What Are The Uses Of Phenoxybenzamine Hydrochloride Biology Essay

What Are The Uses Of Phenoxybenzamine Hydrochloride Biology EssayPhenoxybenzamine Hydrochloride (RS)-benzyl(2-chloroethyl)1-methyl-2-phenoxyethylamine hydrochloride is a alpha-adrenoceptor blocker that covalently binds and irreversibly inhibits the activity of alpha-1 alpha -2 adrenoceptors.3,4 Phenoxybenzamine Hydrochloride is mainly employ to treat episodes of high bank line wardrobe and sweating tie in to phaeochromocytoma. Phaeochromocytoma is a r ar catecholamine-secreting tumour of the adrenal medulla. Patients with phaeochromocytoma atomic number 18 usu completelyy hypertensive and suffer headache, palpitations, and exuberant sweating.3 However it is rarely prescribed due to it unfavourable side effects. one of the side effects of Phenoxybenzamine is that block the ejaculation. Also some studies are at a lower place investigation to use Phenoxybenzamine as male contraceptive pills.11, HClFig. 01 molecular(a) construction of (RS)-N-benzyl-N-(2-chloroethyl)-1- phen oxy-propan-2-aminePhenoxybenzamine hydrochloride is white, odourless, crystalline powder that is sparingly alcohol-soluble in water soluble in ethanol, chloro manakin, and propylene glycol and insoluble in diethyl ether.Neutral and alkalescent solutions are unstable sensitive to oxidation and photo degradation.6,7,8Molecular Weight9 303.82638 g/molMolecular Formula C18H22ClNOMonoIsotopic Mass9 303.138992CAS-No. 63-92-3The stability studies for Phenoxybenzamine injection concentrate were carried out by Zeta uninflected Ltd. for their Clients. Also analytical rule for related marrow of Phenoxybenzamine Injection has been pass by them. During the stability studies, it has been found that some stability batches contain more(prenominal) than 0.1% of enigmatical impurities. Already there are three identified, process related impurities were describe in their clients analytical methods.10Those three impurities A, B, and C are reported as shown below.1. impureness A N-benzyl-N -(2-chloroethyl)-2-phenoxypropan-1-amineFig. 02 Molecular social organisation of N-benzyl-N-(2-chloroethyl)-2- phenoxy-propan-1-amine2. scoria B N-benzyl-N-(2, hydroxyethyl)-1-phenoxypropan-2-amineFig. 03 Molecular structure of N-benzyl-N-(2-hydroxyethyl)-1-phenoxypropan-2-amine3. dross C N-(2-chloroethyl)-1-phenoxypropan-2-amineFig. 04 Molecular structure a of N-(2-chloroethyl)-1-phenoxypropan-2-amineAccording to the International Conference on Harmonization1,2 (ICH) guidelines any component of a pharmaceutical product which is non the chemical entity of fighting(a) substance or excipients, yield at levels higher than 0.1% or 1 mg/ twenty-four hour period intake (whichever is lower) for a maximum daily dose of 2 g/day or less, need to be identified and qualified with countenance toxicological studies. For a daily dose of greater than 2 g of drug substance, the identification threshold is 0.05%.1 ,2 Also British (BP), European (EP) and abbreviate together states (USP) ph armacopeia texts refers the ICH criteria on dross profiling for new drug substance and new drug products.5,6,7Hyphenated proficiencys such as LC-MS and LC-NMR methods as an effective fauna for characterization of impurities and degradation products in drug molecules. therefore, Zeta Analytical proposed this forge to fulfil LC-MS abridgment on Phenoxybenzamine injection for geomorphologic elucidation of unacknow leadged dross. This get word involved method transferring (Tech transfer) from Zeta to capital of Jamaica University and create a LC-MS compatible chromatographicmethod structural identifications of extraterrestrial being impureness.Literature StudyThere are no chromatographic methods have been reported in the literature describing the compend of Phenoxybenzamine and its related substances using UV detection. The chromatographicConditions mentioned in United States Pharmacopoeia (USP) monograph7 (refer Appendix) and Zetas method quiet confusable apart from the det ection wavelength, which is 268nm for USP and 220 nm for Zeta. Both methods are not compatible for LC/MS psychoanalysis. Because orthophosphate devotee is a one component that mobile phase consist in both methods. There are no reports available on the investigation using LC/MS/MS and isolation of related substances in Phenoxybenzamine active pharmaceutical trimmings (API). However, in order to analyse the sample in LC/MS and to get better chromatographic resolution the method has been modified for use in the present investigation.A tax write-off dispatch of Phenoxybenzamine has been mentioned in Vardanyan and Hruby,18 and slightly opposite route of tax write-off for phenoxybenzaime related amines has been reported in Giardink et al.13EXPERIMENTAL stuff and MethodsChemicals use (all anal. Grade )were Phenoxybenzamine Hydrochloride ( Sigma- Aldrich) , scoria B (PBA) from med alchemy S.L Spain, HPLC grade Acetonitrile (Sigma- Aldrich), atomic number 19 phosphate dibasic, Potas sium phosphate monobasic, ammonium ion ion forgeate, Ammonium Hydroxide, MilliQ grade waterSamplesPhenoxybenzamine injection concentrate Each 2 ml ampoule contains degree centigrade mg Phenoxybenzamine Hydrochloride BP and excipients are absolute ethyl alcohol, hydrochloric acid AR and propylene glycol.Sample Preparation for HPLC and LC-MSWhole contents of ampoule was transferred to a 100mL volumetric flask and dissolved with 30 mL of acetonitrile. Then it was shacked for few transactions to mix well and added more acetonitrile to volume up the level of volumetric flask.HPLC analysis at Zeta Analytical Ltd.HPLC analysis was proceeded at these chromatographic coach tug Phenomenex Gemeni-NX 5m C18 110A 250-4.6 mm. Mobile phase used pH 7.5 20mM phosphate relent (Dissolved 2.4g of K2HPO4 in 1L of water and adjusted to pH 7.5 with KH2PO4) Acetonitrile = 30% 70% (Isocratic mode ) . editorial temperature 25C , flow rate 0.9 cm3min-1 , detection at 220nm .HPLC corpse used at Zeta analytical Ltd Pump , railroad car sampler ,UV sensing element and thermoregulator are Agilent 1100 series with Agilent Chemstation for LC info system.HPLC analysis at tutor of chemistry and pharmacy, Kingston UniversityHPLC analysis was proceeded at these chromatographic condition Column Phenomenex Gemeni-NX 5m C18 110A 250-4.6 mm. Mobile phase used pH 7.5 20mM Phosphate lover (Dissolved 2.4g of K2HPO4 in 1L of water and adjusted to pH 7.5 with KH2PO4) Acetonitrile = 30% 70% (Isocratic mode ) .Column temperature 25C , flow rate 0.9 cm3min-1 , detection at 220nm .HPLC system used at Kingston University Pump , Auto sampler ,UV-VIS detector and thermostat are Perkin Elmer series 200 with Totalchrom v6.2 packet.LC- MS analysis at mold of chemistry and pharmacy, Kingston UniversityLC-MS analysis was proceeded at these chromatographic condition Column Phenomenex Gemeni-NX 5m C18 110A 250-4.6 mm. Mobile phase used pH 8.3 20mM Ammonium formate buffer (Dissolved 1.3g o f NH4HCO2 in 1L of water and adjusted to pH 8.3 with NH4OH) Aceotonitrile = 30% 70% (Isocratic mode) .Column temperature 25C , flow rate 0.9 cm3min-1 , detection at 220nm .LC system Pump , Auto sampler ,UV-VIS detector and thermostat are Perkin Elmer series 200 with Totalchrom v6.2 data system.Mass detetectors used Two different fold detectors were employed amnionic fluid Micro plentitude LCT ESI-TOF-MS system with Mass Lynx 4.1 softwareThermo TSQ Quantum Access system ( MSMS ) with Thermo Excalibur softwareRESULTS AND DICUSSIONHPLC outline at Zeta Analytical Ltd.Initially the sample was analyzed in Agilent HPLC in Zeta Analytical Ltd. Based on the Zetas Analytical Method arrangement report for the related substance of Phenoxybenzamine HCl injection,the three identified impurities and one obscure impurity were confirmed. Below the fig.05 (Appendix A-1) shows the percentage points of Phenoxybenzamine and its impurities and the table 01 shows the property prison term of t hose peaks.Fig. 05 HPLC chromatograms of Phenoxybenzamine HCl injections accede 01 Peaks and its retentiveness time of Phenoxybenzamine (zeta)Peaks computer memory time / minImpurity C5.2Impurity B7.3 unsung Impurity8.6Impurity A18.2Phenoxybenzamine19.6HPLC Analysis at Kingston University (Method Transfer)The above results obtained at zeta were replicated again with Perkin-Elmer HPLC system in Kingston University. Same chromatographic condition was employed with same Phenomenex Gemeni-NX 5m C18 110A 250-4.6 mm column . The fig.06 below show chromatogram of Phenoxybenzamine HCl injection analysis iterate at Kingston University(Appendix A-2).The peaks were interested and its retention propagation are shown in the table 02 below.Fig.06 HPLC chromatogram of Phenoxybenzamine HCl Injection (Kingston)Table 02. Peaks and its retention time of Phenoxybenzamine (Kingston)PeaksRetention succession / minImpurity C5.0Impurity B7.0Unidentified Impurity8.4Impurity A17.9Phenoxybenzamine19.5L C-MS Analysis of Phenoxybenzamine Injection ConcentratePhosphate buffers are not compatible for LC-MS due to their non volatile nature. Since it was necessary to replace the phosphate buffer to a volatile buffer. Mean while the chromatographic teaching should not be changed. The ammonium formate buffers are widely used in LC-MS analytical methods and has buffering pH range (8.2-10.2) close to the previous phosphate buffers used which is pH 7.5 . 20mM ammonium phosphate buffer was prepared adjusted the pH to 8.3.The HPLC analysis previously performed was repeated with mobile phase of Ammonium formate buffer Acetonitrile =30 70 instead of mobile phase of phosphate buffer Acetonitrile = 3070 .The isolation of peaks and the resolution obtained in previous analysis was replicated.Fig. 07 Show the HPLC chromatogram of replicated results with Ammonium formate buffer and the table 03 show retention time and its corresponding peaks.Fig.07 HPLC chromatogram of PBA Injection Sample ( Modif ied Mobile phase forLC-MS analysis)Table 03 Peaks and its retention time of Phenoxybenzamine (Ammonium formate asbuffer)The samples were run on HPLC several times and constant chromatographic development was observe. Hence the sample fractions of unknown impurity was collected several time during HPLC run for LC-MS ( ideal spate measurement) and LC-MS/MS (selective ion monitoring) analysis.PeaksRetention Time / minImpurity C5.3Impurity B7.3Unidentified Impurity8.7Impurity A18.5Phenoxybenzamine19.8Accurate green goddess measurement with Time of leakage (ToF) mess hall detectorFeasibility of TOF mass detectors has made it to be used widely for measurement of accurate mass. Several unknown impurity sample fractions were analyzed for the accurate mass measurement on irrigate micromass LCT ESI TOF-MS and obtained the intermediate of the accurate mass value of unknown impurity. Ionization technique is Electron spray Ionization and mass analyzer is Time of Flight analyzer in this in strument. Results were taken on positive mode ( M + H + ). Hence mass of one proton must be deducted from the spiritual mass value to obtain the exact mass value of unknown compound. H1 mass is considered to be 1.007 Da in the calculation below.Table 03 m/z value of Peaks observed its corresponding calculated monoisotopic massM + H + / DaMass of Unknown Compound / Da344.215343.208344.216343.209344.216343.209344.219343.212344.211343.204344.209343.202344.215343.208344.214343.207344.215343.208344.213343.206344.215343.208344.22343.213344.213343.206344.221343.214344.223343.216The average molecular weight and example deviation of results are found to be 343.2086667 and 0.003754363 respectively. Above results were subjected to statistical evaluation using Microsoft excel spread sheet. At 99% confidence level the molecular weight of the unknown impurity is found to be 343.2086667 0.002497.Determination of Elemental Composition of unknown impurity. using Mass Lynx 4.1 MS data management so ftware come-at-able principal(a) composition was obtained for the molecular weight of 343.209 with 200.0 mDa tolerance . It was able to draw a large amount elemental composition to narrow findings. That is excreting of Chlorine in the composition . Because the mass spectrum of unknown impurity does not show the isotopic pattern for chlorine. i.e. When one Chlorine atom is present in a molecule, that will show a n/n+2 ratio of100/32.4 (35Cl/37Cl ratio of 100/32.4). 15,16 Hence yet C,H,N and O elements were limited on search.Still hundreds of composition are oddfield to be examined to find correct elemental composition . The second exception that is Nitrogen rule14 which was used to eliminate many of those composition. Since the unknown impurity molecular weight is odd number, we can eliminate all the composition with the horizontal number of nitrogen in list. The succeeding(a) table shows considerable elemental composition left after above two exclusions .Table 04. potentia l elemental composition and itsMonoisotopic massElemental CompositionCalculated Monoisotopic massC12H25N9O3343.2080C16H29N3O5343.2107C23H25N3343.2048C21H29NO3343.2147C12H29N3O8343.1955C15H29N5O4343.2220C24H25NO343.1936The monoisotopic mass of main active compound is 303.14 and it contains 18 carbon on that molecule. Unknown impurity has the monoisotopic mass value 40 amu higher than the active compound. Therfore if it is been assumed that unknown impurity has more than 18 carbon on its molecule, only two elemental composition would be remained. i.e. C23H25N3 (343.2048) and C24H25NO (343.1936).LC -MS/MS (Tandem mass )Analysis of Phenoxybenzamine HCl Injection ConcentrateThermo TSQ Quantum Access LC-MS/MS was employed for the selective ion monitoring. The unknown impurity fractions , Phenoxybenzamine HCl standard (sigma ) and Impurity B standard were analysed.Analysis of Phenoxybenzamine HCl standardParameter usedParent mass 304.4 skitter time 0.5000 smasher energy 16Collision gas ca rt 1.1 BarrSpray voltage 4000 graze Mode increase Ion surveyFig. 08 Product ion see mass spectrum of Phenoxybenzamine StandardPeaks at m/z 63, 84, 91,107,120, 135 and 212 were observed. The bum peak is observed at m/z 91 benzyl sherd . It is stabilized by the resonance form of benzene.17 The following figure illustrates the fall apart downs and its corresponding mass units observed in the product ion scan spectrum.Fig. 09 good example of commotion downs of PhenoxybenzamineApart from the peaks illustrated on above figure, the peaks at m/z 120 go up due to mass unitCH2NCH2CH2Cl + H+ . This is the oculus portion after the mass unit at m/z 91 and 93 correspond apart from the whole Phenoxybenzamine molecule. i.e M + H+ the molecular ion is m/z 304, but 304 (91+93) = 120 .Analysis of Impurity B standardParameter usedParent mass 286 poop out time 0.5000Collision energy 16Collision gas pressure 1.1 BarrSpray Voltage 4000Scan Mode Product Ion ScanFig. 09 Product ion scan mass spe ctrum of Impurity B StandardProduct Scan spectrum shows peaks at m/z 84, 91,102,107,135,178,194 and 285. The base peak is m/z 91and the molecular ion is m/z 286. The following figure illustrates the break downs and its corresponding mass units observed in the product ion scan spectrum.Fig. 09 Illustration of break downs of Impurity BAs seen in the Phenoxybenzamine product ion scan spectrum the remotion of mass units m/z 91 and 93 also is observed in this Impurity B spectrum. i.e . there is a peak arise at m/z 102 , this is due removal mass units m/z 91 and 93 from the molecular ion m/z 286. 286-(91+93)=102Analysis of Unknown ImpurityParameter usedParent mass 286Scan time 0.5000Collision energy 16Collision gas pressure 1.1 BarrSpray Voltage 4000Scan Mode Product Ion ScanFig. 10 Product ion scan mass spectrum of Unknown ImpurityProduct ion Scan spectrum shows peaks at m/z 84,91,107,119,135,152,160,178,251,267 and 344 .Here the molecular ion peak and base peak are same at m/z 344. This mass spectrum shows quietsimilar fragmentation pattern with Phenoxybenzamine and the impurity B were analysed before.Peaks at m/z 84, 91, 93, 107, and 135 are found in all three , Phenoxybenzamine , Impurity B andunknown impurity product ion scan spectrum. Also as studied previously in the spectrum of PBA and impurity B, the rebate of the mass units m/z 91 and 93 from the molecular ion (m/z 344) results a obvious sharp peak at m/z 160.From the facts studied above in product ion scan mass spectrum and accurate mass measurement for elemental composition using TOF -MS , It can be hypothesized a structure of the unknown impurity . The proposed structure for unknown impurity is shown below in figure.Fig. 11 The Molecular Structure proposed for unknown impurity,N-benzyl-N-(E)-2-phenylethenyl -1- phenoxy-propan-2-amineThe proposed structure can be rationalized with the product ion mass spectrum of unknown impurity. Following fig.12 shows the break downs of the unknown impurity that corr espond to the peaks observed on mass spectrum.Fig. 12 Illustration of break downs of Unknown ImpurityMost of the impurities found in pharmaceutical compounds usually process-related compounds they are most probably structurally similar to the synthesized target drugs. It is prominent to study the tax write-off route of active pharmaceutical ingredient (API), when the unknown impurity of drugs substance is been identified. Unfortunately the original synthesis route followed by the API manufacturer of Phenoxybenzamine is not known. The prediction for synthesis route of Phenoxybenzamine with possibilities for arising of other 3 Identified process related impurities (A, B, and C) is shown in the following scheme below based on Giardink(1995).13+ (a)(ii) (iii) (iv) (b)(v)(c)(d)(vii)(vi)(a)Oxidation (b) 1-phenylmethanamine, HC1/EtOH,molecular sieves 4A, NaBH3CN (c) Br(CH2)2OH, K2CO3, EtOH (d) SOCI 2, HCI (g), Benzene (i) oxybenzene (2)2-methyl oxirane (3)1-phenoxypropan-2-ol(4) 1-phenoxy propan-2-one (5) N-benzyl-1-phenoxypropan-2-amine (6) 2-benzyl(1-phenoxypropan-2-yl)aminoethanol(7) N-benzyl-N-(2-chloroethyl)-1-phenoxypropan-2-amine (Phenoxybenzamine)Scheme 1. Predicted synthesis route for PhenoxybenzamineFormation of Impurity AThe reaction between phenol (i) and 2-methyl oxirane (ii) is SN2 nucleophilic substitution. Nucleophiles are more reactive to most substituted carbon of epoxides under caustic condition and least substituted carbon is favoured under basic condition.19 In this fictitious character carbon position 2 (fig.13) is favoured under basic condition and its form 1-phenoxypropan-2-ol ,which is the precursor molecule for PBA .Fig. 13 Structure of 2-methyl oxiraneTo very(prenominal) few terminus the nucleophiles react at carbon position 3 will form 2-phenoxypropan-1-ol, which will lead to the formation of Impurity A along synthesis process of PBA.Fig.14 Structure of 2-phenoxypropan-1-olFormation of Impurity B and Impurity CImpurity B, 2-benzyl(1-ph enoxypropan-2-yl)aminoethanol is intermediate product during synthesis. Refer structure (vi) of scheme 01 .Impurity C, N-(2-chloroethyl)-1-phenoxypropan-2-amine formed due to chlorination of intermediate product 1-phenoxypropan-2-ol (refer structure (iii) of scheme 01). The unoxidised1-phenoxypropan-2-ol left over is chlorinated by SOCI 2, HCI during last step of the synthesis.Refer (d) of the scheme.CONCLUSIONSPreliminary structural assignments for unknown impurity of Phenoxybenzamine Injection were made on the basis of mass spectral data. Initially the works started with transferring HPLC method from Zeta Lab to Kingston university and developing a LC-MS chromatographic method. Ammonium formate volatile buffer was replaced for phosphate buffer in HPLC method . Same chromatographic development was able to replicated with ammonium formate buffer. Accurate mass measurement was carried out on ESI-TOF LC-MS . Also this studies led to determine possible empirical formula . Then LC-MS/MS analysis was performed. The Product ion Scan mass spectral data are very vital information in final structural elucidation of unknown impurity.The structure deduced from MS/MS confirms the empirical formula C24H25NO that derived with LC-TOF-MS spectral data. eventually the impurity identified in this this preliminary structural assignments , which eluted at retention time of 8.7 minute was predicted as N-benzyl-N-(E)-2-phenylethenyl -1- phenoxy-propan-2-amine . The proposed molecular structure for unknown impurity is shown in fig.11 . The formation of impurities A, B and C those had already identified by manufacture were described based on the predicted synthesis route for Phenoxybenzamine . The formation unknown impurity was not able to explained at this stage of this project since the reaction would have occurred found to be more complicated. This project work was wrapped up at this stage due to time limitation. yet to these preliminary structural assignments various spectrosco pic studies such as LC-NMR and IR need to be carried out to complete the characterization of the unknown compound. at long last the proposed structure can be confirmed by synthesizing N-benzyl-N-(E)-2-phenylethenyl -1- phenoxy-propan-2-amine in future.