Influence of Alkylammonium Acetate Buffers on Protein-Ligand Noncovalent Interactions Using Native Mass Spectrometry.

We investigate the influence of three volatile alkylammonium acetate buffers on binding affinities for protein-ligand interactions determined by native electrospray ionization-mass spectrometry (ESI-MS). Four different types of proteins were chosen for this study. A charge-reduction effect was observed for all the cases studied, in comparison to the ions formed in ammonium acetate solution. When increasing the collision energy, the complexes of trypsin and the ligand were found to be more stable when sprayed from alkylammonium acetate buffers than from ammonium acetate. The determined dissociation constant (Kd) also exhibited a drop (up to 40%) when ammonium acetate was replaced by alkylammonium acetate buffers for the case of lysozyme and the ligand. The prospective uses of these ammonium acetate analogs in native ESI-MS are discussed in this paper as well. Graphical Abstract ᅟ.

Stability of chlorothiazide sodium in polypropylene syringes.

PURPOSE: The stability of a solution of chlorothiazide injection diluted with sterile water and stored in polypropylene syringes under refrigerated conditions was investigated. METHODS: Chlorothiazide solutions were compounded by adding 20 mL of sterile water for injection to a 500-mg vial of chlorothiazide sodium for injection. Six batches of chlorothiazide solution (25 mg/mL) were compounded; 0.5-mL portions were transferred to 1-mL polypropylene syringes, which were sealed with a Luer tip cap and stored at refrigeration temperatures (2-8 °C) protected from light. Three batches were potency tested by stability-indicating high-performance liquid chromatography (HPLC) assay using a 0.5-mL sample from each batch at designated time points. Visual and pH testing were performed using the three remaining batches; the contents of two syringes per batch were combined and visually inspected for container integrity and solution color and clarity, with duplicate pH testing performed at each time point. RESULTS: HPLC analyses showed that the remaining percentage of the initial chlorothiazide content declined at an average daily rate of 1.4%, decreasing to 93% by day 6. All samples remained intact, clear, and colorless, with no visible particulate matter or precipitation observed throughout the study period. For all samples of chlorothiazide solution, pH values remained within the range of 9.2-10.0 throughout the 10-day study period. CONCLUSION: When packaged in 1-mL polypropylene syringes and stored protected from light at refrigerated conditions, a solution of chlorothiazide sodium injection in water was stable for six days.

Impact of alternative solid state forms and specific surface area of high-dose, hydrophilic active pharmaceutical ingredients on tabletability.

In order to investigate the effect of using different solid state forms and specific surface area (TBET) of active pharmaceutical ingredients on tabletability and dissolution performance, the mono- and dihydrated crystalline forms of chlorothiazide sodium and chlorothiazide potassium (CTZK) salts were compared to alternative anhydrous and amorphous forms, as well as to amorphous microparticles of chlorothiazide sodium and potassium which were produced by spray drying and had a large specific surface area. The tablet hardness and tensile strength, porosity, and specific surface area of single-component, convex tablets prepared at different compression pressures were characterized. Results confirmed the complexity of the compressibility mechanisms. In general it may be concluded that factors such as solid-state form (crystalline vs amorphous), type of hydration (presence of interstitial molecules of water, dehydrates), or specific surface area of the material have a direct impact on the tabletability of the powder. It was observed that, for powders of the same solid state form, those with a larger specific surface area compacted well, and better than powders of a lower surface area, even at relatively low compression pressures. Compacts prepared at lower compression pressures from high surface area porous microparticles presented the shortest times to dissolve, when compared with compacts made of equivalent materials, which had to be compressed at higher compression pressures in order to obtain satisfactory compacts. Therefore, materials composed of nanoparticulate microparticles (NPMPs) may be considered as suitable for direct compaction and possibly for inclusion in tablet formulations as bulking agents, APIs, carriers, or binders due to their good compactibility performance.

Impact of process variables on the micromeritic and physicochemical properties of spray-dried porous microparticles, part I: introduction of a new morphology classification system.

OBJECTIVES: This work investigated the impact of spray drying variables such as feed concentration, solvent composition and the drying mode, on the micromeritic properties of chlorothiazide sodium (CTZNa) and chlorothiazide potassium (CTZK). METHODS: Microparticles were prepared by spray drying and characterised using thermal analysis, helium pycnometry, laser diffraction, specific surface area analysis and scanning electron microscopy. KEY FINDINGS: Microparticles produced under different process conditions presented several types of morphology. To systematise the description of morphology of microparticles, a novel morphology classification system was introduced. The shape of the microparticles was described as spherical (1) or irregular (2) and the surface was classified as smooth (A) or crumpled (B). Three classes of morphology of microparticles were discerned visually: class I, non-porous; classes II and III, comprising differing types of porosity characteristics. The interior was categorised as solid/continuous (α), hollow (ß), unknown (γ) and hollow with microparticulate content (δ). Nanoporous microparticles of CTZNa and CTZK, produced without recirculation of the drying gas, had the largest specific surface area of 72.3 and 90.2 m²/g, respectively, and presented morphology of class 1BIIIα. CONCLUSIONS: Alteration of spray drying process variables, particularly solvent composition and feed concentration can have a significant effect on the morphology of spray dried microparticulate products. Morphology of spray dried particles may be usefully described using the morphology classification system.

Impact of process variables on the micromeritic and physicochemical properties of spray-dried microparticles--Part II. Physicochemical characterisation of spray-dried materials.

OBJECTIVES: In this work we investigated the residual organic solvent content and physicochemical properties of spray-dried chlorothiazide sodium (CTZNa) and potassium (CTZK) salts. METHODS: The powders were characterised by thermal, X-ray diffraction, infrared and dynamic vapour sorption (DVS) analyses. Solvent levels were investigated by Karl-Fischer titration and gas chromatography. KEY FINDINGS: Spray-drying from water, methanol (MeOH) and mixes of MeOH and butyl acetate (BA) resulted in amorphous microparticles. The glass transition temperatures of CTZNa and CTZK were ∼192 and ∼159°C, respectively. These materials retained their amorphous nature when stored at 25°C in dry conditions for at least 6 months with no chemical decomposition observed. DVS determined the critical relative humidity of recrystallisation of CTZNa and CTZK to be 57% RH and 58% RH, respectively. The inlet temperature dependant oxidation of MeOH to formaldehyde was observed; the formaldehyde was seen to deposit within the amorphous matrix of spray-dried product. Spray-drying in the open blowing mode coupled with secondary drying resulted in a three-fold reduction in residual BA (below pharmacopoeial permitted daily exposure limit) compared to spray-drying in the closed mode. CONCLUSIONS: Experiments showed that recirculation of recovered drying gas increases the risk of deposition of residual solvents in the spray-dried product.

A novel approach to crystallisation of nanodispersible microparticles by spray drying for improved tabletability.

High-dose API powders which are to be tableted by direct compression should have high compactibility and compressibility. This note reports on a novel approach to the manufacture of crystalline powders intended for direct compaction with improved compactibility and compressibility properties. The poorly compactable API, chlorothiazide, was spray dried from a water/acetone solvent mix producing additive-free nanocrystalline microparticles (NCMPs) of median particle size 3.5 µm. Tablets compacted from NCMPs had tensile strengths ranging from 0.5 to 4.6 MPa (compared to 0.6-0.9 MPa for tablets of micronised CTZ) at compression forces ranging from 6 kN to 13 kN. NCMP tablets also had high porosities (34-20%) and large specific surface areas (4.4-4.8m(2)/g). The time taken for tablets made of NCMPs to erode was not statistically longer (p>0.05) than for tablets made of micronised CTZ. Fragmentation of NCMPs on compression was observed. The volume fraction of particles below 1 µm present in the suspension recovered after erosion of NCMP tablets was 34.8±3.43%, while no nanosized particles were detected in the slurry after erosion of compacted micronised CTZ.

Preparation and characterisation of novel chlorothiazide potassium solid-state salt forms: Intermolecular self assembly suprastructures.

Chlorothiazide (CTZ) is a poorly soluble diuretic agent. The aim of the present work was to produce and characterise a potassium salt form of chlorothiazide which has the potential advantages of improved aqueous solubility and potassium supplementation. A number of novel potassium salt forms of CTZ (CTZK) were prepared: CTZK monohydrate (form I), CTZK dihydrate (form II), anhydrous CTZK (form III), CTZK monohydrate hemiethanolate (form IV) and a desolvate of CTZK monohydrate hemiethanolate (form V). These salt forms were characterised by thermal analysis, PXRD, NMR, elemental analysis, FTIR, Karl Fisher titrimetry, ICP-MS and GC-MS. The ethanol-free CTZK forms were also characterised by dynamic vapour sorption analysis (DVS). CTZK form I was stable (in the DVS) over the range 0-60% RH. The dihydrate form of the salt was stable (in the DVS) over a broader range of relative humidities, 10-90% RH at 25°C. CTZK form II was less hygroscopic at high humidities (70-90% RH) than the previously reported CTZNa dihydrate. Single crystal X-ray analysis indicated that chlorothiazide potassium, crystallised from water or water/acetone mixture, formed a dihydrated polymeric-like intermolecular self-assembly (ISA) suprastructure. The ISA coordination was determined to be: (CTZ)(3)·K·(H(2)O)(2)(CTZ)(2)·(H(2)O)(2)·K·(CTZ)(3) (monoclinic, space group: C2/c, single crystal cell parameters: a=18.328(4)Å, b=7.3662(16)Å, c=19.993(5)Å, α=90°, ß=99.729(3)°, γ=90°). When CTZK was crystallised from ethanol, a monohydrate hemiethanolate ISA was formed, described as (CTZ)(3)·K·CTZ·(H(2)O)(2)·CTZ·K·(CTZ)(2) (triclinic, space group: P-1, single crystal cell parameters: a=7.078(3)Å, b=9.842(5)Å, c=21.994(11)Å, α=87.522(13)°, ß=84.064(14)°, γ=78.822(12)°). The aqueous solubility of CTZK dihydrate, was determined to be 78.71±1.82mg/ml, approximately 400-fold higher than chlorothiazide, indicating a biopharmaceutical advantage associated with the potassium salt form.

Multi-residue analytical method for human pharmaceuticals and synthetic hormones in river water and sewage effluents by solid-phase extraction and liquid chromatography-tandem mass spectrometry.

Pollutants such as human pharmaceuticals and synthetic hormones that are not covered by environmental legislation have increasingly become important emerging aquatic contaminants. This paper reports the development of a sensitive and selective multi-residue method for simultaneous determination and quantification of 23 pharmaceuticals and synthetic hormones from different therapeutic classes in water samples. Target pharmaceuticals include anti-diabetic, antihypertensive, hypolipidemic agents, ß2-adrenergic receptor agonist, antihistamine, analgesic and sex hormones. The developed method is based on solid phase extraction (SPE) followed by instrumental analysis using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with 30 min total run time. River water samples (150 mL) and (sewage treatment plant) STP effluents (100 mL) adjusted to pH 2, were loaded into MCX (3 cm(3), 60 mg) cartridge and eluted with four different reagents for maximum recovery. Quantification was achieved by using eight isotopically labeled internal standards (I.S.) that effectively correct for losses during sample preparation and matrix effects during LC-ESI-MS/MS analysis. Good recoveries higher than 70% were obtained for most of target analytes in all matrices. Method detection limit (MDL) ranged from 0.2 to 281 ng/L. The developed method was applied to determine the levels of target analytes in various samples, including river water and STP effluents. Among the tested emerging pollutants, chlorothiazide was found at the highest level, with concentrations reaching up to 865 ng/L in STP effluent, and 182 ng/L in river water.

Preparation and solid state characterisation of chlorothiazide sodium intermolecular self-assembly suprastructure.

Chlorothiazide (CTZ), unlike other thiazide diuretics, can form salts. An injectable formulation containing the sodium salt is available; however neither the physicochemical characteristics of the salt nor its solid state form have been previously reported. This work reports on the crystal structure of chlorothiazide sodium. The structure was investigated by single crystal X-ray and nuclear magnetic spectroscopy (NMR) analyses and compared to chlorothiazide, while the solid state characteristics were assessed by thermal analysis, powder X-ray diffraction, infrared spectroscopy, dynamic moisture sorption and solubility analysis. The crystal structure of chlorothiazide sodium was determined to be triclinic; the crystal space group type was P-1. Chlorothiazide sodium presented a self-assembly polymeric-type suprastucture, where the unit cell comprised two chlorothiazide molecules bonded together with sodium cations through the water bridges. The coordinate centre comprised the following: (CTZ)(3)·(H(2)O)·Na(H(2)O)(2)Na·(H(2)O)·(CTZ)(3). The crystalline material was determined to be a monosodium dihydrate, stable in the range of 10-90% relative humidity (RH) at 25°C. Additional processing of the salt resulted in a crystalline anhydrous form which was stable in the range 0-20% RH at 25°C. The aqueous solubility of the chlorothiazide sodium dihydrate at 37°C was found to be approximately 400-fold higher than that of chlorothiazide, which may present biopharmaceutical advantages for the salt compared to the non-salt form.

A simple and rapid high-performance liquid chromatographic method for the determination of bisoprolol fumarate and hydrochlorothiazide in a tablet dosage form.

A simple and precise high performance liquid chromatographic method has been developed and validated for the simultaneous determination of bisoprolol fumarate (BF), and hydrochlorothiazide (HCTZ) in a tablet formulation. Chromatography was carried out at 25 degrees C on a 4.6 mm x 250 mm, 5 microm cyano column with the isocratic mobile phase of 0.1M aqueous phosphate buffer, acetonitrile and tetrahydrofuran (85:10:5, v/v/v) at a flow rate of 1.0 ml/min. The UV detection was carried out at 225 nm. HCTZ and BF were separated in less than 10 min with good resolution and minimal tailing, without interference of excipients. The method was validated according to ICH guidelines and the acceptance criteria for accuracy, precision, linearity, specificity and system suitability were met in all cases. The method was linear in the range of 50-150 microg/ml for BF and 125-375 microg/ml for HCTZ.

Accurate molecular structures of chlorothiazide and hydrochlorothiazide by joint refinement against powder neutron and X-ray diffraction data.

The compounds chlorothiazide and hydrochlorothiazide (crystalline form II) have been studied in their fully hydrogenous forms by powder neutron diffraction on the GEM diffractometer. The results of joint Rietveld refinement of the structures against multi-bank neutron and single-bank X-ray powder data are reported and show that accurate and precise structural information can be obtained from polycrystalline molecular organic materials by this route.

Solving molecular crystal structures from X-ray powder diffraction data: the challenges posed by gamma-carbamazepine and chlorothiazide N,N,-dimethylformamide (1/2) solvate.

The crystal structures of gamma-carbamazepine (P1, Z' = 4) and chlorothiazide N,N-dimethylformamide solvate (1/2) (P2(1)/c, Z' = 2) have been determined from synchrotron and laboratory X-ray powder diffraction data, respectively, using simulated annealing. Both structures represent a significant challenge for global optimization and the successful solutions and subsequent refinements highlight the ever-expanding range of applicability of powder diffraction to structural problems of pharmaceutical relevance.

Encapsulation of chlorothiazide in whey proteins: effects of wall-to-core ratio and cross-linking conditions on microcapsule properties and drug release.

A model drug with limited water-solubility, chlorothiazide, was successfully encapsulated in whey protein-based wall systems cross-linked by glutaraldehyde-saturated toluene via an organic phase. The effects of drug content of the core-in-wall suspension and of cross-linking conditions on core retention and on microcapsule size, structure and core release properties were investigated. Spherical, surface cracks-free microcapsules ranging in diameter from approximately 200-1300 microm were obtained. Particle size distribution of microcapsules was affected by core content and cross-linking conditions. Core retention in microcapsules prepared at different cross-linking conditions and different wall-to-core ratios ranged from 48.9-81%, from 42.2-76.1% and from 37.3-67.2% in large (L), medium-size (M) and small (S) microcapsules, respectively. In all cases, drug crystals were physically entrapped and embedded throughout the cross-linked protein matrix. Core release from the microcapsules into enzyme-free simulated gastric fluid was governed by a diffusion-controlled mechanism and did not involve erosion or softening of the wall matrix. Rate of core release was significantly affected by a combined influence of core content, microcapsule size and cross-linking density. Complete core release from L, M and S microcapsule prepared at different wall-to-core ratios and cross-linking conditions ranged from 28.6-81.2 h, from 16.8-28.6 h and from 7.2-15.9 h, respectively. Results suggested that whey protein-based wall matrix cross-linked by GAST may provide significant opportunities in modulating the release of an encapsulated core with a limited water solubility.

Physicochemical properties of liposomes incorporating hydrochlorothiazide and chlorothiazide.

In an attempt to study the effect of hydrophobic drugs on liposome properties, multilamellar liposomes (MLV) consisting of phosphatidylcholine (PC) and incorporating chlorothiazide (CT) or hydrochlorothiazide (HCT), were prepared and characterized. Liposome size, surface charge, stability (in buffer, plasma and sodium cholate) and calcium-induced aggregation were studied for drug-incorporating liposomes and empty liposomes for comparison. Results show that drug incorporation affects liposome size, z-potential and stability in presence of buffer and plasma proteins. Indeed, drug-incorporating liposomes are slightly larger and have a negative surface charge, which increases with the amount of drug incorporated in the lipid membrane. The membrane integrity of drug incorporating liposomes (in absence and presence of plasma proteins) is significantly higher when compared with that of empty liposomes (for both drugs studied). On the contrary, vesicle membrane integrity in presence of sodium cholate and calcium induced vesicle aggregation, are not affected by drug incorporation. Leakage of thiazides from liposomes was demonstrated to be induced by dilution. Low amounts of thiazides (around 10-15%) are released when lipid concentration is over 0.1 mM, while further dilution increased drug leakage exponentially. Concluding, results demonstrate that the presence of HCT or CT in liposome membranes has a significant effect on main vesicle properties, which are known to influence vesicle targeting ability. Thereby, it is very interesting to continue studies in this respect, especially with more lipophilic drugs.

Preparation of albumin microspheres by an improved process.

Albumin is widely used to prepare microspheres and microcapsules. In this study microspheres were prepared by the suspension crosslinking method for the first time in the absence of any surface active agent, using paraffin oil as the dispersion medium and formaldehyde as the crosslinking agent. The microspheres thus obtained were characterized using a Scanning Electron Microscope and found to be spherical and having a particle size distribution in the range 50-400 microns. A preliminary drug release study of chlorothiazide in-vitro indicated a diffusion controlled release of the drug. This method, being simple and cost effective, could be a promising technique for the large scale manufacture of microspheres.

Interactions of diuretics with a neutral temperature-responsive polymer: study by capillary electroporesis and dynamic light scattering.

Interactions between diuretics and a recently synthesized temperature-responsive neutral copolymer, poly(N-isopropyl acrylamide) (PNIPA) grafted with poly(ethyleneoxide) (PEO) (PNIPA-g-PEO) were investigated by capillary electrophoresis and dynamic light scattering (DLS). At ambient temperatures, the copolymer takes an open, random coil conformation, but above the lower critical solution temperature (LCST), the polymer shrinks and forms large, stable aggregates. Among the diuretics studied, hydrochlorothiazide was detected to bind to PNIPA-g-PEO.

Spectrophotometric studies on the photostability of some thiazide diuretics in ethanolic solution.

First derivative and dual-wavelength spectrophotometric methods were used in the quantum yield determination of the photochemical decomposition reactions of three thiazide diuretics (chlorothiazide, hydrochlorothiazide and trichloromethiazide) in ethanolic solution. The radiation absorbed by the compounds was measured using iron(III) oxalate actinometry based on absorption spectrophotometry. An apparatus is described in which the drugs were irradiated in quartz cuvettes cooled by water in a stand built on a magnetic stirrer. The wavelength region available to the reaction cuvette was restricted to 313 nm with chemical potassium chromate filter solutions and a Corning filter plate. Chlorothiazide proved to be more photostable than hydrochlorothiazide and trichloromethiazide in ethanol.

Identification of chlorothiazide and hydrochlorothiazide UV-A photolytic decomposition products.

Methanol solutions of hydrochlorothiazide and chlorothiazide were irradiated with fluorescent UV-A lamps in order to simulate degradation under normal conditions. The degradation products were identified by comparison to synthetic standards featuring electrospray ionization mass spectroscopy, ultraviolet spectroscopy, and high performance liquid chromatography. The standards were characterized by high resolution fast atom bombardment MS and 1H NMR. The photolysis of chlorothiazide resulted in photodehalogenation products exclusively, while the irradiation of hydrochlorothiazide primarily yielded photodehalogenation products with significant yields of photodehydrogenation products and minor amounts of thermal hydrolysis products.

First-derivative UV spectrophotometric and high-performance liquid chromatographic analysis of some thiazide diuretics in the presence of their photodecomposition products.

Ethanolic solutions of three thiazide diuretics, chlorothiazide, hydrochlorothiazide and trichlormethiazide, were irradiated with a high-pressure mercury lamp. The products were isolated and their first-derivative UV spectra in ethanol were recorded and compared to those of the parent compounds. The determination of the parent compounds in the presence of the decomposition products was carried out at wavelengths near 220 nm using the zero-crossing technique. Three reversed-phase HPLC methods were also developed for the analysis of the parent compounds. In parallel analyses of the reaction mixtures a good correlation was achieved between these two methods in the determination of hydrochlorothiazide and trichlormethiazide while there was greater variation in the results of chlorothiazide.

In vivo-in vitro correlations with a commercial dissolution simulator. I: Methenamine, nitrofurantoin, and chlorothiazide.

Dissolution profiles were determined for nine methenamine, 14 nitrofurantoin, and six chlorothiazide dosage forms using a dissolution simulator. Various in vivo-in vitro correlations were examined. The best correlation for methenamine was between the maximum urinary excretion rate and the time for 15% dissolution. A good correlation for the 50-mg nitrofurantoin tablets was also found between cumulative percent of drug excreted in 12 hr and the percent dissolved in 1 hr. There were no significant correlations for the 100-mg nitrofurantoin dosage forms. Good correlations were also observed for the 250- and 500-mg chlorothiazide tablets between the percent of drug dissolved in 1 min or the time for 15% dissolution and the maximum excretion rate.