Isolation and Characterisation of Nateglinide and its impurity in Bulk and Marketed Formulation by HPTLC Method

 

Patil Pallavi M¹*, Mayur Tekade², Samiksha Agarkar³, Mohamad Taleuzzaman⁴

^1,2,3P.E. Society’s Modern College of Pharmacy, Yamunanagar, Nigdi,

Pune-411044  Maharashtra, India, 411044.

⁴Maulana Azad University, Village Bujhawar, Tehsil Luni Jodhpur 342802.

 

ABSTRACT:

The samples were applied on to the plate in the form of band with band length of 5mm. The mobile phase consisted of Chloroform: Methanol in the ratio of 8:2 v/v and 10ml of the mobile phase was used in each chromatographic run. Ascending development technique was carried out in a twin trough chamber saturated with mobile phase vapours for 15 min. The multiple wavelength detector was set at 216 nm, and quantification of the analyte was based on measuring its peak area. Rf for NTG was about 0.59. Calibration curve of NTG was linear in the range 100–600µg/band with correlation coefficient >0.9994. The drug was subjected to forced-degradation conditions of reaction, oxidization and dry heat. Unknown impurity was found in Nateglinide industrial batch stability condition at levels more than 0.1% in HPTLC analysis was characterized preliminarily by ESI-MS/MS studies. The major unknown (unknown-1 and 2) were enriched and isolated by preparative LC and structure was evidenced by 1H NMR spectroscopy, mass spectrometry and FT-IR. This technique will be used for the standard management of each drug substance and drug product. Finally, the projected method made use of hyphenated techniques as a tool for peak identity and purity confirmation.

 

 

 

INTRODUCTION: 

Nateglinideis an amino-acid derivative which reduces the blood glucose levels and stimulates insulin secretion. Chemically, it is formulated as (-N-[(trans-4-isopropylcyclohexyl) carbonyl-D-phenylalanine) salt and is used in the treatment of type two diabetes mellitus (USP, 2010).  Chronic hyperglycemia is an established risk factor for the micro- and macrovascular complications associated with T2DM and especially for cardiovascular disease-the major cause of morbidity and mortality in subjects with diabetes¹. It is a well-known fact that drugs undergo physicochemical degradation upon storage.

 

Thus, stability testing of a drug below numerous temperature and humidness conditions is important throughout the drug development method.In addition, stability testing guidelines issued by International Conference on Harmonization (ICH) and other international agencies ICH2003; WHO 2007; CPMP 2002; TPD 2003 require the reporting, identification and characterization of degradation products (DPs). But, as DPs generated during storage may be in very low levels (∼0.1–0.5%, w/w), stress studies are suggested to generate them in higher amounts². Still sometimes it is very difficult to isolate these species from the stressed mixture due to their low amounts and subject them to spectral analyses for structural information. Therefore, hyphenated techniques like LC–MS and LC–NMR are currently extensively used for this purpose.  Diabetes mellitus is difficult to control with a single oral hypoglycaemic agent and the rate of mono therapy failure is high. Hence combination medical aid with complementary categories of medication that act on completely different aspects of glycemic management would be expected to be an efficient strategy for the management of polygenic disease. Nateglinide is an amino-acid derivative which lowers the blood glucose levels and stimulates insulin secretion. Chemically, it is formulated as(2R)-2-({[trans-4-(1-methylethyl) cyclohexyl] carbonyl} amino)-3-phenylpropanoic acid and is used in the treatment of type two diabetes mellitus USP, 2010. Although it's been within the marketplace for over ten years, solely scant data exists on stability and degradation behaviour of the drug. An investigation was carried Validated assays have been reported for each drug individually. For analysis of Nateglinide from human plasma, high performance liquid chromatographical analysis with ultraviolet illumination detection coumarin- sort fluorescent detection^3-7. spectrophotometric techniques have also been reported for determination of Nateglinide in bulk and tablet dosage forms^8-9 High performance liquid chromatography for metformin individually in human plasma¹⁰ and in combination with other drugs in plasma and pharmaceutical dosage form¹¹

 

As there is no comprehensive report on degradation chemistry of the drug, therefore, the endeavour of the present study was to: (i) Development of a HPTLC method for the separation and determination of NTG potential related impurities i.e. raw materials, by-products and impurities of mentioned synthetic procedure in  Fig. 4(namely Impurities-1-2, and validation as per ICH guidelines¹² and; (ii) (ii) Enhancement and  isolation of major unknown-1 (impurity-1and 2) using preparative LC and characterization by 1H, MS studies and FT-IR. Proposing unknown-2 as secondary degradation impurity based on forced degradation study and MS data.

 

(iii). Many methods have been described for the quantitative determination of Nitaglinate in biological samples   including^13-18

 

MATERIALS AND METHODS:

Chemicals and reagents:

All chemicals and reagents were used analytical grade. Methanol, chloroform and hydrochloric acid (HCL), hydrogen peroxide (H2O2), sodium hydroxide (NaOH) was purchased from Merck Ltd (Mumbai, India). 100% purity pharmaceutical grade NTG obtained from Unicorn pharmachem Pvt Ltd (Mumbai, India). Pharmaceutical formulation, STARLIX® tablets (containing 120mg NTG per tablet) were obtained from Indian market.

 

Instrumentation and chromatographic conditions:

The following criteria are taken into account: data resolution: 100mm/step; band width: 5mm; result output: chromatogram and integrated peak area; slit dimensions: 5×0.45mm; scanning speed: 20mm/s; spraying rate: 10µl/s; data resolution: 100µm/step; band width: 5mm; saturation time is 10 minnute at the 25± 5⁰c; migration distance upto 80mm result output: chromatogram and integrated peak area 0.25mm silica gel 60 F254 was deposited on HPTLC aluminium plates (10x10cm) (Merck, Germany) at the wavelength of  216 nm and retention factor of NTG is 0.59±0.05.

 

Standard solutions preparation:

The stock solution of NTG 1000µg/ml was organized by dissolving 100mg of NTG in (1:1) methanol to make 100mL of solution. The standard working solutions were prepared by diluting aliquots of the stock solution to obtain concentrations ranging from 100–600µg/band. The calibration graph was made by plotting the peak areas obtained at a wavelength of 216nm versus the corresponding applied concentrations.

 

Sample preparation:

The contents of 10 tablets of STARLIX^® 120mg were accurately weighed fine powder. To an accurately weighed portion of the powder equivalent to one tablet, 120mL (1:1) methanol was added then the solution was left in the ultrasonic bath for 10 -20 mins. After that the solution was filtered and the first 10uL was applied then 2mL of filtrate was diluted to 100mL using same solvent µ.

 

Stock solution of degredant:

Accurately weighed quantity (25mg) of degradant was transferred to 25.0mL volumetric flask, dissolved and diluted up to the mark with water. From this solution, 5.0mL was transferred to 50.0mL volumetric flask and diluted to the mark with water (Concentration 100 mg/mL). The solution was mixed and filtered through 0.2µ membrane filter. Results are shown in Figure. 2 and 3.

 

Method validation:

The proposed method's validation was carried out in accordance with the International Conference on Harmonization (ICH) guidelines.¹⁹

 

Table 1: Analytical parameters for the determination of NTG using the proposed HPTLC   method.

Parameter	NTG	Impurity 1	Impurity 2
Wavelegth	216	220	230
Linearity	100-600 µg/band	100-600 µg/band	100-600µg/band
Correlation Coefficient	0.9994	0.9984	0.9941
LOD	11.77 (ng/band)	4.17 (ng/band)	0.28 (ng/band)
LOQ	16.26 (ng/band)	6.21 (ng/band)	1.81 (ng/band)
Precision Inter day	99.25 % Label  claim* 0.2501 S.D. (±) 0.2519 R.S.D.	99.16 %Label claim* 0.2631 S.D. (±)0.2519 R.S.D.	98.12 %Label     claim* 0.5421 S.D. (±)0.4539 R.S.D.
Intra day	98.96  25 %Label  claim* 0.7477S.D. (±)0.7556R.S.D.	99.14 25% Label     claim*0.7333S.D. (±)0.7446R.S.D.	98.07   25 %Label claim* 0.6437 S.D. (±) 0.5636 R.S.D.

 

Figure 1: Pathway of Nateglinide alkaline degradation.

 

 

Degradation pathway for unknown-1 and 2

During the experiment enhancement of unknown impurities, it was observed that intended addition of alkaline stress of Nateglinide gives rise of unknown-1 and 2 impurities (Figure 1). Constructed on stress study and massspectrum fragmentation data the degradation pathway for unknown-1and 2 were proposed.

 

 

Figure 2: typical standard chromatogram of Nateglinide  andImpuriy 1

 

 

Figure 3: typical standard chromatogram of Nateglinide andImpuriy 2

Enhancement of unknown-1 impurity:

Degradation in controlled conditions (i.e., more than 10%) gives a conclusion that unknown-1 and 2 were cumulative in alkaline stress. Based on mass spectral data and degradation studies exertions were made to enrich the impurity using extensive degradation with alkaline condition. The unknown-1 and 2 impurities increased more than 10% in 2 M NaOH stress at 80^oC for 3 hrs and about 87% when extending the stress time to 12h. The stress solutions were injected into MS analysis to generate unknown impurities (unknown-1and 2) mass spectral data and these were matching with unknown impurity mass spectrums of bulk drug stability sample.

 

Isolation of unknown-1, 2 impurity using preparative LC:

In directive to isolate the unknown-1, 2 impurities from alkaline stress of NTG, the isolation conditions for preparative LC used were as mentioned in Section 2.5. Approximately 10mg of sample was applied on plate every time and was continued until the sufficient amounts of unknown-1and 2 were isolated (20 applications). The fractions containing >95% of unknown-1and 2 were collected, concentrated using centrifuged at 4000rpm and then evaporator at 40 ⁰ C under vacuum and finally lyophilized to obtain solid product.

 

Structural elucidation of unknown-1:

In the positive mode MS analysis of the unknown-1 and 2 revealed a protonated molecular ion peak m/z 345.4. The transformation of mass between impurity and NTG is 1 amu, representing deletion and addition on NTG. During degradation studies this was major impurity observed in methanol hydrolysis of NTG. The imine group (-C‚NH) on NTG hydrolysis to carbonyl (-C‚+2 H) leads to increase of 1 amu mass for impurity. This was evidenced by major product ions observed from MS data of NTG m/z 283.15,325.20,456.20 and 306 and of unknown-1(impurity-1 and 2)were 359.2,559.2 and at m/z and impurity 2 325.2, 440.2 and 569.2. The 1H NMR and FT-IR spectral data of NTG and unknown-1 impurity are given in respectively. 1H NMR spectral data of unknown-1 and 2 were showed chemical shift similarity with NTG in all positions except the signals corresponding to A NH in NTG was absent in impurity. From this spectral information the structure was confirmed and shown in Table. 2 as N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine-ethyl ester and N-[{4-(1-methylethyl) phenyl} carbonyl]-D-phenylalanine.

 

Table 2: FT-IR, MS data and NMR of Unknown 1 and Unknown 2

Sr. No	Compound	IR	MS	NMR
1.	Unlnown -1	C-H (strch) 2939.30, C-O-C (strch) 1190.92, NH (def) 1450.36, C=O 1643.23, C-O 1195.78 and O-H 3355.89	m/z Measured EI-MS in Acidic	1H NMR δ 1.35, 13C NMR signal at δ  9.81, NH δ 8.10 and OH δ 7.86
			440.22 m/z, 559.26 m/z
2.	Unlnown -2	C-H (strch) 2877.58, C-O-C (strch) 1458.08, NH (def) 1542.94, C=O 1735.81, C-O 1249.78 and O-H 3741.63	m/z Measured EI-MS in Acidic	1H NMR δ 4.14, 13C NMR signal at δ 44.34 , NH δ δ 8.10and OH δ 5.83
			325.20 m/z, 283.15m/z

 

CONCLUSION:

A HPTLC method was validated for quantitative determination of impurities of NTG drug substance. Effortlessness was illustrated by the minimum requirement of organic solvents since methanol was used as a solvent for the working and sample solutions. This suggests that the proposed method is cost-effective and environment-friendly. To our present knowledge, there is no attempts have been made yet to develop a comprehensive procedure with impurity identified for NTG in bulk form and in pharmaceutical formulations by any analytical methodology. Finally, the proposed method was successfully applied to study the basic degradation processes of NTG. The proposed method ensured a precise and accurate determination of NTG in oral tablet formulation. This study was found impurities 1 and 2 and their characterization. No interference from the excipients was noticed by HPTLC method using   isolation of impurities by LC preparative plate’s method.

 

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Accepted on 19.12.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(5):2343-2346.