The Quantitative Determination of Process Related Genotoxic Impurities in Esomeprazole Magnesium by GC-MS
M. Yogeshwar Reddy1,3*, V. Ramesh2, Ch. Kista Reddy3, N. Venugopal1, G. Saravanana1, M.V. Suryanarayana1, B. Shyam Sunder 1, R.Surendranath Reddy1 and G. Raju2
1Matrix Laboratoires Ltd., R&D Centre, Jinnaram Mandal, Medak, 502325, India.
2National Centre for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad, 500 007, India.
3Department of Chemistry, P.G. College of Science, Saifabad, Hyderabad, India.
*Corresponding Author E-mail: yogeshwar.mamilla@matrixlabsindia.com
ABSTRACT:
A sensitive Gas chromatography-mass spectrometric (GC-MS) method is developed and validated for the determination of residuals 2-Chloromethyl-4-methoxy-3,5-dimethyl pyridine HCl (2), 2,3,5-Trimethyl pyridine N-oxide (3), 4-Nitro-2,3,5-Trimethyl pyridine N-oxide (4) and 4-Methoxy-2,3,5-Trimethyl pyridine N-oxide (5) are genotoxic impurities in Esomeprazole magnesium (1) drug substance. Under the optimal conditions, the recovery for 2, 3, 4, and 5 are between 109.4% to 116.8%, 99.9% to 107.6%, 96.0% to 104.1%, and 87.4% to 105.2% for spiked samples at limits of quantification (LOQ) level in sample preparation. Limits of quantification (LOQ) and detection (LOD) are about 5 ppm and 1.5 ppm for the standard solutions of 2, 3, 4, and 5. The linearity values are 0.9989, 0.9968, 0.9988, and 0.9999 for 2, 3, 4, and 5. Based on these results, the method is applied to determine the residual genotoxic impurities are, 2-Chloro methyl-4-methoxy-3,5-dimethyl pyridine HCl (2), 2,3,5-Trimethyl pyridine N-oxide (3), 4-Nitro-2,3,5-Trimethyl pyridine N-oxide (4) and 4-Methoxy-2,3,5-Trimethyl pyridine N-oxide (5) in Esomeprazole magnesium (1) drug substance
KEYWORDS: Esomeprazole magnesium, Single ion monitoring, Gas chromatography-Mass spectrometry
INTRODUCTION:
Esomeprazole magnesium commonly called as NEXIUM, chemically describes Megnisium (S)-5-methoxy-2-[(4-methoxy-3, 5-dimethylpyridin-2-yl) methyl sulfinyl]-3H-benzoimidazole-1-ide is an antiulcervative agent1, a proton pump inhibitor (PPI) and which is used in the treatment of dyspepsia, peptic ulcer disease (PUD), gastroesophageal reflux disease (GORD/GERD)2, non-steroidal anti-inflammatory3 and Zollinger-Ellison syndrome.
In the synthesis of Esomeprazole magnesium (1), 2-Chloro methyl-4-methoxy-3,5-dimethyl pyridine HCl (2), 2,3,5-Trimethyl pyridine N-Oxide (3), 4-Nitro-2,3,5-Trimethyl pyridine N-Oxide (4) and 4-Methoxy-2,3,5-Trimethyl pyridine N-Oxide (5) are important intermediates.
The chemical structures of the compounds 1, 2, 3, 4 and 5 are given in Scheme 1. Identification and determination of these four impurities in 1 is essential, because their genotoxic nature4. As per the regulatory guidelines5, a threshold of toxicological concern (TTC) value of 1.5 µg day-1 in take of a toxic impurity is permitted. The permitted quantity in ppm is the ratio of TTC in microgram day-1 and dose in gram day-1. Since, 40 mg of Esomeprazole magnesium is administered per day6 in the form of tablets (20 mg to 40 mg with the trade name Nexium), the estimated permissible quantity of these impurities is 37.5 ppm per day.
Recently, Raman and coworkers7 reported that the identification and determination of two carcinogenic impurities viz. methyl camphorsulfonate (MCS) and ethyl camphorsulfonate (ECS) in esomeprazole magnesium (EOM) using gas chromatography-mass spectrometry. The limit of detection and limit of quantitation values for both compounds were found as 3 and 10 ppm, with respect to 50 mg mL-1 of EOM. The method was linear within the range of 10-120 ppm and found to be precise, accurate, specific and robust. High-performance liquid chromatography (HPLC) is well established and robust but it has low sensitivity, requires large amounts of solvents and time consuming process. An approach based on gas chromatography (GC)-mass spectrometry (MS) is feasible within the limits of time and expensive. Despite the importance of the issue no analytical method has been proposed on the quantitation of genotoxic impurities (2, 3, 4 and 5) present in Esomeprazole magnesium (1). Hence, a selective and sensitive GC-MS method was developed for the identification and determination of these impurities in Esomeprazole magnesium.
EXPERIMENTAL:
Chemicals
Esomeprazole magnesium (1), 2-Chloro methyl-4-methoxy-3,5-dimethyl pyridine HCl (2), 2,3,5-Trimethyl pyridine N-Oxide (3), 4-Nitro-2,3,5-Trimethyl pyridine N-Oxide (4) and 4-Methoxy-2,3,5-Trimethyl pyridine N-Oxide (5) were received from the process research department of Matrix laboratories ltd., India. Analytical-grade DMSO was purchased from Merck chemicals (Mumbai, India).
Preparation of standard solutions:
The stock solutions of 2-Chloro methyl-4-methoxy-3,5-dimethyl pyridine HCl (2), 2,3,5-Trimethyl pyridine N-Oxide (3), 4-Nitro-2,3,5-Trimethyl pyridine N-Oxide (4) and 4-Methoxy-2,3,5-Trimethyl pyridine N-Oxide (5) were prepared by dissolving about 10.0 mg each (individually in separate 100 mL volumetric flasks) of the compounds in Dimethyl sulfoxide (DMSO). The diluted stock solution was prepared by pippeting each 1 mL of the stock solution into a 100 mL volumetric flask and diluting to volume with Dimethyl sulfoxide (DMSO). The working standard solutions 5.0 ppm was prepared by further diluting 2.5 mL of the diluted stock solution into 10 mL volumetric flask The sample solution was prepared by accurately weighing about 50 mg of Esomeprazole magnesium (1) into a 2 mL GC vial and adding 1.0 mL of the sample solvent. The concentration of 2, 3, 4 and 5 were about 5.0 ppm with respect to 50 mg mL-1 of 1.
An Agilent 7890A GC system coupled with 5975C single quadrupole mass spectrometer and Agilent 7683B auto sampler was used in the electron ionization (EI) mode (Agilent Technologies, PA, USA). Data acquisition and processing were conducted using the Agilent MSD Chemstation E.02.00 software on a Pentium computer (Digital equipment Co.).
Operating conditions:
The analytes were separated using J&W Scientific DB-5 capillary column (30 m x 0.53 mm I.D. x 5.0 µm). High purity helium (99.999%) was used as the carrier gas at a constant flow rate of 3.0 mL min-1. One microlitre of sample was injected in split mode. The injector and GC-MS interface temperatures were set at 120°C and 230°C, respectively, and the oven temperature is 240 °C, hold for 40 min; the total GC run time, 40 min. The mass spectrometer was operated in the electron ionization mode at 70 eV and the ion source temperature was set at 230°C. Agilent auto sampler was used to inject the samples.
The quantitative analysis was carried out in the selected ion monitoring (SIM) or selected ion-recording (SIR) mode. The mass spectra of 2, 3, 4 and 5 gave fragment ions of m/z 120, m/z 77, and m/z 77and m/z 151 respectively. Thus, fragment ion m/z 120 for 2, m/z 77 for both 3 & 4 and m/z 151 for 5 were chosen to monitor SIM experiments.
RESULTS AND DISCUSSION:
Method development and optimization:
The challenge is to achieve the desired detection and qauntitaion at very low levels using gas chromatography-mass spectrometry (GC-MS). To obtain good separation and the desired sensitivity, one approach is to select either most prominent fragment ion for selected ion monitoring (SIM) experiments in mass spectrometry and if require increase the sample amount injected into the GC-MS system. To decrease the interference of other substances with 2, 3, 4 and 5, other fragments also can be selected for SIM experiments, and suitable gradient column temperature in combination with a moderate inlet temperature (180 0C) may allow a large injection volume without significant deterioration in column efficiency.
The effect of concentration on separation and quantitation of 2, 3, 4 and 5 were investigated by injecting 1 µL of the stock solution and working standard solutions of 5.06 ppm, 5.12 ppm, 5.17 ppm and 5.14 ppm, respectively. Further studies were not done to determine the maximum injection volume.
Method validation:
The validation work was conducted according to the ICH (International Conference on Harmonization) guidelines8-11. The validated method parameters include specificity, limits of detection (LODs), and limits of quantitaion (LOQs), precision, linearity and accuracy.
The specificity for 2, 3, 4 and 5 were showed in a total ion chromatogram (Fig. 1). The specificity for 2, 3, 4 and 5 were recorded in SIM mode. From the SIM chromatograms a signal to noice ratio of 2.9, 3.8, 3.9 and 2.6 was obtained for 2, 3, 4 and 5, respectively. A second instrument (Same instrument manufacturer) was used to repeat the experiments and similar results were obtained. Esomeprazole magnesium at 50 mg mL-1 was spiked with 5.06 ppm, 5.12 ppm, 5.17 ppm and 5.14 ppm for 2, 3, 4 and 5. All four peaks have a signal to noice ratio of near about 3, indicating that this method is capable of detecting about 1.0 ppm level of the 2, 3, and 4 in the drug substance of 1.
In the pharmaceutical industry, the quantitation limit (LOQ) was defined as the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. The LOQ was determined less than or equal to 5.06 ppm, 5.12 ppm, 5.17 ppm and 5.14 ppm for 2, 3, 4 and 5.
Scheme 1: Structures of the studied compounds.
Fig. 1. Total ion chromatogram of genotoxic impurities: 2 at 8.65 min., 3 at 9.58 min., 4 at 15.03 min., and 5 at 16.52 min.
Table 1. Analytical data for the proposed method
|
Parameters |
2 |
3 |
4 |
5 |
|
LOD (ppm) a LOQ (ppm) a Linear range (ppm) a Slope Intercept Correlation coefficient Repeatability (% RSD) b Accuracy |
1.52 5.06 LOQ to 11 1805.2 702.47 0.9989 1.29 109.4 – 116.8 |
1.70 5.12 LOQ to 11 2539.04 2450.46 0.9968 3.08 99.9 – 107.6 |
1.55 5.17 LOQ to 11 4154.96 4149.60 0.9988 1.94 96.0 – 104.1 |
1.54 5.14 LOQ to 11 1689.93 115.6 0.9999 4.11 87.4 – 105.2 |
aLOD and LOQ values are given with respect to 50 mg mL-1 in 1; bSix determinations at LOQ.
Linearity of the method was determined by preparing and analyzing a series of 6 standard solutions to cover the concentration range of LOQ to about 11 ppm for 2, 3, 4 and 5. Regression analysis of the peak area versus concentration data yields R2 > 0.98.
The experimental results also show that this method has excellent precision without using an internal standard. Multiple injections were made for the standard solutions containing 5.06 ppm, 5.12 ppm, 5.17 ppm and 5.14 ppm each of 2, 3, 4 and 5. For six injections of the standard solutions, the percentage of R.S.D of the peak area was 1.29, 3.08, 1.94 and 4.11, respectively.
Accuracy of the method was determined by analyzing a drug substance samples spiked with known amount of the 2, 3, 4 and 5. The spiked levels were 5.06 ppm, 5.12 ppm, 5.17 ppm and 5.14 ppm. The recovery is in the range of 87.4 to 116.8, respectively (Table 1). Because this method uses the dissolve-and-inject approach, for every simple injection, about 1 µL (50 mg mL-1) of the drug substance is introduced into the injection port. The accumulation of drug substance may have minute negative effect on the recovery. Therefore the injection liner cleaned after every sequence of 10-15 injections.
CONCLUSIONS:
A simple and sensitive gas chromatography-mass spectrometric (GC-MS) method has been developed and validated for the trace analysis of 2-Chloro methyl-4-methoxy-3, 5-dimethyl pyridine HCl (2), 2,3,5-Trimethyl pyridine N-Oxide (3), 4-Nitro-2,3,5-Trimethyl pyridine N-Oxide (4) and 4-Methoxy-2,3,5-Trimethyl pyridine N-Oxide (5) in Esomeprazole magnesium (1) drug substances. These four genotoxic impurities (2, 3, 4 and 5) were used during the synthesis of Esomeprazole magnesium. GC-MS method can be conveniently applied to simultaneously low-level determination of 2, 3, 4, and 5 in 1 due to its high sensitivity.
The authors thank the management of Matrix Laboratories Ltd. for supporting this work. Authors wish to acknowledge the Process Research Department for providing the samples and colleagues from the separation science division of Analytical Development for their co-operation in carrying out this work.
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Received on 17.03.2011 Modified on 29.03.2011
Accepted on 07.04.2011 © AJRC All right reserved
Asian J. Research Chem. 4(6): June, 2011; Page 898-901