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World Journal of Analytical Chemistry, 2014, Vol. 2, No. 1, 6-9

Available online at http://pubs.sciepub.com/wjac/2/1/2 © Science and Education Publishing

DOI:10.12691/wjac-2-1-2

Identification and Quantitative Analysis of 2- Phenylethanol as Major Impurity in Bromadol by HPLC

Zhi Li 1,* , Ting Huang 2 , Jia-Hao Guo 1 , Jing-Xiang Zhuang 1

  • 1 Department of Chemistry, School of Science, Beijing Jiaotong University, Beijing, China

  • 2 Division of Chemical Metrology and Analytical Science, National Institute of Metrology, Beijing, China *Corresponding author: zhili@bjtu.edu.cn

Received November 17, 2013; Revised January 17, 2014; Accepted January 25, 2014

Abstract Bromadol can serve as a powerful and versatile agent in medicinal chemistry. In this paper, 2- phenylethanol was first identified as a main impurity in commercial Bromadol sample by HPLC. HPLC quantitative analysis method for impurity 2-phenylethanol in Bromadol has also been established for the first time. By our new HPLC analysis method, the impurity 2-phenylethanol was found to be 9.6% by weight. The identified main impurity 2-phenylethanol can be successfully removed by heating under vacuum, which gave an easy way to purify Bromadol for mass production.

Keywords: HPLC, Bromadol, 2-phenylethanol

Cite This Article: Zhi Li, Ting Huang, Jia-Hao Guo, and Jing-Xiang Zhuang, “Identification and Quantitative Analysis of 2-Phenylethanol as Major Impurity in Bromadol by HPLC.” World Journal of Analytical Chemistry, vol. 2, no. 1 (2014): 6-9. doi: 10.12691/wjac-2-1 -2.

1. Introduction

Bromadol (Figure 1) was first developed by Daniel Lednicer at Upjohn in the 1970s as a powerful narcotic analgesic [1].

World Journal of Analytical Chemistry, 2014, Vol. 2, No. 1, 6-9 Available online at http://pubs.sciepub.com/wjac/2/1/2 ©

Figure 1. Chemical Structure of Bromadol

Initial studies showed that Bromadol was around 10’000 times the strength of morphine [2] in animal models. Recent research [3] assigned a value of x504 morphine for the more active trans Bromadol isomer. The antinociceptive ED 50 in mouse hot plate of Bromadol is 13.4 μg/kg [3] and the binding K i values for μ- opioidreceptor of the Bromadol is 1.49 nM [3]. Recently, Bromadol was prepared and used as regulators for the nociceptin/orphanin FQ ligand ORL-1 receptor system [4]. Bromadol is also the lead compound of 4 -amino -4- arylcyclohexanone derivatives [5,6,7,8,9] which can serve as a potent class of analgesics [3,6,7]. The structure- activity relationships (SAR's) of this series compounds has been discussed [10,11]. p-halo substitutions optimize the ability of the compounds to gain access to the analgesic receptors, whereas m-OH substitution induced narcotic antagonist activity [10]. Trans conformation and hydrophobicity of the substituents at the C atom possessing the OH group in the cyclohexanol nucleus were most important for the analgesic activity [11].

Until now, many literatures [2,4,7,12,13,14] have been reported concerning about the synthesis of Bromadol. The most widely used synthesis route is outlined in scheme 1.

World Journal of Analytical Chemistry, 2014, Vol. 2, No. 1, 6-9 Available online at http://pubs.sciepub.com/wjac/2/1/2 ©

Scheme 1. Most widely used synthesis route of Bromadol

As Bromadol could serve as a powerful and versatile agent in medicinal chemistry, it is of great importance to identify the impurities in Bromadol and establish a quality control methodology for production. However, few literatures have been reported concerning about this issue until now. In this paper, we try to employ HPLC to identify and quantitative analysis of impurities in Bromadol (The Bromadol sample was donated by Beijing Realchem Technology Co., Ltd as a gift).

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2. Experimental

Firstly, the commercial Bromadol sample was dissolved in absolute methanol and the concentration was shown in Table 1.

Table 1. commercial Bromadol sample solution

   

methanol

   

sample

sample

weight

mg

solvent

weight

mg

concentration

(mg/g)

concentration

mg/ml

commercial

       

Bromadol

sample

 

1.08

830.76

1.298

 

1.028

The solution was then subjected to HPLC analysis. The HPLC spectrum and the analysis result were shown in Figure 2 and Table 2 respectively.

1.08 830.76 1.298 1.028 The solution was then subjected to HPLC analysis. The HPLC spectrum and

Figure 2. HPLC spectrum of the commercial Bromadol sample solution

A phase: CH 3 CN (50%), B phase: H 2 O Detection wavelength: 250 nm Injection volume: 10 μL Column temperature: 40°C Column: Inertsil ODS-SP 5um 4.6x250 mm

 

Table 2. HPLC analysis data of the commercial Bromadol sample solution

   

Retention

time

Peak

 

Peak

Peak

 

Symmetry

Peak

No.

(min)

area

height

width

factor

area %

 
  • 1 3.594

7.4

 

1.2

0.084

 

0.458

0.072

 
  • 2 4.385

63.6

 

4.8

0.1857

 

1.499

0.621

 
  • 3 7.317

178.7

 

18.5

0.1468

 

0.764

1.744

 
  • 4 10.113

   
  • 21.4 1.7

0.1923

 

0.753

0.209

 
  • 5 11.398

   
  • 75.7 4.8

0.2401

 

0.74

0.739

 
  • 6 15.012

   
  • 94.5 4.3

0.3262

 

0.852

0.922

 
  • 7 17.485

9804.1

 

264.9

0.5158

 

2.068

95.692

As shown in Figure 2 and Table 2, the retention time of Bromadol was 17.392 min with peak area percentage 95.692% (No.7, Table 2). Many impurity peaks can also be observed. The retention times of the main impurity peaks were 4.379 min, 7.312 min, 11.395 min, 14.972 min respectively.

Subsequently, we tried to identify the main impurities in the commercial Bromadol sample. As impurities may always come from the last synthesis step, the most widely used synthesis route of Bromadol (Scheme 1.) was carefully studied. We speculate that the main impurity may be 2-phenylethanol which may result from hydrolysis of the phenethylmagnesium bromide.

 

Table 3. 2-phenylethanol standard solution

 
 

methanol

 
 

Sample

weight

mg

 

solvent

weight

mg

concentration

(mg/g)

concentration

mg/ml

2-

phenyl-

1.63

1273.89

1.277

 

1.012

sample ethanol
sample
ethanol

A standard solution of 2-phenylethanol in absolute methanol was prepared and the concentration was shown in Table 3. The standard 2-phenylethanol solution was subjected to HPLC under the same analysis conditions which have been employed for the commercial Bromadol sample solution. The HPLC spectrum and analysis result of the standard 2-phenylethanol solution were shown in Figure 3 and Table 4 respectively.

World Journal of Analytical Chemistry 7 2. Experimental Firstly, the commercial Bromadol sample was dissolved in

Figure 3. HPLC spectrum of 2-phenylethanol standard solution

A phase: CH 3 CN (50%), B phase: H 2 O Detection wavelength: 250 nm Injection volume: 10 μL Column temperature: 40°C Column: Inertsil ODS-SP 5um 4.6x250 mm

Table 4. HPLC analysis data of 2-phenylethanol standard solution

No.

Retention

time

Peak

Peak

Peak

Symmetry

Peak

(min)

area

height

width

factor

area %

1

 

5

  • 3.576 0.0711

1

   

0.73

2

 

659.1

  • 4.375 0.1698

55.8

   

96.156

3

 

21.3

  • 4.814 0.1613

1.9

   

3.114

 

0.709

1.817

1.063

As shown in Figure 3 and Table 4, the retention time of 2-phenylethanol was 4.375 min with peak area percentage 96.156% (No. 2, Table 4). The retention time of 2-

phenylethanol was same as one of the main impurities in

the commercial Bromadol sample (see Figure 2). This

result indicated that one of the main impurities in the

commercial Bromadol sample was 2 -phenylethanol.

Next, we tried to analyze the 2-phenylethanol impurity

in the commercial Bromadol sample. The commercial Bromadol sample solution and the standard 2- phenylethanol solution were subjected to HPLC analysis for 3 times under the same conditions which have been employed previously. The peak area of 2 -phenylethanol of the two solutions were shown in Table 5.

 

Table 5. Peak area of 2-phenylethanol in two solutions

No.

peak area of 2-phenylethanol in commercial Bromadol solution

peak area of 2- phenylethanol in 2-phenylethanol standard solution

1

63.6

659.1

2

62.4

658.7

3

66.8

659.6

average

64.3

659.1

With the peak area data of 2 -phenylethanol in Table 1, Table 3 and Table 5, the concentration of impurity 2- phenylethanol in commercial Bromadol sample solution was calculated to be 0.0987 mg/ml and the 2-

phenylethanol accounts for 9.6% of the commercial

Bromadol sample by weight. As 2-phenylethanol is a colorless oil with boiling point of 219°C, we speculate that it could be removed from

8

World Journal of Analytical Chemistry

Bromadol by drying under vacuum without leading to decomposition of Bromadol. 0.5 gram of the commercial Bromadol sample was placed into vacuum drying oven at 45°C for 12 h. After drying under vacuum, the Bromadol sample was dissolved in absolute methanol and the concentration was shown in Table 6.

Table 6. Dried Bromadol sample solution

   

methanol

   

sample

Sample

weight

mg

solvent

weight

mg

concentration

(mg/g)

concentration

mg/ml

Dried

       

Bromadol

1.29

987.76

1.304

1.033

sample

The dried Bromadol sample solution was subjected to HPLC under the same analysis conditions employed previously. The HPLC spectrum and the analysis data were shown in Figure 4 and Table 7 respectively.

8 World Journal of Analytical Chemistry Bromadol by drying under vacuum without leading to decomposition of

Figure 4. HPLC spectrum of dried Bromadol sample solution

A phase: CH 3 CN (50%), B phase: H 2 O Detection wavelength: 250 nm Injection volume: 10 μL Column temperature: 40°C Column: Inertsil ODS-SP 5um 4.6x250 mm

Table 7. HPLC analysis data of dried Bromadol sample solution

 

Retention

Peak

Peak

Peak

Symmetry

Peak

time

area

height

width

factor

area %

1

3.57

8.1

1.2

0.0902

0.449

0.075

     

4.20E-

     

2

4.516

6.2

01

0.2484

4.415

0.058

3

7.344

194.9

19.8

0.1507

0.764

1.815

4

10.307

  • 23.4 1.8

 

0.1955

0.727

0.218

5

11.626

  • 80.3 5.2

 

0.235

0.776

0.748

6

15.254

  • 95.1 4.3

 

0.3283

0.838

0.886

7

17.775

10223.7

270.6

0.5307

2.068

95.215

8

31.629

105.8

2.8

0.467

1.076

0.985

8 World Journal of Analytical Chemistry Bromadol by drying under vacuum without leading to decomposition of

Figure 5. H-NMR spectrum of the dried Bromadol sample

As shown in Figure 4 and Table 7, the peak of impurity 2-phenylethanol (retention time is 4.375 min) disappeared. This result indicated that the impurity of 2-phenylethanol was successfully removed under vacuum at 45°C for 12 h.

The dried Bromadol sample was subsequently subjected to

  • 1 H-NMR analysis and the 1 H-NMR spectrum was shown in Figure 5. As shown in Figure 5, the 1 H-NMR spectrum of the dried Bromadol sample was quite clean which also indicated that the impurity 2-phenylethanol has been successfully removed. The result also gave an easy method to purify Bromadol for mass production.

3. Conclusion

As Bromadol could serve as a powerful and versatile

agent in medicinal chemistry, it is of great importance to identify the impurities in Bromadol in order to give instructions for purification and mass production. It is also important to establish a convenient and reliable quality control methodology for this compound. However, few literatures have been reported concerning about the se issues until now. In this article, 2-phenylethanol has been identified as one of main impurities in commercial Bromadol by HPLC for the first time. This main impurity should come from the last synthesis step. The HPLC quantitative analysis method for 2- phenylethanol in Bromadol has also been established for the first time. The new method can be employed for quality control of Bromadol. By our new analysis method, it was found that the 2-phenylethanol impurity accounted for 9.6% by weight in the commercial Bromadol sample. The identified impurity 2 -phenylethanol can be removed under vacuum at 45°C for 12 h which give an easy method to purify Bromadol for mass production. Further research to identify other impurities in Bromadol is underway in our lab and the result will be reported in the due course.

Acknowledgement

This research was supported by NSFC (21302011) and

the Fundamental Research Funds for the Central

Universities (2011JBM295). The authors thank Beijing

Realchem Technology Co., Ltd for the Bromadol sample.

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