During the past 15 years, liquid chromatography tandem mass spectrometry (LC-MS/MS) has evolved into a vital technology used to perform routine tests in many clinical laboratories. Historically, LC-MS/MS had been used primarily by research, pharmaceutical, or commercial laboratories; however, advances in the technology, decreasing costs for basic systems, intelligible software, an increased number of published protocols and methods, and the release of Food and Drug Administration (FDA)-approved kits has enabled more clinical laboratories to pursue these instruments as viable clinical analyzers.
LC-MS/MS is a powerful qualitative and quantitative analytical technique with a wide range of clinical applications, including therapeutic drug monitoring (TDM), toxicology, endocrinology, pediatrics, microbiology, and the emerging field of proteomics. Interest in the clinical utility of mass spectrometry continues to grow, evidenced by the ever growing number of LC-MS/MS publications and conferences, as well as the recent launch of AACC’s Mass Spectrometry and Separation Sciences Division.
The Principle of Tandem
Tandem mass spectrometry is based on coupling mass spectrometers together in a series to analyze complex mixtures. The method uses two mass filters arranged sequentially with a collision cell between them. The filters can be used in static or scanning mode to select a particular mass-to-charge (m/z) ratio or m/z range. In the collision cell, the precursor ions collide with gas molecules and are fragmented into smaller ions referred to as product ions. Tandem mass spectrometers are versatile in that they can be operated using a variety of different scan modes depending on the clinical application. Figure 1 depicts some of the commonly used MS/MS scan modes.
Advantages of LC-MS/MS
Recognizing LC-MS/MS as a strategic technology, many clinical laboratories are now using it in lieu of other methodologies. Traditionally, immunoassays have been used primarily to measure low molecular weight compounds. However, they suffer from several limitations, including problems with specificity, a lack of concordance between different manufacturers’ assays, and lot-to-lot variation from the same manufacturer due to variable cross-reactivity of antibodies. In addition, heterophilic antibodies and hook effects could limit the dynamic measuring range of many immunoassays.
On the other hand, MS/MS has superior selectivity for many analytes since it recognizes them by at least two physical properties—their precursor and product ion mass. With the introduction of soft ionization techniques, such as electrospray ionization and atmospheric pressure chemical ionization, low molecular weight molecules can be ionized in liquid phase allowing the coupling of high performance LC to MS/MS. When MS/MS is coupled with LC, the retention time adds another property to correctly identify the analyte, resulting in enhanced specificity.
Clinical laboratories have also experienced situations in which manufacturers unexpectedly withdraw immunoassays from the market, leaving labs searching frantically for alternate methods to get test results back to the ordering physician. These experiences have given labs another reason to turn to LC-MS/MS.
MS/MS also exhibits flexibility and versatility in enabling laboratories to offer novel laboratory-developed tests (LDTs) for biomarkers or for newly approved medications before FDA-approved kits or immunoassays to measure them come on the market. In addition, the sensitivity of LC-MS/MS may allow lower limits of detection for some analytes, such as steroids, compared to immunoassays and other methods.
Another benefit of LC-MS/MS is that it gives clinical laboratories the ability to multiplex, identifying and quantifying several analytes of interest simultaneously. Multiplexing lowers the cost per test. LC-MS/MS offers other cost savings and increased throughput via simplified or minimal sample preparation for some applications, such as dilute-and-shoot or protein crash, compared to more time consuming and expensive sample preparation methods like solid phase extraction or derivatization. The derivatization or chemical modification of polar compounds was originally necessary in other methodologies like gas chromatography (GC), because these compounds had to be sufficiently volatile in order to be analyzed. However, the derivatization process added sample preparation time, labor, and expense to an assay.
Despite the numerous benefits of LC-MS/MS, it is not without challenges. As a high complexity system, LC-MS/MS requires a high level of technical expertise to develop and validate LDTs as well as troubleshoot the instruments. In addition, FDA’s recently released draft guidance document on regulating LDTs could affect the majority of tests developed and performed on LC-MS/MS. Currently, laboratories develop and validate LC-MS/MS assays according to CLIA, which established quality standards for lab testing and accreditation programs for clinical labs.
Unlike FDA-approved tests, LDTs have additional analytical validation requirements including an evaluation of imprecision, accuracy, linearity, recovery, sensitivity, specificity, carry-over, ion-suppression, acceptable sample types/collection tubes/anticoagulants, storage/transport conditions, and the establishment of reference ranges. Determining the appropriate sample preparation, column choice, mobile phase, and selection of suitable internal standards are just a few of the many challenges faced during normal method development.
Furthermore, assay-to-assay variations between LC-MS/MS users can arise since LC-MS/MS assays are not standardized or harmonized and commercial calibrators are not available. However, manufacturers have started to deal with these limitations and now offer kits for immunosuppressants, steroids, and 25-hydroxyvitamin D (1). One example is the Waters MassTrak Immunosuppressant kit designed to provide the quantitative measurement of tacrolimus in whole blood for liver and kidney transplant patients.
Initially, mass spectrometry was used primarily in newborn screening and to diagnose organic metabolic disorders in clinical labs. Today, LC-MS/MS is the leading technology employed by pediatric laboratories for newborn screening programs due to its versatility, sensitivity, and specificity (2). For example, it is used to measure acylcarnitines for medium chain acyl coA dehydrogenase deficiency (MCADD) and other amino acid disorders like phenylketonuria. Table 1 shows a list of some of the tests commonly performed via LC-MS/MS.
Therapeutic drug monitoring is another discipline in which LC-MS/MS tests are abundant for the quantitative measurement of medications. Initially, many clinical laboratories adopted LC-MS/MS to measure immunosuppressant drugs like cyclosporine A, tacrolimus, sirolimus, everolimus, and/or mycophenolic acid. In some cases, alternate methodologies didn’t exist to measure sirolimus or everolimus, prompting laboratories to send out those tests to reference labs. This led many laboratories to implement LC-MS/MS as a way to cope with high sendout costs and to meet transplant physicians’ turnaround time (TAT) requirements for patient care.
Another advantage of LC-MS/MS over immunoassays for measuring immunosuppressants is the ability to look for multiple analytes in one analytical run. In patients prescribed multiple drugs—such as sirolimus in combination with cycloporin A or tacrolimus—or as a patient transitions from one immunosuppressant to another, labs using LC-MS/MS can simultaneously measure both drugs in one analysis, saving time and money. Other commonly monitored drugs include: anti-epileptics (i.e. lamotrigine, levetiracetam), anti-fungals (i.e. posaconazole), and anti-depressants (i.e. amitriptyline).
More specialized TDM applications include measuring anti-neoplastic agents like busulfan. For busulfan, blood samples are collected at various time points after administration of the drug to measure its concentration and determine the area under the curve. This pharmacokinetic testing is used to calculate the drug clearance and provide an estimated dose to minimize toxicity while achieving appropriate concentrations to sufficiently ablate the bone marrow prior to hematopoietic stem cell transplantation in both adults and children. LC-MS/MS provides a very cost-effective and rapid solution to meet pharmacists’ and oncologists’ TAT needs for busulfan.
In toxicology, GC-MS has been the most widely utilized technique for confirmatory testing for drugs of abuse and prescription medications like benzodiazepines and opioids. Now, many toxicology laboratories are converting from GC-MS to LC-MS/MS and realizing significant improvements in throughput as well as cost-savings. While the initial cost of a LC-MS/MS system is much higher than a GC-MS system, labs can use fast, simple, inexpensive sample preparation methods without the additional cost and time it takes to derivatize samples. These improvements, along with shorter analytical times and the ability to multiplex, provides extra throughput and capacity for laboratories.
Endocrinology represents another area in which LC-MS/MS plays a major role in clinical testing. Cross-reactivity/specificity issues, matrix effects, and heterophilic antibodies are just a few of the limitations that affect the clinical utility of immunoassays used in endocrinology. For example, dehydroepiandrosterone (DHEAS) is a known interferant in testosterone immunoassays due to the structural similarities between these steroids (1). In addition, LC-MS/MS has been shown to have superior performance and sensitivity at low concentrations for many sex steroids including testosterone in females and estradiol (1, 3). The Endocrine Society and American Urological Association guidelines also have highlighted the limitations of immunoassays for sex steroids and shown that mass spectrometric methods are superior (3).
Other benefits of LC-MS/MS include extended measuring ranges and the ability to measure multiple steroids at once, known as complete steroid profiles, compared to measuring individual steroids in separate immunoassays. Other relevant LC-MS/MS applications include measuring thyroxine, urinary cortisol, and 25-hydroxyvitamin D. Plasma free metanephrines represent another important test routinely performed with LC-MS/MS to presumptively diagnose catecholamine-secreting pheochromocytomas or paragangliomas.
Another expanding area for LC-MS/MS is in the field of proteomics. While LC-MS/MS typically has limits to the size of molecules it can measure (i.e. m/z 2,000), larger proteins or peptides can be digested prior to measurement. Immunoglobulins represent one class of compounds with molecular weights >150 kDa that need to be digested before LC-MS/MS analysis. However, they are key biomarkers for immunity, autoimmunity, cancer detection, and immune system function (4). Pharmaceutical companies are also using monoclonal antibodies as therapeutic agents for a wide variety of diseases. For example, infliximab is used to treat Crohn’s disease and ulcerative colitis, and therapeutic concentrations are associated with clinical response and improved prognosis. Existing immunoassays may be subject to interference by endogenous antibodies directed against infliximab, but this can be overcome using LC-MS/MS methods (5).
Overall, the future of LC-MS/MS is bright as manufacturers continue to improve, automate, and simplify the technology and make this highly complex instrumentation more like automated, FDA-approved chemistry analyzers. Eventually, more simplified, automated LC-MS/MS instruments will enable additional operators. The release of more ready-to-use, FDA-approved reagent kits for LC-MS/MS will also minimize the effort needed for method development and extensive validation, making LC-MS/MS accessible to more labs. Further improvements in sensitivity, specificity, and throughput will also facilitate more clinical applications.
While LC-MS/MS is already a mainstay in several areas of the clinical lab, new applications in metabolomics and metallomics are being investigated. Furthermore, some institutions already are using MS/MS outside of the clinical laboratory—for example, in the operating room at the Imperial College in London where the surgeon’s knife is connected to the MS to differentiate between normal and cancerous tissue (1). Such advances portend an exciting future for LC-MS/MS and for clinical laboratory professionals willing to take on the challenge of grappling with its evolution in science and technology.
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- La Marca G. Mass spectrometry in clinical chemistry: The case of newborn screening. J Pharm Biomed Anal 2014;101:174–82.
- Ketha H, Kaur S, Grebe SK, et al. Clinical applications of LC-MS sex steroid assays: Evolution of methodologies in the 21st century. Curr Opin Endocrinol Diabetes Obes 2014;21:217–26.
- Murray D, Barnidge D. Characterization of immunoglobulin by mass spectrometry with applications for the clinical laboratory. Crit Rev Clin Lab Sci 2013;50:91–102.
- Willrich MAV, Murray DL, Barnidge DR, et al. Serum infliximab quantitation by LC-MS/MS in patients treated for inflammatory disorders. Gastroenterology 2014;146:S243
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Paul J. Jannetto, PhD, DABCC, FACB, MT (ASCP), is a senior associate consultant at the Mayo Clinic in Rochester, Minnesota, where he serves as the director of both the Toxicology and Drug Monitoring Laboratory and the Metals Laboratory.