HS-GC Method Development and Validation for the Estimation of Class-2 Residual Solvents in Ivabradine

 

A. Lakshmi Sai Durga¹, B. Ramya Kuber²*

¹Department of Pharmaceutical Analysis, Institute of Pharmaceutical Technology,

Sri Padmavati Mahila Visvavidyalayam (Women’s university), Tirupati, 517502, Andhra Pradesh, India.

²Department of Pharmacognosy, Institute of Pharmaceutical Technology,

Sri Padmavati Mahila Visvavidyalayam (Women’s university), Tirupati, 517502, Andhra Pradesh, India.

 

ABSTRACT:

A simple, rapid, accurate method for simultaneous estimation of residual solvents like Dichloromethane, Tetrahydrofuran, Toluene and Xylene in Ivabradine was performed by Head space - Gas chromatography. The residual solvents were separated using a 30 m long DB-624 column with an inner diameter of 0.53 mm and a film thickness of 3.0 m, with a flow rate of 1.2 ml/min and a run period of 12 minutes, using Nitrogen as the carrier gas and Dimethyl Sulphoxide as the solvent. All the head space parameters were maintained to achieve a good separation by maintaining temperature of 95°C -105°C and suitable pressure.  Analytical method was also developed by performing various trails by changing various parameters such as chromatographic conditions and head space parameters and an optimized chromatogram was obtained by showing high resolution and accurate peaks. Validation process was carried by using ICH guidelines. All parameters were carried by procedures and results were within limits as per guidelines. Validation parameters such as linearity, precision, accuracy, ruggedness, robustness, LOD, LOQ, Specificity and system suitability were performed and the proposed was said to be more precise, specific, rapid and accurate. The percentage recoveries and peak response of dichloromethane, tetrahydrofuran, toluene and xylene were with range of 90-110% and NMT 15. 

 

 

 

INTRODUCTION: 

Chromatography is a technique which is used to separate mixture of compounds by using stationary phase and mobile phase. Generally, separation is achieved by basing their affinity towards column. Chromatography was discovered by Mikhail Semenovich Tsvett. There are various types of chromatography techniques for better separation of compounds. All organic, inorganic and trace elements can be separated and analyzed by using chromatography¹. Generally, gas chromatography is used to separate volatile solvents.

 

 

But Headspace-Gas chromatography was used to residual solvents present in the sample. Now a days, gas chromatography plays a major role in pharmaceutical industry. Headspace-Gas chromatography was used to determine various residual solvents which are in trace amounts in drugs substance and can help to know the impurity content in the drug substances. It helps to provide the thermodynamic equilibrium in closed system to determine the volatile residual solvents².

 

Residual solvents are the organic volatile chemicals that are present in various pharmaceutical products such as drug substance, excipients and products during the manufacturing and disclosing the substances. These substances as regarded as harmful substances which may effect the health of the humans. As there is no therapeutic role, there should be removes from pharmaceuticals products. They must be completely removed but during synthesis and intermediate stages they can’t be completely retained from the substances. Based on their presences and risk assessment, they are divided into 3 classes. They are:

1)   Class-1: Solvents to be avoided

2)   Class-2: Solvents to be limited

3)   Class-3: Solvents with low toxic potential

 

By using HS-GC, the residual solvents in the given substances can be easily separated and estimated by applying suitable pressure and temperature and also basing their affinity towards substances.

 

Ivabradine drug is chemically 3-[3-[[(7S)-3,4-dimethoxy-7-bicyclo[4.2.0]octa-1,3,5-trienyl]methyl-methylamino]propyl]-7,8-dimethoxy-2,5-dihydro-1H-3-benzazepin-4-one and the its is represented in Fig 1

 

It is an anti-arrhythmic drug used to treat heart intolerance condition during the chronic heart failure. The mechanism involved in treating the heart condition is inhibition of pacemaker ion current inward flow and reduces its activity and reduces mycardial oxygen demand and constant blood flow in the heart.

 

Various analytical techniques were already reported for Ivabradine drug by undergoing literature survey. The reported techniques were HPLC^3-5,HPTLC⁶, LC-MS⁷methods. The present study helps to determine the amount of residual solvents like dichloromethane, tetrahydrofuran, toluene and xylene in Ivabradine pure drug.

 

Figure 1: Structure of Ivabradine

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Chandra labs in Hyderabad provided ivabradine as a free sample. Qualigens provided dichloromethane and tetrahydrofuran (GC grade). Sigma Aldrich provided the toulene and xylene (GC grade). Qualigens provided the dimethyl sulfoxide.

 

Instruments:

The separation of residual solvents was achieved by using column DB-624 column, (30m x0.53mm) 3.0µm with HS-GC make and model is Agilent Technologies and 7697A headspace sampler. The following were the chromatographic conditions and head space parameters were given in Table 1.

 

Table 1: Chromatographic conditions and Headspace parameters

 

Preparation of solutions:

Standard Stock-I Preparation:

In a 100ml container, weigh 374mg of Dichloromethane, 144mg of Tetrahydrofuran, 178mg of Toluene, and 434 mg of Xylene. Make up to volume with diluent in a volumetric flask holding roughly 20ml of diluent and to shaken well.

 

Standard Stock-II Preparation:

Pipette out 5ml of above solution in 50ml volumetric flask containing about 20ml diluent, make up to volume with diluent.

 

Pipette 5ml of above prepared solution in headspace vial containing 0.2gm sodium chloride and seal the vial.

 

Test Sample Preparation:

Weigh accurately about 200mg of test sample (Ivabradine API) and transfer in to 25mL volumetric flask add 15mL of Diluent, vortex it for 5min. Then make up the volume with diluent and mix well.

 

Pipette 5ml of above prepared solution in headspace vial containing 0.2gm sodium chloride and seal the vial.

 

Procedure:

The prepared solutions are placed in a 5ml headspace vial and sealed with a crimper. Headspace analysis is performed on the prepared standard and sample solutions.

 

Analytical method development:

Various trails were performed by changing parameters to obtain an accurate, specific and good resolution peak for determination of residual solvents in the Ivabradine. The Optimized trail peaks can be observed in Fig 2

 

Figure 2: Chromatogram of optimized Trail

 

 

 

Figure 3: Linearity graph of Dichloromethane, Tetrahydrofuran, Toulene and Xylene

 

RESULTS AND DISCUSSION:

Method Validation:

Various validation parameters such as Linearity, Specificity, Accuracy, Precision, LOD, LOQ, Robustness and Ruggedness were performed by following the ICH guidelines.

 

Linearity:

These results were used to Analyze the samples with various concentration of analytes. They can be studied by slope obtained and regression coefficient and its acceptance criteria is not more than 0.999. The results for residual solvents were shown in following Fig 3.

 

Specificity:

It gives information about the residual solvents present in the sample component. In this, first we inject the blank followed by standard residual solvents and finally spiked samples are injected. The results are given in table 2 and chromatogram in fig 4.

 

 

Table 2: Retention time of Individual and Spiked sample

 

Table 3: Data of Accuracy and Precision

 

Accuracy and Precision:

Accuracy studies were carried to know about recovery level of residual solvents and the values for dichloromethane was 103.4%, tetrahydrofuran was 107.3%, Toulene was 104.6%and xylene was 101.4%. The values were in the acceptance criteria which is 90-110%. When coming to precision, it details about the relative standard deviation of individual solvents.

 

Figure 4: Specifiity of Blank, Sample and Spiked sample

 

Figure 5: Chromatogram of Accuracy and Precision

 

 

The values for dichloromethane was 3.4, tetrahydrofuran was 1.5, toulene was 1.1 and xylene was 1.3 and its acceptance criteria is NMT 15. The results were shown in table 3 and chromatograms in Figure 5.

 

LOD and LOQ:

The detection limit of Dichloromethane 0.0001, Tetrahydrofuran 0.00002, Toluene 0.00005 and Xylene 0.0002. The quantification limit of Dichloromethane 0.00045, Tetrahydrofuran 0.00005, Toluene 0.00002 and Xylene 0.0005.

 

Robustness and Ruggedness:

Robustness was carried out to check the flow rate of solvent and its acceptance criteria is less than 2%. While coming to ruggedness, it was studied to observe the various analysts and results obtained and its acceptance criteria is less than 2%. These parameters were carried out and the sample are within the acceptance range.

 

CONCLUSION:

By observing all the parameters, it was concluded that a simple, accurate, rapid and sensitive method was proposed for estimation of residual solvents i.e dichloromethane, tetrahydrofuran, toluene and xylene in Ivabradine. The proposed method was found have good resolution and it was also cost effective.

 

ACKNOWLEDGMENT:

The authors are grateful to the professors at Sri Padmavathi Mahila Visvavidyalayam, Institute of Pharmaceutical Technology.

 

REFERENCE:

1.     Harold M, Nair M, James MM: Basic Gas Chromatography. A Wiley-Interscience publication 1997; 1(1): 2-3.DOI: 10.1016/C2009-0-12062-7.

2.     Ramya kuber B and Swetha Addanki. Novel stability-indicating RP-UPLC method for simultaneous estimation of Sitagliptin and Ertugliflozin in bulk and Pharmaceutical formulations. Future journal of Pharmaceutical Science, 2012; 7(86), 1-10.DOI:10.1186/s43094-021-00231-5.

3.     Seerapu S, Srinivasan. Development and Validation of RP-HPLC Method for the Estimation of Ivabradine Hydrochloride in Tablets. Indian Journal of Pharmaceutical Sciences. 2010; 72 (5): 667-71. DOI10.4103/0250-474x.78545.

4.     Rahman Md, Asaduzzaman Md, Islam M. Development and validation of RP-HPLC method for analysis of Ivabradine Hydrochloride in tablet dosage forms. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2012; 3(3): 1032-1043.DOI :10.29161/PT.v6.i10.2018.26.

5.     Maheshwari S, Amit khandhar. Quantitative Determination and Validation of Ivabradine HCL by Stability Indicating RP-HPLC Method and Spectrophotometric Method in Solid Dosage Form. Eurasian Journal Anl. Chem. 2010; 5(1):53-62.DOI: 10.4103/0250-474X.78545.

6.     Rahman Md, Asaduzzaman Md, Islam M. Development and validation of RP-HPLC method for analysis of Ivabradine Hydrochloride in tablet dosage forms. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2012; 3(3): 1032-1043.DOI; 10.25004/IJPSDR.2017.090505

7.     Patel P, Roshan M.B, Pradip K, Rahul P.G. Characterization of degradation products of Ivabradine by LC-HR-MS/MS: A typical case of exhibition of different degradation behavior in HCl and H₂SO₄ acid hydrolysis. Journal of Mass Spectroscopy. 2015; 50 (2).:344-53. DOI: 10.1002/jms.3533.

 

 

 

 

 

Accepted on 23.06.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):545-549.