Mass Spectrometry for the Clinical Laboratory

Hari Nair, William Clarke, Hari Nair, William Clarke

• Publisher: Academic Press
• Year: 2016
• ISBN:
  9780128009925
• Topic: Physical Sciences
• Subtopic: Spectroscopy & Spectrum Analysis

Mass Spectrometry for the Clinical Laboratory is an accessible guide to mass spectrometry and the development, validation, and implementation of the most common assays seen in clinical labs. It provides readers with practical examples for assay development, and experimental design for validation to meet CLIA requirements, appropriate interference testing, measuring, validation of ion suppression/matrix effects, and quality control. These tools offer guidance on what type of instrumentation is optimal for each assay, what options are available, and the pros and cons of each. Readers will find a full set of tools that are either directly related to the assay they want to adopt or for an analogous assay they could use as an example.

Written by expert users of the most common assays found in a clinical laboratory (clinical chemists, toxicologists, and clinical pathologists practicing mass spectrometry), the book lays out how experts in the field have chosen their mass spectrometers, purchased, installed, validated, and brought them on line for routine testing.

The early chapters of the book covers what the practitioners have learned from years of experience, the challenges they have faced, and their recommendations on how to build and validate assays to avoid problems. These chapters also include recommendations for maintaining continuity of quality in testing. The later parts of the book focuses on specific types of assays (therapeutic drugs, Vitamin D, hormones, etc.). Each chapter in this section has been written by an expert practitioner of an assay that is currently running in his or her clinical lab.

• Provides readers with the keys to choosing, installing, and validating a mass spectrometry platform
• Offers tools to evaluate, validate, and troubleshoot the most common assays seen in clinical pathology labs
• Explains validation, ion suppression, interference testing, and quality control design to the detail that is required for implementation in the lab

Chapter 1

Mass spectrometry in the clinical laboratory: determining the need and avoiding pitfalls

W. Clarke Johns Hopkins University School of Medicine, Baltimore, MD, United States

Abstract

While mass spectrometry (MS) has been available as an analytical tool for more than 100 years and analytical methods based on MS have been used by both scientific researchers and industrial application scientists for quantitative and qualitative analyses routinely, the use of this technology in clinical laboratories is a relatively new development. Based on emerging needs for patient care, the importance of MS in the clinical laboratory is increasing, and doesn’t appear to be slowing down any time soon. Emerging unmet needs for testing often occur because vendors of in vitro diagnostic devices wait until the clinical utility of a particular assay is established and widely accepted prior to the development of a commercial assay. As a result, improvements in patient care from these new clinical tests can be delayed until a commercial assay is available. 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 relative to the clinical decision point. Liquid chromatography (LC)-MS technology shows significant potential as a tool to rapidly develop clinical assays for emerging biomarkers, and advances in instrumentation are likely to enable application of LC-MS technology in routine clinical diagnostic testing.

Keywords

mass spectrometry

liquid chromatography

clinical diagnostic testing

electrospray ionization

quadrupole mass analyzers

1. Clinical mass spectrometry

Historically, the complexity of instrumentation and sample preparation has relegated LC-MS based assays to specialized laboratories with extensive technical expertize. Until recently, applications of MS in the clinical laboratory were limited to gas chromatography (GC)-MS for toxicology confirmation testing and testing for inborn errors of metabolism, some GC-MS applications for steroid analysis in specialty laboratories, and inductively coupled plasma (ICP)-MS for elemental analysis. In most cases, this testing has been restricted to specialized laboratories within a hospital, or to large reference laboratories. However, with the simplification of MS instrumentation and introduction of atmospheric spray ion sources along with the emergence of routine liquid-chromatography tandem MS (LC-MS/MS), MS has become a viable option for routine testing in clinical laboratories.

1.1. Basic mass spectrometry concepts

MS is a powerful analytical technology that can be used to identify unknown organic or inorganic compounds, determine the structure of complex molecules, or quantitate extremely low concentrations of known analytes (down to one part in 10¹²). For MS-based analysis, molecules must be ionized, or electrically charged, to produce individual ions. Thus, MS analysis requires that the atom or molecule of interest has the ability to be ionized and be present in the gas phase. MS instruments analyze molecules by relating the mass of each molecule to the charge; this identifying characteristic is specific to each molecule and is referred to as the mass-to-charge ratio (m/z). Therefore, if the molecule has a single charge (z = 1), the m/z ratio will be equal to the molecular mass.

The analytical power of the mass spectrometer lies in its resolution, or the ability to discern one molecular mass from another. The resolution can be determined by examining the width of an m/z peak or the separation between adjacent peaks; a narrow peak with little overlap indicates greater resolution. For two adjacent peaks of masses m₁ and m₂, the resolving power is defined as m₁/(m₁ – m₂). The expression (m₁ − m₂₎ may also be referred to as ∆m. Higher instrument resolution results in increased mass accuracy and the ability to avoid interference from compounds of similar mass that may also be present in the sample. Mass accuracy is defined as the mass difference that can be detected by the analyzer divided by the observed, or true mass.

Although there are numerous instrument configurations available, MS system operation can be organized into three main segments: (1) generation of ions; (2) separation of ions based on mass and charge in a mass analyzer; (3) detection of ions and instrument output (Fig. 1.1). Depending upon the type of ionization used, these steps fully or partially occur under vacuum pressure to drive ion movement forward through the instrument.

Figure 1.1 Schematic Diagram of Mass Spectrometry (MS)

1.2. Common ion sources for clinical mass spectrometry

There are a variety of ion sources available for mass spectrometers. Some of these ion sources are “direct ionization sources,” in which analytes are directly ionized from a surface or from a solution. Other sources, such as atmospheric pressure ionization sources, produce ions from analytes in solution and these are more commonly used in clinical assays due to their compatibility with liquid chromatography. Common atmospheric pressure ion sources include:

• electrospray ionization (ESI)

• atmospheric pressure chemical ionization (APCI)

• atmospheric pressure photoionization (APPI)

A summary of the strengths and weaknesses for these sources can be found in Table 1.1.

Table 1.1

Overview of Three Ionization Techniques Used in Clinical Mass Spectrometry (MS)

Ionization Technique	Advantages	Limitations
ESI	• Sensitive ionization technique for polar analytes or ions generated in solution • Has broad applicability for relevant analytes in clinical MS • May yield multiply charged ions, which allows for analysis of larger molecules (i.e., >1000 Da)	• May be more sensitive to matrix effects compared to APCI
APCI	• Typically less sensitive to matrix effects than ESI • May provide better sensitivity for less polar analytes	• Typically only singly charged ions are formed, limiting the effective mass range, • May be unsuitable for thermally labile analytes • May yield less absolute signal relative to ESI
APPI	• Works well with nonpolar analytes • In some cases will ionize analytes that do not ionize by either ESI or APCI.	• Demonstrates limited applicability in clinical MS to date.

APCI, Atmospheric pressure chemical ionization; APPI, atmospheric pressure photoionization; ESI, electrospray ionization.

1.2.1. Electrospray Ionization (ESI)

ESI is perhaps the most commonly used ionization technique in clinical MS. It is a sensitive ionization technique for analytes that exist as ions in the LC eluent. In ESI, a solvent spray is formed by the application of a high voltage potential held between a stainless steel capillary and the instrument orifice, coupled with an axial flow of a nebulizing gas (typically nitrogen). Solvent droplets from the spray evaporate in the ion source of the mass spectrometer, releasing ions to the gas phase for analysis in the mass spectrometer. In some ESI sources, heat is used to increase the efficiency of desolvation. While ESI is widely used, it is subject to matrix effects, particularly ion suppression, which must be taken into consideration during method development.

1.2.2. Atmospheric Pressure Chemical Ionization (APCI)

APCI uses heat and a nebulization gas to form an aerosol of the eluent from an LC system. In contrast to ESI, ions are not formed in solution or liquid phase. Instead, ions are formed in the gas phase using a corona discharge (high voltage applied to a needle in the source) to ionize solvent molecules and analytes in the aerosol. Ions released to the gas phase are then analyzed by the mass spectrometer. During...