Stable Isotope-Dilution-LC-MS/MS-based Reference Methods – Suggestion for Description of Methods and Structure of Analytical Series Michael Vogeser (Presenter) University Hospital, LMU Munich Presenter Bio: Professor of Laboratory Medicine at the LMU Munich and Senior Physician at the University Hospital. Co-Editor-in-Chief of CMS. Secretary of the German Association of Clinical Chemistry and Laboratory Medicine.

Authors: Michael Vogeser (1), Carina Schuster (1), Christina Ritter-Sket (2). Anja Kessler (2)
(1) University Hospital, LMU Munich, Institute of Laboratory Medicine, Munich, Germany (2) Referenzinstitut für Bioanalytik), Bonn, Germany

ID-LC-MS/MS methods are increasingly used as reference for routine methods, but actually the characteristics of such reference methods (RM) are so far not conclusively specified. A RM concept specifically for ID-LC/MS was developed, including three key elements: 1. Standardized method description – differentiating between essential characteristics (such as recorded mass transitions) and variable features that realistically can not be standardized over time (such as lot of columns). 2. Standardized structure of analytical series, including several features for performance verification (e.g., addressing matrix effects). 3. Based on this, a series-related validation concept according to pre-defined criteria as essential characteristic of the RM. This novel approach was successfully applied to a RM for the quantification of linezolid in serum.

Introduction

The term and the concept of reference measurement procedures (RMPs) in the context of traceability chains are well described (VIM, vocabulaire international de metrologie; ISO 17511; ISO 15193). RMPs represent a comprehensive system of compound preparations, samples, measurements and assignment of values, typically applied in accredited national reference laboratories. In contrast the term reference method (RM) is widely used in literature and practice but so far not conclusively defined. Basically, a RM can be understood as a very reliable method that is performed as the reference for more practicable, higher-throughput routine diagnostic methods. For this purpose – and not necessarily within the framework of comprehensive traceability systems - ID-LC-MS/MS is increasingly used for RMs, in particular by the diagnostic industry.

ISO 15193 describes a general framework for RMPs, however, in a very generic way. Validation of an RMP is - according to this standard - assessed essentially once and then performance characteristics are expected to be more or less constant for the whole lifecycle of the method. Thus, this standard doesn’t address the high complexity and heterogeneity of ID-LC-MS/MS technology applied in different laboratories over long time. CLSI standard C62 A describes aspects of quality assurance in LC-MS/MS in detail, but not in the specific context of reference methods.

Therefore, the aim of our work was to develop a guidance document that helps to implement ID-LC-MS/MS based RMs with a high level of between-lab and long-term analytical consistency of results.

Methods

A RM concept for ID-LC/MS was developed including three key features:

- Standardized method description – differentiating between essential characteristics (such as recorded mass transitions) and variable features of a method that realistically can not be standardized over time (such as lot of columns and pureness of solvents)

- Standardized structure of analytical series, including several features for performance verification (e.g., addressing matrix effects) and addressing particular technical features of MS/MS

- Based on this, a series-related validation concept according to pre-defined pass-criteria for several analytical variables as essential characteristic of the RM

Results

This novel approach is successfully applied to a RM for the quantification of linezolid in serum in a network of laboratories.

Conclusions & Discussion

A novel approach to ID-LC-MS/MS-based RMs – in particular for application by the diagnostic industry - is suggested for critical evaluation and discussion in the community.