Impurity Identification and Characterization of some Anti-Diabetic Drugs using various Analytical Methods

 

Dhara Vashi1, Suresh Kumar2*

1Assistant Professor, Department of Pharmaceutical Quality Assurance, Shree Dhanvantary Pharmacy College, Kim, Surat-394110, Gujarat, India.

2Professor and HOD, Department of Pharmaceutical Quality Assurance, Shree Dhanvantary Pharmacy College, Nr. Railway Station, Kudsad Road, Kim (E), Surat-394110, Gujarat, India.

*Corresponding Author E-mail: jdrsuresh@gmail.com

 

ABSTRACT:

Various regulatory authorities such as the International Conference on Harmonization (ICH), The United States Food and Drug administration (FDA) are emphasizing on the purity requirements and the identification of impurities in Active Pharmaceutical Ingredients (APIs). Now a day research give great exposure to impurity profiling and forced degradation study of Active pharmaceutical ingredient and combined dosage form used for the treatment of Diabetic. Anti- Diabetic drugs class include: Sulfonylureas, Meglitinides, Thiazolidinediones, Alpha-glucosidases inhibitor, Dipeptidyl-peptidase-4 (DPP-4) inhibitors, Glucagon-like peptide-1 (GLP-1) agonist, Sodium glucose co-transporter 2 (SGLT2) inhibitors. As per ICH, Impurity profiling description of the identified and unidentified impurities present in a typical batch of API produced by a specific controlled production process. There are number of Spectroscopic method like UV(Ultraviolet Spectrometry), MS(Mass spectrometry), IR (infrared spectroscopy), NMR(Nuclear magnetic resonance) and Chromatographic methods like HPLC(High performance liquid chromatography), TLC(Thin layer Chromatography), HPTLC(High Performance thin layer chromatography), GC(Gas chromatography) used for identification, isolation and characterization of Impurities present in pharmaceuticals. Various hyphenated techniques are used for identification and better resolution between impurities having near to similar structure with each other or with APIs like Liquid Chromatography (LC)-Mass Spectroscopy (MS), LC-NMR, LC-NMR-MS, GC-MS, and LC-MS. This leads interest of impurity profiling in pharmaceuticals.

 

KEYWORDS: Anti-Diabetic Drugs, Impurity, Spectroscopic Methods, Chromatographic methods, Characterization.

 

 


 

INTRODUCTION:

Impurities[1-4] in pharmaceuticals are unwanted chemicals that remain with the Active Pharmaceutical Ingredients (APIs) or develop during formulation or develop upon ageing of both APIs and formulated APIs to medicines.

 

Classification according to ICH guidelines:

·       Organic Impurities (Process and drug-related)

·       Inorganic Impurities (Reagent, ligands, catalysts)

·       Residual Solvents (Volatile solvents)

 

1. Organic Impurities[5]:

These type of impurity arise during manufacturing process and/or storage of the drug substance. They are classified by such as,

 

·         Starting Materials or Intermediate Impurities: This type of impurity present in almost every type of API unless a proper care is taken in every step during the multistep synthesis of drug product.

 

·         By-products: During pre-defined controlled process of manufacturing of drug single end product with complete yield is very rare but always a chance of having by products along with desired end product.

 

·         Degradation Products: During improper storage of Bulk drug, there is a chance to degradation of drug and formed degraded product called Degradation product which act like Impurities in pharmaceutical formulation.

 

·         Synthesis Related Impurities: Any additional chemical entity generated during manufacturing process from raw material, solvent, intermediate, byproduct.

 

·         Formulation Related Impurities: During formulation, drugs are exposure to various stages and conditions. Alternation in pre-defined condition leads to degradation of components. Solutions and suspensions are prone to degradation due to hydrolysis.

 

2. Inorganic Impurities:

·         Reagent, Ligands and Catalysts: Rare chances of occurrence of these impurities. If during manufacturing procedure is not followed properly rare chances to occure.

 

·         Heavy Metals: Water is most widely used in different manufacturing processes which provide main source of heavy metals, like Ar, Cd, Cr, Na, Mg, Mn etc. Demineralized water and glass-lined reactors can be used to avoid heavy metals.

 

·         Other Materials (Filter Aids, Charcoal): Centrifuge bags are used on routine basis during manufacturing of Bulk drug in many industries. In some chemical reaction activated carbon is used is also responsible for entering Impurity during synthesis. Regular monitoring of fibers and black particles in the bulk drugs is essential to avoid Contamination.

 

3. Residual Solvents: Residual solvents are organic or inorganic liquids used during the manufacturing process and difficult to remove completely by the work-up process.

Impurities Present in Formulation[6]:

·         Numbers of impurities arise in product formulation due to varieties of conditions that can lead degradation or other deleterious reaction. In the formulation use of water can not only contribute its own impurities but it is also provide a ripe situation for the hydrolysis and catalysis.[6]

 

·         Impurity forms during formulation are :

 

1. Method related:

·         This is the impurity which is related from method. For e.g. Impurity is formed during the sterilization of the parental dosage form of Diclofenac sodium, Indolin-2-one I by Autoclave. Impurity is produced during formation of indolinone derivative and sodium hydroxide takes place due to the intramolecular cyclic reaction of the Diclofenac sodium.

 

2. Environmental related:

These factors are responsible to reduce the stability of the drug substance such as.

 

·         Exposure to adverse temperature: Heat sensitive drug substance produced adverse effect in presence of high temperature. For e.g. vitamins which undergo degradation in liquid formulation that cause the decrease in potency of vitamin.

 

·         Light especially UV light: Some Drug substance become unstable in presence of light. For e.g. Ergometrine and methyl ergometrine injection is unstable or shows the complete degradation when kept in the direct sunlight.

 

·         Humidity: It affect hygroscopic products, which are sensitive in the humid environment. It also affect the bulk powder and solid dosage form for e.g. Aspirin and Ranitidine.

 

3. Dosage form related impurities:

Sometimes the dosage form factors that influence the stability of the drug. In general, liquid dosage forms are much susceptible to both degradation and microbial contamination. In which, water content, pH of the solution, compatibility of the anions and cations, mutual interaction of the ingredients, and the primary containers are the critical factors for the impurities.

 

A)     Mutual interaction amongst ingredients.

Mutual interaction among the ingredient is also the major problem which cause the instability in the drug product. Most of the vitamins are very liable and become unstable on storage specially in liquid dosage form. For e.g. during the formulation, one formulation is in simple distilled water and other is in typical formulated vehicle that include disodium edetate and benzyl alcohol both have similar mutual interaction causing the degradation of the product.

 

B) Functional group- related typical degradation.

·         Ester hydrolysis: It is the reaction when the ester reacts with the water to produce ethanoic acid and ethanol.

 

·         Hydrolysis: It is a reaction which occurs due to water. Mainly this is a reaction of breaking bond in a molecule by using water. For e.g. sodium acetate is a salt which get hydrolyze by adding water and then separate in to the sodium ions and acetate ions.

 

·         Oxidative degradation: It is the degradation in which the cleavage of c=c with the introduction of new carbon and oxygen bond. For e.g. hydrocortisone, methotrexate, conjugated dienes, nitrite derivatives and aldehydes all are susceptible to the oxidative degradation.

 

·         Photolytic cleavage: it occurs due to the direct exposure of the sunlight. For e.g. in the preparation of ciprofloxacin eye drop, sunlight induce the photocleavage reaction producing ethylenediamine analog of ciprofloxacin.

 

·         Decarboxylation: In this case when we heat the p-aminosalicylic acid then it lose the carbon dioxide from the carboxyl group.

 

IMPURITY PROFILE:[7] It is description of the identified and unidentified impurities present in a typical batch of API produced by a specific controlled production process. Process for impurity profiling is given in Fig. 1.

 

Main Reason for identification and characterization of impurities are as below,

·         During manufacturing of new drug or new manufacturing method develop for existing drug, if chemist know about possible impurities present in initial or intermediate drug substance then can be easily reduce at acceptable level or removed by changing in reaction condition.

·         Impurities can be identified and characterized with spectroscopic methods.

·         Identified impurity can be synthesized and used as an ‘impurity standard’ during development of a selective method for the quantitative determination of the impurity in quality control testing of each batch.

·         If any major impurity is found, it is possible to become toxic or hazardous for future purpose. In this case impurities are synthesized and subjected to toxicological studies thus greatly contributing to the safety of drug therapy.

·         For drug authorities the impurity profile of a drug substance is a good fingerprint to indicate the level and constancy of the manufacturing process of the bulk drug substance.

 

Identification and Characterizatiojn of Impurities[8]

The impurities can be identified by the following methods.

1.       Reference standard method: This technique use for identification and detection of active ingredient in dosage form but also for the impurities, degradation product, starting materials, and excipients. In this method reference standard is prepared for use as the standard in an assay, identification, or purity test which can use to evaluate both the process and product performance.

 

2.       Spectroscopic method: Following methods are used for Characterization of Impurities:

 

·         Ultraviolet spectrometry: It is used UV region 200-400 in Electromagnetic spectrum. Individual impurity shows Characteristic absorption of light at specific wavelength, Which is differ then original drug substance.

 

·         Infrared spectroscopy: It is use for solid, liquid or gaseous samples to identify Drug substance and detection of impurities by matching with reference spectra. In this technique samples are not destroy during identification process.

 

·         Mass spectrometers: in this technique, m/e ratio is measure and according to mass abundant impurity can be identified and quantified. Hyphenated technique like GC-MS and LC-MS can use for identifaction with better resolution of impurity with structurally similar substance.

 

GC-MS: Mass spectroscopy able to give detailed structural information but can not be separate with high resolution. It can be overcome gas chromatography which can separate volatile & semi-volatile compound with enough resolution. Vaporized sample injected in to column by heated system which passed through column by inert gaseous mobile phase and detected. So by Combining two method i.e Gas Chromatography and Mass Spectrometry (GC-MS) can identify different substances within a test sample. The sample is transported through the column by the flow of an inert, gaseous mobile phase, the carrier gas. Flow is regulated by the pressure regulators and gas metering valves.

 

 

LC/MS: It is a hyphenated technique, combining the separation power of HPLC, with the detection power of mass spectrometry. LC/MS became really popular with the introduction of the thermo spray interface and the particle beam interface. This is same as GC-MS but removal of liquid carrier from an HPLC eluent before samples are passed in to the MS source. For handle normal eluent flow rate 0.5-2.0 ml/min which is not handled by MS pumping system moving belt inlet systems, jet separators and vacuum nebulizers are used.

 

·         NMR Spectroscopy: by this technique, sample can be determine at its atomic and nuclear level. It is very sensitive in nature.

 

·         Raman spectroscopy: It is used to study vibrational, rotational and other low frequency modes in a system. It have good sensitivity and detect the process related impurities.

 


METHODOLOGY:



Figure 1: Steps for identification and characterization of impurities

 


Separation Methods of Impurity[9]:

The following separation methods are as follows.

 

·         Thin-layer chromatography: In this chromatography the glass, plastic, and aluminum foil which are coated with adsorbent material such as silica gel, aluminum oxide and cellulose is used as stationary phase. Solvent system used as mobile phase which is travel up by capillary action to separate the mixtures and which after the sample has been applied on the plate then a solvent mixture is drawn up by the capillary action. Different analyte have different affinity towards stationary phase and mobile phase according to affinity components are get separated.

 

·         Gas chromatography: In this chromatography sample are used in gaseous form for separation and quantification. This technique is used in combination with other chromatography technique for better result i.e Gas-Liquid chromatography and Gas –Mass chromatography. Most of the time gas chromatography is used for the testing of purity or separating the different components of the mixtures

 

·         High-pressure liquid chromatography: It is a column chromatography used to separate, identify, and quantify the compounds. HPLC have different type’s stationary phases and a pump that moves the mobile phase and also there is a detector to provide the characteristic information about the compound such as API and its impurities. It is helpful to check the quality of the API starting material and also signify the unknown impurities.

 

·         Capillary electrophoresis: It is also known as capillary zone electrophoresis. It is used to separate the ionic species by their charge and size in the small capillary filled inside with an electrolyte. It is based on the different separation principles and also used for the quality control of the pharmaceutical products.

 

·         Supercritical fluid chromatography (SFC): It is the chromatography in which we separate one component from other component by using the super critical fluid. Carbon dioxide is used as a supercritical fluid where ethanol or methanol used as a co-solvent. In this we provide the critical temperature of 31oc with critical pressure of 72 bars.

 

Isolation of Impurities:

Generally chromatographic and non-chromatographic techniques are used for the isolation of the impurities such as.

 

·         Solid-phase extraction method: It is the method which is used to trace the organic compound as well as remove the interfering compound to obtain a clear extract. Mainly this technique is used for the extraction and purification of the compounds. Main use of this method is to clean up the sample before use for the chromatographic technique to quantify the analyte in the sample. This technique is widely applied for the isolation of analytes from a liquid matrix.

 

·         Liquid-liquid extraction method: It is also known as solvent extraction and partitioning method. In this method compounds gets separated according to their relative solubility in two different immiscible liquids usually water and organic solvents. The method is perform in the separating funnel. Commonly solvents used for liquid-liquid extraction are ethyl acetate, methylene chloride and hexanes.

 

·         Accelerated solvent extraction method: It is the better technique used for the extraction of solid and semi-solid samples. All the process done at the elevated temperature and pressure to get the fast and efficient removal of analysts from the samples. It perform the experiment in less time with using smaller quantity of solvent.

 

Table 1: ICH guidelines for Impurities in new drug substance Q3A(R2)[10]

Maximum Daily Dose

Reporting Threshold

Identification Threshold

Qualification Threshold

≤ 2g/day

0.05%

0.10% or 1.0 mg per day intake (whichever is lower)

0.15% or 1.0 mg per day intake (whichever is lower)

> 2g/day

0.03%

0.05%

0.05%

 

Table 2: ICH guidelines for Impurities in new drug Product Q3B(R2)[11-12]

Maximum Daily Dose

Threshold

≤ 1 g

0.1%

> 1 g

0.05%

Identification Thresholds

Maximum Daily Dose

Threshold

< 1 mg

1.0% or 5 µg Total daily intake (TDI), whichever is lower

1 mg - 10 mg

0.5% or 20 µg TDI, whichever is lower

>10 mg - 2 g

0.2% or 2 mg TDI, whichever is lower

> 2 g

0.10%

Qualification Thresholds

Maximum Daily Dose

Threshold

< 10 mg

1.0% or 50 µg TDI, whichever is lower

10 mg - 100 mg

0.5% or 200 µg TDI, whichever is lower

>100 mg - 2 g

0.2% or 3 mg TDI, whichever is lower

> 2 g

0.15%

 

 


 

Table 3: Experimental examples of identification and characterization of some anti-diabetic drugs.

Sr. No.

Title

Method

Impurities Description

1

Seven novel impurities of anti-diabetic drug Repaglinide[13]

UPLC

Column: C8

Mobile Phase:

Gradient program :

Solvent A: 0.05% of Formic acid in water

Solvent B : 0.05% Formic acid in Acetonitrile

Detection Wavelength: 210 nm

Flow Rate: 1 ml/min

Method of Characterization: : IR, 1H NMR,13 C NMR, MS, Crystal X-ray diffraction

Retention Time:

Impurity 8: 2.20 min

Impurity 9: 4.06 min

Impurity 10: 5.21 min

Impurity 11: 5.36 min

Impurity 13: 6.67 min

Impurity 14 : 7.00 min

Impurity 16: 7.55 min

2

Miglitol and its impurities determine by RP-HPLC and Characterization using Mass Spectrometry Techniques[14]

Method: RP- HPLC

Column: prevail carbohydrate ES column (250 x4.6 mm, 5 µm particle size)

Mobile Phase:

Gradient program :

Solvent A: 10 mM Dipotassium hydrogen orthophosphate Solvent B: Acetonitrile

(pH – 8.0)

Detection Wavelength: 210 nm

Flow Rate : 1 ml/min

Method of Characterization: LC/MS

Retention Time:

Miglitol: 24.070 min

Impurity A : 17.037 min

Impurity B : 18.776 min

Impurity C : 19.480 min

Impurity D : 20.604 min

Impurity E: 21.170 min

Impurity F : 26.621 min

3

Identification and characterization of saxagliptin degradation related impurities [15]

Method: HPLC

Column: C 18

Mobile Phase:

 Gradient program :

Solvent A :Water

Solvent B: Acetonitrile

Detection Wavelength: 213 nm

Flow Rate : 1 ml/min

Method of Characterization: MS

Degradation related impurities (DRI) :

DRI 1 (Acid Hydrolysis, HCl 1N, 60°C for 6 hours )

DRI 2 (Acid Hydrolysis, HCl 1N, 60°C for 6 hours )

DRI 3 (Base Hydrolysis, NaOH 1N, 60°C)

4

Potential process-related impurity and its degradation product in Vildagliptin[15]

UHPLC

Column: C 18

Mobile Phase:

 Solvent A :10 mM Sodium dihydrogen phosphate monohydrate

(pH :7.0)

Solvent B: Solvent A + Acetonitrile , (7:3 v/v).

Detection Wavelength: 210 nm

Flow Rate: 1 ml/min

Method of Characterization: IR, 1H NMR,

13C NMR, MS

Retention Time:

Vildagliptin : 18.55 min

Impurity E : 21.92 min

Impurity F : 6.01 - 6.69 min

5

Alogliptin Benzoate and its potential impuritiesn in bulk drug and tablets[16]

Method: RP-HPLC

Column: C18

Mobile Phase:

Solvent A: Water/Acetonitrile/Trifluoro-acetic acid (1900:100:1 v/v/v)

Solvent B:

Acetonitrile/Water/Trifluoro-acetic acid (1900:100:1 v/v/v) Detection Wavelength: 278 nm

Flow Rate: 1 ml/min

Method of Characterization: 1H-NMR ,ESI-MS

Impurity A

Impurity B

Impurity C

Impurity D

Impurity E

Impurity F

Impurity G

6

Impurity profile study of Repaglinide[17]

HPLC

Column: C18

Mobile Phase:

0.01 M Pottasium dihydrigen phosphate : Acetonitrile

50:50 (v/v) (pH 3.5)

Detection Wavelength: 200 nm

Flow Rate : 1 ml/min

Method of Characterization: IR, 1H NMR

13 C NMR, MS

Retention Time:

Repaglinide: 24 - 26 min

Impurity 1: 10 – 12 min

Impurity 2: 13 – 15 min

Impurity 3: 17 – 19 min

Impurity 4: 30-32 min

 


REFERENCES:

1.        S. Ahuja, K. M. Alsante. Handbook of Isolation and Characterization of Impurities in Pharmaceuticals, Vol. 5, Separation Science and Technology, Academic press, 2003.

2.        S. Ahuja. Impurities Evaluation of Pharmaceuticals, Marcel Dekker, Inc. New York, 2006.

3.        S. Ahuja, S. Scypinski. Handbook of Modern Pharmaceutical Analysis, Vol. 3, Separation Science and Technology, Academic press, 2003

4.        J. Roy. Pharmaceutical Impurities–a mini review, AAPS PharmSciTech 3(2): 1-8 (2002). ICH Harmonized Triplicate Guideline: Impurities in New Drug Substances Q3A (R2), ICH Steering Committee, Step 4 of ICH process, 25th Oct. 2006.

5.        Venkatesan P. Impurity profiling: Theory and practice. Journal of pharmaceutical science & Reaserch. 2014; 6(7): 254-259.

6.        Pawale S. Impurity Profile in Bulk Drugs and Pharmaceutical Preparation; International Journal of Pharmaceutical and Chemical Sciences. 2012; 1(4): 1227-1237.

7.        Tegeli S. Significance of Impurity Profiling; International Journal of Drug Formulation and Research, 2011; 2: 174-194.

8.        Bari S. Impurity profile significance in Active Pharmaceutical Ingredient; Eurasian journal of Analytical Chemistry.2007; 2: 32-53.

9.        “Impurities in new drug products Q3A(R2),” October 2006, www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3A_R2/Step4/Q3A_R2 Guideline.pdf

10.      “Impurities in new drug products Q3B(R2),” June 2006, www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3B_R2/Step4/Q3B_R2_Guideline.pdf

11.      ICH Harmonized Triplicate Guideline: Impurities in New Drug Products Q3B (R2), ICH Steering Committee, Step 4 of ICH process, 2nd June 2006. 6. ICH Harmonized Triplicate Guideline: Guideline for Residual Solvents Q3C (R3), ICH Steering Committee, Step 4 of ICH process, Nov 2005.

12.      Prasad K, Srinivas K and Pallavi A,” Identification, isolation, and synthesis of seven novel impurities of anti-diabetic drug Repaglinide. Drug Testing and Analysis. 2017;10(1):212-221.

13.      Balakumaran K, Janagili M, Rajana N, and Papureddy S. Development and validation of Miglitol and its impurities by RP-HPLC and characterization using mass spectrometry techniques. Scientia Pharmaceutica. 2016; 84(4): 654-670.

14.      Farooqui FI and Kakde R. Reversed-phase liquid chromatography with mass detection and characterization of Saxagliptin degradation related impurities. Journal of Chemical and Pharmaceutical Reaserch. 2016; 8(7):509-514.

15.      Neeraj K, Rao DS, Singh G, and Kadirappa A, Identification, isolation and characterization of potential process-related impurity and its degradation product in Vildagliptin. Journal of Pharmaceutical and Biomedical Analysis. 2016; 119: 114-121.

16.      Zhang K, Panqin M, Jing W and Zhang X, A developed HPLC method for the determination of Alogliptin Benzoate and its potential impurities in bulk drug and tablets.” Asian Journal of Pharmaceutical Science. 2015; 10(2): 152-158.

17.      Reddy K, Babu JM, Mathad VT, Reddy MS, Dubey PK and Vyas K. Impurity profile study of Repaglinide Journal of Pharmaceutical and Biomedical Analysis. 2003; 32(3): 461-467.

 

 

 

 

 

Received on 12.07.2019         Accepted on 14.08.2019

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2019; 9(4):243-248.

DOI: 10.5958/2231-5691.2019.00039.X