What Is Related Substances?
Related substances is the chromatographic test used in pharmacopoeia monographs targeting impurities structurally related to the active substance. The monograph names the impurities (Impurity A, B, C...), defines a response factor and acceptance limit for each, and also gives a limit for any unspecified single impurity and the total.
Purpose of Related Substances
The purpose is to show the product meets pharmacopoeial quality requirements for its impurity profile. This test is a core component of a pharmacopoeia compliance claim.
Which Products Is Related Substances Applied To?
- Active pharmaceutical ingredients
- Finished dosage forms
- Generic products
- Stability specimens
- Imported raw materials
In Which Sectors Is Related Substances Used?
Standards Related to Related Substances
- Ph. Eur. relevant substance monographs
- Ph. Eur. 2.2.29 (liquid chromatography)
- Ph. Eur. 5.10 (control of impurities)
- USP-NF relevant monographs
Related Substances Conditions
- Method
- The chromatographic conditions defined in the monograph are applied exactly as given
- System suitability
- The resolution and repeatability criteria in the monograph must be met
- Response factors
- The response factors given in the monograph are used in the calculation
Equipment for Related Substances
- HPLC / UPLC (with DAD detector)
- Analytical balance
- Ultrasonic bath
- Impurity reference standards
Sample Requirements for Related Substances
- Sufficient specimen quantity
- The impurity reference standards specified in the monograph
- Batch number and monograph reference must be provided
- The specimen must be sent per its storage condition
Duration of Related Substances
Analysis depends on the chromatographic run time and number of specimens; it is usually completed within a few working days.
Related Substances Procedure
- 1The chromatographic conditions in the monograph are set up.
- 2The system suitability solution is prepared and the criteria verified.
- 3Specimen and reference solutions are prepared.
- 4Chromatographic separation is performed.
- 5Named impurities are calculated using retention and response factors.
- 6Individual and total impurity results are reported.
Related Substances Acceptance Criteria
The acceptance criterion is not given on this page: for every active substance and every product, the limit comes from the relevant pharmacopoeia monograph or the approved specification in the marketing authorisation dossier. The pharmacopoeia's general chapter defines the method; the monograph sets the limit.
Evaluation of Results
The monograph method must be applied exactly; changing the column, mobile phase or gradient alters the method's resolving power and can invalidate the result. If an alternative method is to be used, its equivalence to the monograph method must be demonstrated.
Related Substances Report
- Specimen identification and batch information
- Monograph and edition applied
- System suitability results
- Result for each named impurity
- Unspecified single impurity and total
- Chromatograms
Accreditation
EUROLAB holds internationally recognised accreditation obtained from accreditation bodies party to the IAF and ILAC multilateral recognition arrangements, under ISO/IEC 17025 for laboratory testing, ISO/IEC 17020 for inspection and surveillance, and ISO/IEC 17021-1 and ISO/IEC 17065 for system and product certification. This chain ensures the reports and certificates issued are accepted worldwide.
Whether this test can be offered within accredited scope depends on your product and the standard applied; since accreditation is method-based, the scope is confirmed in writing at the quotation stage.
Scope and authorisationsRelated Regulations
- 2001/83/EC Directive on Medicinal Products for Human Use
- GMP Guide (EudraLex Volume 4)
Pharmaceutical Analysis — What Does This Testing Field Cover?
The section below describes the field that the Related Substances test belongs to; the conditions and acceptance criteria of this particular test are given in the sections above. The scope specific to your product is confirmed in writing at the quotation stage.
Pharmaceutical analysis determines the identity, purity, quantity and behaviour of an active substance and of the finished product. What sets this family apart is that the methods are largely defined in PHARMACOPOEIAS: the European Pharmacopoeia (Ph. Eur.) and the United States Pharmacopeia (USP) give both the method and the acceptance framework together.
The binding nature of the pharmacopoeial method shapes how the field works. In other sectors a laboratory can choose a suitable method, whereas here the method in the monograph governs; if an alternative method is to be used, its equivalence must be demonstrated. This is what makes results comparable worldwide.
Analyses are carried out in several groups. Identity and assay (HPLC, UV, titration) show what the product is and how much active substance it contains. The impurity profile and residual solvents look for unwanted compounds arising from the manufacturing process. Elemental impurities address metals originating from catalysts and equipment. Dissolution and disintegration behaviour is the most important in vitro indicator of how the drug will be released in the body.
The DISSOLUTION TEST is one of the most critical measurements in the field. It is not enough for a tablet to contain the right amount of active substance; that substance must dissolve at a suitable rate. The dissolution profile is used to assess the effect of formulation changes on bioavailability and holds a central place in generic drug licensing.
METHOD VALIDATION is a mandatory step in this family. If a pharmacopoeial method is used, it is verified that the method works properly under that laboratory's conditions. If an in-house method has been developed, it is validated against parameters such as linearity, precision, accuracy, specificity, limits of detection and quantification, and robustness. A result produced with an unvalidated method cannot be used in a licence dossier.
Stability studies determine shelf life and are the longest-running work in the field. The product is held under defined temperature and humidity conditions, analysed at set intervals, and the results become the basis of the shelf life and storage condition claims. Accelerated conditions give preliminary information but real-time data is what counts.
The microbiological side is a separate body of work: microbial enumeration, testing for specified microorganisms, sterility testing for sterile products and the bacterial endotoxin test. For sterile products, packaging integrity and sterilisation validation also come into play.
Data integrity has moved to the centre of inspections in recent years. It is not enough for the result to be correct; the raw data must be traceable, unalterable and auditable. Validation of electronic record systems has therefore become an inseparable part of analysis.
Method groups in this field
Identity and assay
HPLC, UPLC, UV-Vis spectrophotometry, titration, identity confirmation by FTIR.
Impurities and residues
Related impurities, degradation products, residual solvents (GC headspace), nitrosamine screening.
Elemental impurities
Determination of elemental impurities by ICP-MS and ICP-OES and the associated risk assessment.
Physicochemical
Dissolution and disintegration, hardness, friability, uniformity of dosage units, water content (Karl Fischer), particle size.
Microbiological
Microbial enumeration, specified microorganisms, sterility, bacterial endotoxin, preservative efficacy.
Stability
Real-time and accelerated stability studies; photostability and in-use stability.
Commonly confused in this field
- The pharmacopoeial method is binding; an alternative method may be used only by demonstrating its equivalence.
- A result produced without method validation cannot be used in a licence dossier.
- Accelerated stability does not replace real-time data; shelf life is supported by real-time results.
- The dissolution profile is sensitive to apparatus type, agitation rate and medium; the conditions must be complete in the report.
- A shortcoming in data integrity produces a non-conformity in an inspection even when the result is correct.
- A change of formulation or supplier requires analytical comparison and, where necessary, a new stability study.
Which decisions these results support
- The licence dossier and batch release
- Determining shelf life and storage conditions
- Method transfer and method validation
- Incoming raw material acceptance control from suppliers
- Assessing the effect of a formulation change on bioequivalence
Related Tests
Frequently Asked Questions About Related Substances
We don't have a reference standard for Impurity A — can the analysis be done?
In most cases the monograph specifies a reference standard or system suitability solution. Without it, identifying and correctly calculating named impurities may not be possible; this is stated as a limitation in the report.