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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
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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
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2500
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500
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ps
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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
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ps
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10
0
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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
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y, c
ps
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0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
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3.71
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ps
Inte
nstty,
cps
1200
1000
800
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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
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500
00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.54.0
3.72
4000
3500
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3.714500
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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
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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
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Time, min
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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
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tensi
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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
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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
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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.
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