Characterization of a novel impurity in bulk drug of lisinopril by multidimensional NMR technique

Characterization of a novel impurity in bulk drug of lisinopril by multidimensional NMR technique 1 2 first-author 1 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:suncuirong@zju.edu.cn †E-mail:suncuirong@zju.edu.cn suncuirong@zju.edu.cn 1 Department of Chemistry, Zhejiang University, Hangzhou 310027, China Department of Chemistry, Zhejiang University, Hangzhou 310027, China Zhejiang Huahai Pharmaceutical Co., Ltd., Linhai 317024, China Zhejiang Huahai Pharmaceutical Co., Ltd., Linhai 317024, China During the routine impurity profile of lisinopril bulk drug by HPLC (high-performance liquid chromatography), a potential impurity was detected. Using multidimensional NMR (nuclear magnetic resonance) technique, the trace-level impurity was unambiguously identified to be 2-(-2-oxo-azocan-3-ylamino)-4-phenyl-butyric acid after isolation from lisinopril bulk drug by semi-preparative HPLC. Formation of the impurity was also discussed. To our knowledge, this is a novel impurity and not reported elsewhere. Lisinopril Impurity Structural characterization NMR INTRODUCTION 1 Lisinopril is a lysine analog of enalaprilat, the active metabolite of enalapril and is a long-acting, nonsulfhydryl angiotensin-converting enzyme (ACE) inhibitor that used for treating hypertension and congestive heart failure (Lancaster and Todd, 1988 1988 ; Goa et al., 1996 1996 ; Simpson and Jarvis, 2000 2000 ). The impurity profile of a drug substance is critical to its safety assessment, is important for monitoring manufacturing process, and it is a stringent regulation that all impurities at level ≥0.1% should be identified (Krishna Reddy et al., 2002 2002 ; Zhou et al., 2005 2005 ; Cao et al., 2005 2005 ). Presently, structural identification is difficult as these compounds represent low-level components of a mixture. In this study, a trace-level impurity in the bulk drug of lisinopril was analyzed using multidimensional NMR technique to identify the structure. EXPERIMENTAL DETAILS 1 Materials 2 The bulk drug of lisinopril under investigation was obtained from Zhejiang Huahai Pharmaceutical Co. Ltd. (Taizhou, China). HPLC (high-performance liquid chromatography) grade methanol and acetonitrile were obtained from Merk Co. (Darmstadt, Germany) and water was purified using a Milli-Q purification system (Millipore, Bedford, USA). All other chemicals were of analytical grade. High-performance liquid chromatography (HPLC) 2 Analytical HPLC was performed on an Agilent 1100 series HPLC system. A Waters Symmetry C18 column (4.6 mm×150 mm, 5 μm, Waters) was used for the separation. The mobile phase was a mixture of acetonitrile and buffer (1:9, v/v), and buffer consisting of 20 mmol/L KH 2 PO 4 adjusted with KOH to pH 4.9. The flow rate was set at 0.8 ml/min, and detection was carried out at 210 nm wavelength. A ZORBAX C18 column (9.4 mm×250 mm, 5 μm, Agilent) was used for the semi-preparation on a Waters 600 HPLC system. The mobile phase consisted of methanol and water (1:9, v/v) at flow rate of 3.0 ml/min. Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) 2 High resolving power and accuracy mass measurements were performed on a Bruker APEXIII 7.0T FTICR mass spectrometer (Billerica, MA, USA) equipped with an Apollo ESI source to obtain the element composition of target impurity. Nuclear magnetic resonance (NMR) spectroscopy 2 1D ( 1 H NMR, 13 C NMR) and 2D ( 1 H- 1 H COSY, DEPT135, HMQC, HMBC) NMR experiments were performed on a Bruker Advance DMX 500 instrument with a quadranuclear probe (QNP) head at ambient temperature, using D 2 O as solvent. The data were acquired on silicons graphics O 2 workstations using XWINNMR version 2.1 (Bruker Analytik, GmbH, Germany). RESULTS AND DISCUSSION 1 Detection and isolation 2 Seven batches of bulk drug were analyzed by analytical HPLC, and potential impurity at a level 0.05% to 0.3% based on HPLC peak area percentage was detected. A typical HPLC chromatogram was recorded (Fig. 1 1 ). The target impurity was isolated by semi-preparative HPLC. Then the collected fraction was concentrated under high vacuum on a Buchi Rotavapor Model R250 to dryness for further NMR experiments to generate spectral data. Structural elucidation 2 The target impurity was isolated as white powder. Its molecular formula was determined to be C 16 H 22 N 2 O 3 by FTICRMS ( m / z 291.1719 [M+H] + , calcd 291.1703). The FT-IR spectrum exhibited a characteristic stretching absorption band at 3448 cm −1 indicating the presence of NH group. And the strong C=O stretching (amide band-I) and bending (amide band-II) bands at 1670 and 1541 cm −1 indicated the presence of amide group. In addition, strong adsorption band attributed to benzene ring at 1627 cm −1 was observed. These adsorption bands clearly indicated the presence of C=O, NH, benzene ring groups, etc. in the molecular structure of impurity. The 1 H and 13 C NMR spectra, together with data from DEPT and HMQC experiments, revealed the presence of six methylenes (C-3, C-4, C-4′, C-5′, C-6′, C-7′), five sp 2 -hybridized methines (C-6, C-7, C-8, C-9, C-10), two sp 3 -hybridized methines (C-2, C-3′) and three quaternary sp 2 -carbons (C-1, C-5, C-2′) (Table 1 1 ). The presence of mono-substituted benzene ring in the 1 H NMR spectrum was indicated by the proton signals at δ 7.30 (2H, m, H-6, H-10), 7.23 (2H, m, H-7, H-9), 7.19 (1H, m, H-8), with corresponding carbon signals at δ 130.3, 129.9, 128.0 (Table 1 1 ). In the up-field region, 1 H- 1 H COSY spectrum defined two spin systems: one involving two methylenes and a methine group at δ 3.60 (1H, t, J =5.8 Hz, H-2), 2.68 (2H, m, H-4), 2.12 (2H, m, H-3), and the other involving four methylenes and a methine group at δ 1.70 (1H, m, H-4′), 2.10 (1H, m, H-4′), 1.97 (1H, m, H-5′), 1.64 (1H, m, H-5′), 1.75 (1H, m, H-6′), 1.28 (1H, m, H-6′), 3.16 (2H, m, H-7′) and 4.05 (1H, d, J =10.3, H-3′). In the HMBC spectrum, long-range correlations between δ 3.16 (H-7′) and δ 174.1 (C-2′), together with their chemical shifts, indicated that both C-2′ and C-7′ were connected to the same nitrogen atom. Further, 3 J correlations between δ 2.12 (H-3) and δ 174.7 (C-1), δ 2.68 (H-4) and δ 129.9 (C-6, C-10), unambiguously determined the positions of the carboxyl group at C-1 and the mono-substituted benzene group (Fig. 2 2 ). In addition, cross peaks were observed between δ 4.05 (H-3′) and δ 174.1 (C-2′), δ 64.0 (C-2) in the HMBC spectrum. Taking into consideration the chemical shifts of H-3′ and H-2, the positions of C-3′ and C-2 were proposed (Fig. 2 2 ). Based on the above evidence, the structure of the target impurity was identified as 2-(2-oxo-azocan-3-ylamino)-4-phenyl-butyric acid. To our knowledge, this is a novel compound and not reported anywhere. C/H δ H δ C DEPT HMBC 1 / 174.7 C H-2, H-3 2 3.60, t (5.8) 64.0 CH H-3, H-4, H-3′ 3 2.12, m 33.5 CH 2 H-2, H-4 4 2.68, m 32.2 CH 2 H-2, H-6, H-10 5 / 142.1 C H-3, H-4, H-7, H-9 6, 10 7.23, m 129.9 CH H-4, H-8 7, 9 7.30, m 130.3 CH / 8 7.19, m 128.0 CH H-6, H-10 2′ / 174.1 C H-3′, H-7′ 3′ 4.05, d (10.3) 61.5 CH H-2 4′ 1.70, m; 2.10, m 29.3 CH 2 H-3′ 5′ 1.64, m; 1.97, m 28.2 CH 2 H-3′, H-7′ 6′ 1.28, m; 1.75, m 28.5 CH 2 H-4′ 7′ 3.16, m 42.5 CH 2 Formation of impurity 2 According to the synthesis route of lisinopril (Sun and Zhi, 1997 1997 ), the formation of impurity could be explained by the reaction of intermediate II with NaOH to give intermediate III, which might be further cyclized to yield the impurity (Fig. 3 3 ). CONCLUSION 1 Using multiple dimensional NMR technique, we successfully identified the structure of a novel trace-level impurity in bulk drug of lisinopril. The formation of the impurity was discussed. 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Separation and characterization of synthetic impurities of triclabendazole by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry 10.1631/jzus.2006.B0310 Project (No. 20375036) supported partly by the National Natural Science Foundation of China 2005-09-15 2005-11-08 2006-03-13 2022-01-05 Copyright © 2006, Journal of Zhejiang University Science 2006 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 07