Development and validation of a simple chromatographic method for simultaneous determination of clindamycin phosphate and rifampicin in skin permeation studies.

Rifampicin (RIF) and clindamycin phosphate (CDM) are the main drugs currently used in combination to treat severe infectious diseases in hair follicles. This work describes a simple, rapid and sensitive method for simultaneous analysis of RIF and CDM in the different skin layers using high performance liquid chromatography (HPLC). The efficient chromatographic separation of CDM and RIF was succeeded using a C18 column (150 mm x 4.6 mm, 5 µm) with gradient elution using a mobile phase composed of 0.01 M phosphoric acid and methanol at a flow rate of 1 mL min-1. Determinations were performed using UV-vis detector at 200 nm and 238 nm for CDM and RIF, respectively. The method was precise, accurate and linear (r2 > 0.999) with regression curve in the concentration range from 0.5 to 20.0 µg mL-1 and recovery rates from the skin layers higher than 85%. The retention times for CDM and RIF were approximately 7.4 and 12.2 min, respectively. The presence of skin components did not interfere with the analysis. The validated method was therefore appropriate for quantification of both CDM and RIF and thus may be feasible to be used in skin permeation studies.

Novel electrochemical biosensor based on PVP capped CoFe2O4@CdSe core-shell nanoparticles modified electrode for ultra-trace level determination of rifampicin by square wave adsorptive stripping voltammetry.

This work introduces a new electrochemical sensor based on polyvinyl pyrrolidone capped CoFe2O4@CdSe core-shell modified electrode for a rapid detection and highly sensitive determination of rifampicin (RIF) by square wave adsorptive stripping voltammetry. The new PVP capped CoFe2O4@CdSe with core-shell nanostructure was synthesized by a facile synthesis method for the first time. PVP can act as a capping and etching agent for protection of the outer surface nanoparticles and formation of a mesoporous shell, respectively. Another important feature of this work is the choice of the ligand (1,10-phenanthroline) for precursor cadmium complex that works as a chelating agent in order to increase optical and electrical properties and stability of prepared nanomaterial. The nanoparticles have been characterized by field emission scanning electron microscopy (FESEM), transmission electron microscope (TEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), UV-vis, photoluminescence (PL) spectroscopy, FT-IR, and cyclic voltammetry techniques. The PL spectroscopy study of CoFe2O4@CdSe has shown significant PL quenching by the formation of CoFe2O4 core inside CdSe, this shows that CoFe2O4 NPs are efficient electron acceptors with the CdSe. It is clearly observed that the biosensor can significantly enhance electrocatalytic activity towards the oxidation of RIF, under the optimal conditions. The novelty of this work arises from the new synthesis method for the core-shell of CoFe2O4@CdSe. Then, the novel electrochemical biosensor was fabricated for ultra-trace level determination of rifampicin with very low detection limit (4.55×10-17M) and a wide linear range from 1.0×10-16 to 1.0×10-7M. The fabricated biosensor showed high sensitivity and selectivity, good reproducibility and stability. Therefore, it was successfully applied for the determination of ultra-trace RIF amounts in biological and pharmaceutical samples with satisfactory recovery data.

Absolute Quantification of Rifampicin by MALDI Imaging Mass Spectrometry Using Multiple TOF/TOF Events in a Single Laser Shot.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R2) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%. Graphical Abstract ᅟ.

Nickel hydroxide nanoparticles-reduced graphene oxide nanosheets film: layer-by-layer electrochemical preparation, characterization and rifampicin sensory application.

Electrochemical deposition, as a well-controlled synthesis procedure, has been used for subsequently layer-by-layer preparation of nickel hydroxide nanoparticle-reduced graphene oxide nanosheets (Ni(OH)2-RGO) on a graphene oxide (GO) film pre-cast on a glassy carbon electrode surface. The surface morphology and nature of the nano-hybrid film (Ni(OH)2-RGO) was thoroughly characterized by scanning electron and atomic force microscopy, spectroscopy and electrochemical techniques. The modified electrode appeared as an effective electro-catalytic model for analysis of rifampicin (RIF) by using linear sweep voltammetry (LSV). The prepared modified electrode exhibited a distinctly higher activity for electro-oxidation of RIF than either GO, RGO nanosheets or Ni(OH)2 nanoparticles. Enhancement of peak currents is ascribed to the fast heterogeneous electron transfer kinetics that arise from the synergistic coupling between the excellent properties of RGO nanosheets (such as high density of edge plane sites, subtle electronic characteristics and attractive π-π interaction) and unique properties of metal nanoparticles. Under the optimized analysis conditions, the modified electrode showed two oxidation processes for rifampicin at potentials about 0.08 V (peak I) and 0.69 V (peak II) in buffer solution of pH 7.0 with a wide linear dynamic range of 0.006-10.0 µmol L(-1) and 0.04-10 µmol L(-1) with a detection limit of 4.16 nmol L(-1) and 2.34 nmol L(-1) considering peaks I and II as an analytical signal, respectively. The results proved the efficacy of the fabricated modified electrode for simple, low cost and highly sensitive medicine sensor well suited for the accurate determinations of trace amounts of rifampicin in the pharmaceutical and clinical preparations.

LC-MS based assay to measure intracellular compound levels in Mycobacterium smegmatis: linking compound levels to cellular potency.

Dihydrofolate reductase (DHFR) plays a central role in maintaining cellular pool of tetrahydrofolic acid, a cofactor necessary for DNA, RNA and protein synthesis. The clinical validation of DHFR as antibacterial target was established by the success of trimethoprim (TMP). DHFR is also an attractive target for identifying anti-tuberculosis molecules however, due to observed weak cellular potency, no DHFR inhibitors have been developed as drugs so far. TMP and its analogs have poor cellular potency on Mycobacterium tuberculosis and Mycobacterium smegmatis cells. We found a mutant strain of M. smegmatis, mc²155 to be sensitive to TMP whereas wild type strain was not inhibited by TMP. We utilized this system to probe if poor or lack of activity of TMP is a consequence of poor intracellular compound levels. An LC-MS based method was developed for measuring TMP and rifampicin (RIF) in M. smegmatis. Using the assay, equivalent RIF levels were observed in both strains however, TMP was detected only in mc²155 cells, hence proving a positive correlation between potency and compound levels. To the best of our knowledge this is the first time LC-MS method has been used to measure compound levels in mycobacterial cells. We propose it to be a valuable tool to understand the lack of potency or resistance mechanisms in antimycobacterial drug development.

Formulation and in vitro characterization of inhalable rifampicin-loaded PLGA microspheres for sustained lung delivery.

The solvent evaporation method with premix membrane homogenization was applied, with class-3 ethyl acetate as organic solvent, to produce narrowly size-distributed rifampicin (RIF)-loaded poly(lactide-co-glycolide) (PLGA) microspheres for sustained lung delivery as aerosol. Microsphere formulations (simple or multiple emulsions, different PLGA and RIF concentrations) and process parameters (transmembrane pressure, SPG membrane pore diameter) were investigated as their effects on RIF content, microsphere size, aerodynamic properties of the freeze-dried powder and in vitro release profiles. Narrowly size distributed microspheres with diameters from 2 to 8 µm, satisfactory RIF contents (from 4.9 to 16.5%), 80% RIF release from 12h to 4 days, and adequate aerodynamic properties were prepared from a multiple emulsion and using SPG membrane pore diameter of 19.9 µm. The premix membrane homogenization appeared to be a rapid and efficient method to prepare monodisperse drug-loaded microspheres suitable for lung delivery as sustained-release microsphere aerosol.

Gastroretentive delivery of rifampicin: in vitro mucoadhesion and in vivo gamma scintigraphy.

Rifampicin, a first line anti-tubercular drug, has maximum solubility and permeability in the stomach. An oral multi-particulate formulation with site specific sustained delivery of rifampicin was developed. This oral gastroretentive rifampicin formulation consisted of rifampicin pellets for immediate release as the loading dose and a bio/mucoadhesive rifampicin tablet for extended release. Immediate release pellets of rifampicin were prepared by extrusion-spheronization process and were evaluated for physico-mechanical properties: usable yield, size, shape, abrasion resistance, mechanical crushing force, residual moisture and drug release. For the mucoadhesive rifampicin formulation, statistical experimental strategy was utilized to simultaneously optimize the effect of two independent variables namely amount of Carbopol and MCC. The two dependent responses selected were, work of adhesion; estimated using Texture Analyzer and T(50%); determined from dissolution studies. Graphical and mathematical analysis of the results allowed the identification and quantification of the formulation variables influencing the selected responses. To study the gastrointestinal transit of the optimized gastroretentive formulation, the in vivo gamma scintigraphy was carried out in six healthy human volunteers, after radiolabeling the formulation with (99m)Tc. The transit profiles demonstrated that the dosage form was retained in the stomach for more than 320 min. The human data validates the design concept and signifies the potential of the developed system for stomach targeted delivery of rifampicin for improved bioavailability.

A new respirable form of rifampicin.

The aim of this research was to investigate a novel dry powder formulation of rifampicin (RF) that presents an improved lung deposition profile by means of a polymorphic transformation into a flake-like crystal hydrate. Rifampicin dihydrate (RFDH) was prepared by recrystallization of RF in anhydrous ethanol. A control formulation, amorphous RF (RFAM) was prepared by spray drying. The physicochemical properties of the RFDH and the RFAM were characterized. Aerosol performances of RFDH and RFAM were studied with two dry powder inhalers (DPIs), an Aerolizer and a Handihaler, using a Next Generation Impactor (NGI). The RFDH powder was successfully prepared using simple recrystallization process and had a MMAD of 2.2 µm. The RFDH powders were characterized as having a very thin flaky structure; this unique morphology provided improved aerosolization properties with a decreased device dependency upon aerosolization. The flaky morphology of RFDH resulted in a reduced agglomeration tendency than that of spherical RFAM particles. The maximum fine particle fraction (FPF(TD)) of 68% for the RFDH was achieved with the Aerolizer device. Significant chemical degradation was not observed from the RFDH, while the RFAM showed significant chemical degradation at 9 months. The excipient-free formulation of the RFDH offers the benefit of delivering a maximum potency formulation, of the antibiotic, directly to the site of infection, the lung.

Development and validation of indirect RP-HPLC method for enantiomeric purity determination of D-cycloserine drug substance.

A new chiral purity method was developed for D-cycloserine (D-cys) by reverse phase HPLC and validated. Chiral derivatizing reagents, viz., o-phthalaldehyde and N-acetyl-L-cysteine were utilized in this method. The resultant diastereomers were resolved using Zorbax SB Phenyl HPLC column under isocratic elution. A mobile phase of 95:05 (v/v), 20mM Na(2)HPO(4) (pH 7), and acetonitrile, respectively, was used with the flow rate of 1.0 mL/min and UV detection at 335 nm. The method development with different chiral stationary phases and chiral derivatization reagents were also investigated. The stability of diastereomer derivative and influence of organic modifier and pH of the mobile phase were studied and optimized. The stability-indicating capability of the method was established by performing stress studies under acidic, basic, oxidation, light, humidity and thermal conditions. The detection and quantitation limit of L-cycloserine (L-cys) were 0.015 and 0.05% (w/w), respectively. A linear range from 0.05 to 0.30% (w/w) was obtained with the coefficient of determination (r(2)) 0.998. The recovery obtained for L-cys was between 92.9 and 100.2%. This method was applied successfully in pharmaceutical analysis to determine the content of L-cys in D-cys bulk drug.

Simultaneous determination of isoniazid, rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography-tandem mass spectrometry.

A rapid and selective high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of isoniazid (INH), rifampicin (RFP) and levofloxacin (LVX) in mouse tissues and plasma has been developed and validated, using gatifloxacin as the internal standard (I.S.). The compounds and I.S. were extracted from tissue homogenate and plasma by a protein precipitation procedure with methanol. The HPLC separation of the analytes was performed on a Welch materials C4 column (250mmx4.6mm, 5.0microm, USA) at 25 degrees C, using a gradient elution program with the initial mobile phase constituting of 0.05% formic acid and methanol (93:7, v/v) at a flow-rate of 1.0ml/min. For all the three analytes, the recoveries varied between 83.3% and 98.8% in tissues and between 75.5% and 90.8% in plasma, the accuracies ranged from 91.7% to 112.0% in tissues and from 94.6% to 108.8% in plasma, and the intra- and inter-day precisions were less than 13.3% in tissues and less than 8.2% in plsama. Calibration ranges for INH were 0.11-5.42microg/g in tissues and 0.18-9.04microg/ml in plasma, for RFP were 0.12-1200microg/g in tissues and 4.0-200microg/ml in plasma, and for LVX were 0.13-26.2microg/g in tissues and 0.09-4.53microg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11, 0.12 and 0.13microg/g and for those in mouse plasma were 18.1, 20.0 and 21.8ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04, 0.05 and 0.05microg/g and for those in mouse plasma were 5.5, 6.0 and 6.6ng/ml, respectively. The established method was successfully applied to simultaneous determination of isoniazid, rifampicin and levofloxacin in mouse plasma and different mouse tissues.

LC-MS/TOF and UHPLC-MS/MS study of in vivo fate of rifamycin isonicotinyl hydrazone formed on oral co-administration of rifampicin and isoniazid.

The formation and fate of 3-formylrifamycin isonicotinyl hydrazone (HYD) was investigated following oral co-administration of rifampicin (RIF) and isoniazid (INH) in Sprague-Dawley (SD) rats (n=5) using advanced analytical modalities. The study was carried out with 20 and 5mg/kg doses of RIF and INH, respectively. The plasma, urine and faeces samples were collected at different time points up to 48h, which were qualitatively and quantitatively evaluated for the presence of HYD after proper sample preparation. For the same, initially liquid chromatography-mass spectrometry/time-of-flight (LC-MS/TOF) method was developed in electrospray ionization (ESI) positive mode, wherein separation was achieved on a C18 column (4.6mmx250mm, 5microm), using a volatile mobile phase in a gradient mode. The presence of HYD was confirmed by accurate mass study, spiking with the standard and UV-visible spectra matching. For quantitative evaluation of HYD, a selective and sensitive ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for all the three matrices. In this case, elution of HYD was achieved on a small C18 column (4.6mmx50mm, 1.8microm) using a short gradient method. The quantitation was done by selective reaction monitoring (SRM) in ESI positive mode. The validation parameters like linearity, accuracy, precision, selectivity, matrix effect, recovery and stability were assessed as per regulatory guidelines. The calibration range was established between 1 and 200ng/ml, with r(2)>0.99 in all the cases. The back calculated values for three quality control (QC) samples, and at lower limit of quantitation (LLOQ) were within 15 and 20%, respectively, of the nominal values. Similarly, the intra- and inter-day precisions were found within 15% at the four tested levels. The HYD was found to be stable for the duration of sample preparation and analysis in the controlled experimental conditions. The analysis of in vivo samples showed a significant extent of HYD in faeces, however, the interaction product was not found in plasma and urine. To verify the results, 5mg/kg oral dose of HYD standard was given to rats separately, and its presence was studied in all the three matrices. Further, in vitro plasma stability of HYD was also carried out to explain its absence in plasma and urine, which showed approximately 55% disappearance of HYD in 2h.

Tuberculosis multirresistente en la infancia / Multiresistant tuberculosis in childhood

No disponible

Development of a liquid chromatographic method for the determination of related substances and assay of d-cycloserine.

d-cycloserine or d-4-amino-3-isoxazolidinone is an antibiotic produced by Streptomyces garyphalus and Streptomyces orchidaceus. d-Cycloserine is used in the second line treatment of tuberculosis and is often used in developing countries. Therefore, expensive high-tech techniques are not recommended for analysis. Here, a liquid chromatography method with ultraviolet detection (LC-UV) is described using a base deactivated column (Hypersil BDS column; 25 cm x 4.6 mm I.D.) kept at 45 degrees C. The gradient method uses mobile phases containing acetonitrile (ACN), 20mM sodium octane sulphonate (SOS), 0.2M potassium dihydrogen phosphate buffer pH 2.8, water: A: (4:70:10:16v/v/v/v) and B: (17:70:10:3v/v/v/v). The method proved to be robust, linear, repeatable, sensitive, selective and easy to perform. For the related substances test 50 microl of a 0.5 mg/ml d-cycloserine solution is injected. For assay, a concentration of 0.1 mg/ml is proposed to avoid overloading of the detector.

In vitro analysis of rifampicin and its effect on quality control tests of rifampicin containing dosage forms.

The chemical stability of rifampicin both in solid state and various media has widely been investigated. While rifampicin is appreciably stable in solid-state, its decomposition rate is very high in acidic as well as in alkaline medium and a variety of decomposition products were identified. The literature reports on highly variable rifampicin decomposition in acidic medium. Hence, the objective of this investigation was to study possible reasons responsible for this variability. For this purpose, filter validation and correlation between rifampicin and its degradation products were developed to account for the loss of rifampicin in acidic media. For analysis of rifampicin with or without the presence of isoniazid, a simple and accurate method was developed using high performance chromatography recommended in FDC monographs of the United States Pharmacopoeia. Using the equations developed in this investigation, the amount of rifampicin degraded in the acidic media was calculated from the area under curve of the degradation products. Further, it was proved that in a dissolution study, the colorimetric method of analysis recommended in the United States Pharmacopoeia provides accurate results regarding rifampicin release. Filter type, time of injection as well as interpretation of data are important factors that affect analysis results of rifampicin in in vitro studies and quality control.

UV spectroscopy and reverse-phase HPLC as novel methods to determine Capreomycin of liposomal fomulations.

Capreomycin (CS) is an antitubercular drug active against several Mycobacterium strains, in particular, against M. Avium. In spite of its activity, it is considered a second line drug because it can induce severe renal and hepatic damages when administered as free drug. However, it is possible to employ drug delivery systems, such as liposomes, to reduce the toxicity of the peptide without loss of its biological activity. For this purpose, appropriately validated time and money saving analytical methods are needed for a careful capreomycin dosage. In the present paper, UV spectroscopy and a reverse-phase HPLC (RP-HPLC) were investigated as alternative methods for capreomycin quantitative analysis. These techniques were validated against the USP XXVI microbiological turbidimetric assay and the normal-phase HPLC (NP-HPLC) method reported in the British Pharmacopoeia 2003. The results obtained showed that either UV spectrophotometry or RP-HPLC are techniques having higher accuracy and reproducibility with respect to the microbiological assay. Moreover, the RP-HPLC method provided improved performances if compared to NP-HPLC. In fact, RP-HPLC showed: (i) enhanced sensitivity and (ii) increased resolution. Thus we propose RP-HPLC and UV as valid alternative methods to the conventional procedures for capreomycin quantitative analysis.

High-performance liquid-chromatographic determination of rifampicin in plasma and tissues.

A HPLC-UV method has been developed for assaying rifampicin in plasma and liver. The assay involved a liquid-liquid extraction procedure with dichloromethane-pentane (1:1). An Ultrabase-C18 column and a simple mobile phase consisting of a water (pH 2.27)-acetonitrile (40:60, v/v) mixture were used. The flow-rate was 1 ml/min and the effluent was monitored at 333 nm. Results from the HPLC analyses showed that the assay method is linear in the ranges 0.1-1 and 1-50 microg/ml for plasma, and 0.6-40 microg/g for liver. Intra- and inter-day R.S.D. were below 15% for all the sample types. Recoveries averaged 83 and 95% for plasma and liver, respectively. The method is being successfully applied to determine rifampicin in plasma and liver samples taken during pharmacokinetic studies in rats.

Evaluation of the recently reported USP gradient HPLC method for analysis of anti-tuberculosis drugs for its ability to resolve degradation products of rifampicin.

The recently notified USP gradient HPLC method for quantitative determination of rifampicin, isoniazid and pyrazinamide in fixed dose combination (FDC) formulations was evaluated to determine its ability to resolve major degradation products of rifampicin, viz. 3-formylrifamycin SV, rifampicin N-oxide, 25-desacetyl rifampicin, rifampicin quinone, and the newly reported isonicotinyl hydrazone, an interaction product of 3-formylrifamycin and isoniazid. The first observation was that the requirements of theoretical plates listed in the given method were met for rifampicin, but not for isoniazid and pyrazinamide, even on columns of different makes. The resolving power of the method was also dependent upon make of the column. On two of the three columns of the three tested, it was able to resolve most degradation products, except rifampicin N-oxide and 25-desacetylrifampicin, which were overlapping. The method was modified and an overall satisfactory resolution for all components was obtained by changing the buffer: organic modifier ratio of solution B in the gradient from 45:55 to 55:45 and decreasing the flow rate from 1.5 to 1.0 ml/min, keeping all other conditions constant.

Electrochemical behavior and differential pulse polarographic determination of rifampicin in the pharmaceutical preparations.

Differential pulse polarographic (DPP) analytical procedure for the rifampicin antibiotic, which can be applied to monitor its synthetic process from the starting antibiotic of rifamycin B or rifamycin SV, has been developed based on the electrochemical reduction of an azomethine group. Rifampicin exhibited a cathodic peak due to the azomethine group in the side chain of 3-[(4-methyl-1-piperazinyl)imino]methyl moiety and another cathodic peak due to the carbonyl group in rifamycin SV by DPP The experimental peak potential shift of an azomethine reduction was -73 mV/pH in the pH range between 3.0 and 7.5, agreeing with involvement of 4 e- and 5 H+ in its reduction. By the cyclic voltammetric(CV) studies, the azomethine and the carbonyl reductions in rifampicin were processed irreversibly on the mercury electrode. The plot of peak currents vs. concentrations of rifampicin ranging 1.0 x 10(-7) M tp approximately 1.0 x 10(-5) M yielded a straight line with a correlation coefficient of 0.9996. The detection limit was 1.0 x 10(-8) M with a modulation amplitude of 50 mV. DPP has been successfully applied for the determination of rifampicin in the pharmaceutical preparations.

Characterization of the solubility and dissolution properties of several new rifampicin polymorphs, solvates, and hydrates.

Based on reports that tuberculosis is on the increase, this investigation into the physicochemical properties of rifampicin when recrystallized from various solvent systems was undertaken. Rifampicin is an essential component of the currently recommended regimen for treating tuberculosis, although relatively little is known about its solubility and dissolution behavior in relation to its solid-state properties. A rifampicin monohydrate, a rifampicin dihydrate, two amorphous forms, a 1:1 rifampicin:acetone solvate, and a 1:2 rifampicin:2-pyrrolidone solvate were isolated and characterized using spectral, thermal, and solubility measurements. The crystal forms were relatively unstable because except for the 2-pyrrolidone solvate, all the hydrated or solvated materials changed to amorphous forms after desolvation. Fourier transform infrared (FTIR) analysis confirmed the favorable three-dimensional organization of the pharmacophore to ensure antibacterial activity in all the crystal forms except the 2-pyrrolidone solvate. In the 2-pyrrolidone solvate, the strong IR signals of 2-pyrrolidone interfered with the vibrations of the ansa group. The 2-pyrrolidone solvate was the most soluble in phosphate buffer at pH 7.4. This solvate also had the highest solubility (1.58 mg/ml) and the fastest dissolution in water. In 0.1 M HCl, the dihydrate dissolved the quickest. A X-ray amorphous form (amorph II) was the least soluble and had the slowest dissolution rate because the powder was poorly wettable and very electrostatic.