Low-Level Determination of Residual 4-Bromo Methyl-2'-Cyanobiphenyl in Valsartan by Liquid Chromatography-Mass Spectrometry
Yogeshwar Reddy M1,3*, Ramesh V2, Kista Reddy Ch3, Venugopal N1, Saravanan G1, Suresh Y1, Suryanarayana M1, Debashish Datta1 and Raju B2
1Matrix Laboratories Ltd., R and 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: yogeshwarreddy@yahoo.com
ABSTRACT:
A Liquid chromatographic (LC) method using mass spectrometric (MS) detection was developed and validated for the trace analysis (ppm level) of 4-Bromo methyl-2'-Cyanobiphenyl in Valsartan drug substances. LC analysis of 4-Bromo methyl-2'-Cyanobiphenyl was done on Necleosil C18 (100 mm x 4.6 mm, 3 μm) column and the mobile phase is in the ratio of 50:50:1 containing water, acetonitrile, and acetic acid. The flow rate was 1.0 mL min-1 and the elution was monitored at 225 nm. The method was validated as per International Conference on Harmonization (ICH) guidelines. LC-MS is able to quantitate up to 1.0 ppm of 4-Bromo methyl-2'-cyanobiphenyl.
KEYWORDS: Liquid chromatography- mass spectrometry Valsartan
1.0. INTRODUCTION:
Valsartan, chemically describes (S)-3-methyl-2- [N- ({4-[2-(2H-1, 2,3,4-tetrazol-5-yl) phenyl] phenyl} methyl) pentanamido] butanoic acid is an antihypertensive [1]. In the synthesis of Valsartan 4-Bromo methyl-2'-cyanobiphenyl is an important raw material. Identification and determination of 4-Bromo methyl-2'-cyanobiphenyl is essential because of its genotoxic nature [2]. As per regulatory guidelines [3], 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, 50 mg of Valsartan is administered per day [4] in the form of tablets (40 mg, 80 mg, 160 mg, and 320 mg with the trade name Diovan), the estimated permissible quantity of 4-Bromo methyl-2'-cyanobiphenyl is 4.6 ppm per day. Therefore, it is of great importance to develop analytical methods that are sensitive enough and meet all the regulatory requirements. Hence, a LC-MS method was developed for identification and low level determination of 4-Bromo methyl-2'-cyanobiphenyl in valsartan.
The 4-Bromo methyl-2'-cyanobiphenyl is solid at ambient temperature and it is feasible to separate and quantify these compounds by liquid chromatography mass spectrometry (LC-MS). The analysis of 4-Bromo methyl-2'-cyanobiphenyl using gas-chromatography (GC)-mass spectrometry (MS) is not straightforward because of the specific nature and physical properties of the compound. This manuscript describes a simple and sensitive method for the determination of 4-Bromo methyl-2'-cyanobiphenyl in Valsartan using LC-MS in selective ion monitoring mode (SIM) or selective ion recording (SIR) mode.
2.0. EXPERIMENTAL:
Chemicals:
Samples of API and 4-Bromo Methyl-2'-Cyanobiphenyl were received from the Process Research Department of Matrix Laboratories Limited, Hyderabad, India. HPLC-grade acetonitrile, methanol and acetic acid were purchased from Merck chemicals (Mumbai, India) and were used without further purification. High pure water was prepared by using the Millipore Milli-Q Plus purification system.
Preparation of solutions:
The stock solution of 4-Bromo methyl-2'-cyanobiphenyl was prepared by dissolving 10.0 mg of the compound in sample solvent (100 mL). The diluted stock solution was prepared by pippeting 1 mL of the stock solution in to a 100 mL volumetric flask and diluting to volume with the sample solvent. The working standard solution (1.0 ppm was prepared by further diluting 1.5 mL of the diluted stock solution into 100 mL volumetric flask. The sample solution was prepared by accurately weighing about 5 mg of the drug substance into a 2 mL HPLC-vial and adding 1.0 mL of the sample solvent.
Instrumentation:
LC-MS experiments were acquired using Applied Bio systems/MDS Sciex (Foster City, USA) API 3000 model equipped with an electrospray ionization source. The data acquisition was under the control of Analyst QS software (Foster City, CA) on a Pentium computer (Digital equipment Co).
Operating conditions:
Necleosil C18 (100 mm x 4.6 mm, 3 μm) column (Waters Co., USA) in isocratic mode used for separation of the impurities under LC conditions using water, acetonitrile and acetic acid are the mobile phase in the ratio of 50:50:1. The flow rate was 1.0 mL min-1, with the flow rate split down to 0.2 mL min-1 into the MS source. The column was monitored at 30 °C and the wavelength was set to 225 nm, repecively. The injection volume was 10 µL. Selective ion monitoring (SIM) mode used in positive electrospary ionization mass spectrometric conditions for quantification of 4-Bromo methyl-2'-cyanobiphenyl in drug substances. In this method 4-Bromo methyl-2'-cyanobiphenyl is monitored by its molecular ion 192 (Protonated). The ion spary voltage 5.00 kV, focusing potential 260 V, declustering potential 60 V, resolution 8000 (full-width half-maximum) and entrance potentials were 4500, 267, 69 and 10, repectively. The Curtain gas flow and nebulization pressure are 8 and 9 psi, respectively.
3.0. RESULTS AND DISCUSSION:
Method development and optimization:
The challenge was to achieve the desired detection and qauntitation at very low level under the given LC-MS conditions, i.e., liquid chromatography with mass spectrometry. To obtain good separation and the desired sensitivity, one approach is to select either most prominent fragment ion as selective ion monitoring mode (SIM) in MS and if require increase the sample amount injected in to the LC-MS system. To decrease the interference of other substances with the 4-Bromo methyl-2'-cyanobiphenyl can also be selected for SIM experiments.
The effect of concentration on separation and quantitation of the 4-Bromo methyl-2'-cyanobiphenyl was investigated by injecting 10 µL of the stock solution and working standard solutions of 1.0 ppm. Further studies were not done to determine the maximum injection. An injection volume of 10 µL was chosen for this method. This method utilizes a dissolve-and-inject approach for the residual analysis (4-Bromo methyl-2'-cyanobiphenyl). Several factors were considered in selection of a sample solvent, including the purity, its ability to dissolve the analyte, and its chemical compatibility with compounds of interest. To detect the 4-Bromo Methyl-2'-Cyanobiphenyl at about 1.0 µg g-1 level, the purity of the sample solvent is critical. In each case 10 µL of the sample was injected. The tested sample concentration of drug substances was 5 mg mL-1. The 4-Bromo methyl-2'-cyanobiphenyl showed reasonable ability and well separation in this method methanol as diluent. This is important because many pharmaceuticals are in salt forms, which sometimes show limited solubility in pure organic solvents.
Scheme 1: Structures of the studied compound
Method validation:
The validation work was conducted according to the ICH (International Conference on Harmonization) guidelines (5-8). The validated method parameters include specificity, limits of detection (LOD), limits of quantitation (LOQ), precision, linearity and accuracy. The detection limit (LOD) for the 4-Bromo methyl-2'-cyanobiphenyl was estimated from a total ion chromatogram of a solution containing about 0.34 ppm for 4-Bromo methyl-2'-cyanobiphenyl, and S/N (signal to noice) ratio was obtained as 3.4. A second instrument (Same instrument manufacturer) was used to repeat the experiment and similar results were obtained. A drug substance sample at 5 mg mL-1 was spiked with 1.0 ppm of the 4-Bromo methyl-2'-cyanobiphenyl. It has a signal to noice ratio of near about 3, indicating that this method is capable of detecting about 0.3 ppm level of the 4-Bromo Methyl-2'-Cyanobiphenyl in the drug substance.
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 to be less than or equal to 1.0 for 4-Bromo Methyl-2'-Cyanobiphenyl bases on the precision and accuracy data discussed below.
Linearity of the method was determined by preparing a analyzing a serious of 5 standard solutions to cover the concentration range of LOQ to 5 ppm for 4-Bromo methyl-2'-cyanobiphenyl. Regression analysis of the peak area versus concentration data yields an R2 > 0.99 for each of the three calibration curves.
The experimental results also show that this method has excellent precision without using an internal standard. Multiple injections were made for the standard solution containing 1.0 ppm, 4-Bromo methyl-2'-cyanobiphenyl. For six injections of the standard solution, the percentage of R.S.D of the peak area was 3.26.
Fig. 1. Mass spectrum of the 4-Bromo methyl-2'-cyanobiphenyl
Fig. 2. TIC of the 4-Bromo methyl-2'-cyanobiphenyl and Valsartan
Accuracy of the method was determined by analyzing a drug substance samples spiked with known amount of the 4-Bromo methyl-2'-cyanobiphenyl. The recovery was in the range of 99.2 to 103.1, respectively, when the spiked level is 1.0 ppm. Because this method uses the dissolve-and-inject approach, for every simple injection, about 5 mg of the drug substance is introduced in the injection port. The accumulation of drug substance may have negative effect on the recovery.
4.0. CONCLUSIONS:
A simple and sensitive LC-MS method has been developed and validated for the trace analysis of 4-Bromo methyl-2'-cyanobiphenyl in Valsartan. The validation has been conducted according to ICH guidelines, compared with the previously reported methodologies; this method utilizes a Liquid chromatograph mass spectrometer, which is available in the pharmaceutical industry. This method is sensitive enough to detect 0.3 µg g-1 and quantify 1.0 µg g-1 levels of the 4-Bromo methyl-2'-cyanobiphenyl in Valsartan.
The limit of detection and limit of quantitation were determined to be 0.33 ppm and to be 1.0 ppm with respect to 5 mg mL-1 of API, respectively. The method utilizes a dissolve-and-inject approach for sample preparation and introduction. Factors affecting method development and validation will be discussed and the work also includes the partial validation and method development.
5.0. ACKNOWLEDGEMENTS:
The authors wish to thank the management of Matrix Laboratories Ltd. for supporting this work. Authors wish to acknowledge the Process Research Department for providing the samples for this research. We also would like to thank colleagues from the separation science division of Analytical Development for their co-operation in carrying out this work.
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Received on 04.02.2010 Modified on 22.02.2009
Accepted on 02.03.2009 © AJRC All right reserved
Asian J. Research Chem. 3(2): April- June 2010; Page 407-410