What Are The Uses Of Phenoxybenzamine Hydrochloride Biology Essay

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Phenoxybenzamine 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 used to treat episodes of high blood pressure and sweating related to phaeochromocytoma. Phaeochromocytoma is a rare catecholamine-secreting tumour of the adrenal medulla. Patients with phaeochromocytoma are usually hypertensive and suffer headache, palpitations, and excessive 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 under investigation to use Phenoxybenzamine as male contraceptive pills.11

, HCl

Fig. 01 - Molecular structure of (RS)-N-benzyl-N-(2-chloroethyl)-1- phenoxy-propan-2-amine

Phenoxybenzamine hydrochloride is white, odourless, crystalline powder that is sparingly soluble in water; soluble in ethanol, chloroform, and propylene glycol; and insoluble in diethyl ether.

Neutral and alkaline solutions are unstable; sensitive to oxidation and photo degradation.6,7,8

Molecular Weight9: 303.82638 [g/mol]

Molecular Formula : C18H22ClNO

MonoIsotopic Mass9: 303.138992

CAS-No. : 63-92-3

The stability studies for Phenoxybenzamine injection concentrate were carried out by Zeta Analytical Ltd. for their Clients. Also analytical method for related substance of Phenoxybenzamine Injection has been validated by them. During the stability studies, it has been found that some stability batches contain more than 0.1% of unknown impurities. Already there are three identified, process related impurities were reported in their client's analytical methods.10

Those three impurities A, B, and C are reported as shown below.

1. Impurity A: N-benzyl-N-(2-chloroethyl)-2-phenoxypropan-1-amine

Fig. 02 - Molecular structure of N-benzyl-N-(2-chloroethyl)-2- phenoxy-propan-1-amine

2. Impurity B: N-benzyl-N-(2, hydroxyethyl)-1-phenoxypropan-2-amine

Fig. 03 - Molecular structure of N-benzyl-N-(2-hydroxyethyl)-1-phenoxypropan-2-amine

3. Impurity C: N-(2-chloroethyl)-1-phenoxypropan-2-amine

Fig. 04 - Molecular structure a of N-(2-chloroethyl)-1-phenoxypropan-2-amine

According to the International Conference on Harmonization1,2 (ICH) guidelines any component of a pharmaceutical product which is not the chemical entity of active substance or excipients, present at levels higher than 0.1% or 1 mg/day intake (whichever is lower) for a maximum daily dose of 2 g/day or less, need to be identified and qualified with appropriate 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 United states (USP) pharmacopeia texts refers the ICH criteria on impurity profiling for new drug substance and new drug products.5,6,7

Hyphenated techniques such as LC-MS and LC-NMR methods as an effective tool for characterization of impurities and degradation products in drug molecules. Therefore, Zeta Analytical proposed this project to perform LC-MS analysis on Phenoxybenzamine injection for structural elucidation of unknown impurity. This project involved method transferring (Tech transfer) from Zeta to Kingston University and developing a LC-MS compatible chromatographic

method structural identifications of unknown impurity.

Literature Study:

There are no chromatographic methods have been reported in the literature describing the analysis of Phenoxybenzamine and its related substances using UV detection. The chromatographic

Conditions mentioned in United States Pharmacopoeia (USP) monograph7 (refer Appendix) and Zeta's method quiet similar apart from the detection wavelength, which is 268nm for USP and 220 nm for Zeta. Both methods are not compatible for LC/MS analysis. Because phosphate buffer 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 ingredient (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 synthesis route of Phenoxybenzamine has been mentioned in Vardanyan and Hruby,18 and slightly different route of synthesis for phenoxybenzaime related amines has been reported in Giardink et al.13


Material and Methods

Chemicals used (all anal. Grade )were: Phenoxybenzamine Hydrochloride ( Sigma- Aldrich) ,

Impurity B (PBA) from med alchemy S.L Spain, HPLC grade Acetonitrile (Sigma- Aldrich), Potassium phosphate dibasic, Potassium phosphate monobasic, Ammonium formate, Ammonium Hydroxide, MilliQ grade water


Phenoxybenzamine injection concentrate ; Each 2 ml ampoule contains 100 mg Phenoxybenzamine Hydrochloride BP and excipients are absolute ethyl alcohol, hydrochloric acid AR and propylene glycol.

Sample Preparation for HPLC and LC-MS

Whole contents of ampoule was transferred to a 100mL volumetric flask and dissolved with 30 mL of acetonitrile. Then it was shacked for few minutes 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 condition : Column Phenomenex Gemeni-NX 5µm C18 110A 250-4.6 mm. Mobile phase used : pH 7.5 20mM Phosphate buffer (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 Zeta analytical Ltd : Pump , Auto sampler ,UV detector and thermostat are Agilent 1100 series with Agilent Chemstation for LC data system.

HPLC analysis at School of chemistry and pharmacy, Kingston University:

HPLC analysis was proceeded at these chromatographic condition : Column Phenomenex Gemeni-NX 5µm C18 110A 250-4.6 mm. Mobile phase used : pH 7.5 20mM Phosphate buffer (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 software.

LC- MS analysis at School of chemistry and pharmacy, Kingston University:

LC-MS analysis was proceeded at these chromatographic condition : Column Phenomenex Gemeni-NX 5µm C18 110A 250-4.6 mm. Mobile phase used : pH 8.3 20mM Ammonium formate buffer (Dissolved 1.3g of 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 mass detectors were employed :

Waters Micromass LCT ESI-TOF-MS system with Mass Lynx 4.1 software

Thermo TSQ Quantum Access system ( MS\MS ) with Thermo Excalibur software


HPLC Analysis at Zeta Analytical Ltd.

Initially the sample was analyzed in Agilent HPLC in Zeta Analytical Ltd. Based on the Zeta's Analytical Method validation report for the related substance of Phenoxybenzamine HCl injection,

the three identified impurities and one unidentified impurity were confirmed. Below the fig.05 (Appendix A-1) shows the peaks of Phenoxybenzamine and its impurities and the table 01 shows the retention time of those peaks.

Fig. 05 - HPLC chromatograms of Phenoxybenzamine HCl injections

Table 01: Peaks and its retention time of Phenoxybenzamine (zeta)


Retention Time / min

Impurity C


Impurity B


Unidentified Impurity


Impurity A




HPLC 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 5µm C18 110A 250-4.6 mm column . The fig.06 below show chromatogram of Phenoxybenzamine HCl injection analysis repeated at Kingston University(Appendix A-2).

The peaks were interested and its retention times 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)


Retention Time / min

Impurity C


Impurity B


Unidentified Impurity


Impurity A




LC-MS Analysis of Phenoxybenzamine Injection Concentrate:

Phosphate 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 development 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 = 30:70 .

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 ( Modified Mobile phase for

LC-MS analysis)

Table 03: Peaks and its retention time of Phenoxybenzamine (Ammonium formate as

buffer)The samples were run on HPLC several times and constant chromatographic development was observed. Hence the sample fractions of unknown impurity was collected several time during HPLC run for LC-MS (accurate mass measurement) and LC-MS/MS (selective ion monitoring) analysis.


Retention Time / min

Impurity C


Impurity B


Unidentified Impurity


Impurity A




Accurate mass measurement with Time of Flight (ToF) mass detector

Feasibility 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 Waters micromass LCT ESI TOF-MS and obtained the average of the accurate mass value of unknown impurity. Ionization technique is Electron spray Ionization and mass analyzer is Time of Flight analyzer in this instrument. Results were taken on positive mode ( M + H + ). Hence mass of one proton must be deducted from the spectral 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 mass

M + H + / Da

Mass of Unknown Compound / Da































The average molecular weight and standard 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 software possible elemental composition was obtained for the molecular weight of 343.209 with 200.0 mDa tolerance . It was able to exclude a large amount elemental composition to narrow findings. That is elimination 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 of

100/32.4 (35Cl/37Cl ratio of 100/32.4). 15,16 Hence only C,H,N and O elements were limited on search.

Still hundreds of composition are left to be examined to find correct elemental composition . The second exclusion 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 even number of nitrogen in list. The following table shows considerable elemental composition left after above two exclusions .

Table 04. Possible elemental composition and its

Monoisotopic mass

Elemental Composition

Calculated Monoisotopic mass















The 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 Concentrate

Thermo 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 standard

Parameter used:

Parent mass: 304.4

Scan time: 0.5000

Collision energy: 16

Collision gas pressure: 1.1 Barr

Spray Voltage: 4000

Scan Mode: Product Ion Scan

Fig. 08 Product ion scan mass spectrum of Phenoxybenzamine Standard

Peaks at m/z 63, 84, 91,107,120, 135 and 212 were observed. The base peak is observed at m/z 91 benzyl fragment . It is stabilized by the resonance form of benzene.17 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 Phenoxybenzamine

Apart from the peaks illustrated on above figure, the peaks at m/z 120 arise due to mass unit

CH2NCH2CH2Cl + H+ . This is the middle portion after the mass unit at m/z 91 and 93 break 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 standard

Parameter used:

Parent mass: 286

Scan time: 0.5000

Collision energy: 16

Collision gas pressure: 1.1 Barr

Spray Voltage: 4000

Scan Mode: Product Ion Scan

Fig. 09 Product ion scan mass spectrum of Impurity B Standard

Product 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 B

As seen in the Phenoxybenzamine product ion scan spectrum the removal 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)=102]

Analysis of Unknown Impurity

Parameter used:

Parent mass: 286

Scan time: 0.5000

Collision energy: 16

Collision gas pressure: 1.1 Barr

Spray Voltage: 4000

Scan Mode: Product Ion Scan

Fig. 10 Product ion scan mass spectrum of Unknown Impurity

Product 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 quiet

similar 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 and

unknown impurity product ion scan spectrum. Also as studied previously in the spectrum of PBA and impurity B, the deduction 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-amine

The 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 correspond to the peaks observed on mass spectrum.

Fig. 12 Illustration of break downs of Unknown Impurity

Most 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 synthesis 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)






(a)Oxidation ;(b) 1-phenylmethanamine, HC1/EtOH,molecular sieves 4A, NaBH3CN; (c) Br(CH2)2OH, K2CO3, EtOH; (d) SOCI 2, HCI (g), Benzene (i)phenol; (2)2-methyl oxirane ;(3)1-phenoxypropan-2-ol;(4) 1-phenoxypropan-2-one; (5) N-benzyl-1-phenoxypropan-2-amine; (6) 2-[benzyl(1-phenoxypropan-2-yl)amino]ethanol;(7) N-benzyl-N-(2-chloroethyl)-1-phenoxypropan-2-amine (Phenoxybenzamine)

Scheme 1. Predicted synthesis route for Phenoxybenzamine

Formation of Impurity A

The reaction between phenol (i) and 2-methyl oxirane (ii) is SN2 nucleophilic substitution. Nucleophiles are more reactive to most substituted carbon of epoxides under acidic condition and least substituted carbon is favoured under basic condition.19 In this case 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 oxirane

To very few extent 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-ol

Formation of Impurity B and Impurity C

Impurity B, 2-[benzyl(1-phenoxypropan-2-yl)amino]ethanol 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 unoxidised

1-phenoxypropan-2-ol left over is chlorinated by SOCI 2, HCI during last step of the synthesis.

Refer (d) of the scheme.


Preliminary 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. Further to these preliminary structural assignments various spectroscopic studies such as LC-NMR and IR need to be carried out to complete the characterization of the unknown compound. Ultimately the proposed structure can be confirmed by synthesizing N-benzyl-N-[(E)-2-phenylethenyl] -1- phenoxy-propan-2-amine in future.