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.