Clinical Chemistry 62:1 92–98 (2016) Reviews Effective Use of Mass Spectrometry in the Clinical Laboratory Paul J. Jannetto1* and Robert L. Fitzgerald2 BACKGROUND: Historically the success of mass spectrom- (4). This evolution, driven by continuous improvements etry in the clinical laboratory has focused on drugs of in analytical platforms, is anchored by the analytical spec- Downloaded from https://academic.oup.com/clinchem/article/62/1/92/5611766 by guest on 30 September 2021 abuse confirmations, newborn screening, and steroid ificity of MS. Conclusive identification of molecules that analysis. Clinical applications of mass spectrometry con- range in size from tens of daltons (small molecules) to tinue to expand, and mass spectrometry is now being hundreds of thousands of daltons (biomolecules) is based used in almost all areas of laboratory medicine. on different principles. For example, small molecules are currently identified using LC-MS/MS. LC-MS/MS CONTENT: A brief background of the evolution of mass identifications are based on several unique characteristics, spectrometry in the clinical laboratory is provided with a including retention time, parent ion, and ratios of frag- discussion of future applications. Prominent examples of ment ions. In the case of newborn screening, samples are mass spectrometry are covered to illustrate how it has directly infused into the mass spectrometer with identi- improved the practice of medicine and enabled physi- fications based on specific transitions of precursor and cians to provide better patient care. With increasing eco- product ions. In the microbiology laboratory, identifica- nomic pressures and decreasing laboratory test reim- tions are based on patterns of ions generated from micro- bursement, mass spectrometry testing has been shown to bial proteins ionized by laser ablation. In all cases, the provide cost-effective solutions. In addition to pointing analytical specificity of the analysis is based on the ability out the numerous benefits, the challenges of implement- of a mass spectrometer to “weigh on the molecular scale” ing mass spectrometry in the clinical laboratory are also by determining the mass-to-charge ratio (m/z) of the ions covered. of interest. In the simplest form, MS provides some type of a molecular fingerprint of the analyte of interest. This SUMMARY: Mass spectrometry continues to play a prom- minireview provides a brief background on the evolution inent role in the field of laboratory medicine. The ad- of MS in the clinical laboratory (Fig. 1) and summarizes vancement of this technology along with the develop- how MS is being used to improve patient care in a cost- ment of new applications will only accelerate the effective manner. incorporation of mass spectrometry into more areas of A major impetus that moved MS from the research medicine. laboratory to the clinical laboratory was the accident on © 2015 American Association for Clinical Chemistry the aircraft carrier Nimitz. On May 26, 1981, an aircraft crashed while landing on the Nimitz, killing 14 and in- juring 45 (5). Subsequent immunoassay tests demon- 3 Mass spectrometry (MS) provides unique capabilities in strated that a large percentage of urine samples from ser- the clinical laboratory and is rapidly transitioning from vicemen were positive for marijuana metabolites. This specialized testing to being broadly applied. Historically, prompted President Reagan to develop a zero tolerance major impacts of MS include confirmation of for drugs of abuse in the military (6). Due to a large immunoassay-positive drug screens (1), identification of number of false-positive immunoassay results, antibody- inborn errors of metabolism (2), and analysis of steroid based drug screens began to be considered “presumptive” hormones (3). More recently, MS has dramatically im- until confirmed by GC-MS (7). It was clear that the early proved the time required for microbial identifications use of drug testing was effective at reducing the number of positive employee drug test results, because positive rates dropped from 18% to 8% over a 10-year time span 1 Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN; 2 Department of Pa- (8). Several studies also demonstrated that urine drug thology, University of California-San Diego, San Diego, CA. testing was cost-effective (8, 9). The requirement for * Address correspondence to this author at: Mayo Clinic, Laboratory Medicine and Pathol- GC-MS confirmation drove the development of MS in ogy, 200 First St. SW, Rochester, MN 55905. E-mail [email protected]. Received August 19, 2015; accepted September 21, 2015. toxicology laboratories, where it also began to be used for Previously published online at DOI: 10.1373/clinchem.2015.248146 therapeutic drug monitoring. © 2015 American Association for Clinical Chemistry As the clinical laboratory became more familiar 3 Nonstandard abbreviations: MS, mass spectrometry; ESI, electrospray ionization; FDA, US Food and Drug Administration; LDT, laboratory-developed test; LIS, laboratory infor- with GC-MS, the limitation of immunoassays for ste- mation system. roids became evident, especially when measuring low 92 Effective Use of MS in the Clinical Lab Reviews Downloaded from https://academic.oup.com/clinchem/article/62/1/92/5611766 by guest on 30 September 2021 Fig. 1. Approximate timeline of some important events in the evolution of clinical MS. concentrations of testosterone in women and children different LC systems are staggered so that the mass spec- (3, 10). A primary limitation of GC-MS was that ana- trometer is always measuring peaks of interest (Fig. 2). It lytes needed to be volatile and thus most clinical assays can also be used to analyze different compounds, poten- required multiple extraction/purification steps along tially enabling the technique to be applied in a random with a chemical derivatization to render the analytes access mode as opposed to more traditional batch analy- sufficiently volatile for analysis. The extensive sample sis. The basic principle that makes multiplexing effective preparation schemes required for GC-MS analysis lim- is that peaks of interest typically elute over a several sec- ited the widespread application of MS in the clinical ond time period during a chromatographic run, which laboratory because of low throughput and high cost. can last several minutes. Without multiplexing, most of Atmospheric pressure ionization techniques such as the time the mass spectrometer is “waiting” for peaks to electrospray ionization (ESI) combined with high- elute. By staggering injections, a multiplex with 4 LC performance LC-MS/MS were the next major analyt- systems can increase the productivity of the MS several ical improvements that enabled MS as a viable plat- fold. form for routine clinical laboratories. In the microbiology laboratory, the development of ESI LC-MS/MS eliminated the need for volatile MALDI combined with TOF mass analyzers allowed for analytes and thus helped simplify sample preparation the rapid identification analysis of microbes (12). Before schemes. Simplified sample preparation equates to im- implementation of MALDI-TOF, microbiology labora- proved sample throughput and lower costs. GC-MS as- tories depended on gram stain, culture, biochemical tests, says that took a technologist8htoprepare 50 samples and susceptibility testing. US Food and Drug Adminis- could now be done by the same technologist at much tration (FDA)-approved approaches using MALDI- higher throughput in a couple of hours when using LC- TOF have decreased the mean time to identification by MS/MS. The rate-limiting step for GC-MS analysis has 1.45 days compared with conventional techniques (13). been the sample preparation time, whereas the rate- Tan et al. estimated that implementing MALDI-TOF limiting step in LC-MS/MS typically is the LC run time would save more than 50% of the costs of reagents and of the assay. By simplifying sample preparation schemes labor compared with standard culture techniques (13). LC-MS/MS has enabled MS to be a cost-effective analyt- These authors reported on the laboratory savings, but did ical tool in the clinical laboratory. not comment on the overall healthcare savings associated A particularly effective tool for improving through- with rapid pathogen identification, which likely are sub- put of MS has been the development of multiplex assays stantial. The commercialization of MALDI-TOF con- (11). From an initial capital outlay perspective, the mass tinues for clinical microbiology and systems consisting of spectrometer is the highest-cost component (typically the MS, software, and databases of microorganisms by $200 000 to $500 000). By interfacing several (com- some manufacturers (bioMerieux Inc. and Bruker Dal- monly 2–4) LC systems (which cost $50 000 each) into tonics Inc.) have been FDA cleared. Although the FDA- a single MS, the cost-effectiveness of the entire process cleared list of organisms is not exhaustive and is primarily can be improved several fold. Multiplex LC works best limited to gram-negative and gram-positive bacteria and for single-analyte assays for which the injections of the yeast, research use–only libraries are also available (14). Clinical Chemistry 62:1 (2016) 93 Reviews and practice, MALDI imaging has been used to iden- tify proteins, peptides, drugs or metabolites, lipids, and other analytes in tissue (17). Specifically, MALDI imaging has allowed the label-free, multiplex measure- ment of a wide variety of molecules in tissue sections