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Vol. 6, No. 2, 2015 ISSN 2233-4203/ e-ISSN 2093-8950 www.msletters.org Mass Spectrometry Letters Identification of Degradation Products in the Phosphodiesterase (PDE-4)Inhibitor Roflumilast Using High Resolution Mass Spectrometry and DensityFunctional Theory Calculations Saroj Kumar Paul* and Upendra N. Dash Department of Chemistry, Institute of Technical Education and Research (ITER), Siksha ‘O' Anusandhan University, Bhubaneswar,Odisha, India Received March 18, 2015; Revised April 12, 2015; Accepted April 20, 2015First published on the web June 30, 2015;o DOI: 10.5478/MSL.2015.6.2.38 Abstract: Roflumilast analogs are a group of drugs which act as selective photodiesterase (PDE-4) inhibitor for the treatment severechronic pulmonary disease associated with chronic brochnonities. Structural identification of degradation products using high resolu-tion mass spectrometry and theoretical investigation by density functional theory have been successfully carried out on roflumilast toidentify four degradation products namely, 3,5-dichloropyridin-4-amine, N-(3,5-dichloropyridin-4-yl)-4-(difluoromethoxy)-3-hydroxybenzamide, N-(3,5-dichloropyridin-4-yl)-3-(cyclopropylmethoxy)-4-(difluoromethoxy) benzamide and 3-(cyclopropylmethoxy)-N-(3,5-dichloro-1-oxidopyridin-4-yl)-4-(difluoro methoxy) benzamide, generated in alkali, acidic and oxidative conditions.
Keywords: Roflumilast, Mass Spectrometry, Degradation products, DFT The International Conference on Harmonisation of Phosphodiesterases (PDEs) are a group of enzymes that Technical Requirements for Registration of Pharmaceuticals catalyze the breakdown of cyclic adenosine monophosphate for Human Use (ICH) defines a degradation product as an and cyclic guanosine monophosphate to their inactive impurity resulting from a chemical change in the drug form. PDE4 is the principal selective cyclic adenosine substance brought about during manufacture and/or storage monophosphate metabolizing enzyme in inflammatory and of the new drug product by the effect of, for example, light, immune cells which is highly expressed in leukocytes and other temperature, pH, water, or by reaction with an excipient inflammatory cells involved in the pathogenesis of inflammatory and/or the immediate container closure system.4 ICH lung diseases, such as asthma and chronic obstructive pulmonary guidelines also necessitate the drugs to be subjected to disease (COPD).1 Roflumilast (RFL) is a second generation stress decomposition studies followed by identification and selective phosphodiesterase (PDE-4) inhibitor approved for the characterization of the degradation products (DP) which treatment of severe chronic obstructive pulmonary disease are formed 0.1%.5,6 associated with chronic bronchitis. The IUPAC name for A thorough literature survey has revealed that only a few roflumilast is 3-(cyclopropylmethoxy)-N-(3, 5-dichloropyridin- studies have been reported for the degradation behaviour of 4-yl)-4-(difluoromethoxy) benzamide; CAS 162401-32-3).2 roflumilast. Tarek S. Belal et al. reported significant Roflumilast has been approved in the EU (as Daxas) and in degradation of roflumilast under acidic, alkali and the US (as Daliresp) for treatment of severe COPD oxidative conditions7 but none have been identified or associated with chronic bronchitis and a history of characterized. The drug has been investigated by Barhateet al. to be stable under neutral, thermal and photolyticconditions but unstable to acidic, alkaline and oxidative *Reprint requests to Saroj Kumar Paul E-mail: paul.saroj@gmail.com conditions at 80°C. But no attempt has been directed forthe identification of DPs.8 All MS Letters content is Open Access, meaning it is accessible online to However, there have been reports of process related everyone, without fee and authors' permission. All MS Letters content ispublished and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org /licenses/by/3.0/). Under this license, authors reserve the copyright for their content; however, they permit anyone to unrestrictedly use, distribute, and reproduce the content in any medium as far as the original authors and source are cited. For any Density Functional Theory (DFT) has been known to reuse, redistribution, or reproduction of a work, users must clarify the predict the fragmentation profile of protonated molecular license terms under which the work was produced.
Identification of Degradation Products in the Phosphodiesterase (PDE-4) Inhibitor Roflumilast ions with reasonable accuracy.12 P. Wright et al. have interface at 250oC. Nitrogen was used both as sheath gas reported a rationalized approach for fragmentation profile and auxiliary gas. The electro spray and tube lens were set of maraviroc using DFT.13 Use of DFT for modelling of 15 at 4.5 kv and 90 V respectively. The mass spectrometer molecules with respect to protonation-induced bond length was operated in full scan MS with data dependent MS2 changes and subsequent prediction of their fragmentation mode in positive polarity.
profiles during collision induced dissociation (CID) has The selected range was from 100 to 1000 m/z and the also been reported.14 resolution was 70,000 full width half maximum (FWHM) In this current study the chemical structures of the with an isolation window applied, followed by a data different DPs produced under acidic, alkali and oxidative dependent scan at a resolution of 17,500 FWHM with the stress conditions are investigated by liquid chromato- fragmentation energy applied. The target capacity of the C- graphy-high resolution mass spectrometry. Efforts have also trap was defined at 1×106 charges and the maximum been made to rationalize the fragmentation profile of the injection time was limited to 50 ms.
parent drug molecule by identifying the most favourableposition of protonation and the changes in bond length Density functional theory consequent to it, DFT. The DPs were characterized by Gas-phase basicity, 3D structure and bond length calculations comparing their collision induced dissociation (CID) mass were performed using DFT, calculations at the B3LYP level spectral data with that of the parent drug molecule of RFL.
using the 6-31G* basis set, with Gaussian09. The optimised This rationalized fragmentation profile can also be geometry for the neutral molecule was calculated, basic sites extended to identify any other unknown transfor-mation were then protonated and the relating minimum energy products of RFL by comparing their daughter ions geometry calculated for each possible structure. The energy obtained under similar experimental condition.
differences between the most favourable cation (highest negativeenergy value in Hartrees) and all others were converted from Hartrees into kcal/mol using the conversion factor of 627.503.
Materials and reagents Sample preparation and degradation studies HPLC grade acetonitrile and methanol were purchased Standard solution of roflumilast (100 µg/mL) was from Merck India limited (Mumbai, India). Ultrapure prepared by dissolving it in acetonitrile. Stress degradation water (18.2 MΩ) was prepared using a Milli-Q plus water sample, acidic (1 N HCl, 80oC, 6 h), alkaline (1 N NaOH, purification system from Millipore (Bedford, MA, USA).
80oC, 6 h) and oxidative (30% H2O2, 80oC, 6 h) were Formic acid and standard of RFL were obtained from prepared by dissolving 10 mg of sample in 1 mL of Sigma-Aldrich Corporation (Bangalore, India). Analytical acetonitrile, followed by the addition of 2 mL 1 N HCl, reagent grade ammonium acetate, sodium hydroxide, 3 mL 1 N NaOH and 3 mL 30% H2O2 respectively. All the hydrochloric acid and hydrogen peroxide were obtained sample solutions were neutralized and volume made up to from Qualigens India Limited (Mumbai, India) 10 mL with acetonitrile prior to their injection into themass spectrometer.
Liquid chromatography All compound solutions were introduced into the ESI Results and discussion MS (electrospray ionisation mass spectrometry) source byhigh performance liquid chromatography (HPLC), Dionex The drug exhibited degradation under acidic, alkali and ultimate 3000 (Thermo scientific, USA), using a hypersil oxidative stress conditions. The results of the degradation BDS C18 column (150×4.6 mm, 5 µm, Thermo scientific, study have been summarized in Table 1.
USA). A mobile phase consisting of A, 10 mM ammonium In an attempt to identify the DPs by mass spectral acetate adjusted to pH 3.2±0.05 with acetic acid and B, analysis it is highly desirable to have a clear understanding acetonitrile in gradient mode; T(min)/%B: 0/25, 10/60, 15/85, of the fragmentation pathway of parent drug RFL. Under 20/85, 25/25, 30/25. Column temperature was maintained ESI conditions RFL underwent protonation at the nitrogen at 30 C and the flow rate was 1.0 mL/min. The samples atom of the pyridine ring and the position of protonation were injected (10 µL) into the HPLC system in acetonitrile.
has also been supported by DFT calculations. The resultsof DFT calculation for the energies of different protonation High-resolution mass spectrometry sites has been depicted in Figure 1. The protonated The MS and MS/MS studies were performed on molecular ion of RFL measured accurately to be m/z Thermofisher Q-exactive mass spectrometer (Thermo 403.0449 Da and produced key fragment ions at m/z Electron, Bremen, Germany) using electrospray ionization 367.0686 Da, 348.9975 Da, 241.0687 Da, 187.0213 Da source and orbitrap mass analyzer. Heated electrospray and 163.9676 Da. The most abundant product ion formed ionization source was used for ionization. The temperature at m/z 241.0687 Da due to loss of 3, 5-dichloropyridine of the heater was kept at 450oC and capillary of the ESI (C5H3Cl2N, 146.9643) by the cleavage of C-N, which further Mass Spectrom. Lett. 2015 Vol. 6, No. 2, 38–42



Saroj Kumar Paul and Upendra N. Dash Table 1. The retention times (RT), measured masses, predicated elemental compositions, theoretical exact masses, mass errorsand major fragment ions of degradation products (DP) *RFL is not a DP and stands for the parent molecule of Roflumilast.
Figure 1. The energies of roflumilast molecule at different Figure 2a. DFT optimized structure of RFL indicating bond potential protonation sites in hartee units. (Hartree is the atomic length before protonation.
unit of energy; Hartrees are converted into kcal mol-1 bymultiplying by 627.503).
underwent loss of methylene cyclopropane (C4H6, 54.047Da) to generate fragment ion at m/z 187.0213 Da. DFToptimized structure of the protonated molecular ion ofroflumilast also indicated elongation of C-N bond asdepicted in Figure 2. The formation product ion at m/z348.9975 Da is due to loss of methylene cyclopropane(C4H6, 54.047 Da), which in turn under goes rearrangementwith a neutral loss of 185 Da to produce the cation of 3,5-dichloropyridin-4-ol.The schematic presentation of thefragmentation profile of roflumilast is shown in Figure 3a.
DP-1 formed during alkaline degradation showed its protonated molecular ion at m/z 162.9821 Da and underwent Figure 2b. DFT optimized structure of RFL indicating change in fragmentation by losing a molecule of hydrochloric acid to bond length after protonation.
produce a product ion at m/z 90.9777 Da, as shown inFigure 3b. Based on the mass spectra data DP-1 identifiedas 3, 5-dichloropyridin-4-amine.
evidently supported it to be N-(3,5-dichloropyridin-4-yl)-4- DP-2 with a measured accurate mass of m/z 348.9946 Da (difluoro methoxy)-3-hydroxybenzamide, formed by the loss formed during acidic as well as oxidative stress conditions.
of methyl cyclopropane moiety from roflumilast.
The characteristic isotopic pattern of two chlorine atoms in the DP-3 was formed under alkaline as well as oxidative stress mass spectra and product ion spectra containing diagnostic conditions and showed its protonated molecular ion at m/z fragment ions of m/z 163.9676 Da, 313.0208 Da (Figure 3c) 353.0453 Da with characteristic isotopic pattern of two Mass Spectrom. Lett. 2015 Vol. 6, No. 2, 38–42 Identification of Degradation Products in the Phosphodiesterase (PDE-4) Inhibitor Roflumilast Figure 3a. Plausible fragmentation pathway of roflumilast.
with that of RFL and exhibited similar neutral losses. Basedon this the most possible structure of DP-4 is proposed to bethe N-oxide of roflumilast. The pathway depicting theformation of product ions is shown in Figure 3e.
Figure 3b. Plausible fragmentation pathway of DP-1.
Roflumilast was subjected to stress study and found to be chlorine atoms. The diagnostic product ions formed at m/z sensitive in alkaline, acidic and oxidative environment. A 162.9838 Da and 191.0718 Da by the neutral loss of 3- rationalized approach based on DFT and comparative high resolution mass spectral analysis with product ion profiling and 3,5-dichloropyridin-4-amine evidently supported it to be was used for rapid identification of the degradation formed by the loss of CHF2 side chain from the molecule of products. The four degradation products have been identified roflumilast. Based on this mass spectral analysis as discussed above and depicted in Figure 3d, the most plausible structure of DP-3 has been proposed as N-(3,5-dichloropyridin-4-yl)-3- During oxidative stress study degradant formed with an accurate mass of m/z 419.0361 Da which is 16 Da higherthan that of RFL and showed isotopic pattern confirming the presence of two chlorine atoms, is assigned the codename DP-4. The product ions formed in DP-4 are identical The authors acknowledge the support of Dr. Naresh K.
Figure 3c. Plausible fragmentation pathway of DP-2.
Mass Spectrom. Lett. 2015 Vol. 6, No. 2, 38–42 Saroj Kumar Paul and Upendra N. Dash Figure 3d. Plausible fragmentation pathway of DP-3.
Figure 3e. Plausible fragmentation pathway of DP-4.
Jena of Stockholm University for DFT calculations, Mr.
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Mass Spectrom. Lett. 2015 Vol. 6, No. 2, 38–42

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