Liquid chromatography coupled with tandem mass spectrometry (LC–MS-MS) is emerging as a complementary method to traditional methodology used for clinical applications. Reduced sample preparation and high-throughput capabilities are providing significant benefits to clinical scientists conducting routine analyses. This technology is expected to expand rapidly as scientists focus on more complicated challenges that can be solved efficiently by adding LC–MS-MS to their arsenal of techniques.

The use of mass spectrometry (MS) in clinical diagnosis goes back to the early 1970s with the application of gas chromatography (GC)–MS to the determination of a variety of biologically significant molecules. Because GC requires a certain level of analyte volatility, and since most biologically active molecules are polar, thermolabile, and involatile, elaborate extraction and derivatization protocols needed to be devised to make GC–MS useful for the analysis of clinically relevant samples. To make sample analysis less difficult by MS there had been a significant amount of R&D invested over several decades aimed at coupling high performance liquid chromatography (HPLC) with MS since HPLC is a much better separation technology than GC for polar thermolabile biologically relevant molecules. This coupling was not without significant challenges; most of the LC–MS coupling techniques that evolved during the 1970s and 1980s were not very successful, and many of those that enjoyed some widespread popularity, such as thermospray, flow-fast atom bombardment (FAB), and particle beam, are now virtually extinct. All of these techniques were quickly displaced by interfaces involving ionization techniques that separated the ionization process from the high vacuum analyzer portion of the instruments, namely atmospheric pressure ionization (API), including electrospray (ESI), ion-spray (nebulizer-assisted electrospray, ISP), atmospheric pressure chemical ionization (APCI) using a heated nebulizer, and atmospheric pressure photoionization (APPI), also using a heated nebulizer. Not until the development of API did there become a much more rapid adoption of LC–MS to the analysis of clinically important samples.

An exceptionally good review of the development of API techniques was written by Thomson (1). As the name implies, API creates ions at atmospheric pressure, outside the pristine analyzer, thereby making LC–MS very robust and relatively free of analyzer contamination. Coupling of HPLC to API-MS is trivial since the MS vacuum system is not directly involved; this relative ease-of-use permitted those researchers more familiar with biochemistry than with analytical technologies to actually use LC–MS more routinely. This caused an enormous increase in applications for LC–MS across a wide range of biologically significant molecules, including biopolymers such as polysaccharides, DNA, and proteins as well as small molecule analytes such as nucleotides, amino acids, acylcarnitines, sphingolipids, phospholipids, and biogenic amines. The vast majority of LC–MS applications for clinical research today are being run using API.

One of the features of API techniques is that it is very "soft," creating primarily intact molecular ions, which, unless very high resolution and mass accuracies are employed, are not very analytically specific. This has necessitated the use of tandem mass spectrometry, or MS-MS, to enable the analysis of trace analytes from complex biological matrices typically encountered in clinical research samples. There have been some excellent reviews on this subject (2,3).

Examples of the most prevalent uses of MS-MS in clinical research include the screening of newborns for congenital metabolic diseases such as aminoacidopathies, organic acidurias, and fatty acid oxidation disorders (4,5); multi-analyte therapeutic drug monitoring (TDM), especially for the administration of cocktail therapies involving immunosuppressants (6–8), oncology drugs (9), and anti-retrovirals (10,11); toxicant and drugs-of-abuse screening, in which samples can be screened and validated in a single run (12,13); the analysis of endogenous peptides, especially where different isoforms exist; and the analysis of steroid hormones (14). With respect to the latter, there has been a recent growing level of interest in the application of LC–MS-MS to clinical research in endocrinology to the point where the American Endocrine Society has issued a statement recommending LC–MS-MS for the determination of endogenous levels of steroid hormones such as testosterone over more traditional technologies such as immunoassays (15). The rationale for this position has been the superiority of analytical results by LC–MS-MS, especially for low levels of these analytes (16,17). The reader is referred to a recent review article on the use of LC–MS-MS for a variety of endocrinology applications (18). Despite these useful applications and the rapid growth of LC–MS-MS in clinical research, the number of LC–MS-MS systems in use in routine diagnostic laboratories is actually relatively small compared to more traditional biochemical or immunological analyzers.