Available online on 15.12.2020 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

© 2011-20, publisher and licensee JDDT, This is an Open Access article which permits unrestricted non-commercial use (CC By-NC), provided the original work is properly cited

Open Access                                                                                              Research Paper

Cost Effective Accurate and Precise Analytical Method Development of Content Estimation of N-Nitrosodimethyl Amine and N-Nitrosodiethyl Amine in Olmesartan Medoxomil by GC-MS

Baskar Venkatesan¹*, Pushpendra Sharma¹, Asheesh Singh²

¹ Department of Science, Sri Satya Sai University of Technology and Medical Sciences, Sehore- (MP) – 466001, India

² Faculty of pharmacy, VNS group of Institutions, Neelbud Bhopal, India- (MP) – 462044, India

__________________________________________________________________________________________________________________________________________________

Abstract

A simple, precise and accurate GCMS method was developed for estimation the content estimation of N-Nitrosodimethylamine (NDMA) & N-Nitrosodiethylamine (NDEA) in olmesartan medoxomil (OLM) in drug substances. The content was determined by GCMS on DB-CAM 30.0 m Χ 0.32 mm, 0.5µm Capillary column and helium was used as carrier gas, using methanol as diluent at column flow rate of 2.0 mL/min and Ion source temperature & Interface temperature at 200ºC and Detector gain mode relative to tuning file with acquisition mode Q3 SIM. The method was developed and evaluated for validation parameter as per ICH guidelines for Specificity, linearity, accuracy and precision. The method shows good linearity over the range of 10%-150% for NDMA and NDEA for olmesartan. The average percentage recoveries were found within predefined acceptance criteria (10% and 100%) for N-Nitrosodimethylamine (NDMA) & N-Nitrosodiethylamine (NDEA) in olmesartan medoxomil, respectively. Therefore, the proposed method can be applied for routine analysis of the bulk drugs as well as combined pharmaceutical dosage forms of olmesartan medoxomil.

Keywords: Olmesartan medoxomil (OLM), N-Nitrosodimethylamine (NDMA) &, N-Nitrosodiethylamine (NDEA) GCMS, Validation, Analytical validation.

__________________________________________________________________________________________________________________________________________________

Article Info: Received 03 Oct 2020;       Review Completed 16 Nov 2020;        Accepted 27 Nov 2020;      Available online 15 Dec 2020

Cite this article as:

Venkatesan B, Sharma P, Singh A,Cost Effective Accurate and Precise Analytical Method Development of Content Estimation of N-Nitrosodimethyl Amine and N-Nitrosodiethyl Amine in Olmesartan Medoxomil by GC-MS, Journal of Drug Delivery and Therapeutics. 2020; 10(6-s):37-44      http://dx.doi.org/10.22270/jddt.v10i6-s.4444         

*Address for Correspondence:

Bask ar Venkatesan, Research Scholar (PhD), Department of Science, Sri Satya Sai University of Technology & Medical Sciences, Sehore- (MP) - 466001

__________________________________________________________________________________________________________________________________________________

INTRODUCTION:

Olmesartan medoxomil (OLM), (5-methyl-2oxo-1,3-dioxol-4-yl)methylester of 4-(1hydroxy-1-methylethyl)-2-propyl-1-{[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl}-1H-imidazole-5-carboxylic acid (Figure 1), is a novel selective angiotensin II type 1 (AT₁) receptor antagonist having antihypertensive efficacy. It is an ester prodrug which is completely and rapidly hydrolysed to the active form, olmesartan. It works by blocking the binding of angiotensin II to the AT₁ receptors in vascular smooth muscle and as a result of this blockade olmesartan reduces vasoconstriction. This lowers blood pressure by decreasing total peripheral resistance in hypertensive individuals. Olmesartan medoxomil is obtained as colourless crystalline powder, practically insoluble in water and sparingly soluble in methanol ^1-3.

Figure 1: Chemical structures of olmesartan

Nitrosamines in Drug Substances

European Medicine Agency in March 2020 describes the currently identified sources of nitrosamine impurities. Recently, many countries have banned the use of sartans due to reports that carcinogenic N-nitrosodimethylamine (NDMA) or/and. N-nitrosodiethylamine (NDEA) are present as impurities in drug substances^4-7. NDMA impurities were also recently observed in ranitidine tablets that belong to the class of drugs known ashistamine-2 blockers. It has been found that the NDMA impurities of ranitidine products increase over time and during storage at temperatures above room temperature, and that a large amount of NDMA is produced, especially when heated to high temperatures^8-9. The U.S. FDA has reported that low concentrations of NDMA have been detected in some metformin products, and no sample of metformin exceeds the acceptable daily intake for NDMA¹⁰.

GC–MS/MS is recognized as efficient analytical equipment for NA analysis due to its advantages, including low interference, high sensitivity and reasonable price. Because ranitidine thermally degrades to NDMA in a GC instrument, GC analysis is inappropriate for analysis of NDMA in ranitidine. In order to prevent interference and thermal degradation, a clean-up method is required to completely remove the drug sub-stances such as sartans, ranitidine and metformin. If a clean-up method for completely removing drug substances is developed, it can be applied to analyze NAs in different types of pharmaceutical products with an analysis method using GC–MS/MS. The aim of this study was to develop a method for the simultaneous analysis of NDMA and NDEA in drug substances such olmesartan by GC–MS/MS. This study focused on optimizing the extraction and clean-up methods for NDMA and NDEA from olmesartans^11-12.

MATERIALS AND METHODS

Instruments/ chemicals & reagents /standards & samples:

Methodology:

Identification of Compound: Initially 100ppm of Impurity solution was injected in full scan mode & optimizes the SIM method (74 & 102) of N-Nitrosodimethyl amine and N-Nitrosodiethyl amine impurity was determined in GCMS.

Experiment No 1: Optimize the method for N-Nitrosodimethyl amine and N-Nitrosodiethyl amine impurity in Olmesartan Medoxomil by GCMS.

Name of diluent:    Methanol

Chromatographic Conditions:                                                           

Preparation of Blank: Diluent is used as blank.

Preparation of Standard stock solution-1: Pipetted 50µL of NDMA & NDEA into a 50mL volumetric flask, and made upto the volume with MDC and mixed well.

Preparation of Standard stock solution-2: Pipetted 50µL of NDMA & NDEA into a 50mL volumetric flask, and made upto the volume with DMSO and mixed well.

Preparation of Standard stock solution-3: Pipetted 50µL of NDMA & NDEA into a 50mL volumetric flask, and made upto the volume with MTBE and mixed well.

Preparation of Standard stock solution-4: Pipetted 50µL of NDMA & NDEA into a 50mL volumetric flask, and made upto the volume with Methanol and mixed well.

Preparation of Standard solution: Pipetted 1mL of above stock solution into a 10mL volumetric flask, and made upto the volume with Methanol and mixed well.

Experiment No 2:

Chomatographic conditions:

Preparation of Blank: Diluent is used as blank.

Preparation of Standard stock solution: Weighed and transferred 244.15mg of NDMA & 66.09mg of NDEA into a 10mL volumetric flask, and made upto the volume with Methanol and mixed well.

Preparation of Intermediate standard stock solution-1: Pipetted 0.5mL of Standard stock solution into a 10mL volumetric flask, and made upto the volume with diluent and mixed well.

Preparation of Intermediate standard stock solution-2: Pipetted 0.5mL of intermediate standard stock solution-1 into a 10mL volumetric flask, and made upto the volume with diluent and mixed well.

Preparation of Intermediate standard stock solution-3: Pipetted 1mL of intermediate standard stock solution-2 into a 10mL volumetric flask, and made upto the volume with diluent and mixed well.

Preparation of Standard solution (2.4ppm & 0.6ppm, 100%): Pipetted 1mL of intermediate standard stock solution-3 into a 10mL volumetric flask, and made upto the volume with Methanol and mixed well.

Preparation of Standard solution (10%): Pipetted 1mL of standard solution (2.4 ppm, 0.6ppm) into a 10mL volumetric flask, and made upto the volume with Methanol and mixed well.

Preparation of as such sample solutions: Below samples mixed well and filtered through 0.45µ filters, transferred to AOC vial and placed in GC MS system.

Table 1: Preparation of as Such Sample Solution

Preparation of 10% Spiked sample solutions: Below samples mixed well and filtered through 0.45µ filters, transferred to AOC vial and placed in GC MS system.

Table 2: Preparation of as 10% Spike Sample Solution

Preparation of 100% Spiked sample solutions: Below samples mixed well and filtered through 0.45µ filters, transferred to AOC vial and placed in GC MS system.

Table 3: Preparation of as 100% Spike Sample Solution

Preparation of Linearity level solutions: Below linearity solutions transferred to AOC vial and placed in GC MS system.

Table 4: Preparation of Linearity level Solution

Specification Limit:    Content of NDMA is 2.39µg/g and NDEA is 0.66µg/g.

Chomatographic conditions:

Preparation of Blank: Diluent is used as blank.

Preparation of Standard stock solution: Weighed and transferred 244mg of NDMA & 66mg of NDEA into a 10mL volumetric flask, and made upto the volume with Methanol and mixed well.

Preparation of Intermediate standard stock solution-1: Pipetted 0.5mL of Standard stock solution into a 10mL volumetric flask, and made upto the volume with diluent and mixed well.

Preparation of Intermediate standard stock solution-2: Pipetted 0.5mL of intermediate standard stock solution-1 into a 10mL volumetric flask, and made upto the volume with diluent and mixed well.

Preparation of Intermediate standard stock solution-3: Pipetted 1mL of intermediate standard stock solution-2 into a 10mL volumetric flask, and made upto the volume with diluent and mixed well.

Preparation of Standard solution (2.4ppm & 0.6ppm): Pipetted 1mL of intermediate standard stock solution-3 into a 10 mL volumetric flask, and made upto the volume with Methanol and mixed well.

Preparation of as such sample solutions: Weighed and transferred 250 mg of sample in 5 mL vial and add 1mL diluent and mixed well filter the solution through PVDF filter.

Injection sequence:

Procedure: Inject blank, standard and sample solution into the GCMS system and record the chromatograms. Disregard the peaks due to blank. The retention time of N-Nitrosodimethylamine about 7.6 and N-Nitrosodiethylamine peak is about 8.4 minutes.

System Suitability Requirements: The %RSD for N-Nitrosodimethylamine and N-Nitrosodiethylamine peak areas from six replicate standard injections are NMT 15.0.

Calculation: Content of N-Nitrosodimethylamine / N-Nitrosodiethylamine in Olmesartan Medoxomil

                               A_T           C_S          P     

                       =­­­­­­­­­­­­­­­­­­­­­­­     ––––   X  –––- X  ––––  X  10⁶          

                                A_S           C_T        100

Where,

A_T    = Peak area of N-Nitrosodimethylamine /N-Nitrosodiethylamine obtained in sample solution.

A_S    = Average peak area of N-Nitrosodimethylamine /N-Nitrosodiethylamine obtained with standard solution

C_S    = Standard concentration                

C_T    = Sample concentration

P     = Purity of Standard

Specification: Content of N-Nitrosodiethylamine should be NMT 0.66µg/g and N-Nitrosodimethylamine in Olmesartan Medoxomil should be NMT 2.39µg/g.

RESULT AND DISCUSSION:

Observations & Results of experiement 1: No blank interference is observed at the retention time of standard in MDC diluent. Blank interference was observed in DMSO diluent. No blank interference was observed at NDMA standard RT-7.6 in Methanol diluent. Blank interference was observed at NDEA standard RT-8.4. NDMA standard Rt at 3.5min. NDEA standard RT at 4.5 min. Recovery results was not meeting the criteria. Further optimization changed to split ratio.

Observations & Results of experiement 2: No blank interference is observed at the retention time of NDMA & NDEA. System suitability found satisfactory. NDMA peak eluted at 7.5min, NDEA peak eluted at 8.6min. Correlation coefficient for linearity of NDMA is found: 0.99 and Correlation coefficient for linearity of NDEA is found: 0.99. Based on below mentioned recovery results method was meets the system suitability, Linearity, Accuracy criteria. Method is suitable for proceeding to validation. Based on the above experiments, the following method parameters are finalised for Content estimation of N-Nitrosodimethyl amine and N-Nitrosodiethyl amine in Olmesartan medoxomil.

Recovery Results (10%) :

Table 5: Recovery of 10% Spike solution

Recovery Results (100%) :

Table 6: Recovery of 100% Spike solution

Figure 2: Typical chromatogram of Blank

Figure 3: Typical chromatogram of Standard

Figure 4: Typical chromatogram of As such sample

CONCLUSION:

The test method for the estimation of NDMA & NDEA in Olmesartan Medoxomil by GCMS has been developed. The proposed method is found to be Precise, Specific, Linear, Accurate at LOQ Level and can be used for routine analysis. The method was free from Interferences. Therefore, this method may be useful for routine analysis of olmesertan in bulk drugs.

ACKNOWLEDGMENTS:

The authors are thankful to performics Analytical labs LLP, Hyderabad for providing necessary Laboratory facilities to carry out the work.

CONFLICT OF INTEREST:

Author has no conflict of interest during preparation of this research manuscript.

Abbreviations:

REFERENCES:

1. Martindale, K.P. The Extra Pharmacopoeia, the Complete Drug Reference, thirty fourth edition volume 3, Royal Pharmaceutical Society, 2005; 455-467.
2. British pharmacopoeia: The Stationery Office, London. 2007; 1036-103
3. Huber, L. Validation of analytical methods, in: Validation and Qualification in the Analytical Laboratories, Interpharm Press, Buffalo Grove, IL, 1998; 11:107-115.
4. Text on Validation of Analytical Procedures, in: International Conference on Harmonization of Technical Requirements for Registration of Pharmaceutical for Human Use, Geneva 2000.
5. Validation of Analytical Procedure: Methodology, in: International Conference on Harmonization of Technical Requirements for Registration of Pharmaceutical for Human Use, Geneva 2005.
6. Nitrosamines impurities in human medicinal products and related guidance. European medicine Agency. CHMP/428592/2019. 2020.
7. French National Agency for Medicines and Health Products Safety Laboratory Controls Division – French OMCL Method reference: 19A0416-01, Determination of NDMA and NDEA in SARTAN drug substances by HPLC/UV,www.ansm.sante.fr. April, 2020.
8. Schmidtsdorff, S., Schmidt, A.H. Simultaneous detection of nitrosamines and other sartan-related impurities in active pharmaceutical ingredients by supercritical fluid chromatography, J. Pharm. Biomed. Anal. 2019; 174:151–160.
9. The General European OMCL Network, LC-MS/MS Method for the determination of NDEA and NDMA in Valsartan, Irbesartan and Losartan APIs and finished dosage form 2020.
10. Sorgel, F, Kinzig M, Abdel-Tawab, M, Bidmon, C, Schreiber, A, Ermel, S, Wohlfart, j, Besa, A, Scherf-Clavel, O, Holzgrabe, U. The contamination of valsartanof valsartan and other sartans, part 1: new findings, J. Pharm. Biomed. Anal. 2019; 192:395–405.
11. US FDA. Combined Direct Injection N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) Impurity Assay by GC/MS 2018.
12. Combined direct injection method: a GC-MS/MS method that allows for determination of both NDMA and NDEA simultaneously (https://www.fda.gov/media/123409/download) OTR has been asked to develop a gas chromatography-tandem mass spectrometry (GC-MS/MS) method utilizing liquid injection to look for all these nitrosamine impurities. www.fda.gov/media/123409.April, 2020.