The Changing Face of LC–MS: From Experts to Users - Researchers and practitioners from various disciplines and subdisciplines within chemistry, biochemistry, and physics regularly depend on mass

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The Changing Face of LC–MS: From Experts to Users

Researchers and practitioners from various disciplines and subdisciplines within chemistry, biochemistry, and physics regularly depend on mass spectrometric analysis. Pharmaceutical industry workers involved in drug discovery and development rely upon the specificity, dynamic range, and sensitivity of mass spectrometry (MS). Particularly in drug discovery, where compound identification and purity from synthesis and early pharmacokinetics are determined, MS has proved indispensable. Biochemists expand the..

Nov 1, 2008
By: Robert S. Plumb, Michael P. Balogh
Special Issues

Figure 1: Daughter ion scans obtained with (top) and without (bottom) the ScanWave function.

Two decades ago, MS was the preserve of experts and skilled technicians as the instrumentation required constant attention and adjustment. At that time, liquid chromatography (LC)–MS was in its infancy and atmospheric pressure ionization (API) source interfacing was just beginning. Samples requiring analysis were passed from the requesting scientist to these "experts for analysis." The samples would be analyzed, processed, and interpreted, and the results returned via a written report. Two decades later, the users and capabilities of LC–MS have changed significantly. Now mass spectrometers and LC–MS systems are ubiquitous in the analytical laboratory, especially in the pharmaceutical industry. These instruments are used by a wide variety of scientists for a diverse range of tasks, from purity screening in medicinal chemistry, to the quantification of drugs in blood and the identification of proteins for biomarker discovery. The usability of the current MS platforms has improved dramatically, with scientists able to operate the systems remotely via the internet, carry out complex data-dependent tasks, such as purification and peptide fragmentation to open-access systems where nonanalytical chemists can queue their samples for analysis and have the results e-mailed to them without ever having to know or concern themselves about the LC–MS process.

Figure 2: Mass spectra of alprazolam and a plasma sample.

Recent reports put the number of LC–MS systems sold per year in excess of 2500 units. This large number of units sold each year is also reflected in the increased number of users. In 1980, the number of scientists attending ASMS was around 1250. By 2002, this number had risen to greater than 4000, with a growth rate of 10% per year. This growth in LC–MS users has occurred because of the increase in the number of samples analyzed each year per user, creating larger and larger amounts of high-quality data. More and more, this data is being turned directly into information or knowledge so that decisions are made in real time. Many of these new users have little interest in becoming expert mass spectroscopists and are instead looking for the instrumentation itself to decide the appropriate experiments to be performed as well as to interpret the data automatically and recommend a course of action (for example, pass/fail, pure/impure).

The interfacing of LC with MS allowed analytical chemists access to about 80% of the chemical universe unreachable by gas chromatography (GC). It is also responsible for the phenomenal growth and interest in mass spectrometry in recent decades. A few individuals are singled out for coupling LC with MS. Beginning arguably in the 1970s, LC–MS as we know it today reached maturation in the early 1990s. Many of the devices and techniques we use today in practice are drawn directly from that time.

In its simplest form, LC relies upon the ability to predict and reproduce with great precision competing interactions between analytes in solution (the mobile or condensed phase) being passed over a bed of packed particles (the stationary phase). Development of columns packed with a variety of functional moieties in recent years and the solvent delivery systems able to precisely deliver the mobile phase has enabled LC to become the analytical backbone for many industries. Continued advances in performance since then, including development of smaller particles and greater selectivity, also saw the meaning of the acronym change to high-performance liquid chromatography. In 2004, further advances in instrumentation and column technology achieved significant increases in resolution, speed, and sensitivity in liquid chromatography. Columns packed with smaller particles (1.7 μm) and instrumentation with specialized capabilities designed to deliver the mobile phase at pressures up to 15,000 psi (1000 bar) came to be known as ultrahigh-pressure liquid chromatography (UHPLC). Much of what is embodied in this current technology was predicted by investigators such as Prof. John Knox in the 1970s.

Mass spectrometers can be smaller than a coin, or they can fill very large rooms. Although the various instrument types serve in vastly different applications, they nevertheless share certain operating fundamentals. The unit of measure has become the dalton (Da), displacing other terms such as amu. 1 Da = 1/12 of the mass of a single atom of the isotope of carbon-12 (¹² C). Once employed strictly as qualitative devices — adjuncts in determining compound identity — mass spectrometers were once considered incapable of rigorous quantitation. But in more recent times, they have proved themselves as both qualitative and quantitative instruments. A mass spectrometer can measure the mass of a molecule only after it converts the molecule to a gas-phase ion. To do so, it imparts an electrical charge to molecules and converts the resultant flux of electrically charged ions into a proportional electrical current that a data system then reads. The data system converts the current to digital information, displaying it as a mass spectrum.

The ions required in MS can be created in a number of ways suited to the target analyte in question. These methods include laser ablation of a compound dissolved in a matrix on a planar surface, such as by matrix-assisted laser desorption ionization (MALDI); by interaction with an energized particle or electron, such as in electron ionization (EI); or as part of the transport process itself, as we have come to know electrospray ionization (ESI), where the eluent from a liquid chromatograph receives a high voltage, resulting in ions from an aerosol. The ions are separated, detected, and measured according to their mass-to-charge ratios (m/z). Relative ion current (signal) is plotted versus m/z, producing a mass spectrum. Small molecules typically exhibit only a single charge. The m/z is therefore some mass (m) over 1, with the "1" being a proton added in the ionization process (represented by M+H+ or M-H+ if formed by the loss of a proton), or if the ion is formed by loss of an electron, it is represented as the radical cation [M+.]. Larger molecules can capture charges in more than one location within their structure. Small peptides typically might have two charges (M+2H+), while very large molecules have numerous sites, allowing simple algorithms to deduce the mass of the ion represented in the spectrum.

The general term atmospheric pressure ionization includes the most notable technique, ESI, which itself provides the basis for various related techniques capable of creating ions at atmospheric pressure rather than in a vacuum. The sample is dissolved in a polar solvent (typically less volatile than that used with GC) and pumped through a stainless steel capillary, which carries between 500 and 4000 V. The liquid forms an aerosol as it exits the capillary at atmospheric pressure, and the desolvating droplets shed ions that flow into the mass spectrometer, induced by the combined effects of electrostatic attraction and vacuum. The mechanism by which potential transfers from the liquid to the analyte, creating ions, remains a topic of controversy. In 1968, Malcolm Dole first proposed the charge residue mechanism, in which he hypothesized that as a droplet evaporates, its charge remains unchanged. The droplet's surface tension, ultimately unable to oppose the repulsive forces from the imposed charge, explodes into many smaller droplets. These coulombic fissions occur until droplets containing a single analyte ion remain. As the solvent evaporates from the last droplet in the reduction series, a gas-phase ion forms. In 1976, Iribarne and Thomson proposed a different model, the ion evaporation mechanism, in which small droplets form by coulombic fission, similar to the way they form in Dole's model. It is possible that the two mechanisms might actually work in concert, with the charge residue mechanism dominant for masses higher than 3000 Da, and ion evaporation dominant for lower masses.