INTRODUCTION:

Today, the pharmaceutical business is an important part of the global economy. The industry has had and will continue to have a significant impact on people's lives. The R&D sector is one of the most important areas of attention for pharmaceutical companies in order to discover new medication compounds. A new medicine molecule costs $1 billion and takes 10 to 15 years to produce.¹

A suitable analytical method can determine the purity of the drug product based on the percentage of the API label contained in it. If the efficacy of certain impurities is equal to or higher than the efficacy of the bulk drug itself, they may not have a negative impact on the quality of the drug. On the other hand, drug substances can be regarded as relapses.

As a result, the purity of the API must be tested independently of these undesired foreign elements in order to ensure that the correct amount of API is administered to the patient (such as inert, toxic or pharmacologically superior impurities).

Organic and inorganic impurities, as well as residual solvents, are detected, identified/structured, and quantitatively determined in APIs and pharmaceutical formulations using impurity analysis. Various pharmacopoeias, such as the British Pharmacopoeia (BP) and the United States Pharmacopoeia (USP), are gradually regulating the use of all pharmaceuticals.³

Designation of Impurities:

A. Common terms of impurities:^4,5

Impurities have been named by various regulatory bodies and ICH such as:

i. By products

ii. Degradation products

iii. Interaction products

iv. Intermediates

v. Transformation products

vi. Related products

i. By products:

The molecules created in the process other than the requisite intermediates. They can happen as a result of a variety of adverse effects, such as overreaction, incomplete reaction, undesired reaction, demonization, and rearrangement interactions of starting elements or intermediates with each other catalysts or chemical reagents.

ii. Degradation products:

They are formed when an active ingredient or other material of interest decomposes under the influence of external factors such as heat, light, and moisture.

iii. Interaction products:

These products resulted from intentional or unintentional chemical interactions.

iv. Intermediates:

The compounds generated during the synthesis of a desired material or as part of the synthesis route.

v. Transformation products:

They have something to do with theorised and non-theorized products that may appear in their action. They're similar to by-products, but there's more information about reaction products.

vi. Related products:

These have a chemical structure that is similar to that of a drug substance and may even have biological activity.

B. Commonly discovered impurities in medicinal preparations:³

· Activity depressing Impurities

· Because of colouring or flavouring agents, such as Sodium Salicylate.

· Humidity

· Reduce the shelf life of the product.

· Physical and chemical characteristics

· Impurities are impurities that cause substances to become incompatible.

C. Classification of impurities ^1,2,6

Impurities are classified into three types as below:

I. Organic impurities

II. Inorganic impurities

III. Residual solvents

I. Organic impurities:

Organic contaminants may develop during the production and/or storage of a novel medicinal ingredient. They can be identifiable or unknown, volatile or non-volatile, and include the following:

Fig 1: Classification of Organic Impurity

II. Inorganic impurities:

The manufacturing process can produce inorganic contaminants. They are usually well-known and identifiable, and include the following:

Fig 2: Classification of Inorganic Impurity

III. Solvents:

In the synthesis of a new drug substance, solvents are inorganic or organic liquids that are used as vehicles to prepare solutions or suspensions. Because their toxicity is well-known.

Residual solvent:

These are undesirable substances. They may alter the properties of certain compounds or pose a health risk to humans. These solvents also have an impact on the bulk drug's physicochemical properties, such as crystallinity.

According to ICH guidelines, solvents are divided into three categories:

a. Class 1 solvents:

Because of their unacceptable toxicity or detrimental effects, these solvents are not used in the manufacturing of pharmacological compounds, excipients, or formulations. Example: Benzene, carbon tetrachloride, Dichloromethane, etc Table.1.

b. Class 2 solvents:

These solvents are only used in a limited number of pharmaceutical goods, Because of their inherent toxicity. Example: acetonitrile, chlorobenzene, chloroform, etc. Table.2.

c. Class 3 solvents:

These are the ones that are less toxic and have a lower risk of harming human health. There are no serious risks associated with these solvents. Example: Acetone, Acetic acid, Heptanes.

Table 1: Class 1 Solvents to be avoided in pharmaceutical products.⁶

Solvent	Concentration limit(ppm)	Concern
Benzene	2	Carcinogen
CCl₄	4	Toxic and environmental hazard
1,2-Dichloroethane	5	Toxic
1,1-Dichloroethane	8	Toxic
1,1,1-Trichloroethane	1500	Environmental hazard

Table 2: Class 2 solvents to be limited in pharmaceutical products.⁶

Solvent	PDE (mg/day)	Concentration limit(ppm)
Acetonitrile	4.1	410
Chloroform	0.6	60
Cyclohexane	38.8	3880
Ethylene glycol	6.2	620
Formamide	2.2	220
Hexane	2.9	290
Methanol	30.0	3000

D. Regulatory guidelines on impurities in an active pharmaceutical ingredient^7-10

Impurities in drug products must be monitored for ethical, economic, and competitive reasons, as well as for safety and efficacy. Monitoring and controlling impurities, on the other hand, can mean different things to different people, or even the same people at different times. Even those in the pharmaceutical sciences and related fields have struggled at times industry. To ensure that everyone is on the same page, a common vocabulary is required. To ensure that everyone uses the same terminology when discussing impurities, a unified terminology is required .The guidance prepared under the guidance of the International Conference on Harmonization (ICH) has been endorsed by the United States Food and Drug Administration (US FDA).The ICH guideline for impurities in pharmaceuticals was developed with the help of regulators and industry representatives from the European Union (EU), Japan, and the United States, and it has helped to ensure that data submitted to various regulatory agencies is consistent across regions. The deadlines not only help sponsors of New Drug Applications (NDA) or Abbreviated New Drug Applications (ANDA) understand what information should be included in their applications, but they also help FDA reviewers and field investigators interpret and apply regulations in a consistent manner1-2.

The following are the various regulatory guidelines for impurities:

1. ICH guidelines “stability testing of new drugsubstances and products"- Q1A

2. ICH guidelines “Impurities in New Drug Substances”- Q3A

3. ICH guidelines “Impurities in New Drug Products”-Q3B

4. ICH guidelines “Impurities: Guidelines for residual solvents”- Q3C

5. US-FDA guidelines “NDAs -Impurities in New Drug Substances”

6. US-FDA guidelines “ANDAs – Impurities in New Drug Substances”

7. Australian regulatory guideline for prescriptionmedicines, Therapeutic Governance Authority (TGA), Australia

E. Analytical Method Development:

At various stages of the development of a new medicine, meaningful and trustworthy analytical data is required.⁵

a) Sample set selection for analytical method development.

b) Typically, chromatographic conditions and phases are screened. gradient elution using the linear solvent-strength model.

c) Method optimization to fine-tune factors linked to

ruggedness and robustness.

The following approaches can be used to identify impurities:⁷

1. Reference standard method

2. Spectroscopic method

3. Separation method

4. Isolation method

5. Characterization method

1. Reference standard method:⁶

It's the procedure for preparing a reference standard for use as a standard in an assay, identification, or purity test. We may assess both the process and the product's performance using this way. It includes information on contaminants, degradation products, starting materials, and excipients, as well as the active ingredient in dosage form.

2. Spectroscopic method:^6,11

Impurities are frequently identified using UV, IR, MS, NMR, and Raman spectroscopy approaches.

a) Ultraviolet spectrometry:

This is a spectroscopy technique that makes use of light. It is used to determine the absorber concentration in a solution. We can determine the impurity by observing quick changes in absorbance.

b) Infrared spectroscopy:

It is a technique for identifying samples, performing quantitative analysis, and detecting contaminants in the laboratory. It can be used on solid, liquid, or gaseous samples without destroying them in the process.

c) Mass spectrometers:

These instruments provide precise mass measurements, sample identification, and sample quantitation. It works with both GC-MS and LC-MS systems. It combines gas and liquid chromatography features.

d) NMR Spectroscopy:

This is a highly sophisticated system that employs nuclear magnetic resonance technology. It is used to determine the sample's atomic and molecular properties. It has a delicate nature.

e) Raman spectroscopy:

It is a technique for studying low-frequency vibrational, rotational, and other modes in a system. It has a good sensitivity and can detect impurities that are related to the process.

3. Separation method:¹²

Chromatographic techniques such as TLC, HPTLC, HPLC, Gas Chromatography (GC), Supercritical Fluid Chromatography (SFC), Electrophoresis techniques such as Capillary electrophoresis, Gel permeation chromatography, and others are used in the separation method.

a) Thin-layer chromatography:^6,11

It is a type of chromatography that is used to separate mixtures. It's done on glass, plastic, and aluminium foil sheets that have been coated with adsorbent materials like silica gel, aluminium oxide, and cellulose. The capillary action draws up a solvent mixture after the sample has been applied to the plate. After a while, the mixture separates.

b) High pressure Liquid chromatography:

It is a type of column chromatography that is used to separate, identify, and quantify substances. HPLC has several types of stationary phases, a pump that moves the mobile phase, and a detector that provides information about the compound, such as API and impurities.

c) Gas chromatography:

It is a type of chromatography that is commonly used to separate and analyse compounds. There are two types of chromatography: Gas-Liquid chromatography and Gas chromatography. The majority of the time, gas chromatography is used to test purity or separate the various components of a mixture. It is beneficial in the preparation of pure compounds from mixtures.

d) Supercritical fluid chromatography (SFC):

It is a type of chromatography in which a supercritical fluid is used to separate one component from another. As a supercritical fluid, carbon dioxide is used, with ethanol or methanol as a co-solvent. We use a critical temperature of 31°C and a critical pressure of 72 bars in this example.

e) Capillary electrophoresis:

It also known as capillary zone electrophoresis, is a type of electrophoresis that uses capillaries to separate proteins. It is used to separate ionic species in a small capillary filled with an electrolyte based on their charge and size. It is based on various separation principles and is also used for pharmaceutical product quality control.

4. Isolation method:¹²

Impurities can be isolated and characterized using a variety of methods. However, the effectiveness of any method is determined by the impurity's nature (i.e., its structure, physicochemical properties, and availability). For the isolation of impurities, chromatographic techniques are commonly used, but non-chromatographic techniques are also occasionally used.

The following methods are generally used:

· Solid-phase extraction methods

· Liquid-liquid extraction methods

· Accelerated solvent extraction methods

· Column chromatography

· Flash chromatography

· TLC

· GC

· HPLC

· HPTLC

· Capillary electrophoresis (CE)

· Supercritical fluid chromatography (SFC).

a) Solid-phase extraction method:⁶

This is a method for tracing organic compounds and removing interfering compounds in order to obtain a clear extract. This technique is primarily used for compound extraction and purification. This method is primarily used to clean the sample before using chromatographic techniques to quantify the analyte in the sample. This technique is commonly used to separate analytes from a liquid matrix.

b) Liquid-liquid extraction:

It is also known as solvent extraction and partitioning, is a technique for extracting liquids from liquids. It's a technique for separating compounds based on their relative solubility in two immiscible liquids, typically water and organic solvents. The procedure is carried out in a separating funnel. Ethyl acetate, methylene chloride, and hexanes are common solvents for liquid-liquid extraction.

c) Accelerated solvent extraction method:

It is the preferred method for solid and semi-solid sample extraction. All of the steps are carried out at a high temperature and pressure to ensure that the analysts are removed quickly and efficiently from the samples. It completes the experiment in less time and with a smaller amount of solvent.

5. Characterization method:^7,13

In the identification of minor components (drugs, impurities, degradation), highly sophisticated instrumentation, such as MS attached to a GC or HPLC, is an unavoidable tool. In a variety of matrices (e.g., products, metabolites) For different techniques are used to characterise impurities.

The following are some of the terms that have been used:

NMR's capacity to reveal information about a substance's particular bonding structure and stereochemistry. A common mixture of real materials containing both monomers and dimers was used to validate the capacity of NMR-based diffusion coefficient determination to distinguish between monomeric and dimeric compounds.¹³

Unfortunately, NMR has a reputation for being less sensitive than other analytical techniques. NMR samples are typically on the order of 10 mg, whereas MS samples are on the order of 1 mg.

MS:

Over the last few decades, it has had an increasingly major impact on the pharmaceutical development process. Advances in the design and efficiency of interfaces that connect separation processes to mass spectrometers have opened up new possibilities for research.

Drug-related monitoring, characterization, and quantification of compounds found in active pharmaceutical components, as well as formulations for pharmaceuticals

Hyphenated Methods:

• LC-MS-MS

• HPLC-DAD-MS

• HPLC-DAD-NMR-MS

• GC-MS

• LC-MS

Determining which of the various potential impurities are, in fact, produced within the manufacturing process and which occur under a given set of storage conditions may be a common goal for researchers looking into both process and product degradation-related impurities.

F. Limits of impurity^8,12

According to the ICH recommendations on impurities in new drug products, unless potential impurities are expected to be unusually potent or toxic, identification of impurities below the 0.1 percent level is not considered necessary.¹⁴ The maximum daily dose qualification threshold to be considered, according to the ICH, is in Table-3.

APPLICATION OF IMPURITIES¹⁴

Profiling of impurities in drugs is a regulatory requirement to ensure the safety and efficacy of pharmaceutical products. Several applications in the areas of drug design and monitoring have been sought.

Pharmaceutical compounds' quality, stability, and safety, whether synthesised, extracted from natural products, or produced through recombinant methods. Alkaloids, amines, amino acids, analgesics, antibacterial, anticonvulsants, antidepressants, tranquillizers, antineoplastic agents, local anaesthetics, macromolecules, steroids, and other compounds are used in various applications.

CONCLUSION:

Impurities in drug substance and drug product are discussed in this review. The impurity profile of pharmaceuticals is becoming increasingly important, and public and media attention to drug safety is increasing. This article provides the valuable information about the impurity’s types and its classification, Regulatory guidelines on impurities in an API, analytical techniques for the determination. Knowing the impurities present in APIs is now a mandatory requirement in various pharmacopoeias. As a result, impurity profiling can be used as a quality assurance tool. It can provide critical information on the toxicity, safety, and various detection and quantification limits of a variety of organic and inorganic impurities that are commonly found in APIs and finished products. With regard to impurities, there is a strong need for unique specifications/standards.

Maximum Daily Dose a	Reporting Threshold b, c	Identification Threshold c	Qualification Threshold
≤2 gm/day	0.05%	0.1% or 1.0mg /day intake (whichever is lower)	0.15% or1.0mg/day intake whichever is lower
>2 gm/day	0.03%	0.05%	0.05%