Characterization of impurities in the bulk drug lisinopril by liquid chromatography/ion trap spectrometry

Characterization of impurities in the bulk drug lisinopril by liquid chromatography/ion trap spectrometry 1 first-author 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:suncuirong@zju.edu.cnzhiquan_shen@163.com †E-mail:suncuirong@zju.edu.cnzhiquan_shen@163.com suncuirong@zju.edu.cn 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:suncuirong@zju.edu.cnzhiquan_shen@163.com †E-mail:suncuirong@zju.edu.cnzhiquan_shen@163.com suncuirong@zju.edu.cn Department of Chemistry, Zhejiang University, Hangzhou 310027, China Department of Chemistry, Zhejiang University, Hangzhou 310027, China Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China Two trace impurities in the bulk drug lisinopril were detected by means of high-performance liquid chromatography coupled with mass spectrometry (HPLC/MS) with a simple and sensitive method suitable for HPLC/MS n analysis. The fragmentation behavior of lisinopril and the impurities was investigated, and two unknown impurities were elucidated as 2-(6-amino-1-(1-carboxyethylamino)-1-oxohexan-2-ylamino)-4-phenylbutanoic acid and 6-amino-2-(1-carboxy-3-phenylpropylamino)-hexanoic acid on the basis of the multi-stage mass spectrometry and exact mass evidence. The proposed structures of the two unknown impurities were further confirmed by nuclear magnetic resonance (NMR) experiments after preparative isolation. Lisinopril Impurities High-performance liquid chromatography (HPLC) Multi-stage mass spectrometry (MS n ) INTRODUCTION 1 Lisinopril, (1-[N-[(S)-1-carboxy-3-phenylpropyl]-L-lysyl] -L-proline dehydrate), is an angiotensin-converting enzyme (ACE) inhibitor, used for the treatment of hypertension, heart failure, and acute myocardial infarction (Eveson et al., 2007 2007 ; Wang et al., 2006 2006 ). A wide variety of separation and detection techniques have been applied to the analysis of lisinopril, such as gas chromatography coupled with mass spectrometry (GC/MS) (Leis et al., 1998 1998 ; 1999 1999 ), high-performance liquid chromatography (HPLC) (Wong and Charles, 1995 1995 ; Beasley et al., 2005 2005 ), and high-performance liquid chromatography coupled with mass spectrometry (HPLC/MS) (Shinde et al., 2007 2007 ; Pei et al., 2006 2006 ). The impurity profile of a drug substance is critical to its safety assessment and manufacturing process. For safety reasons, the impurities that exceed 0.1% in a drug must be identified prior to clinical trials (International Conference on Harmonization, 2003 2003 ). This paper presents the identification of unknown impurities in trace level by on-line and off-line multi-stage mass spectrometry (MS n ) analyses. To ascertain the structures of the unknown impurities clearly, 1D and 2D nuclear magnetic resonance (NMR) techniques were applied after preparative isolation. MATERIALS AND METHODS 1 Materials 2 The sample of lisinopril was obtained from Jianyuan Inc. (Hangzhou, China). Ammonium acetate and glacial acetic acid of analytical grade were purchased from Guangzhou Chemical Co. (Guangzhou, China) and Hangzhou Chemical Co. (Hangzhou, China), respectively. Acetonitrile of HPLC grade was obtained from Merck Co. (Darmstadt, Germany), and water was purified by a Milli-Q purification system (Millipore, Bedford, MA, USA). Analytical HPLC 2 Analytical HPLC was performed on an Agilent 1100 series HPLC equipped with a G1312A Binary pump and a G1314A variable wave detector (VWD). A model 7725 injection valve fitted with a 20 µl sample loop was used, along with an Agilent ChemStation data system. The separation was achieved on a Waters symmetry C18 column (4.6 mm×150 mm, 5 µm). The mobile phase consisted of a mixture of 5% (v/v) acetonitrile and 95% (v/v) aqueous buffer. The aqueous buffer was prepared by dissolving 20 mmol/L ammonium acetate in purified water adjusted with glacial acetic acid to pH 4.5. The flow rate was set at 1.0 ml/min, and the effluent was monitored at 210 nm. Preparative HPLC 2 Preparation was conducted with a Waters 600 semi-preparative HPLC system on a Zorbax C18 column (9.4 mm×250 mm, 5 μm, Agilent, USA). The mobile phase consisted of methanol and water (5:95, v/v) at flow rate of 3.0 ml/min. MS 2 HPLC/ESI-MS n analyses were carried out on the Agilent HPLC system described above combined with a Bruker Esquire 3000 plus ion trap mass spectrometer (Bruker-Franzen Analytik GmbH, Bremen, Germany) which is equipped with an electrospray ionization (ESI) source. Instrument control and data acquisition were performed with Esquire 5.0 software. The samples were infused into the source chamber from the HPLC system with a T-junction delivering approximately 1/3 of the flow to the mass spectrometer. The ion source temperature was 250 °C and the ESI needle voltage was always set at 4.0 kV. Nitrogen was used as drying gas at a flow rate of 10 ml/min and the nebulizer gas at a back-pressure of 2.0685×10 5 Pa. Helium was introduced into the ion trap with an estimated pressure of 6.3×10 −9 Pa to improve trapping efficiency, and act as the collision gas for both on-line and off-line MS n experiments. The collision energy was set between 0.60 and 0.70 V to maximize the ion current in the spectra. The accurate MS experiments were performed on an Apex III 7.0 Tesla FTICR mass spectrometer (Bruker, Daltonic, Billerica, MA, USA) combined with an ESI source in the positive ion mode. Solution introduction was accomplished by using a Cole-Parmer syringe pump at a rate of 3 μl/min. Accurate mass measurements were performed using NaI as an external calibrant. XMASS software version 6.1.1 was used for instrument control, data acquisition and processing. The spray voltage was 4.5 kV. The temperature of the capillary was 250 °C. Nebulizing gas and drying gas (N 2 ) were set 2.41325×10 5 Pa and 30 units, respectively. Products ions were generated in the collision cell and argon was used as the collision gas. NMR 2 NMR spectra were recorded with a Bruker Advance DMX 500 instrument (Bruker, Billerica, MA, USA) with a QNP probe head at ambient temperature. The data were acquired on Silicons Graphics O2 workstations by using XWINNMR version 2.1 (Bruker Analytik, GmbH, Germany). RESULTS AND DISCUSSION 1 HPLC/MS analysis 2 During the routine impurity profiling of bulk lisinopril, two unknown trace impurities were detected with molecular masses of 308 and 379 by their positive ESI mass spectra (Fig. 1 1 ). Identification of the unknown impurities 2 1. Impurity 1 3 Impurity 1 had the molecule mass of 379. The collision-induced dissociation (CID) spectrum for the protonated impurity 1 was dominated by the ion at m / z 291 (Fig. 2a 2a ), which was also observed in the product ion spectrum (Fig. 2b 2b ) of protonated lisinopril. The MS 3 spectrum of the m / z 291 product ion of impurity 1 was indistinguishable from that of lisinopril (Figs. 3a and 3b 3a and 3b ). The dissociation spectra showed that the m / z 291 ion gave rise to a major dissociation product at m / z 245, which corresponded to the loss of HCOOH. No other significant fragmentation channels were observed. The same structure of the two ions at m / z 291 was further supported by the fragmentation experiments of the ion at m / z 245. When the respective ion of m / z 245 was further collisionally activated under the same condition, the product ion spectra (Figs. 4a and 4b 4a and 4b ) were also very similar. Since the impurity was generated as a by-product when the bulk was synthesized, the results above suggest that the impurity 1 probably shared some common structural moieties with lisinopril. A detailed study of the fragmentation patterns of the parent drug served as a template to elucidate the structures of unknown impurities by comparison of their fragmentation pathways and neutral losses (Lee and Kerns, 1999 1999 ). Analysis of product ion spectra of protonated lisinopril clearly indicated the precursor ion underwent a distinct loss of proline to form an abundant dipeptide N-terminal b 1 ion at m / z 291, with subsequent loss of HCOOH to form the ion at m / z 245 (Roepstorff and Fohlman, 1984 1984 ). The product ion at m / z 263 corresponded to the dipeptide N-terminal a 1 ion with the combined loss of proline and CO, while ion at m / z 246 was formed from subsequent loss of NH 3 . The product ion at m / z 309 was formed through a rearrangement (Florêncio et al., 1998 1998 ; Hiserodt et al., 2007 2007 ), with the elimination of dihydropyrrole and CO, and could be used to characterize the 6-amino-2-(1-carboxy-3-phenylpropyl-amino)-hexanoic acid structure. The product ion at m / z 227 was formed by the neutral loss of 2-amino-4-phenylbutanoic acid (179 Da). The CID spectrum of protonated lisinopril was similar to those obtained in previous studies (Burinsky and Sides, 2004 2004 ; Pei et al., 2006 2006 ). The ion at m / z 309 from impurity 1 also suggested the probable structure of 6-amino-2-(1-carboxy-3-phenylpropylamino)-hexanoic acid. The product ion at m / z 201 would arise from the same formation mechanism (loss of 2-amino-4-phenylbutanoic acid, 179 Da) as that responsible for the product ion at m / z 227 from lisinopril. The molecular formula for the impurity 1 (C 19 H 29 N 3 O 5 ) was confirmed from the accurate mass ( m / z ) of the precursor ion in positive mode. These data suggest that the most plausible structure of impurity 1 would be 2-(6-amino-1-(1-carboxyethylamino)-1-oxohexan-2-ylamino)-4-phenylbutanoic acid (Fig. 5 5 ), which was a by-product of the lisinopril synthesis. The proposed fragmentation mechanism for impurity 1 is shown in Fig. 6 6 . To confirm the fragmentation mechanism, we further studied the exact mass of these fragments using FT-ICRMS (fourier transform ion cyclotron resonance mass spectrometry) (Table 1 1 ). These mass values were determined in triplicate and were less than 2×10 −6 from the calculated mass. Precursor ions Product ions Measured mass Calculated mass Formula Error (×10 −6 ) 380 (Impurity 1 ) − 380.2181 380.2180 C 19 H 30 N 3 O 5 + 0.26 363 363.1920 363.1914 C 19 H 27 N 2 O 5 + 1.65 334 334.2128 334.2125 C 18 H 28 N 3 O 3 + 0.90 317 317.1859 317.1860 C 18 H 25 N 2 O 3 + −0.32 309 309.1811 309.1809 C 16 H 25 N 2 O 4 + 0.65 291 291.1704 291.1703 C 16 H 23 N 2 O 3 + 0.34 246 246.1489 246.1489 C 15 H 20 NO 2 + 0 245 245.1647 245.1648 C 15 H 21 N 2 O + −0.41 201 201.1234 201.1234 C 9 H 17 N 2 O 3 + 0 309 (Impurity 2 ) − 309.1806 309.1809 C 16 H 25 N 2 O 4 + −0.97 292 292.1542 292.1543 C 16 H 22 NO 4 + −0.34 291 291.1703 291.1703 C 16 H 23 N 2 O 3 + 0 246 246.1489 246.1489 C 15 H 20 NO 2 + 0 245 245.1647 245.1648 C 15 H 21 N 2 O + −0.41 130 130.0863 130.0863 C 6 H 12 NO 2 + 0 2. Impurity 2 3 Impurity 2 exhibited an abundant ion at m / z 309 in the positive ion mass spectrum. The CID spectra for the protonated lisinopril also showed a fragment ion at m / z 309, a rearrangement ion which could be used to characterize the 6-amino-2-(1-carboxy-3-phenylpropylamino)-hexanoic acid structure unit. The structural relationships were investigated further by comparing the MS 2 and MS 3 CID spectra for the protonated impurity 2 (Fig. 7a 7a ) and the product ion of m / z 309 derived from the protonated lisinopril (Fig. 7b 7b ). Analysis of the product ion spectrum of protonated impurity 2 clearly showed that its fragmentation pathway was indistinguishable from that of protonated lisinopril ( m / z 309). The ion at m / z 292 suggested the loss of NH 3 from the ion of m / z 309, with subsequent loss of HCOOH to form the ion at m / z 246. The ion at m / z 291 was a b 1 ion, which could lose HCOOH to form the fragment ion at m / z 245 (Pei et al., 2006 2006 ). The ion at m / z 263 was thought to be formed by the direct elimination of HCOOH from m / z 309. Therefore, impurity 2 was proposed as 6-amino-2-(1-carboxy-3-phenylpropylamino)-hexanoic acid, which was further supported by off-line FT-ICRMS data (Table 1 1 ). NMR analysis 2 The impurity 1 and impurity 2 were obtained by preparative chromatography and their structures were further confirmed by NMR experiments. 1. Selected data for impurity 1 3 1 H-NMR (500 MHz, D 2 O), δ : 1.26 (d, 3H), 1.48 (m, 2H), 1.64 (m, 2H), 1.91 (m, 2H), 2.10 (m, 2H), 2.69 (m, 2H), 2.96 (t, 2H), 3.36 (t, 1H), 3.83 (t, 1H), 4.10 (q, 1H), 7.26 (m, 5H); 13 C-NMR (125 MHz, D 2 O), δ : 15.10, 19.27, 24.27, 27.96, 28.83, 30.14, 37.12, 49.48, 58.53, 59.83, 124.66~126.90 (5C), 138.51, 165.60, 171.05, 177.39. 2. Selected data for impurity 2 3 1 H-NMR (500 MHz, D 2 O), δ : 1.43 (m, 2H), 1.62 (m, 2H), 1.84 (m, 2H), 2.13 (m, 2H), 2.70 (m, 2H), 2.94 (t, 2H), 3.50 (t, 1H), 3.54 (t, 1H), 7.27 (m, 5H); 13 C-NMR (125 MHz, D 2 O), δ : 19.59, 24.26, 27.66, 28.86, 30.31, 37.09, 60.63, 60.72, 124.58~126.89 (5C), 138.75, 171.41, 171.42. CONCLUSION 1 An HPLC/MS n method was developed for the identification of trace level impurities in the bulk drug lisinopril. In combination with the FT-ICRMS and NMR data, the structures of two unknown impurities were confirmed to be 2-(6-amino-1-(1-carboxyethylamino)-1-oxohexan-2-ylamino)-4-phenyl-butanoic acid and 6-amino-2-(1-carboxy-3-phenylpropylamino)-hexanoic acid. References CA Beasley , J Shaw , Z Zhao , RA Reed . Development and validation of a stability indicating HPLC method for determination of lisinopril, lisinopril degradation product and parabens in the lisinopril extemporaneous formulation . 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DOI: 10.1016/0378-4347(95)00268-4 http://doi.org/10.1016/0378-4347(95)00268-4 . Determination of the angiotensin-converting enzyme inhibitor lisinopril in urine using solid-phase extraction and reversed phase high performance liquid chromatography 10.1631/jzus.B0820031 Project (No. 20772109) supported by the National Natural Science Foundation of China 2008-01-24 2008-04-06 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 09