A Review on Impurity Profiling Using Q-TOF & Orbital Trap Mass Spectroscopy

The impurity profile has become essential as per various regulatory requirements. For the pharmaceutical industries impurity is considered as organic material or unwanted chemicals which remain with Active Pharmaceutical Ingredient (API’s).  The impurity be developed either during formulation or ageing of both API’s and Formulation.  The presence of these unwanted chemicals may influence the efficacy and the safety of the pharmaceutical products. Impurity profiling gaining more attention from regulatory authorities. The impurity profiling is the description of identified and unidentified impurities present in new drug substance.  Some impurities have been named as per International Conference on Harmonization (ICH) such as [1].

· By-products.

· Degradation products.

· Interaction products.

· Intermediates.

· Related products.

· Transformation products.

Impurity: “It is defined as any substance coexisting with the original drug, such as starting

material, intermediate, and any side reactions”. It is the unwanted chemicals substance which influences the safety and efficacy of the pharmaceutical products. [2]

Profiling: It is the process of the identification through which we get the characteristic of the substance. [3]

Impurity Profiling: It is the process of evaluating the data that establish biological safety of an individual impurity.  For maintaining the stability or efficacy of the API we do the profiling of impurities. It helps in identifying and quantifying the impurities in API. It gives maximum possible types of impurities present in drug substance (API) and in pharmaceutical formulations. The impurity control in the pharmaceutical product is the main goal of the drug development and for controlling the impurity, there are various regulatory guidelines which monitored the impurities in API [4]

Classification of impurities in API

As per the ICH guidelines, the impurities are classified in three types as below.

1. Organic impurities (process and drug related).

2. Inorganic impurities.

3. Residual solvents.

1. Organic impurities:  These impurities are classified as starting material, By-product, degradation product, reagents and chiral impurities

a) Starting materials: The impurities from the starting material or by product are found in every drug substance, so for that proper care should be done to remove that impurities before it effect the end product. For e.g. In the synthesis of baclofen, the last step is carried out with gutarimide, after which on reaction with sodium hydroxide solution at room temperature it yield a potential impurity i.e. p-chloro phenyl gluteric acid.

b)  By-products:  There is always a chance of having by-products. Because they can be formed through a variety of side reactions, such as incomplete reaction, over reaction, isomerization, dimerization, rearrangement or unwanted reaction between starting materials or intermediate with the chemical reagents or catalysts. For e.g. In the case of Paracetamol bulk production, diacetylated Paracetamol may form as a by-product.

2. Inorganic impurities:  Inorganic impurities are derive from the manufacturing process and excipients. Inorganic impurities mainly include water, salts from buffers, reagents, ligands, catalysts, heavy metals, or other residual metals and also the inorganic compounds used in the processing such as filter aids and charcoal.

· Reagent, ligand and catalyst: The chances of these impurities are rare but these impurities could create a problem so proper care has to be taken during production. For e.g. chloride.

· Heavy metals: The main sources of the heavy metals are, the reactors where acidification and acid hydrolysis takes place. The impurities which are arising due to the heavy metals, that can easily be avoided by demineralized water and by using glass lined reactors. For e.g. water.

· Filter aids and charcoal:  These are the centrifuge bags which are routinely used in the bulk drugs manufacturing plants. For e.g. activated carbon.

3. Residual solvents: These are the undesirable substance. They modify the properties of certain compounds or may be hazardous for the human health.  These solvents also affect

the physicochemical properties of the bulk drug such as crystallinity of the bulk drug. As per ICH guidelines, the solvents are classified into three categories:

1. Class 1 solvent: These solvents are not employed in the manufacture of drug substances, excipients and formulations because of their unacceptable toxicity or their deleterious effects Table.1.

2. Class 2 solvent: these solvents have limited use in the pharmaceutical products because of their inherent toxicity.

3. Class 3 solvents: these are the less toxic and possess lower risk to the human health. These solvents do not have any serious hazardous

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

Table:2 Class 2 Solvents to be limited in pharmaceutical products

Table:3: Solvents for which no adequate toxicological data was found

Analytical Techniques Used in Impurity Profiling

Various analytical techniques are employed for impurity profiling, including chromatography and spectroscopy. Commonly used methods are High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Thin Layer Chromatography (TLC), Nuclear Magnetic Resonance (NMR), Infrared Spectroscopy (IR), and Mass Spectrometry (MS). Among these, High-Resolution Mass Spectrometry (HRMS) techniques such as Quadrupole Time-of-Flight (Q-TOF) and Orbitrap Mass Spectrometry have gained significant importance due to their high sensitivity, selectivity, and accurate mass measurement capabilities.

Role of High-Resolution Mass Spectrometry (HRMS) in Impurity Profiling

HRMS plays a crucial role in identifying known and unknown impurities at trace levels. It provides exact mass measurements, elemental composition, isotopic patterns, and structural information, which are essential for impurity characterization. HRMS is particularly valuable in:

• Identification of unknown and genotoxic impurities
• Degradation pathway studies
• Stability testing
• Non-targeted impurity analysis
• Compliance with regulatory guidelines

Quadrupole Time-of-Flight (Q-TOF) Mass Spectrometry

Principle

Q-TOF mass spectrometry combines a quadrupole mass analyzer with a time-of-flight (TOF) analyzer. The quadrupole acts as a mass filter to select precursor ions, while the TOF analyzer measures ion flight time to determine the mass-to-charge ratio (m/z) with high accuracy.

Instrumentation

A typical Q-TOF system consists of:

• Ion source (ESI/APCI/APPI)
• Quadrupole mass analyzer
• Collision cell for MS/MS experiments
• Time-of-flight analyzer
• Detector and data system

Applications in Impurity Profiling

• Identification of process-related and degradation impurities
• Structural elucidation using MS/MS fragmentation
• Non-targeted impurity screening
• Metabolite identification

Advantages

• High mass accuracy (≤5 ppm)
• Fast acquisition speed
• Suitable for LC–MS/MS analysis
• Good sensitivity for trace impurities

Limitations

• Slightly lower resolving power compared to Orbitrap
• Requires frequent calibration
• Limited performance in very complex matrices

Orbitrap Mass Spectrometry

Principle

Orbitrap mass spectrometry measures ion frequencies as they orbit around a central electrode. These frequencies are converted into m/z values using Fourier transform, providing ultra-high resolution and mass accuracy.

Instrumentation

An Orbitrap system includes:

• Ion source (usually ESI)
• Quadrupole for ion selection
• C-trap
• Orbitrap mass analyzer
• High-performance data processing system

Applications in Impurity Profiling

• Detection of trace-level and unknown impurities
• Identification of elemental composition
• Forced degradation and stability studies
• Genotoxic impurity assessment

Advantages

• Extremely high resolving power (>100,000 FWHM)
• High mass accuracy (<2 ppm)
• Excellent isotopic pattern recognition
• Ideal for complex impurity profiling

Limitations

• Higher cost of instrumentation
• Longer scan times compared to Q-TOF
• Requires skilled operation and data interpretation

Analytical Workflow for Impurity Profiling Using Q-TOF and Orbitrap

1. Sample Preparation – Extraction, dilution, and filtration
2. Chromatographic Separation – LC or UHPLC
3. HRMS Detection – Accurate mass measurement
4. Data Acquisition – Full scan and MS/MS
5. Data Processing – Software-assisted impurity detection
6. Structural Elucidation – Fragmentation and database matching
7. Quantification and Reporting

Regulatory Perspective

Regulatory authorities such as ICH, FDA, and EMA emphasize impurity identification and qualification. Guidelines such as:

• ICH Q3A (Impurities in New Drug Substances)
• ICH Q3B (Impurities in New Drug Products)
• ICH M7 (Genotoxic Impurities)

Recent Trends and Advancements

• Use of HRMS impurity databases.
• Software-assisted structural prediction.
• Artificial intelligence and machine learning in impurity identification.
• Integration of Q-TOF and Orbitrap with UHPLC.
• Increased focus on genotoxic and nitrosamine impurities.

Comparison Between Q-TOF and Orbitrap