Calibration, qualification and validation

Certified Reference Materials for Calibration and Method Validation

Reference materials are substances or materials with established properties that are used for example for calibration, method validation, and quality control in chemical analysis. The intended use varies by reference material quality grades as shown below. They ensure the accuracy and reliability of measurements by providing a standard against which other materials can be compared. Additionally, the use of RMs facilitates the comparability of results from different laboratories around the globe, promoting consistency and confidence in analytical outcomes. With various quality grades available, selecting the appropriate one for your specific application can be challenging. This article aims to guide you in determining the best quality grade for your needs.

Strict requirements and procedures must be followed for the manufacturing of reference materials, as they differ from standard lab reagents in terms of certification and data traceability. These requirements are defined in the ISO 17034 and related ISO standards for reference material producers.

Depending on the method requirements or product availability, reference materials are commonly used in three different formats ‒ neat, solution, or matrix.

Reference materials hold an essential role in analytical workflows. They are necessary for various analytical techniques, including chromatography, spectroscopy, and physical property measurements, and are utilized across all analytical sectors, such as pharmaceuticals, clinical testing, environmental monitoring, food and beverage, and cosmetics.

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Reference material quality grades and their features

There are key differences between the definitions of reference materials according to ISO and those established by regulatory bodies in the pharmaceutical industry. To illustrate these distinctions, we present two parallel hierarchical pyramids representing the quality grades of reference materials. The ISO quality grades align with the International System of Units (SI), whereas the pharmaceutical quality grades are based on definitions specific to the pharmaceutical sector described in the different Pharmacopoeias. This visualization highlights the unique frameworks each system employs to ensure the quality and reliability of reference materials in their respective contexts.

Choosing the Correct Reference Material for your Testing Purpose

Figure 1.Quality hierarchy structure for Reference Materials and pharmaceutical standards

National metrology standard (e.g. NIST, JRC, NMI Australia)
Compendial standard (e.g. USP, EP, BP, JP, IP)

Issued by an authorized body
Considered to provide the highest level of accuracy and traceability

Certified reference material (CRM) (ISO 17034, 17025)

Considered to provide the highest level of accuracy, uncertainty, and traceability to an SI unit of measurement
Manufactured by an accredited Reference Material Producer

Reference material (RM) (ISO 17034)

Fulfilling ISO requirements which are less demanding than for CRMs
Manufactured by an accredited Reference Material Producer

Analytical standard (ISO 9001)

Certificate of Analysis available
Level of certification varies

Reagent grade / research chemical

May come with a Certificate of Analysis
Are not characterized for use as reference materials

The main characteristics that describe and differentiate the different quality grades are traceability, stability, homogeneity and measurement uncertainty. These will be further evaluated in the following.

Traceability

A crucial concept for understanding reference materials is metrological traceability. This means that a value of a material is traceable to a universally accepted basic unit through an unbroken chain of measurements.

Certified Reference Materials (CRMs) must be traceable to the SI. They may be manufactured by metrology institutes or by commercial reference material producers holding an ISO 17034 accreditation. Note that for the lower quality grades in Fig. 1 (the left), i.e. Reference Material (RM) and Analytical Standard, metrological traceability is not a mandatory requirement.

In pharmaceutical quality control, specific compendial (e.g. USP, EP, BP etc.) primary standards have to be used. Alternatively, secondary or working standards that are traceable to the corresponding pharmacopoeia primary standards are also accepted. While SI traceability is not a requirement, Compendial primary or secondary standards should only be used for the monograph methods they are intended for, as defined by the Pharmacopeia.

Homogeneity

Homogeneity is a critical property for reference materials (RMs) that ensures consistency across all units of the material. According to ISO 17034, Reference Material Producers must specify acceptance criteria for and assess the homogeneity of their materials. This includes appropriate sampling procedures to evaluate homogeneity. A homogeneous reference material allows users to obtain reliable and comparable results regardless of which unit they use, which is essential for quality control and method validation purposes.

Stability

Stability is another crucial characteristic of reference materials. Ensuring stability means that the properties of the reference material remain constant over time within specified limits. This is vital because users need to trust that the certified values of the RM remain valid throughout its shelf life. This is typically ensured by monitoring samples at different temperatures (accelerated stress test, real-time stability test).

Measurement uncertainties

Understanding and quantifying uncertainties is fundamental in the production and use of certified reference materials (CRMs). Reference Material Producers must establish uncertainty budgets and estimate uncertainties of certified value(s). This is crucial because every measurement has some degree of uncertainty, and users of the reference material need to know the range in which the true value is likely to lie.

All factors that contribute to the overall uncertainty must be considered which can be visualized in a so-called fish bone or Ishikawa diagram.

Properly evaluated and reported uncertainties allow users to make informed decisions about the suitability of the reference material for their specific application.

By addressing these 4 key aspects –traceability, homogeneity, stability, and uncertainties - reference material producers ensure the quality and reliability of their products, which in turn supports accurate and comparable measurements across various fields in science and industry.

The table below shows the different quality grades of reference materials and their requirements with respect to the parameters just described. As mentioned before, certified reference materials (CRMs) need to be traceable to the SI and the values must be reported with an uncertainty; stability and homogeneity has to be assessed. The requirements for the RM (Reference Material) quality grade are less demanding, where metrological traceability and uncertainty are not mandatory. The same is true for Analytical Standards while stability and homogeneity tests are optional.

For Pharmaceutical primary standards, values are also reported with no uncertainty and the values are not traceable to SI.

Notably, our PHR products are traceable not only to the current lot of one or more pharmacopoeia primary standards, but also carry an SI-traceable certified value. Thus, these products are also certified reference materials in addition to pharma secondary standards and can be used in either the ISO or the pharmacopoeia context.

Selection of reference materials based on testing purpose

Selecting the right reference material is of critical importance to a laboratory's testing application, as results are only as accurate as the reference used. The choice of reference material depends on the specific task at hand.

For instrument qualification and calibration, traceability must be established and maintained. The selected reference material plays a key role in helping a laboratory achieve this.

In daily routine system suitability testing, it is important to choose a reference material that is practical, easy to use, reliable, and cost-effective for everyday use.

In method validation, the use of highly accurate and precise reference materials is essential to demonstrate that the laboratory method is fit for purpose.

For identity confirmation and screening purposes, key attributes of reference materials include proven authenticity and well-established identity.

In applications involving quantitation, assays, or stability assessments, stable and accurate Certified Reference Materials (CRMs) are required.

The selection of Fit-for-purpose reference materials can depend on several factors such as regulatory requirements, availability, type of testing application, level of accuracy, and sample matrix.

Additional information: Key ISO documents for Reference Material Producers (RMPs)

Below you can find an overview of the relevant ISO documents describing the procedures that must or may be followed by Reference Material Producers accredited under ISO 17034. Our company holds ISO 17034 accreditation at three manufacturing sites globally.

ISO 17034 - general requirements for the competence of reference material producers

Purpose: This is the primary normative document specifying the general requirements for the competence and consistent operation of RMPs.
Scope: It covers all types of reference materials (RMs), including certified reference materials (CRMs).
Benefits: Implementing ISO 17034 demonstrates a producer's commitment to quality, accuracy, and consistency, building user confidence and meeting regulatory requirements. 17034 has to be followed for accreditation, the other ones are not mandatory but help the RMPs to fulfill ISO 17034.

ISO Guide 30 - Reference materials — selected terms and definitions

Purpose: This guide provides essential definitions and terminology for reference materials and their associated concepts, ensuring clear and consistent communication within the field.

ISO 33401:2024 (Formerly ISO Guide 31) - Reference materials — contents of certificates, labels and accompanying documentation

Purpose: This document outlines the information that must be included on the certificates and labels for reference materials. This ensures that comprehensive and standardized information about the RM's properties and intended use is provided.

ISO 33403:2024 (Formerly ISO Guide 33) - Reference materials — requirements and recommendations for use

Purpose: This document describes good practice in using reference materials (RMs), and certified reference materials (CRMs) in particular, in measurement processes including the assessment of precision and trueness of measurement methods; quality control; assigning values to materials; calibration and establishing conventional scales.

ISO 33405:2024 (Formerly ISO Guide 35) - Reference materials — approaches for characterization and assessment of homogeneity and stability

Purpose: This guide provides a framework for the processes involved in certifying reference materials, including principles for characterization, homogeneity and stability assessment, and the statistical principles used for assigning certified values and uncertainties.

ISO 33406:2024 Approaches for the production of reference materials with qualitative properties

Purpose: This document provides concepts to the following aspects of the production of reference materials (RMs): the assessment of homogeneity; the assessment of stability and the management of the risks associated with possible stability issues related to the properties of interest; the characterization and value assignment of properties of an RM; the evaluation of uncertainty for certified values; the establishment of the metrological traceability of certified values.