Journal of Chromatographic Science Advance Access published February 1, 2015 Journal of Chromatographic Science 2015;1– 6 doi:10.1093/chromsci/bmu211
Article
Isolation and Structural Elucidation of an Unknown Impurity in Prasugrel by Semi-Preparative Liquid Chromatography Chao Liu1,2, Zhangxin Yu2, Fujun Wang2, Xing Zhong2, Li Jiang2, Feifei Zhang2, Yinxia Tang2, Zhaohua Yan2, Su Zeng1 and Tong Pu2* 1
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang Province, China, and 2Zhejiang Charioteer Pharmaceutical Co. Ltd., Tongyuanxi, Dazhan, Xinju County, Zhejiang Province, China
*Author to whom correspondence should be addressed. Email: [email protected] Received 23 April 2014; revised 19 November 2014
A brand-new impurity was detected by RP-HPLC in the prasugrel. The impurity was named as Impurity X. Impurity X was isolated by using semi-preparative HPLC followed by characterization using nuclear magnetic resonance spectroscopy and liquid chromatography–mass spectrometry. The functional mechanism of Impurity X was speculated. Impurity X could be controlled in the manufacture process of the prasugrel active pharmaceutical ingredient effectively.
Introduction Prasugrel hydrochloride is a newly generated thienopyridine organic compound. It is used to control the agglomeration of the hematoblast. It is an inactive pro-drug. After taken orally, it is turned into the active metabolite by the cytochrome P450 enzyme. It is found to block the adenosine diphosphate receptor so as to restrain the activation of the hematoblast (1 –4). There are several methods for the determination of the prasugrel and its impurities in bulk drug and pharmaceutical dosage forms reported in the literature (5 – 9). The high-performance thin layer chromatography (HPTLC) method has been reported for the determination on prasugrel (5). The UV-VIS spectroscopic method was reported for the determination for the prasugrel assay (6, 7). The reversed-phase high-performance liquid chromatography (RP-HPLC) method such as the different mobile phases in isocratic mode cannot be used for determination on the related substances of prasugrel active pharmaceutical ingredient (API) (8, 9). A brand-new RP-HPLC method was developed for the determination of the related substances of prasugrel in our laboratory. The gradient elution was developed especially on prasugrel API. Determination of unknown organic impurities is the key to the production of high-quality drug substances (10). ICH guidelines indicate that impurities at or above 0.1% in the drug substance require identification (11). An unknown impurity was detected in a laboratory sample of prasugrel when analyzed as own-developed RP-HPLC method. The semi-preparative HPLC was performed to isolate the unknown impurity followed by characterization using nuclear magnetic resonance spectroscopy and liquid chromatography –mass spectrometry (LC-MS). To the best of our knowledge, this impurity has not been previously reported. Experimental Materials and reagents Samples of prasugrel (batch no. 201308004-crude and 20140102-crude) were obtained from Zhejiang Charioteer
Pharmaceutical Co., Ltd. De-ionized water was prepared using a Milli-Q plus water purification system from Millipore (Bradford, PA, USA). HPLC grade methanol, acetonitrile, potassium dihydrogen phosphate and triethylamine were purchased from Merck China Limited (Shanghai, China).
High-performance liquid chromatography Samples were analyzed on a SHIMADZU LC-20A (Shimadzu Corporation, Kyoto, Japan) equipped with diode-array detector using an Agilent ZORBAX SB-C18 column (250 4.6 mm i.d., 5 mm particles; Agilent Corporation, USA). For chromatographic separations, mobile phase A was 0.02 M potassium dihydrogen phosphate (containing 0.20% triethylamine, pH adjusted to 4.50 with phosphoric acid) and mobile phase B was acetonitrile. The HPLC gradient program was set as time (min) /%A(v/v):0/ 50,25/50,50/30,60/30,61/50 and 70/50. The flow rate was 1.0 mL/min, and the photo-diode array detector wavelength was 240 nm. The column oven temperature was maintained at 208C. The prasugrel sample was prepared in diluent (acetonitrile) at 1 mg/mL concentration, and 10 mL of sample solution was injected into HPLC system. Semi-preparative HPLC Impurity X was isolated from the crude sample of prasugrel using a Shimadzu semi-preparative HPLC system consisting of a LC-8A binary gradient pump, a SPD-10AVP UV detector, a SIL-10AP autosampler and a FRC-10A fraction collector (Shimadzu Corporation, Kyoto, Japan). A Shim-pack PREP-ODS(H) KIT column (250 20 mm i.d., particle size 10 mm) (Shimadzu Corporation, Kyoto, Japan) was used for semi-preparative isolation. Mobile phase A was water and mobile phase B was acetonitrile. The HPLC gradient program is set as time (min)/ %A(v/v):0/50, 25/50, 50/30, 60/30, 61/50 and 70/50. The flow rate was 16.0 mL/min. A sample solution of 20 mg/mL was prepared using acetone as the diluent. The injection volume was 1 mL, and the detection was monitored at 240 nm. The collected fractions (between a retention time of 48 and 51 min) were evaporated by a rotory evaporator at 508C. LC-MS The MS study was performed on LC-MS2020 (Shimadzu Corporation, Kyoto, Japan) using an electrospray ionization source and a Q mass spectrometer. The temperature of heat block and decreased liquid were 400 and 2008. Nitrogen was used as both sheath and auxiliary gas. The mass-to-charge ratio
# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. Chromatogram of prasugrel in the HPLC method.
Figure 2. Chromatogram of prasugrel in the semi-preparative HPLC method.
Figure 3. MS spectrum of impurity X.
2 Liu et al.
Figure 4. MS spectrum of prasugrel.
Figure 5. MS spectrum fragmentation of prasugrel.
Table I Analysis on 1H-NMR of Impurity X and Its Resonance Peak of Correlation Spectroscopy
dH
HSQC dC
1
H– 1H COSY dH
HMBC dC
Position
0.735, 0.758, 0.867, 0.877 2.08 2.288 2.329 2.61 2.988 3.108 3.259 3.586 3.689 4.346 4.583 6.492 6.559 7.186, 7.203, 7.217, 7.233 7.374, 7.386, 7.396, 7.413
9.987, 10.409, 10.723, 11.574 18.051 19.906 16.288 23.232 52.903 36.109 35.811 41.835 43.613 52.535 70.126 114.276 114.422 115.441, 115.579, 122.907, 122.976 126.709, 128.893, 129.453, 129.69
2.08 0.735, 0.758
11.574, 10.723, 10.409, 9.987, 23.232, 70.126, 201.066, 202.737 9.987, 10.409, 115.579, 201.066 168.766 10.723, 11.574 40.616, 123.068, 124.133
20, 21, 31, 32 19 1 30 4 15 14 5 16 6 28 17 7 11 24, 25, 37, 38 26, 27, 35, 36
0.867, 0.877 3.586, 3.689 3.108 2.988 2.61 2.61
7.374, 7.386, 7.396, 7.413 7.186, 7.203, 7.217, 7.233
122.907, 135.845 122.907, 135.845, 166.965 124.133, 166.965 23.232, 126.709 122.907, 129.69 120.676, 122.976, 206.202 124.133, 126.709 128.188 158.534, 160.174 52.903, 158.534, 160.174
Isolation and Structural Elucidation of an Unknown Impurity in Prasugrel 3
was scanned across the range of m/z 50 –1,000. Impurity X was prepared in diluent (methanol) at 0.01 mg/mL concentration, and 1 mL of sample solution was directly injected into LC-MS system. The prasugrel sample was prepared in diluent (acetonitrile) at 0.01 mg/mL concentration, and 1 mL of sample solution was directly injected into the LC-MS system. NMR spectroscopy 1 H- and 13C-NMR spectra were recorded at 600 and 125 MHz, respectively, using a Bruker AVANCE 600 MHz spectrometer (Bruker, Fallanden, Switzerland) equipped with a 5-mm broadband probe and a z-gradient shim system. The 1H spectra were recorded with a 1-s pulse repetition time using 308 flip angle, while 13C spectra were recorded with power-gated decoupling using 308 flip angle with a repetition time of 2 s. Samples were dissolved in dimethyl sulphoxide-d6. The 1H and 13C chemical shift values were reported on the chemical shift scale in ppm relative to DMSO-d6 (2.50 ppm). All spectra were recorded with sample spinning. Results Detection of impurities by HPLC From Figure 1, the presence of Impurity X was 0.05% (by the area normalization method), which was marked as Impurity X (retention time 52.7 min). As shown in Supplementary Material, Figure S1 and S2, in the bulk trials of prasugrel, the amount of Impurity X was 0.232%, which was beyond 0.1% according to the ICH guidelines. Semi-preparative HPLC From Figure 2, Impurity X was collected between retention times 48 and 51 min. Identification of impurity and prasugrel by LC-MS Mass spectral data showed a protonated molecular ion peak at m/z 705 for Impurity X (Figure 3). In Figure 3, the ion peak of 743 was
Figure 6. Chemical structure of Impurity X.
4 Liu et al.
the molecular ion peak of 705 combined with K atom. In Figures 4 and 5, the mass spectrum and fragmentation prasugrel were shown.
Structural confirmation of Impurity X by NMR The structural confirmation of Impurity X by NMR was established using the 1H-NMR, 13C-NMR, H-H COSY, DEPT 1358, HSQC and HMBC experiments. All hydrogen atoms and carbon atoms could be confirmed clearly, and there were ten methenes according to DEPT 1358. The detailed data of NMR are shown in Tables I and II. The chemical structure of Impurity X is shown in Figure 6.
Discussion In Figure 5, Impurity A, Impurity B and Impurity C have been reported in the literature (5). Impurity B and Impurity C were raw materials used to synthesize prasugrel. Impurity A was brought by Impurity B because the Br atom was replaced. In our RP-HPLC Table II Analysis on 13C-NMR of Impurity X and Its Resonance Peak of Correlation Spectroscopy
dC
DEPT
HSQC dH
HMBC dH
Position
9.987, 10.409, 10.723, 11.574 18.051 19.906 16.288 23.232 52.903 36.109 35.811 41.835 43.613 52.535 70.126 114.276 114.422 115.441, 115.579, 122.907, 122.976 126.709, 128.893, 129.453, 129.69
CH2
0.735, 0.867, 2.08 2.288 2.329 2.61 2.988 3.108 3.259 3.586 3.689 4.346 4.583 6.492 6.559 7.186, 7.217, 7.374, 7.396,
0.867, 0.87, 0.735, 0.758 0.735, 0.758
20, 21, 31, 32
CH CH3 CH CH2 CH2 CH2 CH2 CH2 CH2 CH CH CH CH CH CH
0.758, 0.877
0.867, 0.87 3.259 3.108 2.988 2.61 2.61
7.203, 7.233 7.386, 7.413
7.374, 7.396, 7.186, 7.217,
7.386, 7.413 7.203, 7.233
19 1 30 4 15 14 5 16 6 28 17 7 11 24, 25, 37, 38 26, 27, 35, 36
Figure 7. Chromatogram of prasugrel added with Impurity A, Impurity B and Impurity C.
Figure 8. Formation of Impurity X.
conditions, Impurity X was isolated with Impurity A, Impurity B and Impurity C completely. The chromatogram is shown in Figure 7. The synthetic process of prasugrel is shown in Supplementary Material, Figure S3. During the synthesis of prasugrel, the reaction temperature was 608. Therefore, cyclization
reaction was occurred between two molecular of prasugrel at which was formatted as Impurity X according to the structure of Impurity X. The drying temperature should be reduced properly to decrease the formation of Impurity X, which is shown in Figure 8. Isolation and Structural Elucidation of an Unknown Impurity in Prasugrel 5
Conclusion Our study revealed a major process-related impurity while analyzing prasugrel through the RP-HPLC method. The LC-MS study reported the molecular weight, and the NMR spectroscopic study confirmed the structure of the unknown impurity. The impurity is not reported at present. Supplementary Material Supplementary materials are available at Journal of Chromatographic Science (http://chromsci.oxfordjournals.org). References 1. Angiolillo, D.J.; Antiplatelet therapy in type 2 diabetes mellitus; Current Opinion in Endocrinology, Diabetes and Obesity, (2007); 14(2): 124– 131. 2. Cairns, J.A., Theroux, P., Lewis, H.D., Ezekowitz, M., Meade, T.W.; Antithrombotic agents in coronary artery disease; Chest, (2001); 120(4): 1427. 3. Jakubowski, J.A., Winters, K.J., Naganuma, H., Wallentin, L.; Prasugrel: a novel thienopyridine antiplatelet agent. A review of preclinical and clinical studies and the mechanistic basis for its distinct antiplatelet profile; Cardiovascular Drug Reviews, (2007); 25(4): 357–374. 4. Donahoe, S.M., Stewart, G.C., McCabe, C.H., Mohanavelu, S., Murphy, S.A., Cannon, C.P., et al. ; Diabetes and mortality following acute coronary syndromes; Journal of the American Medical Informatics Association, (2007); 298(7): 765–775.
6 Liu et al.
5. Damle, M.C., Borole, T.C., Mehendre, R., Bothara, K.G.; Development and validation of stability indicating HPTLC method for determination of prasugrel; Journal of Chemical and Pharmaceutical Research, (2010); 2(4): 907–913. 6. Ashok Kumar, A., Anil Kumar, A., Gowri Shanka, D.; Development, estimation and validation of prasugrel in bulk and its pharmaceutical formulation by UV-VIS spectroscopic method; Pharmanest, (2011); 2(1): 37 –39. 7. Viriyala, R.K., Jena, F.M., Ravi Kumar, B.V.V., Annapurna, M.M., Bisht, S.P.S.; Validated new spectrophotometric methods for the estimation of prasugrel in bulk and pharmaceutical dosage forms; International Journal of Clinical Pharmacy, (2011); 2(6): 1– 3. 8. Mohammed Ishaq, B., Vanitha Prakash, K., Krishna Mohan, G.; Analytical method development and validation of prasugrel in bulk and its pharmaceutical formulation using the RP-HPLC method; Journal of Chemical and Pharmaceutical Research, (2011); 3(4): 404–409. 9. Sriram, V., Sriram, K., Angirekula, J., Tripathi, U.M., Nayakanti, D.; Development and validation of reverse phase HPLC method for the determination of impurities in prasugrel hydrochloride; International Journal of PharmTech Research, (2012); 4(4): 1407– 1416. 10. Sheldon, E.M., Downar, J.B.; Development and validation of a single robust HPLC method for the characterization of a pharmaceutical starting material and impurities from three suppliers using three separate synthetic routes; Journal of Pharmaceutical and Biomedical Analysis, (2000); 23: 561–572. 11. ICH Guidelines. (2006) Impurities in New Drug Substances Q3A (R2). October 25.
Article
Isolation and Structural Elucidation of an Unknown Impurity in Prasugrel by Semi-Preparative Liquid Chromatography Chao Liu1,2, Zhangxin Yu2, Fujun Wang2, Xing Zhong2, Li Jiang2, Feifei Zhang2, Yinxia Tang2, Zhaohua Yan2, Su Zeng1 and Tong Pu2* 1
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang Province, China, and 2Zhejiang Charioteer Pharmaceutical Co. Ltd., Tongyuanxi, Dazhan, Xinju County, Zhejiang Province, China
*Author to whom correspondence should be addressed. Email: [email protected] Received 23 April 2014; revised 19 November 2014
A brand-new impurity was detected by RP-HPLC in the prasugrel. The impurity was named as Impurity X. Impurity X was isolated by using semi-preparative HPLC followed by characterization using nuclear magnetic resonance spectroscopy and liquid chromatography–mass spectrometry. The functional mechanism of Impurity X was speculated. Impurity X could be controlled in the manufacture process of the prasugrel active pharmaceutical ingredient effectively.
Introduction Prasugrel hydrochloride is a newly generated thienopyridine organic compound. It is used to control the agglomeration of the hematoblast. It is an inactive pro-drug. After taken orally, it is turned into the active metabolite by the cytochrome P450 enzyme. It is found to block the adenosine diphosphate receptor so as to restrain the activation of the hematoblast (1 –4). There are several methods for the determination of the prasugrel and its impurities in bulk drug and pharmaceutical dosage forms reported in the literature (5 – 9). The high-performance thin layer chromatography (HPTLC) method has been reported for the determination on prasugrel (5). The UV-VIS spectroscopic method was reported for the determination for the prasugrel assay (6, 7). The reversed-phase high-performance liquid chromatography (RP-HPLC) method such as the different mobile phases in isocratic mode cannot be used for determination on the related substances of prasugrel active pharmaceutical ingredient (API) (8, 9). A brand-new RP-HPLC method was developed for the determination of the related substances of prasugrel in our laboratory. The gradient elution was developed especially on prasugrel API. Determination of unknown organic impurities is the key to the production of high-quality drug substances (10). ICH guidelines indicate that impurities at or above 0.1% in the drug substance require identification (11). An unknown impurity was detected in a laboratory sample of prasugrel when analyzed as own-developed RP-HPLC method. The semi-preparative HPLC was performed to isolate the unknown impurity followed by characterization using nuclear magnetic resonance spectroscopy and liquid chromatography –mass spectrometry (LC-MS). To the best of our knowledge, this impurity has not been previously reported. Experimental Materials and reagents Samples of prasugrel (batch no. 201308004-crude and 20140102-crude) were obtained from Zhejiang Charioteer
Pharmaceutical Co., Ltd. De-ionized water was prepared using a Milli-Q plus water purification system from Millipore (Bradford, PA, USA). HPLC grade methanol, acetonitrile, potassium dihydrogen phosphate and triethylamine were purchased from Merck China Limited (Shanghai, China).
High-performance liquid chromatography Samples were analyzed on a SHIMADZU LC-20A (Shimadzu Corporation, Kyoto, Japan) equipped with diode-array detector using an Agilent ZORBAX SB-C18 column (250 4.6 mm i.d., 5 mm particles; Agilent Corporation, USA). For chromatographic separations, mobile phase A was 0.02 M potassium dihydrogen phosphate (containing 0.20% triethylamine, pH adjusted to 4.50 with phosphoric acid) and mobile phase B was acetonitrile. The HPLC gradient program was set as time (min) /%A(v/v):0/ 50,25/50,50/30,60/30,61/50 and 70/50. The flow rate was 1.0 mL/min, and the photo-diode array detector wavelength was 240 nm. The column oven temperature was maintained at 208C. The prasugrel sample was prepared in diluent (acetonitrile) at 1 mg/mL concentration, and 10 mL of sample solution was injected into HPLC system. Semi-preparative HPLC Impurity X was isolated from the crude sample of prasugrel using a Shimadzu semi-preparative HPLC system consisting of a LC-8A binary gradient pump, a SPD-10AVP UV detector, a SIL-10AP autosampler and a FRC-10A fraction collector (Shimadzu Corporation, Kyoto, Japan). A Shim-pack PREP-ODS(H) KIT column (250 20 mm i.d., particle size 10 mm) (Shimadzu Corporation, Kyoto, Japan) was used for semi-preparative isolation. Mobile phase A was water and mobile phase B was acetonitrile. The HPLC gradient program is set as time (min)/ %A(v/v):0/50, 25/50, 50/30, 60/30, 61/50 and 70/50. The flow rate was 16.0 mL/min. A sample solution of 20 mg/mL was prepared using acetone as the diluent. The injection volume was 1 mL, and the detection was monitored at 240 nm. The collected fractions (between a retention time of 48 and 51 min) were evaporated by a rotory evaporator at 508C. LC-MS The MS study was performed on LC-MS2020 (Shimadzu Corporation, Kyoto, Japan) using an electrospray ionization source and a Q mass spectrometer. The temperature of heat block and decreased liquid were 400 and 2008. Nitrogen was used as both sheath and auxiliary gas. The mass-to-charge ratio
# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. Chromatogram of prasugrel in the HPLC method.
Figure 2. Chromatogram of prasugrel in the semi-preparative HPLC method.
Figure 3. MS spectrum of impurity X.
2 Liu et al.
Figure 4. MS spectrum of prasugrel.
Figure 5. MS spectrum fragmentation of prasugrel.
Table I Analysis on 1H-NMR of Impurity X and Its Resonance Peak of Correlation Spectroscopy
dH
HSQC dC
1
H– 1H COSY dH
HMBC dC
Position
0.735, 0.758, 0.867, 0.877 2.08 2.288 2.329 2.61 2.988 3.108 3.259 3.586 3.689 4.346 4.583 6.492 6.559 7.186, 7.203, 7.217, 7.233 7.374, 7.386, 7.396, 7.413
9.987, 10.409, 10.723, 11.574 18.051 19.906 16.288 23.232 52.903 36.109 35.811 41.835 43.613 52.535 70.126 114.276 114.422 115.441, 115.579, 122.907, 122.976 126.709, 128.893, 129.453, 129.69
2.08 0.735, 0.758
11.574, 10.723, 10.409, 9.987, 23.232, 70.126, 201.066, 202.737 9.987, 10.409, 115.579, 201.066 168.766 10.723, 11.574 40.616, 123.068, 124.133
20, 21, 31, 32 19 1 30 4 15 14 5 16 6 28 17 7 11 24, 25, 37, 38 26, 27, 35, 36
0.867, 0.877 3.586, 3.689 3.108 2.988 2.61 2.61
7.374, 7.386, 7.396, 7.413 7.186, 7.203, 7.217, 7.233
122.907, 135.845 122.907, 135.845, 166.965 124.133, 166.965 23.232, 126.709 122.907, 129.69 120.676, 122.976, 206.202 124.133, 126.709 128.188 158.534, 160.174 52.903, 158.534, 160.174
Isolation and Structural Elucidation of an Unknown Impurity in Prasugrel 3
was scanned across the range of m/z 50 –1,000. Impurity X was prepared in diluent (methanol) at 0.01 mg/mL concentration, and 1 mL of sample solution was directly injected into LC-MS system. The prasugrel sample was prepared in diluent (acetonitrile) at 0.01 mg/mL concentration, and 1 mL of sample solution was directly injected into the LC-MS system. NMR spectroscopy 1 H- and 13C-NMR spectra were recorded at 600 and 125 MHz, respectively, using a Bruker AVANCE 600 MHz spectrometer (Bruker, Fallanden, Switzerland) equipped with a 5-mm broadband probe and a z-gradient shim system. The 1H spectra were recorded with a 1-s pulse repetition time using 308 flip angle, while 13C spectra were recorded with power-gated decoupling using 308 flip angle with a repetition time of 2 s. Samples were dissolved in dimethyl sulphoxide-d6. The 1H and 13C chemical shift values were reported on the chemical shift scale in ppm relative to DMSO-d6 (2.50 ppm). All spectra were recorded with sample spinning. Results Detection of impurities by HPLC From Figure 1, the presence of Impurity X was 0.05% (by the area normalization method), which was marked as Impurity X (retention time 52.7 min). As shown in Supplementary Material, Figure S1 and S2, in the bulk trials of prasugrel, the amount of Impurity X was 0.232%, which was beyond 0.1% according to the ICH guidelines. Semi-preparative HPLC From Figure 2, Impurity X was collected between retention times 48 and 51 min. Identification of impurity and prasugrel by LC-MS Mass spectral data showed a protonated molecular ion peak at m/z 705 for Impurity X (Figure 3). In Figure 3, the ion peak of 743 was
Figure 6. Chemical structure of Impurity X.
4 Liu et al.
the molecular ion peak of 705 combined with K atom. In Figures 4 and 5, the mass spectrum and fragmentation prasugrel were shown.
Structural confirmation of Impurity X by NMR The structural confirmation of Impurity X by NMR was established using the 1H-NMR, 13C-NMR, H-H COSY, DEPT 1358, HSQC and HMBC experiments. All hydrogen atoms and carbon atoms could be confirmed clearly, and there were ten methenes according to DEPT 1358. The detailed data of NMR are shown in Tables I and II. The chemical structure of Impurity X is shown in Figure 6.
Discussion In Figure 5, Impurity A, Impurity B and Impurity C have been reported in the literature (5). Impurity B and Impurity C were raw materials used to synthesize prasugrel. Impurity A was brought by Impurity B because the Br atom was replaced. In our RP-HPLC Table II Analysis on 13C-NMR of Impurity X and Its Resonance Peak of Correlation Spectroscopy
dC
DEPT
HSQC dH
HMBC dH
Position
9.987, 10.409, 10.723, 11.574 18.051 19.906 16.288 23.232 52.903 36.109 35.811 41.835 43.613 52.535 70.126 114.276 114.422 115.441, 115.579, 122.907, 122.976 126.709, 128.893, 129.453, 129.69
CH2
0.735, 0.867, 2.08 2.288 2.329 2.61 2.988 3.108 3.259 3.586 3.689 4.346 4.583 6.492 6.559 7.186, 7.217, 7.374, 7.396,
0.867, 0.87, 0.735, 0.758 0.735, 0.758
20, 21, 31, 32
CH CH3 CH CH2 CH2 CH2 CH2 CH2 CH2 CH CH CH CH CH CH
0.758, 0.877
0.867, 0.87 3.259 3.108 2.988 2.61 2.61
7.203, 7.233 7.386, 7.413
7.374, 7.396, 7.186, 7.217,
7.386, 7.413 7.203, 7.233
19 1 30 4 15 14 5 16 6 28 17 7 11 24, 25, 37, 38 26, 27, 35, 36
Figure 7. Chromatogram of prasugrel added with Impurity A, Impurity B and Impurity C.
Figure 8. Formation of Impurity X.
conditions, Impurity X was isolated with Impurity A, Impurity B and Impurity C completely. The chromatogram is shown in Figure 7. The synthetic process of prasugrel is shown in Supplementary Material, Figure S3. During the synthesis of prasugrel, the reaction temperature was 608. Therefore, cyclization
reaction was occurred between two molecular of prasugrel at which was formatted as Impurity X according to the structure of Impurity X. The drying temperature should be reduced properly to decrease the formation of Impurity X, which is shown in Figure 8. Isolation and Structural Elucidation of an Unknown Impurity in Prasugrel 5
Conclusion Our study revealed a major process-related impurity while analyzing prasugrel through the RP-HPLC method. The LC-MS study reported the molecular weight, and the NMR spectroscopic study confirmed the structure of the unknown impurity. The impurity is not reported at present. Supplementary Material Supplementary materials are available at Journal of Chromatographic Science (http://chromsci.oxfordjournals.org). References 1. Angiolillo, D.J.; Antiplatelet therapy in type 2 diabetes mellitus; Current Opinion in Endocrinology, Diabetes and Obesity, (2007); 14(2): 124– 131. 2. Cairns, J.A., Theroux, P., Lewis, H.D., Ezekowitz, M., Meade, T.W.; Antithrombotic agents in coronary artery disease; Chest, (2001); 120(4): 1427. 3. Jakubowski, J.A., Winters, K.J., Naganuma, H., Wallentin, L.; Prasugrel: a novel thienopyridine antiplatelet agent. A review of preclinical and clinical studies and the mechanistic basis for its distinct antiplatelet profile; Cardiovascular Drug Reviews, (2007); 25(4): 357–374. 4. Donahoe, S.M., Stewart, G.C., McCabe, C.H., Mohanavelu, S., Murphy, S.A., Cannon, C.P., et al. ; Diabetes and mortality following acute coronary syndromes; Journal of the American Medical Informatics Association, (2007); 298(7): 765–775.
6 Liu et al.
5. Damle, M.C., Borole, T.C., Mehendre, R., Bothara, K.G.; Development and validation of stability indicating HPTLC method for determination of prasugrel; Journal of Chemical and Pharmaceutical Research, (2010); 2(4): 907–913. 6. Ashok Kumar, A., Anil Kumar, A., Gowri Shanka, D.; Development, estimation and validation of prasugrel in bulk and its pharmaceutical formulation by UV-VIS spectroscopic method; Pharmanest, (2011); 2(1): 37 –39. 7. Viriyala, R.K., Jena, F.M., Ravi Kumar, B.V.V., Annapurna, M.M., Bisht, S.P.S.; Validated new spectrophotometric methods for the estimation of prasugrel in bulk and pharmaceutical dosage forms; International Journal of Clinical Pharmacy, (2011); 2(6): 1– 3. 8. Mohammed Ishaq, B., Vanitha Prakash, K., Krishna Mohan, G.; Analytical method development and validation of prasugrel in bulk and its pharmaceutical formulation using the RP-HPLC method; Journal of Chemical and Pharmaceutical Research, (2011); 3(4): 404–409. 9. Sriram, V., Sriram, K., Angirekula, J., Tripathi, U.M., Nayakanti, D.; Development and validation of reverse phase HPLC method for the determination of impurities in prasugrel hydrochloride; International Journal of PharmTech Research, (2012); 4(4): 1407– 1416. 10. Sheldon, E.M., Downar, J.B.; Development and validation of a single robust HPLC method for the characterization of a pharmaceutical starting material and impurities from three suppliers using three separate synthetic routes; Journal of Pharmaceutical and Biomedical Analysis, (2000); 23: 561–572. 11. ICH Guidelines. (2006) Impurities in New Drug Substances Q3A (R2). October 25.
