommended drug for the prevention of heart disease, but its long action (>4 h) dosage form is lacking in the commercial market and development of modified-release formulation may have industrial significance. Regulatory agencies emphasize Quality by Design based approach for product development to entrust quality in the product. OBJECTIVE: The current research aimed to develop a quality product profile of HCTZ modifiedrelease tablets (MRT; ~14 h) by applying Response Surface Methodology using the computational QbD approach. METHODS: Three independent factors were identified by qualitative and quantitative risk assessment. Statistical terms like p-value, lack of fit, the sum of square, R-squared value, model F value, and linear equations were determined. Graphical tools like normal plot of residual, residual vs predicted plot and box cox plot were used to verify the model selection. The graphical relationship among the critical, independent variables was represented using the Contour plot and 3-D surface plot. Design space was identified by designing an overlay plot using response surface design. RESULTS: Excellent correlation was observed between actual and predicted values. Similarity Factor (F2) of reproducible trials was 78 and 79, and content uniformity was 100.9% and 100.4%. Average weight, hardness, thickness, diameter, and friability were within acceptable limits. CONCLUSION: QbD approach, along with a quality risk management tool, provided an efficient and effective paradigm to build quality MRT of HCTZ.

Application of polyvinyl acetate in an innovative formulation strategy for lyophilized orally disintegrating tablets.

Orally disintegrating tablets (ODTs) manufactured by freeze-drying, also called oral lyophilizates, are a patient-centred dosage form. They possess ultra-fast disintegration dynamics, have acceptable mechanical strength and have a smooth mouth texture. In this study, polyvinyl alcohol (PVA) was investigated as an alternative polymeric binder to gelatin for ODT formulations. A low- and high-dose formulation were in-scope with mannitol as filler and xanthan gum as a viscosity enhancer. Design of experiments (DoE) methodology was applied to study the formulation effects on several quality attributes. Sedimentation during the initial phases of freeze-drying was successfully measured using Raman spectroscopy and could be minimized by adjusting the xanthan gum concentration. Multiple linear regression models were subsequently applied to establish design spaces and robust optimised formulations. A 19 mg hydrochlorothiazide (HCT) as low-dose and 500 mg paracetamol as high-dose ODT were developed in this study. The work displayed the use of PVA as a viable polymeric binder, and alternative for gelatin, in lyophilized ODTs.

High-Silica Zeolites as Sorbent Media for Adsorption and Pre-Concentration of Pharmaceuticals in Aqueous Solutions.

The present work focused on the use of high-silica commercial zeolites as sorbent media for pharmaceuticals in an aqueous matrix. As drug probes, ketoprofen, hydrochlorothiazide, and atenolol were selected, because of their occurrence in surface waters and effluents from wastewater treatment plants. Pharmaceuticals adsorption was evaluated for two Faujasite topology zeolites with Silica/Alumina Ratio 30 and 200. The selected zeolites were demonstrated to be efficient sorbents towards all investigated pharmaceuticals, thanks to their high saturation capacities (from 12 to 32% w/w) and binding constants. These results were corroborated by thermal and structural analyses, which revealed that adsorption occurred inside zeolite's porosities, causing lattice modifications. Finally, zeolites have been tested as a pre-concentration media in the dispersive-solid phase extraction procedure. Recoveries higher than 95% were gained for ketoprofen and hydrochlorothiazide and approximately 85% for atenolol, at conditions that promoted the dissolution of the neutral solute into a phase mainly organic. The results were obtained by using a short contact time (5 min) and reduced volume of extraction (500 µL), without halogenated solvents. These appealing features make the proposed procedure a cost and time saving method for sample enrichment as well as for the regeneration of exhausted sorbent, rather than the more energetically expensive thermal treatment.

Validated LC-MS/MS method for the simultaneous determination of enalapril maleate, nitrendipine, hydrochlorothiazide, and their major metabolites in human plasma.

Hypertension is a major risk factor for atherosclerosis and ischemic heart disease. Most hypertensive patients need a combination of antihypertensive agents to achieve therapeutic goals. A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometric method was developed and validated for simultaneous determination of enalapril maleate (ENA) and its major metabolite enalaprilat (ENAT), nitrendipine (NIT) and its major metabolite dehydronitrendipine (DNIT), and hydrochlorothiazide (HCT) in human plasma using felodipine as an internal standard (IS). The drugs were extracted from plasma using one-step protein precipitation. Chromatographic separation was performed on a Symmetry C18 column, with water and acetonitrile (10:90, v/v) as mobile phase. The detection was carried out using multiple reaction monitoring mode and coupled with electrospray ionization source. Multiple reaction monitoring transitions were m/z 377.1 → 234.1 for ENA, m/z 349.2 → 206.1 for ENAT, m/z 361.2 → 315.1 for NIT, m/z 359 → 331 for DNIT, m/z 295.9 → 205.1 for HCT, and m/z 384.1 → 338 for felodipine (IS). The method was linear over concentration ranges of 1-200, 20-500, 5-200, 2-100, and 5-200 ng/mL for ENA, ENAT, NIT, DNIT, and HCT, respectively, with r2 ≥ 0.99. Method validation was performed according to U.S. Food and Drug Administration guidelines. The validated method showed good sensitivity and selectivity and could be applied for therapeutic drug monitoring and bioequivalence studies.

Photostability study of multicomponent drug formulations via MCR-ALS: The case of the hydrochlorothiazide-amiloride mixture.

The kinetics and photodegradation mechanism of the pharmaceutical mixture of hydrochlorothiazide (HCT) and amiloride (AML) has been studied in depth using a chemometric approach. Water solutions of HCT and AML, separately or in binary mixtures, were irradiated with forced light at different pH values (3, 7, 9 and 12). Multivariate Curve Resolution - Alternating Least Squares (MCR-ALS) modelling has been applied to the experimental data recorded by UV spectrophotometry and HPLC-UV/MS. 78 data sets were collected and their chemometric processing has allowed the simultaneous determination of the behaviour of the two drugs in the mixture when exposed to light and the dependence of their photodegradation kinetics on pH. MCR-ALS has been applied using three different implementations. Soft-MCR-ALS and hybrid Hard/Soft-MCR-ALS have been used to resolve the experimental data and to get the equilibrium and kinetic parameters of the investigated chemical processes. A third implementation of the MCR-ALS method has been used in the analysis of the incomplete data sets obtained when UV spectrophotometric and HPLC-UV/MS data were simultaneously analysed, using a row- and column-wise incomplete augmented data matrix arrangement. In these matrices, information from HPLC-UV detector was used as a bridge between the data recorded by UV spectrophotometry (acid-base and kinetic reactions monitoring) and the data obtained by HPLC-MS.

Blend of guar gum and Eudragit shows excellent drug release retarding behavior in sustained release tablets.

The objective of the current study was to compare sustained release behavior of natural and synthetic polymers in matrix tablets of lisinopril and hydrochlorothiazide combination. Guar gum was used as a hydrophilic natural polymer while Eudragit L 100-55 was used as synthetic polymer. Tablets were formulated by direct compression method using different ratios and combinations of both polymers. Various physical tests were performed. After that, in vitro drug release patterns were investigated by performing dissolution in pH 6.8 phosphate buffer. Results indicated that tablets with combination of both guar gum and Eudragit L 100-55 (formulation F10) were having the best drug release retarding behavior. All formulations followed zero order kinetics indicating the drug release was independent of the concentration. Higuchi model revealed drug release by diffusion mechanism while Korsmeyer Peppas model suggested that formulations followed the non-fickian release behavior.

New orodispersible mini-tablets for paediatric use - A comparison of isomalt with a mannitol based co-processed excipient.

The development of orodispersible mini-tablets (ODMTs) for paediatric use has gained importance within recent years as European authorities set up regulations for developing suitable and palatable dosage forms for paediatric patients. Polyols like mannitol and isomalt are frequently used in the manufacture of tablets where sensory properties have to be taken into account. In literature, ODTMs based on a commercialized co-processed excipient based on mannitol (Ludiflash®) have been already described. Isomalt is known for its pleasant sensory properties and therefore appears to be a good candidate for ODMTs. The feasibility of the direct compression grade of isomalt for the manufacture of ODMTs was assessed and compared to Ludiflash®. Hydrochlorothiazide and enalapril maleate were chosen as model drugs and compressed to 2 mm mini-tablets. ODMTs could be obtained fulfilling the criteria of Ph.Eur. with disintegration times of 180 s or even the FDA limit of 30 s. Dissolution studies and mass variation were fulfilled for all mini-tablets. Acceptance values (AV) ≤ 15 were achieved for formulations based on both isomalt and Ludiflash®. Stability data showed the change of disintegration time and tensile strength as a function of storing time, condition and excipient. Both excipients showed their potential for ODMTs for paediatric use.

Mass-customization of oral tablets via the combination of 3D printing and injection molding.

The new frontier of medicine is the personalization of treatment to match a patient's individual needs. Fused-filament fabrication (FFF) offers a platform for the personalization of drug dosage forms, but one of its chief shortcomings compared to other tablet production methods such as dry compression and wet granulation is relatively low throughput. Conversely, injection molding (IM) is a manufacturing technique for the high-volume production of parts, but in which individual part customization is both expensive and slow requiring the modification of expensive mold tooling. Mass-customization is the manufacture of custom products that match the needs of individual consumers but which are produced at the low unit cost associated with high-volume production. We successfully integrated for the first time FFF with IM in a multi-step manufacturing process for the production of custom bilayer tablets loaded with two active pharmaceutical ingredients used in the treatment of cardiovascular disease. The FFF layer was loaded with the diuretic hydrochlorothiazide, while the IM layer was loaded with lovastatin. Infill percentage was varied for the FFF layer as a means to modify drug release. The IM injection pressure was evaluated for its effect on drug release and layer-layer adhesion. The bilayer tablets obtained offered different combinations of drug release profiles, which were governed by a combination of factors, including surface area to volume ratio; IM injection volume penetration into the FFF layer; FFF infill percentage; layer tortuosity and porosity. These different parameters could be utilized to modify the individual release of both drugs from the bilayer tablet. Thus for the first time, we have demonstrated a viable method for the mass-customization of oral tablets which could hasten the rollout of personalized medicine.

Inhibiting or Accelerating Crystallization of Pharmaceuticals by Manipulating Polymer Solubility.

Polymers play a central role in controlling the crystallization of pharmaceuticals with effects as divergent as amorphous form stabilization and the acceleration of crystallization. Here, using pyrazinamide and hydrochlorothiazide as model pharmaceuticals, it is demonstrated that the same functional group interactions are responsible for these opposing behaviors and that whether a polymer speeds or slows a crystallization can be controlled by polymer solubility. This concept is applied for the discovery of polymers to maintain drug supersaturation in solution: the strength of functional group interactions between drug and polymer is assessed through polymer-induced heteronucleation, and soluble polymers containing the strongest-interacting functional groups with drug are shown to succeed as precipitation inhibitors.

Preparation of submicron drug particles via spray drying from organic solvents.

Manufacturing poorly water-soluble active pharmaceutical ingredients (API) with sufficient bioavailability is a significant challenge in pharmaceutical research. A higher bioavailability can reduce both the applied dosage and the side effects for the patient. One method of increasing the bioavailability is to reduce the particle size of the drug down to the nanoscale. An innovative procedure for the preparation of particles in the submicron size range is spray drying with aerosol conditioning, followed by subsequent separation of the particles in an electrostatic precipitator (ESP). This process has been tested before in an earlier work with aqueous model substances at high production rates (1 g/h) and narrow particle-size distributions (mannitol: d50,0 = 455 nm, span = 0,8) in the submicron range. Spray drying from an aqueous solution with low drug concentrations (<1 wt-%) leads to particles in the lower nanosize range, but the low concentrations make this process inefficient. A custom-made plant was modified in order to handle the organic spray-drying process. In addition, explosion protection had to be considered. This work focuses on the spray drying of submicron particles from organic solvents for the purpose of increasing the dissolution rate of the API griseofulvin. API particles were successfully produced in the submicron size-range, characterized and the dissolution behavior was investigated. The dissolution time to dissolve 80% of the drug, t80, was reduced from 21.5 min for the micronized grade API to 8.5 min for the submicron product.

Molecular conformation, vibrational spectroscopic and NBO analysis of atenolol and atenolol-hydrochlorothiazide cocrystals.

Geometry optimization of atenolol (ATN) in the gas phase was carried out using B3LYP-D3BJ/6-31++G(d,p), CAM-B3LYP/6-31++G(d,p) and M06-2X/6-31++G(d,p) levels of DFT. The computed structural parameters were compared with the data obtained by single crystal X-ray diffraction experiment. Chemical reactivity (electronegativity, electrophilicity, hardness, chemical softness and chemical potential) was predicted with the help of HOMO- LUMO energy values. Experimental FT-IR was recorded and the calculated values were also analyzed using the same level of DFT. A complete vibrational spectrum was made to analyze the potential energy distribution (PED). Stability of the molecule arising from hyperconjugative interaction was analyzed by the natural bond orbital (NBO) analysis. The molecular electrostatic potential map was used to detect the possible electrophilic and nucleophilic sites in ATN molecule. Cocrystallization of atenolol-hydrochlorothiazide (ATN-HCTZ) was performed and the structure was analyzed by powder X-ray diffraction. NBO analysis was carried out on the ATN-HCTZ cocrystal for the elucidation of inter and intra-molecular hydrogen bonding interactions in the structure. Atenolol interaction with human serum albumin (HSA) was investigated by a molecular docking study.

Hydrochlorothiazide binding to human serum albumin induces some compactness in the molecular structure of the protein: A multi-spectroscopic and computational study.

The interaction between hydrochlorothiazide (HCTZ), a diuretic drug, with human serum albumin (HSA) was investigated by different biophysical approaches such as UV absorption, circular dichroism (CD), Fourier transform infrared (FT-IR), and fluorescence spectroscopy in 50 mM sodium phosphate buffer, pH 7.4. The results of fluorescence titration experiments revealed that HCTZ strongly quenches the intrinsic fluorescence of HSA through a static quenching mechanism. Binding constants and the number of binding sites were calculated using Stern-Volmer plots. Thermodynamic analysis of the binding data elucidated that hydrogen bonding and van der Waals interactions played the major role in the binding process. Computation of the protein surface hydrophobicity (PSH) index using 1-anilinonaphtalene-8-sulfonate indicated that considerable decrement in PSH value of the protein happened upon drug binding. The binding site of HCTZ in HSA was identified using warfarin and ibuprofen as site markers, a result confirmed by molecular docking study. The results of CD experiments showed that some alterations occurred in the structure of the protein upon ligation. Also, the results of FT-IR experiments were in good agreement with CD experiments. It looks like that both secondary and tertiary structures of HSA have been affected upon HCTZ binding.