Detection and structure elucidation of the new degradation impurities in the pharmaceutical formulations of ruxolitinib hydrobromide.

New degradation impurities at m/z 327.15 and m/z 311.16 using gradient UHPLC method with UV detection and highly selective QDa mass detection were observed during the ruxolitinib hydrobromide (RUX.HBr) : excipient binary mixture degradation study. High mass resolution LC-MS and nuclear magnetic resonance (NMR) techniques were employed to identify and fully characterize the degradation compounds. The degradation impurities were unambiguously identified as (R)-4-amino-6-(1-(2-cyano-1-cyclopentylethyl)-1H-pyrazol-4-yl)pyrimidine-5-carboxylic acid and (R)-3-(4-(6-amino-5-formylpyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile and mechanism of their formation was proposed. It has been confirmed that the degradation products are formed in mixtures of RUX.HBr with some excipients in the presence of oxygen. Based on the forced degradation study, the chemically stable of pharmaceutical formulations were prepared to eliminate the formation of these impurities.

Identification and structure elucidation of a new degradation impurity in the multi-component tablets of amlodipine besylate.

New unknown impurity at m/z 421.15 was observed during the accelerated stability analysis (40 °C/75% relative humidity) in the multi-component tablets of amlodipine besylate by reversed-phase ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). UHPLC-MS and nuclear magnetic resonance (NMR) techniques were employed to identify and fully characterize the degradation compound. The degradation product was unambiguously identified as 3-ethyl 5-methyl 4-(2-chlorophenyl)-6-methyl-2-(morpholin-2-yl)-1,4-dihydropyridine-3,5-dicarboxylate and mechanism of its formation was proposed. It was confirmed that the degradation product was formed by the reaction of amlodipine with formaldehyde originating from the excipients present in the dosage form.

Esterification of Ibuprofen in Soft Gelatin Capsules Formulations-Identification, Synthesis and Liquid Chromatography Separation of the Degradation Products.

Unknown impurities were identified in ibuprofen (IBU) soft gelatin capsules (SGCs) during long-term stability testing by a UHPLC method with UV detection and its chemical formula was determined using high resolution/accurate mass (HRAM) LC-MS. Reference standards of the impurities were subsequently synthesized, isolated by semi-preparative HPLC and characterized using HRAM LC-MS, NMR and IR. Two impurities were formed by esterification of IBU with polyethylene glycol (PEG), which is used as a fill of the SGCs, and were identified as IBU-PEG monoester and IBU-PEG diester. Two other degradants arised from reaction of IBU with sorbitol and sorbitan, which are components of the shell and serves as plasticizers. Thus, IBU sorbitol monoester (IBU-sorbitol) and IBU sorbitan monoester (IBU-sorbitan ester) were identified. An UHPLC method was further optimized in order to separate, selectively detect and quantify the degradation products in IBU SGCs.

A novel approach for HPLC determination of 2-cynaoacetamide using derivatization procedure with 2-hydroxyacetophenone as a new useful derivatization reagent.

A novel and sensitive derivatization procedure for the determination of 2-cynaoacetamide in pharmaceutical samples using liquid chromatography with the fluorescence detection was discovered. The method is based on derivatization of 2-cynaoacetamide using 2-hydroxyacetophenone as a new derivatization reagent. The product of derivatization reaction was isolated and characterized using spectroscopic techniques namely LC-MS, NMR and IR. The structure of 2-cyanoacetamide derivative was unambiguously assigned as a 2-amino-4-phenylfuran-3-carboxamide. Two derivatization systems were optimized in terms of reaction temperature, reaction time, pH and concentration of 2-hydroxyacetophenone, and a new pre- and post-derivatization HPLC methods were developed. The separations on HPLC with pre-column derivatization were accomplished using stationary phase based on a XBridge C18 column (100×4.6, 3.5µm) and isocratic elution using the mobile phase acetonitrile - 0.1% formic acid (30:70, v/v). The separations on the HPLC with post-column derivatization were performed on stationary phase on a TSKgel Amide-80 column (150×4.6mm, 3µm). The mobile phase was a mixture of acetonitrile, methanol and 10mM sodium formate buffer at pH=4.5 in ratio 3:2:95 (v/v). Both HPLC methods were fully validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy and precision according to ICH guidelines. The pre-column derivatization method was linear in the range 1.1-2000µg/l with method accuracy≥98.2% and method precision RSD≤4.8%. The post-column derivatization method was linear in the range 12-2000µg/l. Method accuracy≥96.3% and method precision RSD≤3.5%. Proposed new methods were proved to be highly sensitive, simple and rapid, and were successfully applied to the determinations of 2-cynaoacetamide in pregabalin.

Identification, characterization, synthesis and HPLC quantification of new process-related impurities and degradation products in retigabine.

Two new impurities were described and determined using gradient HPLC method with UV detection in retigabine (RET). Using LC-HRMS, NMR and IR analysis the impurities were identified as RET-dimer I: diethyl {4,4'-diamino-6,6'-bis[(4-fluorobenzyl)amino]biphenyl-3,3'-diyl}biscarbamate and RET-dimer II: ethyl {2-amino-5-[{2-amino-4-[(4-fluorobenzyl) amino] phenyl} (ethoxycarbonyl) amino]-4-[(4-fluorobenzyl)amino] phenyl}carbamate. Reference standards of these impurities were synthesized followed by semipreparative HPLC purification. The mechanism of the formation of these impurities is also discussed. An HPLC method was optimized in order to separate, selectively detect and quantify all process-related impurities and degradation products of RET. The presented method, which was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and selectivity is very quick (less than 11min including re-equilibration time) and therefore highly suitable for routine analysis of RET related substances as well as stability studies.

Interaction of ionic liquids ions with natural cyclodextrins.

The interaction of natural α-, ß-, and γ-cyclodextrins (CDs) with 14 hydrophobic ionic moieties of ionic liquids (ILs) was systematically examined in dilute aqueous solutions using isothermal titration microcalorimetry (ITC) and NMR spectroscopy. The studied cationic and anionic moieties involved some recently developed heavily fluorinated structures, as well as some others of common use. To isolate the effect of a given ion, the measurements were performed on salts containing the hydrophobic IL ion in question and a complexation-inactive counterion. Additional ITC experiments on ILs whose both cation and anion can interact appreciably with the CD cavity demonstrated that to resolve the effect of individual ions from such data is generally a tricky task and confirmed the superiority of the isolation strategy adopted for the purpose throughout this work. The binding constant, enthalpy and entropy determined at 298.15 K for the 1:1 (ion:CD) inclusion complex formation range in broad limits, being 0 < K < 2 × 10(5), 0 < -Δ(r)H°/(kJ·mol(-1)) < 44, and -28 < TΔ(r)S°/(kJ·mol(-1)) < 14, respectively. The stabilities of complexes of perfluorohexyl bearing ions with ß-CD belong to the highest ever observed with natural CDs in water. The established binding affinity scales were discussed in both thermodynamic and molecular terms. The concepts of hydrophobic interaction and guest-host size matching supported by simple molecular modeling proved useful to rationalize the observed widely different binding affinities and suggest possible binding modes. Enthalpy and entropy contributions to the stability of the ion-CD complexes were found to compensate each other considerably obeying more or less the linear compensation relationship marked by existing literature data on binding other guests to natural CDs. As outliers to this pattern, the most stable complexes of -C(6)F(13) bearing ions with ß-CD were found to receive an enhanced inherent entropy stabilization due to extraordinarily high extent of desolvation occurring in the course of binding.

Crystallization products of risedronate with carbohydrates and their substituted derivatives.

The gastrointestinal absorption of bisphosphonates is in general only about 1%. To address this problem mixtures of risedronate monosodium salt with twelve varied sugar alcohols, furanoses, pyranoses and eight gluco-, manno- and galactopyranoside derivatives as counterions were designed in an effort to prepare co-crystals/new entities with improved intestinal absorption. Crystalline forms were generated by means of kinetically and/or thermodynamically controlled crystallization processes. One hundred and fifty-two prepared samples were screened by means of FT-NIR and FT-Raman spectroscopy. No co-crystal was prepared, but noteworthy results were obtained. A new solid phase of risedronate monosodium salt generated in the presence of phenyl-ß-d-galactopyranoside under thermodynamically controlled crystallization conditions was found and also characterized using solid state NMR spectroscopy, X-ray powder diffraction and differential scanning calorimetry. This new polymorph was named as form P. Interactions between risedronate monosodium salt and both carbohydrates were confirmed by means of molecular dynamics simulation. In the present study the relationships between the chemical structures of the studied compounds required for crystalline form change are discussed.

Drug-excipient compatibility testing-Identification and characterization of degradation products of phenylephrine in several pharmaceutical formulations against the common cold.

Different pharmaceutical preparations against the common cold containing phenylephrine (PHE) and saccharose were studied. New impurities were discovered in these preparations after exposure using isocratic ion-pair chromatography separation on a C18 column. LC-MS and NMR techniques were employed to identify and to fully characterize these new compounds. The products were identified as 1-[5-(hydroxymethyl)-2-furyl]-2-methyl-1,2,3,4-tetrahydroisochinolin-4,8-diol and 1-[5-(hydroxymethyl)-2-furyl]-2-methyl-1,2,3,4-tetrahydroisochinolin-4,6-diol. Identification of these degradation products allowed to understand and to confirm their formation mechanism. The developed HPLC method separates of all known impurities and impurities originated from PHE as well.

Preparation and properties of new co-crystals of ibandronate with gluco- or galactopyranoside derivatives.

Mixtures of ibandronate monosodium salt with eleven gluco- and/or galacto-pyranoside derivatives as counterions were designed to prepare co-crystals with improved intestinal absorption. In general, gastrointestinal absorption of bisphosphonates after oral administration is approximately 1%. Co-crystals were generated by means of thermodynamically and/or kinetically controlled crystallization processes. Seventy-seven prepared samples were analyzed by means of FT-NIR, FT-Raman spectrometry and solid state NMR spectroscopy. New entities of ibandronate monosodium salt with phenyl-ß-D-galactopyranoside were found and characterized. The absorption of these potential new co-crystals was investigated by means of PAMPA experiments. In the present study the relationships between the chemical structures of the studied compounds required for co-crystal generation are discussed.

Ring-substituted benzohydroxamic acids: 1H, 13C and 15N NMR spectra and NH-OH proton exchange.

NMR spectra (1H, 13C, 15N) of para- and meta-substituted benzohydroxamic acids were studied in dry dimethyl sulfoxide solutions. The 13C chemical shifts were very close to those found by cross-polarization magic angle spinning in solids, the hydroxamic (not hydroximic) structure of which is unambiguous. The hydroxamic structure of these acids in DMSO solutions was proved independently by their 15N chemical shifts. The 15N and 1H chemical shifts of the NH-OH fragment showed excellent mutual dependences and dependences on the nature of the ring substituent. According to these dependences and ab initio energy calculations, all the acids assume the same Z conformation. Proton exchange between hydroxamic OH and NH groups in DMSO proceeded by both intra- and intermolecular exchange and the rates did not exhibit any simple relationship to the substituent constants.

The complexation of metal cations by D-galacturonic acid: a spectroscopic study.

Solid complexes of D-galacturonic acid (GalA) with cobalt(II), copper(II), nickel(II) and oxovanadium(IV) (1-4) were prepared and characterised. The metal-to-ligand molar ratio was 1:2 for complexes 1-3 and 1:1 for complex 4. The alpha- and beta-anomers of GalA were detected in all the complexes in solid state and in solutions. An addition of small amounts of the paramagnetic complexes to the D2O solution of pure ligand led to NMR line broadening of some 1H and 13C nuclei. This broadening was sensitive to the anomeric state of GalA in the case of complexes 1 and 4. NMR and vibrational spectroscopic data indicate the formation of carboxylate complexes of all the cations, while noncarboxylic oxygens are also involved into the metal bonding in some cases. VCD spectra of complexes 1-4 in D2O and Me2SO-d6 solutions confirm that GalA carboxylic group may participate in the formation of optically active species around the metal cation. Possible ways of GalA coordination by metal cations of this study were proposed and discussed.

The function of the blood aqueous barrier in eyes with emulsified silicone oil.

PURPOSE: To evaluate the function of the blood aqueous barrier(BAB) in the eyes with silicone oil emulsification (SOE). METHODS: Protein concentrations, expressed in albumin equivalents,were determined in aqueous humor of the eyes with SOE in 11 consecutive patients by means of proton nuclear magnetic resonance (1H NMR) spectroscopy. Correlations with various clinical factors were studied. RESULTS: Normal function of the BAB (albumin equivalents 1mg/ml and less was found in 8 eyes (73%) independently on underlying disease, early postoperative reaction after pars plana vitrectomy with SO implantation, degree of SOE and late postoperative complications. Increased permeability of the BAB (albumin equivalents equal 2, 3 and 6.5 mg/ml) was found in 3 eyes (27%) with recent acute complication (retinal detachment after SO removal in 2 eyes, and secondary angle closure glaucoma in 1 eye). CONCLUSION: SOE in vivo was associated with increased permeability of the BAB in the minority of the eyes. Other factors should be studied to explain the variability of SOE. 1H NMR spectroscopy might be a valuable method for the study of SOE.