Research Article

OPEN ACCESS Freely available online

doi:10.4172/1948-593X.1000029

JBABM/Vol.2 Issue 4

J Bioanal Biomed ISSN:1948-593X JBABM, an open access journal

Volume 2(4) : 091-095 (2010) - 091

Journal of Bioanalysis & Biomedicine - Open Access

Quantifi cation of 4-Oxiranyl Methoxy-9h-Carbazole a Genotoxic Impurity in Carvedilol Drug Substances by Lc-MsM. Srinivasa Rao1*, Sumathi V. Rao1, U.K.Ray1, G.Sri Siva Kumar1, Hemant Kumar Sharma1 and K.Mukkanti2

1Department of Analytical research, Aurobindo Pharma Research Centre, 313 Bachupally, Qutubullapur Mandal, Hyderabad - 500090, India2Head of Centre for Pharmaceutical sciences, JNTU Institute of sciences & Technology, Kukatpally, Hyderabad - 500085, India

Keywords: HPLC; LC-MS; Genotoxins; Carvedilol; 4-Oxiranylmethoxy-9H-Carbazole

Introduction

4-Oxiranylmethoxy-9H-Carbazole is generally used as an important intermediate in the preparation of pharmaceutical compounds such as antihipertensives. However, this 4-Oxiranylmethoxy-9H-Carbazole is found to be a genotoxic product. This Genotoxic compounds affects the human genes, and the presence of these genotoxins in the pharmaceutical compounds can be controlled. In current regulatory practice (ICH Guidelines 2002), genotoxic compounds are usually considered to operate by a non-threshold mode of action and, thus any level of exposure carries at least theoretically a risk. This precautions view implies that pharmaceutical measurements should be guided by the so called ‘ALARA’ principle (As Low As Reasonably Achievable), i.e., where avoidance is not possible, genotoxic impurities must be kept to a low level. However, the draft guidelines from European agency (Guidelines on the limits of Genotoxic impurities CPMP/SWP/5199/02, EMEA/CHMP/QMP/251344/2006, EMEA/CHMP/SWP/431994/2007) and feedback from the US food and drug administration (USFDA) (USFDA, Guidance for Industry 2008) to pharmaceutical industries via drug application has enabled the pharmaceutical industries to establish interim strategies. Generally the daily intake of these genotoxins has been limited to a daily dose of 1.5 g/day. Therefore it is preferable for the potential genotoxins to be controlled during the synthesis; where the levels cannot be controlled and no safety data yet exists it may be preferable for the pharmaceutical company to change the route of synthesis of the drug substances. As 4-Oxiranylmethoxy-9H-Carbazole is also a genotoxic compound, the regulatory team may be expected to estimate the levels of 4-Oxiranylmethoxy-9H-Carbazole to be controlled to 15 ppm in the drug substance. It was felt necessary to develop simple, sensitive validated method for 4-Oxiranylmethoxy-9H-Carbazole.

Experimental

Sample, chemicals and reagents

Carvedilol was synthesized from the CRD research department

in APL Research centre, (A Division of Aurobindo Pharma Ltd.) (Bachupally, Quthubullapur, Hyderabad-90, INDIA). Acetonitrile (HPLC grade), Ammonium acetate (BDH grade), High pure water was prepared by using the Milli-Q plus purifier.

Preparation of solution

Preparation of standard stock solution: Accurately weigh and transfer about 10 mg of 4-Oxiranylmethoxy-9H-Carbazole working

standard into a 100 ml volumetric flask (0.10 g/ml). Dilute 10 ml of this solution to 100 ml with diluents (0.01 g/ml). Further dilute 10 ml of this solution to 100 ml with diluents (0.001 g/ml).

Preparation of standard stock solution: Transfer accurately 7.5 ml of 4-Oxiranylmethoxy-9H-Carbazole Stock solution in 100ml clean (15 g/gm with respect to sample concentration 5 mg/ml). Spectrogram is shown in the Figure 1.

Instrumentation

Mass spectrometry: The LC-MS/MS system was used for method

*Corresponding author: M. Srinivasa Rao, APL Research Centre (A Division of

Aurobindo Pharma Ltd.), 313, Bachupally, Quthubullapur, Hyderabad 500090, India, Tel: +91 40 23040261; Fax: +91 40 23042932; E-mail: metta_cnu@yahoo.co.in

Received July 02, 2010; Accepted August 23, 2010; Published August 23, 2010

Citation: Srinivasa Rao M, Rao SV, Ray UK, Siva Kumar GS, Sharma HK, et

al. (2010) Quantifi cation of 4-Oxiranyl Methoxy-9h-Carbazole a Genotoxic Impurity in Carvedilol Drug Substances by Lc-Ms. J Bioanal Biomed 2: 091-095.

doi:10.4172/1948-593X.1000029

Copyright: © 2010 Srinivasa Rao M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which

permits unrestricted use, distribution, and reproduction in any medium, provided

the original author and source are credited.

Abstract

LC-MS multiple reaction monitoring (MRM) method had been evaluated for the determination of the very low level of 4-Oxiranylmethoxy-9H-Carbazole in drug substances such as Carvedilol (U.S. Pharmacopoeia (USP), 32NF-27,Britishpharmacopoeia (BP), 2009, Martindale 35th edition) This 4-Oxiranylmethoxy-9H-Carbazole have been identifi ed aspotential genotoxic impurity. LC-MS was found to be more promising and the limit of quantifi cation was 15 μg/gm.

Concentration (μg/g) Area Statistical analysis

22.5 31715

18 25327 Slope 1381

15 20813

12.5 16380 Intercept -191

6 8407

1.5 1884Residual Sum of Squares 371

1 1383

0.2 330Correlation Coefficient 0.9995

Table 1: Results of linearity study.

28330

24330

20330

16330

12330

8330

4330

330

0.206 3.206 6.206 9.206 12.206 15.206 18.206 21.206

Concentration ( g/g)

Linearity plot

Citation: Srinivasa Rao M, Rao SV, Ray UK, Siva Kumar GS, Sharma HK, et al. (2010) Quantifi cation of 4-Oxiranyl Methoxy-9h-Carbazole a Genotoxic Impurity in Carvedilol Drug Substances by Lc-Ms. J Bioanal Biomed 2: 091-095. doi:10.4172/1948-593X.1000029

J Bioanal Biomed ISSN:1948-593X JBABM, an open access journal

Volume 2(4) : 091-095 (2010) - 092

development and validation was done in Applied Biosystems Sciex API 2000 model coupled with Shimadzu HPLC system with mass detector and auto sampler was used in the experiment. Data acquisition and processing were conducted by using the Analyst software.

LC-MS/MS analysis

LC-MS/MS analysis was carried out using Perkin Elmer triple

Inte

nstty,

cps

3000

2500

2000

1500

1000

500

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Time, min

3.71

Inte

nstt

y, c

ps

3000

2500

2000

1500

1000

500

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Time, min

3.71

Inte

nstty,

cps

3000

2500

2000

1500

1000

500

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Time, min

3.71

Inte

nstt

y, c

ps

3000

2500

2000

1500

1000

500

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Time, min

3.72

Inte

nstt

y, c

ps

3000

2500

2000

1500

1000

500

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Time, min

3.72

Inte

nstt

y, c

ps

3000

2500

2000

1500

1000

500

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Time, min

3.72

Figure 1: System Precision.

Area

4-Oxiranylmethoxy-9H-Carbazole Injection ID

LOD LOQ

1 1148 3426

2 988 3693

3 1140 3655

4 1044 3922

5 1115 3833

6 1273 4153

Mean 1118 3780

SD 98 249

% RSD 8.8 6.6

Conc. (μg/g) 0.825 2.68

Note: SD: Standard deviation, RSD: Relative Standard deviation

Table 2: Results of LOD and LOQ study.

Inte

nsity,

cps

Time, min

140

120

100

60

50

40

20

01 2 3 4 5 6 7 8 9 10

0.33 0.72 1.58 2.25 2.88 3.1 5.757.30 9.42 9.91

8.928.077.896.56 6.745.02 5.32

SPECIFICITY - A

Inte

nsity,

cps

1 2 3 4 5 6 7 8 9 10Time, min

SPECIFICITY - B

800

700

600

500

400

300

200

100

0

Inte

nsity,

cps

Time, min

0.28 1.15 1.41 1.96 2.75 3.12 4.23 5.364.90

3.75

6.36 6.63 7.40 8.50 9.02 9.65 9.858.467.01

1 2 3 4 5 6 7 8 9 10

SPECIFICITY - C

3000

2500

2000

1500

1000

500

0

Figure 2: Specifi city-A: Mass Spectrogram of Carvedilol Drug Substance (Control sample). Specifi city-B: Mass Spectrogram of Carvedilol Drug

Substance spiked with all related Compounds without 4 -Oxiranylmethoxy-9H-

Carbazole at specifi cation level. Specifi city-C: Mass Spectrogram of Carvedilol Drug Substance spiked with all related. Compounds with 4 -Oxiranylmethoxy-

9H-Carbazole at specifi cation level.

Research Article

OPEN ACCESS Freely available online

doi:10.4172/1948-593X.1000029

JBABM/Vol.2 Issue 4

J Bioanal Biomed ISSN:1948-593X JBABM, an open access journal

Volume 2(4) : 091-095 (2010) - 093

Journal of Bioanalysis & Biomedicine - Open AccessIn

tenstty,

cps

Inte

nstt

y, c

ps

90

80

70

60

50

40

30

20

10

0

300

250

200

150

100

50

0

200

150

100

50

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Time, min

Time, min

Inte

nstty,

cps

Time, min

3.71

3.71

0.310.49

0.220.320.630.701.03 1.23 1.651.99 2.112.192.54 2.852.91 3.34 4.114.20

0.310.53 1.04 1.81 1.97 2.612.89 3.53 4.074.124.444.68

4.784.87

0.78 0.83 1.50 1.852.04

2.29 2.892.91

2.983.17

3.50 3.864.04 4.60 4.72

0.140.22 0.63 0.911.101.43 1.571.75 2.05

2.232.482.70 3.053.273.804.134.44 4.504.78

3.72

Inte

nstt

y, c

ps

Inte

nstt

y, c

ps

Inte

nstt

y, c

ps

2500

2000

1500

1000

500

0

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Time, min

Time, min

3.71

Inte

nstt

y, c

ps

Inte

nstty,

cps

1200

1000

800

600

400

200

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Time, min

Time, min

Time, min

3.72

3000

2500

2000

1500

1000

500

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.54.0

3.72

4000

3500

3000

2500

2000

1500

1000

500

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

3.714500

4000

3500

3000

2500

2000

1500

1000

500

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

3.71

quadrupole mass spectrometer (API 2000, PE SCIEX) coupled with

Shimadzu HPLC equipped with SPD 10 A VP UV-VIS detector and LC

10 AT VP pumps. Analyst software was used for data acquisition and

data processing. The turbo ion spray voltage was maintained at - 4.5

Kv and temperature was set at 375°C. The auxiliary gas and sheath

gas used was high pure Nitrogen. Zero air was used as Nebulizer

gas. The analysis was carried out using YMC PACK C8 250 X 4.6 mm

column with 5 m particle diameter with mobile phase consisting of

a mixture of 0.01M Ammonium acetate pH-5 in water and acetonitrile

in the ratio of 15:85 v/v. The flow rate was 1ml/min with flow rate

split down to 0.2 ml/min in to the LC-MS system. The column was

monitored at 55°C.The injection volume was 20 l. Electrospray

ionization in negative mode was used with a multiple reaction

monitoring (MRM) mode was used as MS method for quantification of

4-Oxiranylmethoxy-9H-Carbazole in drug substances. In this method

4-Oxiranylmethoxy-9H-Carbazole is monitored by its molecular

ion value of 238.10 (M-H) and its daughter ion 181.0 with focusing

potential-270, declustering potential -25, entrance potential -10 , and

the curtain gas flow 25 (psi) respectively.

Results and Discussion

The main target of LC-MS/MS method was to quantification of

4-Oxiranylmethoxy-9H-Carbazole impurity in the Carvedilol active

ingredient. During the method development we used different

reversed phase stationary and mobile phase was used and finally

chromatographic separation was achieved on a YMC PACK C8

250*4.6,5mic column (YMC) in isocratic mode using with 0.01M

Ammonium acetate pH-5 in water and Acetonitrile in the ratio of

15:85 v/v. The flow rate was 1.0 ml/min split down to 0.2 ml/min in

to LC-MS system.

Figure 3: Linearity mass spectrogram of 4 -Oxiranylmethoxy-9H-Carbazole.

Citation: Srinivasa Rao M, Rao SV, Ray UK, Siva Kumar GS, Sharma HK, et al. (2010) Quantifi cation of 4-Oxiranyl Methoxy-9h-Carbazole a Genotoxic Impurity in Carvedilol Drug Substances by Lc-Ms. J Bioanal Biomed 2: 091-095. doi:10.4172/1948-593X.1000029

J Bioanal Biomed ISSN:1948-593X JBABM, an open access journal

Volume 2(4) : 091-095 (2010) - 094

Validation of the method for 4-oxiranylmethoxy-9H-carbazole in carvedilol

Specificity of 4-Oxiranylmethoxy-9H-Carbazole: Solutions are

prepared using Carvedilol drug substance (Control Sample), Spiked

with all the related substances in Carvedilol drug substance except

4-Oxiranylmethoxy-9H-Carbazole (Specificity-A) and Spiked with all

the related substances including 4-Oxiranylmethoxy-9H-Carbazole in

Carvedilol at specification level and subjected for LC-MS/MS study

for the evaluation of specificity. The sample spiked with all other

impurities without 4-Oxiranyl methoxy-9H-Carbazole at specification

level, do not show any relevant response in spectrogram. The sample

spiked with all other impurities along with 4-Oxiranylmethoxy-

9H-Carbazole (Specificity-B) at specification level shows response

equivalent to standard in spectrogram at retention time of

4-Oxiranylmethoxy-9H-Carbazole (m/z-238.10 M-H and 181.0) hence

the method is specific and selective. Spectrogram is shown in the

Figure 2.

Linearity of 4-Oxiranylmethoxy-9H-Carbazole: By selecting

ion monitoring, the linearity of 4-Oxiranylmethoxy-9H-Carbazole

was satisfactorily done a series of solutions were prepared using

4-Oxiranylmethoxy-9H-Carbazole at concentration levels from around

detection level to 150% and the concentration levels are 22.5 (g/g),

18 (g/g), 15 (g/g), 12.5 (g/g), 6 (g/g), 1.5 (g/g), 1 (g/g) and 0.2

(g/g) respectively. The peak area versus concentration data was

done by linearity plot slop, intercept, and residual sum of squares

analysis. The calibration curve was given based on response over

the concentration range for 4-Oxiranylmethoxy-9H-Carbazole. The

correlation coefficients for 4-Oxiranylmethoxy-9H-Carbazole were

0.999. Linearity of the 4-Oxiranylmethoxy-9H-Carbazole spectrogram

was shown in the Figure 3 and the results are tabulated in Table 1.

Limit of detection (LOD) and Limit of Quantification (LOQ)

for 4-Oxiranylmethoxy-9H-Carbazole: The LOD and LOQ values of

4-Oxiranylmethoxy-9H-Carbazole were predicted from the linearity

data. Each predicted concentration was verified for precision by

preparing the solutions at about predicted concentration and

injecting each solution six times for LC-MS/MS study and the predicted

concentration for LOQ was 2.68 (g/g) and LOD was 0.825 (g/g). The

Sample ID Amount added (μg/g)

Amount found (μg/g)

% Recovery

Statistical Analysis

LOQ Level Sample 1

2.679 2.7600 103.0 Mean 96.1

LOQ Level Sample 2

2.681 2.4240 90.4 SD 6.39

LOQ Level Sample 3

2.680 2.5410 94.8 % RSD 6.6

100% Level Sample 1

15.445 15.5480 100.7 Mean 97.7

100% Level Sample 2

15.491 14.2500 92.0 SD 4.91

100% Level Sample 3

15.454 15.5000 100.3 % RSD 5.0

150% Level Sample 1

23.181 24.4420 105.4 Mean 101.5

150% Level Sample 2

23.204 22.5190 97.0 SD 4.24

150% Level Sample 3

23.195 23.6960 102.2 % RSD 4.2

Overall Statistical Analysis

Mean 98.4 SD 5.16 % RSD 5.2 95%

Confidence Interval

± 12.8

Note: SD: Standard deviation, RSD: Relative Standard deviation

Table 3: Results of recovery study.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

0.250.17

200

180

160

140

120

100

80

60

40

20

0

0.50 1.08 1.47 1.74 1.85 2.35 2.45 2.782.97 3.17 3.27

3.72

3.89 4.25 4.83

Inte

nsi

ty,c

ps

Inte

nsi

ty,c

ps

Inte

nsi

ty,c

ps

Inte

nsi

ty,c

ps

Time, min

Time, min

Time, min

Time, min

Time, min

200

180

160

140

120

100

80

60

40

20

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

0.17 0.31 0.75 1.14 1.411.75 2.13 2.49 2.57 2.96 3.30 3.58

3.74

4.04 4.18 4.59 4.85

Inte

nsi

ty,c

ps

Time, min

200

180

160

140

120

100

80

60

40

20

0In

tensi

ty,c

ps

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

200

180

160

140

120

100

80

60

40

20

0

0.020.35 0.520.701.02 1.181.48 1.72

2.11 2.32 2.492.88 3.17 3.50

3.79

4.20 4.42 4.75

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

0.33 0.430.70

1.031.26 1.63 2.002.19 2.402.73 2.86 2.94 3.42

3.73

4.08 4.38 4.874.96

220

200

180

160

140

120

100

80

60

40

20

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

0.30 0.71 0.94 1.26 1.56 1.882.09 2.442.63 2.81 3.27 3.51 4.03 4.14 4.87

3.73

0.37

200

180

160

140

120

100

80

60

40

20

00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

0.31 0.430.74 1.17 1.32 1.501.92 2.10 2.312.65

2.713.003.10

3.60

3.74

3.874.44 4.584.74

Figure 4: LOD precision mass spectrogram of 4 -Oxiranylmethoxy-9H-

Carbazole.

Research Article

OPEN ACCESS Freely available online

doi:10.4172/1948-593X.1000029

JBABM/Vol.2 Issue 4

J Bioanal Biomed ISSN:1948-593X JBABM, an open access journal

Volume 2(4) : 091-095 (2010) - 095

Journal of Bioanalysis & Biomedicine - Open Access

spectrograms are shown in Figure 4 and Figure 5 (Figure 5 is included in supplementry data). Further the results are tabulated in Table 2.

Recovery of 4-oxiranylmethoxy-9H-carbazole from the API: The accuracy of the method was evaluated in sample solutions were prepared in triplicate by spiking 4-Oxiranylmethoxy-9H-Carbazole at LOQ level to 150% with Carvedilol drug substance and injected each solution in to LCMS as per methodology. The percentage of recovery was calculated. A satisfactory value of 4-Oxiranylmethoxy-9H-Carbazole (96.10%, 97.70% and 101.5%) was found. At such low levels these recoveries and % RSD were satisfactory. Accuracy at LOQ to 150% level spectrogram is shown in Figure 6 and Figure 7 (Figure 6 and Figure 7 is included in supplementry data), further the results are tabulated in Table 3.

Conclusion

LC-MS/MS method has been suitable for quantification of 4-Oxiranylmethoxy-9H-Carbazole for highly sensitive method ofanalysis with limit of detection 0.825 ppm. The methodology has oneof the restrictions of 4-Oxiranylmethoxy-9H-Carbazole in Carvedilolsample.

Acknowledgement

The authors gratefully acknowledge the management of Aurobindo Pharma

Limited, in general and APL Research Centre in particular for allowing us to carry

out the present work. The authors are also thankful to the colleagues of Analytical

research department and Chemical research department.

References

1. British pharmacopoeia (BP), pg374 2009.

2. EMEA Q&A EMEA/CHMP/SWP/43994/2007.

3. Guidelines on the limits of Genotoxic impurities EMEA-CIIMP/

QMP/251344/2006, 28 June, 2006.

4. International conference on Harmonization (ICH) Guidelines, Q3A(R):

Impurities in new drug substance February 2003.

5. Martindale 35th edition pg1114.

6. The European medicines agency (EMEA), committee for medical products for

Human use (CHMP), Guidelines on the limits of Genotoxic impurities CPMP/

SWP/5199/02.

7. U.S. Pharmacopoeia. (USP32 NF-27) (2007) 4036.

8. US FDA, Guidence for industry: genotoxic and carcinogenic impurities in drug

substances and products: recommended approaches (draft), December 2008.