Choosing and effectively employing analytical technology is the broader discussion that underlies the diversity of topics that appear in this installment of "MS—The Practical Art." Over the last five years, we have presented the reasoning of the practitioners we consult, to disseminate their wisdom and further our own knowledge in a number of areas such as accurate mass measurement, sample preparation, modern small-particle ultrahigh-pressure liquid chromatography (UHPLC) technology, and evaluating the viability of emerging non-LC ionization prospects.

This month, Drs. Kind and Fiehn discuss small-molecule structure elucidation (excluding peptides) using hyphenated chromatographic techniques, mass spectrometers, and other spectroscopic detectors. Their opinions help us glimpse the hardware and software challenges that we face in the near future.

In the last 20 years, no major discussion of large-scale efforts on the structure elucidation of small molecules has found its way into high-impact journals like Science and Nature. Innovative, breakthrough technologies like comprehensive gas chromatography (GC) (GC×GC), Orbitrap mass spectrometry (MS) from Thermo Fisher Scientific (San Jose, California), DART ionization from JEOL (Peabody, Massachusetts), and UHPLC separations developed from Waters Corporation (UPLC, Milford, Massachusetts) appeared largely in method-oriented journals. Yet high-impact journals targeting broad readerships frequently showcase techniques like gene sequencing and expression, which are relatively mature. Why? Results obtained by genomic analysis can be linked directly to biological activity, gene names, and protein structures and their functions. Because they can deliver results that answer biological questions, such techniques attract major research interest.

There is also the problem of difficulty. Structure elucidation of small molecules using only hyphenated techniques like GC–MS and LC–MS without nuclear magnetic resonance (NMR) spectroscopy is extremely difficult to perform. The failure 40 years ago of the Dendral project (1) is an apt example. In that project, scientists pioneered computer-assisted structure elucidation (CASE) techniques. In doing so, they laid the foundation for most of the subsequent research in the field. Yet since then, no similar project has attracted funding, this despite the increasing sophistication of software technology and computer design.

Hyphenated Chromatographic Techniques Deliver Multidimensional data

Let us first address the pure chromatographic technologies: orthogonal techniques like LC×LC, GC×GC, ultraperformance techniques like UHPLC, new separation principles, and new phases like monolithic capillary phases, hydrophilic interaction, and aqueous normal-phase chromatography. Unfortunately, those separation techniques cannot solve all of our current problems regarding structure elucidation of small molecules. Nevertheless, we must use them in conjunction with spectroscopic and MS detectors to separate complex mixtures and, finally, to obtain the true isomer structures of molecules.

We can say the same for pure MS technologies: that ultrahigh resolving power, high mass accuracy, and high isotopic abundance accuracy alone cannot solve all our structure elucidation problems. Only together with chromatographic separations do they unfold their power. The process of structure elucidation requires use of all dimensions of acquired multidimensional data. Running a GC–MS analysis on the basis of a mass spectral library search only without retention-index information is not the best approach to confirming identity when an added informational dimension is easily available. Nor is acquiring LC–MS chromatograms and neglecting MSⁿ and MS^e information because it lacks software concepts. MS^e is the practice of using high and low collision energies to enhance the spectral quality of an analyte in MS-MS while MSⁿ is a technique popularized in ion traps of successively fragmenting ions which are themselves products of a fragmentation experiment to enhance spectral quality.