SAMPLE. Liquid Chromatography-Mass Spectrometry Methods; Approved Guideline

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1 October 2014 Liquid Chromatography-Mass Spectrometry Methods; Approved Guideline This document provides guidance to the clinical laboratorian for the reduction of interlaboratory variance and the evaluation of interferences, assay performance, and other pertinent characteristics of clinical assays. This guideline emphasizes particular areas related to assay development and presents a standardized approach for method verification that is specific to mass spectrometry technology. A guideline for global application developed through the Clinical and Laboratory Standards Institute consensus process.

2 Clinical and Laboratory Standards Institute Setting the standard for quality in clinical laboratory testing around the world. The Clinical and Laboratory Standards Institute (CLSI) is a not-for-profit membership organization that brings together the varied perspectives and expertise of the worldwide laboratory community for the advancement of a common cause: to foster excellence in laboratory medicine by developing and implementing clinical laboratory standards and guidelines that help laboratories fulfill their responsibilities with efficiency, effectiveness, and global applicability. Consensus Process Consensus the substantial agreement by materially affected, competent, and interested parties is core to the development of all CLSI documents. It does not always connote unanimous agreement, but does mean that the participants in the development of a consensus document have considered and resolved all relevant objections and accept the resulting agreement. Commenting on Documents CLSI documents undergo periodic evaluation and modification to keep pace with advancements in technologies, procedures, methods, and protocols affecting the laboratory or health care. CLSI s consensus process depends on experts who volunteer to serve as contributing authors and/or as participants in the reviewing and commenting process. At the end of each comment period, the committee that developed the document is obligated to review all comments, respond in writing to all substantive comments, and revise the draft document as appropriate. Comments on published CLSI documents are equally essential, and may be submitted by anyone, at any time, on any document. All comments are addressed according to the consensus process by a committee of experts. Appeals Process If it is believed that an objection has not been adequately addressed, the process for appeals is documented in the CLSI Standards Development Policies and Process document. All comments and responses submitted on draft and published documents are retained on file at CLSI and are available upon request. Get Involved Volunteer! Do you use CLSI documents in your workplace? Do you see room for improvement? Would you like to get involved in the revision process? Or maybe you see a need to develop a new document for an emerging technology? CLSI wants to hear from you. We are always looking for volunteers. By donating your time and talents to improve the standards that affect your own work, you will play an active role in improving public health across the globe. For further information on committee participation or to submit comments, contact CLSI. Clinical and Laboratory Standards Institute 950 West Valley Road, Suite 2500 Wayne, PA USA P: F: standard@clsi.org

3 ISBN (Print) ISBN (Electronic) ISSN (Print) ISSN (Electronic) Vol. 34 No. 16 Liquid Chromatography-Mass Spectrometry Methods; Approved Guideline Volume 34 Number 16 William Clarke, PhD, MBA, DABCC Ross J. Molinaro, PhD, MLS(ASCP) CM, DABCC, FACB Lorin M. Bachmann, PhD, DABCC Julianne Cook Botelho, PhD Zhimin Cao, MD, PhD, DABCC Deborah French, PhD, DABCC Seema Garg, MS, MBA John M. Gawoski, MD Russell P. Grant, PhD Abstract Andrew N. Hoofnagle, MD, PhD Bagyalakshmi Iyer, PhD Vathany Kulasingam, PhD, FCACB Donald S. Mason, MS Brian Rappold Danyel H. Tacker, PhD, DABCC Steven M. Truscott, PhD, DABCC Chunli Yu, MD, FACMG Yusheng Zhu, PhD, DABCC, FACB Clinical and Laboratory Standards Institute document Liquid Chromatography-Mass Spectrometry Methods; Approved Guideline provides guidance for the development and verification of liquid chromatography-mass spectrometry (LC-MS) methods in the clinical laboratory. The document is intended to reduce interlaboratory variance for clinical assays through guidance for evaluating interferences, assay performance, and other pertinent characteristics. It emphasizes particular areas related to assay development and presents a standardized approach for method verification that is specific to mass spectrometry (MS) technology. This document is intended for laboratorians responsible for development and verification of MS-based assays, physicians who may use these assays for patient care decisions, external quality assessment programs, and manufacturers of MS instrumentation and reagent kits designed to be paired with a particular mass spectrometer. This document is limited to discussion of LC-MS and is focused on the steps for development of a method, eg, whether the analyte is a drug, hormone, protein, or peptide. Clinical and Laboratory Standards Institute (CLSI). Liquid Chromatography-Mass Spectrometry Methods; Approved Guideline. CLSI document (ISBN [Print]; ISBN [Electronic]). Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania USA, The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in the CLSI catalog and posted on our website at If you or your organization is not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: ; Fax: ; customerservice@clsi.org; Website:

4 Number 16 Copyright 2014 Clinical and Laboratory Standards Institute. Except as stated below, any reproduction of content from a CLSI copyrighted standard, guideline, companion product, or other material requires express written consent from CLSI. All rights reserved. Interested parties may send permission requests to permissions@clsi.org. CLSI hereby grants permission to each individual member or purchaser to make a single reproduction of this publication for use in its laboratory procedure manual at a single site. To request permission to use this publication in any other manner, permissions@clsi.org. Suggested Citation CLSI. Liquid Chromatography-Mass Spectrometry Methods; Approved Guideline. CLSI document C62- A. Wayne, PA: Clinical and Laboratory Standards Institute; Approved Guideline October 2014 ISBN (Print) ISBN (Electronic) ISSN (Print) ISSN (Electronic) ii

5 Volume 34 Contents Abstract... i Committee Membership... iii Foreword... vii 1 Scope Standard Precautions Terminology A Note on Terminology Definitions Abbreviations and Acronyms Instrumentation High-Performance Liquid Chromatography and Ultra-High Performance Liquid Chromatography Ion Sources for Mass Spectrometry Mass Analyzers: Quadrupole, Time-of-Flight, and Ion Traps Instrumentation Performance Parameters Preexamination Considerations Analyte of Interest Internal Standard Selection Reagents and Sample Preparation Assay Development Ion Transitions High-Performance Liquid Chromatography Stationary Phase High-Performance Liquid Chromatography Mobile Phase Examination Variables Sources of Error Assay Calibration Preverification Assay Verification Limit of Detection and Lower Limit of the Measuring Interval Linearity and Dilution Imprecision Assay Interferences Trueness Quality Assurance and Quality Control of Liquid Chromatography-Mass Spectrometry Assays Selection of Quality Control Materials Quality Control Concentration and Frequency Additional Quality Assurance/Quality Control Samples for Liquid Chromatography-Mass Spectrometry Methods Evaluation of Quality Control Acceptability Corrective Action for Failed Quality Control Quality Control Considerations for Multicomponent or Multiplexed Assays Periodic Quality Assurance Procedures v

6 Number 16 Contents (Continued) 8.8 Additional Quality Assurance Aspects Postimplementation Monitoring Proficiency Testing System Performance Monitoring References The Quality Management System Approach Related CLSI Reference Materials vi

7 Volume 34 Foreword The importance of mass spectrometry (MS) in the clinical laboratory is increasing, and this CLSI document was developed in response to the need for increased robustness and harmonization of liquid chromatography-mass spectrometry (LC-MS) methods. Vendors of in vitro diagnostic devices often wait until the clinical utility of a particular assay is established and widely accepted before beginning development of a commercial assay. As a result, improvements in patient care can be delayed until a commercial assay is available. These delays can be significant if there are difficulties in the development of the assay. For laboratories with the capability to develop and implement laboratory-developed tests, MS provides an attractive alternative solution based on the ability to rapidly develop an analytically robust assay with excellent analytical sensitivity and specificity. In some cases, it offers an attractive alternative to commercially available assays that have already been developed, but do not offer acceptable performance in the current clinical context. LC-MS technology shows promise as a tool to rapidly develop clinical assays for emerging biomarkers coming from research in proteomics. Advances in instrumentation are likely to enable application of LC-MS technology in routine clinical diagnostic testing. Despite the significant advantages that can be gained from incorporating MS into the clinical laboratory, considerable challenges exist. Among these is the fact that for some assays, significant laboratory-tolaboratory variability for the same analyte has been observed. There is currently limited assay standardization for MS-based methods, and much of the assay variability can be attributed to the lack of commercially available calibrators; that is, each clinical laboratory must formulate its own calibrators. For example, some sites may use powdered or commercially lyophilized material, some may use organic solvent solutions, and some may even use formulations obtained from their institution s pharmacy to make calibrators. Moreover, the preparation of the calibrators also varies from using solutions made in buffer, from using analyte-free serum or plasma as the matrix, or from using patient specimen remnants as the calibrator matrix a problem for endogenous analytes. Differences in chromatographic methods from site to site lead to variable matrix effects during analysis. In addition, many laboratories verify their assays using various protocols in accordance with different regulatory or industry standards. This document addresses these issues by providing guidance for the development and verification of LC-MS methods in the clinical laboratory. This document outlines many important elements for successful implementation of LC-MS technology for clinical analyses. The basic instrument components needed both for chromatography and MS are discussed, along with instrument parameters that must be optimized for development of robust LC-MS methods. In addition, the document contains a discussion of preexamination considerations that must be addressed during the method development process. Various elements of method development are summarized, along with best practice recommendations for addressing those elements during the process. Guidance is provided for verification of an LC-MS method, including a recommendation for preverification evaluation before full method verification. Finally, the document provides guidance for QA, including assay QC and postimplementation monitoring. Key Words Chromatography, liquid chromatography-mass spectrometry, mass spectrometry, method verification, postimplementation monitoring, quality control vii

8 Volume 34 Liquid Chromatography-Mass Spectrometry Methods; Approved Guideline 1 Scope This document provides an introduction to, and guidance for, method development, verification, and postimplementation monitoring of quantitative clinical applications using liquid chromatography-mass spectrometry (LC-MS). While LC-MS may also be used for qualitative analyses, the focus of this 3 document is on the use of this technology for quantification of clinical analytes. In addition, while there are commercial and research methods that allow direct injection without chromatography for rapid analyses, this guideline is exclusively focused on liquid chromatography (LC) coupled to mass spectrometry (MS). The purpose of this guideline is to educate both clinical LC-MS practitioners and health care providers (including physicians) who may use these assays for patient care decisions on the benefits and limitations of LC-MS methods used in the clinical laboratory, as well as provide a practical guide for the development and implementation of LC-MS based clinical applications. It is intended to serve not only as a companion to CLSI document C50, 1 which serves as excellent general guidance for MS in the clinical laboratory, but also to provide an enhanced focus on methods, best practices, and instrumentation related to LC-MS, which is emerging as the most common approach to clinical analyses. This document is also intended to be a resource for instrument manufacturers, manufacturers of LC-MS reagents, regulatory agencies, and educators, as well as individuals responsible for developing laboratory standards and policy. A description of all current clinical applications of LC-MS, as well as all of the pertinent information regarding development and verification of these methods, is beyond the scope of this document. As such, this guideline directs the reader to appropriate existing resources wherever possible. In providing guidance for LC-MS method development, verification, and implementation, this document focuses on: Important features of LC-MS instrumentation Preexamination factors that can impact assay performance and utility Assay calibration Analytical variables important in method development Assay verification QA and QC Postimplementation monitoring of clinical methods 2 Standard Precautions Because it is often impossible to know what isolates or specimens might be infectious, all patient and laboratory specimens are treated as infectious and handled according to standard precautions. Standard precautions are guidelines that combine the major features of universal precautions and body substance isolation practices. Standard precautions cover the transmission of all known infectious agents and thus are more comprehensive than universal precautions, which are intended to apply only to transmission of bloodborne pathogens. The Centers for Disease Control and Prevention (CDC) address this topic in published guidelines that address the daily operations of diagnostic medicine in human and animal medicine while encouraging a culture of safety in the laboratory. 2 For specific precautions for preventing the laboratory transmission of all known infectious agents from laboratory instruments and materials and for recommendations for the management of exposure to all known infectious diseases, refer to CLSI document M29. Clinical and Laboratory Standards Institute. All rights reserved. 1

9 Number 16 3 Terminology 3.1 A Note on Terminology CLSI, as a global leader in standardization, is firmly committed to achieving global harmonization wherever possible. Harmonization is a process of recognizing, understanding, and explaining differences while taking steps to achieve worldwide uniformity. CLSI recognizes that medical conventions in the global metrological community have evolved differently in the United States, Europe, and elsewhere; that these differences are reflected in CLSI, International Organization for Standardization (ISO), and European Committee for Standardization (CEN) documents; and that legally required use of terms, regional usage, and different consensus timelines are all important considerations in the harmonization process. In light of this, CLSI s consensus process for development and revision of standards and guidelines focuses on harmonization of terms to facilitate the global application of standards and guidelines. In order to align the usage of terminology in this document with that of ISO, the term accuracy, in its metrological sense, refers to the closeness of the agreement between the result of a (single) measurement and a true value of a measurand, and comprises both random and systematic effects. Trueness is used in this document when referring to the closeness of agreement between the expectation of a test result or a measurement result and a true value ; the measurement of trueness is usually expressed in terms of bias. Precision is defined as the closeness of agreement between independent test/measurement results obtained under stipulated conditions. As such, it cannot have a numerical value, but may be determined qualitatively as high, medium, or low. For its numerical expression, the term imprecision is used, which is the dispersion of independent results of measurements obtained under specified conditions. In addition, different components of precision are defined in C62, primarily repeatability, ie, the closeness of the agreement between results of successive measurements of the same measurand carried out under the same conditions of measurement; while Reproducibility describes the closeness of agreement of results of measurements under changed conditions. Measuring interval has replaced reportable range when referring to a set of values of quantities of the same kind that can be measured by a given measuring instrument or measuring system with specified instrumental uncertainty, under defined conditions. An interval [a;b] is delineated by two limits, a and b (b > a), whereas a range (r[a;b]) is expressed as the difference between b and a (b a). Thus, the range of the interval [a;b] is the difference (b a) that is denoted by r[a;b]. Similarly, the term analytical measuring interval has replaced analytical measuring range. The term measurand is used when referring to the quantity intended to be measured instead of analyte (component represented in the name of a measurable quantity); the term measurement procedure replaces analytical method for a detailed description of a measurement according to one or more measurement principles and to a given measurement method, based on a measurement model and including any calculation to obtain a measurement result. Verification focuses on whether specifications of a measurement procedure can be achieved, whereas validation verifies that the procedure is fit for purpose. Both concepts can describe procedures of varying complexity. 3.2 Definitions acceptability based on individual criteria that set the minimum operational characteristics for a measurement procedure. accuracy (measurement) closeness of agreement between a measured quantity value and a true quantity value of a measurand (JCGM 200:2012). 4 2 Clinical and Laboratory Standards Institute. All rights reserved.

10 Number 16 The Quality Management System Approach Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system (QMS) approach in the development of standards and guidelines, which facilitates project management; defines a document structure via a template; and provides a process to identify needed documents. The QMS approach applies a core set of quality system essentials (QSEs), basic to any organization, to all operations in any health care service s path of workflow (ie, operational aspects that define how a particular product or service is provided). The QSEs provide the framework for delivery of any type of product or service, serving as a manager s guide. The QSEs are as follows: Organization Personnel Process Management Nonconforming Event Management Customer Focus Purchasing and Inventory Documents and Records Assessments Facilities and Safety Equipment Information Management Continual Improvement addresses the QSE indicated by an X. For a description of the other documents listed in the grid, please refer to the Related CLSI Reference Materials section, beginning on page 70. Organization Customer Focus Facilities and Safety M29 Personnel Purchasing and Inventory Equipment C50 GP31 NBS04 Process Management X C24 EP05 EP06 EP07 EP09 EP10 EP14 EP15 EP17 EP21 EP23 EP32 GP27 GP29 Documents and Records Information Management Nonconforming Event Management NBS04 Assessments EP10 GP27 GP29 Continual Improvement GP27 QMS06 QMS12 68 Clinical and Laboratory Standards Institute. All rights reserved.

11 Volume 34 Path of Workflow A path of workflow is the description of the necessary processes to deliver the particular product or service that the organization or entity provides. A laboratory path of workflow consists of the sequential processes: preexamination, examination, and postexamination and their respective sequential subprocesses. All laboratories follow these processes to deliver the laboratory s services, namely quality laboratory information. does not address any of the clinical laboratory path of workflow steps. For a description of the documents listed in the grid, please refer to the Related CLSI Reference Materials section on the following page. Examination ordering Sample collection Preexamination Examination Postexamination Sample transport Sample receipt/processing NBS04 Examination EP23 NBS04 Results review and follow-up C50 EP23 NBS04 Interpretation Results reporting and archiving Sample management C50 EP14 EP23 NBS04 Clinical and Laboratory Standards Institute. All rights reserved. 69

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