Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen "enormous growth" in clinical labs during the last 10-15 years, according to a February 2011 article in Clinical Biochemist Reviews. The authors say LC-MS/MS's analytical specificity is superior to that of immunoassays and conventional high-performance pressure liquid chromatography (HPLC) for low-molecular-weight analytes and has higher throughput than gas chromatography-mass spectrometry (GC-MS).
Lisa Sapp, senior product manager of clinical research business at AB SCIEX, says her company has seen an increased interest in mass spectrometry in general because of its ability to accurately identify/quantify minute amounts of compounds in complex matrices such as blood, urine and oral fluid, and due to its low cost per sample since only small quantities of reagents are needed.
Mass spectrometers are considered general-purpose laboratory instruments for research use only, but this may change in the not-too-distant future. "Our customers are saying, 'We need regulatory-approved mass spectrometers,' and so we're putting the infrastructure in place to get our instruments cleared for use by regulatory bodies," Sapp says.
Initially, only large reference laboratories had the resources to bring in mass spectrometry, but small to mid-size laboratories are becoming adopters of the technology for the long-term cost benefits and because easier-to-use, more automated software is making this possible. AB SCIEX's Cliquid® software is a four-step approach to getting samples acquired and reports generated.
In addition to cost benefits, mass spectrometry offers many operational advantages, says Neil Leaver, consultant head of the Immunosuppression Monitoring Service and UK National Monitoring Service for Sirolimus at Royal Brompton & Harefield NHS Foundation Trusts, whose lab uses Triple Stage Quadrupole LC-MS systems from Thermo Scientific. Leaver says 25-hydroxy Vitamin D test requests "seem to be arriving exponentially," and mass spectrometry can provide more information to clinicians than traditional commercial immunoassay. For example LC-MS/MS offers data to differentiate the concentration of Vitamin D patients are producing naturally from the concentrations of supplements.
In addition to Vitamin D, analysis of trace hormones, steroid panels, drugs of abuse and immunosuppressant drugs are key applications for many new users of mass spectrometry.
Newborn screening continues to be one of the most prevalent uses of LC-MS/MS1-particularly for investigation of inborn error in metabolism that can lead to serious problems (e.g., enlarged heart/liver and brain swelling) down the road if not treated. Advantages to using LC-MS/MS for newborn screening include the ability to analyze all 46 amino acid and acylcarnitine markers in a rapid, inexpensive and specific way.
Leaver's lab implemented the first therapeutic drug monitoring program for immunosuppressives using mass spectrometry in the UK in the late 1990s. "In those days, HPLC was the reference method, and one commercial immunoassay method was well-documented as having over 45% positive bias relative to the reference method, so it was cross-reacting with non-active metabolites, and there was a slight calibration bias. This resulted in overestimation of the levels of immunosuppressant [activity], and it also meant that you could never really compare results between hospitals using different assays because it all depended on the specificity of the monoclonal antibody used in each institution. So mass spectrometry (LC-MS/MS) was quite a big thing for us because we could pick out the active parent drug from everything else."
More recently, Leaver's lab expanded into vitamin D and antifungal drugs and is exploring new monitoring strategies. Leaver likes that mass spectrometry allows him total control material over his assay system. "The only thing we contract out to a third party is the manufacture of calibration and quality control material," Leaver says. "We're totally MS based."
One disadvantage of LC-MS/MS has been that, though sample throughput is higher than conventional HPLC or GC-MS, it lags behind automated immunoassays primarily because of upfront sample cleanup and due to the fact that it requires samples to be analyzed sequentially. Helping to speed up analysis are ultra-high performance LC methods and automated sample preparation. Techniques improving throughput are said to be direct sample preparation (eliminating the need for sample dilution), LC multiplexing (or using multiple LC systems to stagger runs) and sample multiplexing (automatically detecting multiple reaction monitoring transitions in the run).
Another obstacle-the need for greater specificity and sensitivity-has been aided by pre-analysis HPLC sample cleanup to remove potential interferences, and chromatography optimization for another dimension of separation and selectivity.
Peptide/protein analysis may be the next application on the horizon for LC-MS/MS. Due to their role in structural, catalytic and signaling functions, peptides and proteins have value for discovery/validation of potential biomarkers to signal disease onset or progression.
Jill Hoffman is managing editor.
Grebe S, Singh RJ. LC-MS/MS in the Clinical Laboratory - Where to From Here? Clin Biochem Rev 2011;32(1):5-31.
Cliquid® Software for Routine Screening and Quantitation from AB SCIEX is designed to work in conjunction with Analyst® Software Version to provide a simple four-step workflow for routine analysis by LC/MS/MS. The software can be used with any AB SCIEX QTRAP® or triple quadrupole LC/MS/MS. While Cliquid® Software itself does not include any preconfigured tests, a number of preconfigured iMethod™ Tests, including libraries and MRM catalogues, are available for clinical research analysis.
Thermo Fisher Scientific Inc.
Thermo Fisher Scientific Inc. offers clinical research laboratories the Thermo Scientific Exactive high performance benchtop LC-MS system powered by Orbitrap™ technology. The Exactive™ can be combined with Thermo Scientific TraceFinder software to achieve ease of integration into laboratory workflows with minimal user training; accurate mass spectral data and multiple forms of compound identification integrated into a single software solution. Advanced software algorithms include parameter-less peak detection, automated component detection, isotope pattern matching with intelligent elemental composition calculation and reverse library search. The product is for research use only.
No matter the application or matrices, Perkin Elmer's liquid chromatography and mass spectrometry systems deliver the speed, sensitivity, wide dynamic range and mass accuracy needed for confidence in results. A suite of solutions is specifically designed to provide rapid and accurate mass identification and quantification across a broad range of samples. The Flexar™ SQ 300 MS detector was designed for HPLC and UHPLC applications and incorporates proprietary ion source and ion guide technologies, yielding high performance for LC/MS applications.