INTRODUCTION:

When pancreas does not able to produce enough insulin or when the body cannot effectively use the insulin it produces a chronic disease named Diabetes occurs. Millions of people in world are suffering from Diabetes mellitus with type-1 or type-2.^1-3 This disease is the disease that precludes body from appropriately consuming energy from the food eaten. It has marked as long lasting disease in world with disturbing value of life of the patients severely which employees a major fiscal burden on the world.^4-5

Treatment and prevention of diabetes and its complications is decisive. Glyburide (also known as Glibenclamide: IUPAC Name: (A) 1-[4-[2-(chloro-2-methoxybenzamide) ethyl]-benzene sulfonyl]-cyclohexylurea, (B)5-chloro-N-[2-(4-[[(cyclohexylcarbamoyl)amino] sulfonyl] phenyl) ethyl]-2-methoxybenzamide is known for anti-diabetic drug which belongs to sulfonyl urea group and used to control diabetes mellitus with mild to soberly severe type II which does not require insulin and can be controlled sufficiently by diet alone.⁶ In recent years HPLC is developed as a sensitive tool to estimate and identify API or drug molecule.^7-8 Some method other then HPLC are also developed to validate and estimate glibenclamide.⁹ High performance Liquid Chromatograph (HPLC) analysis of sulfonamide intermediate of Glyburide drug saw an impurity content at 1.65 RRT with more than 0.1% level. Impurities are unexpected contents present in the Active Pharmaceuticals Ingredient (API) coming from normal synthesis process during manufacture, degradation on stability and trace (forced) degradation study.^10-12 Impurities have no beneficial importance and are actively and potentially harmful to the body. Therefore, they need to be controlled. As per the International Conference on Harmonisation of technical requirement for registration of pharmaceuticals for human use (ICH) guideline and regulatory bodies^13-15 from the world the threshold for impurity in drug products is ≤ 0.15% for known impurities and ≤ 0.10 for unknown impurities per maximum daily dose of drug product is ≤ 1gm and is ≤ 0.05% per maximum, daily dose of drug product is > 1gm. To meet these stringent regulatory requirements, the impurity profile study has to be carried out for final API to identify and characterize the impurity that is present at > 0.1%. This paper demonstrates the isolation and structure elucidation of impurity present in the sulfonamide intermediate of Glyburide API using preparative HPLC.^16-18

Figure 1 Chemical structure of Glyburide and Sulfonamide of Glyburide

MATERIALS AND METHODS:

Chemicals and reagents:

HPLC grade Acetonitrile, Methanol and Water were purchased from Merck (Mumbai, India). Crude sample of Isomeric impurity at 1.65 RRT for impurity isolation and Sulphonamide of Glyburide Reference standard were obtained as gratis sample from Cadila Pharmaceuticals Ltd. Research Laboratories (Ahmedabad, India).

High performance Liquid Chromatography (Conventional):

This impurity was analyzed by RP-HPLC for purity analysis. A Waters alliance quaternary e2695 chromatography system equipped with Waters 2998 PDA detector was used (Waters Corporation, USA). A Inertsil C8 (250 x 4.6)mm, 5µ particle size (GL Sciences Inc., Japan) was employed for separation. The mobile phase consisted of Water: Acetonitrile: Methanol in the ratio of 60:15:25 (v/v). The flow rate was set at 1 ml/min. Detection was carried out at 300nm. 10µL of 2 mg/mL concentration of sample in methanol was injected. The column oven temperature was at 25°C. Empower3 software was used to record data.

High performance Liquid Chromatography (Preparative):

A Waters quaternary 2535Q preparative chromatography system equipped with Waters 2998 PDA detector (Waters Corporation, USA), and Rheodyne Injector Model 7725i (IDEX Health & Science, USA) with 5.0ml fixed loop was used. A Luna C8 (250 x 21.2) mm, (Phenomenex, USA) was employed for separation. The mobile phase consisted of Water: Acetonitrile in the ratio of 70:30 (v/v). The flow rate was set at 8.0ml/min. Detection was carried out at 300nm. Empower3 software was used to record data.

NMR spectroscopy:

The 1H NMR¹⁹ spectra were recorded on Bruker 400 Ultra shield spectrometer. The 1H (400 MHz) was recorded using TMS and DMSO-d6 as internal standards and diluent, respectively. Topspin 3.1 was used to record data.

Mass spectrometry:

Mass spectrometer Waters Xevo TQD was used with ESI (72eV) and The ion spray voltage (V), Curtain energy (CE), entrance potential and declustering potential were kept as 3500V, 42V, 19V and 117V, respectively to record mass spectra. The sample was introduced to mass spectrometer with particle beam interface using LC. The source manifold and quadrupole temperatures were maintained at 230°C and 90°C respectively. Nitrogen was used as a reagent gas for chemical ionization (CI) mode.^20-22 The ESI mass spectra were recorded on Waters Xevo mass spectrometer with mass-lynx software.

FT-IR spectroscopy:

FT-IR^23-24 spectra were recorded on Shimadzu 8400 series FT-IR as KBr powder.

RESULT AND DISCUSSION:

Detection of Impurity:

During a conventional related substance RP-HPLC analysis of sulfonamide intermediate of Glyburide API, a consistence impurity at 1.65 RRT was recorded. This synthesized impurity shows itself impure which need to make pure by isolation process using preparative HPLC. This objective impurity under study was manifested as impurity of sulfonamide intermediate of Glyburide at 1.65 RRT. Analytical chromatogram of 1.65 RRT impurity of sulfonamide intermediate of Glyburide was recorded using related substance HPLC method.

Fig. 2. Conventional HPLC chromatogram of impurity of sulfonamide intermediate of Glyburide

Isolation of impurity by preparative HPLC:

A reverse phase isocratic solvent delivery system was developed (High performance Liquid Chromatography - Preparative) and used for the isolation of this crude impurity of sulfonamide intermediate of Glyburide that was mixture of two or more different content. All segments of impurity were collected separately, concentrated and solidify by evaporation of solvent using rotavapour. These isolated solids received from different fractions were then analyzed by related substance HPLC method to check its purity and to use for further analytical exercise for structural elucidation.

Structure elucidation:

The ESI mass spectra of 1.65 RRT impurity of sulfonamide intermediate of Glyburide was displayed the molecular ion peak at m/z 383.12. The ES⁺ mass spectra further confirmed this with the presence of protonated molecular ion peak as base peak at m/z 383.12 amu (i.e. M+H), which is equivalent mass unit 382.08 amu to that of N-methyl sulfonamide intermediate of Glyburide. This can be recognized as an impurity of parent substance sulfonamide intermediate of Glyburide. The IR spectra of this isolated impurity shows the presence of all wave number equal to sulfonamide intermediate of Glyburide a parent drug which also further confirms with 1H NMR spectra presented below.

Mass spectra:

Fig. 3. Mass spectra of 1.65 RRT impurity of sulfonamide intermediate of Glyburide

Mass Spectra
	1.65 RRT Impurity of Sulfonamide of Glyburide
Exact Mass	382.08
Molecular Ion (m/z)	383.12

1H NMR Spectra

Fig. 3. 1H NMR spectra of 1.65 RRT impurity of sulfonamide intermediate of Glyburide

Assignment of Proton by 1H NMR:

1H-NMR (DMSO-d6, 400 MHz): dH. (ppm): 2.369-2.400 (3H, d, CH3), 2.915-2.950 (2H, t, CH2), 3.526-3.576 (2H, q, CH2), 3.813 (3H, s, CH3), 7.145-7.167 (1H, d, CH aromatic), 7.414 (1H, s, NH), 7.484-7.520 (3H, m, CH aromatic), 7.613-7.631 (1H, m, CH aromatic), 7.713-7.734 (2H, m, CH aromatic), 8.275-8.303 (1H, t, NH).

Structure of 1.65 RRT impurity of sulfonamide intermediate of Glyburide

5-Chloro-2-methoxy-N-[2-(4-methylsulfamoyl-phenyl)-ethyl]-benzamide

Empirical Formula : C₁₇H₁₉ClN₂O₄S

Mol. Wt. : 382.86

Exact Mass : 382.08

CONCLUSION:

In conclusion, this process related impurity of sulfonamide intermediate of Glyburide formed during process of sulfonamide intermediate of Glyburide bulk drug, was isolated by preparative HPLC and characterized as N-methyl impurity of sulfonamide intermediate of Glyburide API. Structural elucidations of this content was carried out by using 1H NMR and Mass spectral data along with HPLC analysis. The regulatory requirement was therefore fulfilled by characterizing this impurity and prepared impurity standard. This impurity standard was further used for analytical method validation studies. This work also supported the process development optimization of sulfonamide stage of Glyburide and facilitated to control forming this impurity during the process.

ACKNOWLEDGEMENT:

The authors are very grateful to the Faculty of Science and Pharmacy for providing necessary facilities for the completion of research work.

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Received on 21.07.2020 Modified on 23.12.2020

Research J. Pharm.and Tech 2021; 14(12):6471-6474.

DOI: 10.52711/0974-360X.2021.01118