Driving high sensitivity in biomolecular MS: improving this key parameter enables novel applications and powers additional techniques.

Mass spectrometry has become a key tool for the analysis of biomolecules in applications that range from discovery of biomarkers to the analysis of drug metabolites. While many of the analytical benchmarks of the mass spectrometer - limit of quantitation or LOQ, signal-to-noise or S/N, dynamic range, mass resolution - are important for these applications, the absolute sensitivity is a key factor that drives these characteristics.

As each new product with added sensitivity is introduced to the marketplace, new applications are enabled, and existing applications can be done more rapidly or with more confidence and better precision. High sensitivity also enables the use of additional techniques that provide improved analytical specificity - techniques such as higher mass resolution, ion mobility/mass spectrometry combinations, or added levels of tandem mass spectrometry (MS/MS/MS).

Although LC/MS systems were introduced in the 1970s by some vendors, the technique was not widely adopted until atmospheric pressure ion sources such as electrospray and atmospheric pressure chemical ionication became available in the 1980s.

Introduced in 1982, the TAGA 6000 was the first commercial triple quadrupole, and ion evaporation was the first electrospray-like LC/MS interface. Together they enabled LC/MS at liquid flow rates that were compatible with standard LC systems, with ion sources that were optimised for the analysis of polar biomolecules.

The technique of LC/MS/MS with multiple-reaction monitoring (MRM) began to be adopted as a standard method of quantitative analysis by the pharmaceutical industry during the late 1980s and early 1990s, and from that time onward the continual need for greater sensitivity began to drive development of improved methods.

Improved Sensitivity

Figure 1: The growth in sensitivity of high-flow LC/MS mass spectrometer systems over the last 30 years.

Figure 1 charts the growth of sensitivity, beginning in 1982, for high-flow LC/MS instruments (at flow rates of ~1 mL/min) from AB SCIEX. Plotted as absolute sensitivity on the logarithmic vertical axis (normalised to the data point at 1982), each data point represents another product or product platform, and it shows an increase by a factor of nearly one million over this time period. This is a rate that exceeds the technology benchmark growth rate of Moore’s Law of transistors (doubling the density approximately every two years).

In 1982, it typically required an injection of nanogram amounts of a compound to obtain good signal-to-noise. Today, as shown in Figure 2, we are able to quantitatively measure injected amounts of drugs or metabolites at the sub-femtogram level. This represents only ~0.001 part-per-trillion (ppt) concentration in the 1 mL/minute of solvent flowing from the LC into the ion source. Considering the challenges in ionising, desolvating, sampling ions through an orifice, focusing, mass filtering, and detecting, this is an exquisite level of sensitivity that would not have been imagined during the early days of LC/MS development.

Figure 2: Signal from 500 attograms of verapamil injected on-column monitored in MRM mode using the transition 455/165.

The evolution is a result of developments in many areas of the instrument. Significant improvements have been made in creating more ions in the source region by more effectively evaporating the droplets and desolvating the ions in the atmosphere-to-vacuum interface. However, ions in the source need to be sampled through a small aperture into the vacuum system, where they are focused into the entrance of the mass spectrometer.

The sampling aperture typically represents the largest area of ion losses. Consequently, increasing the size of the aperture results in better ion transfer and increased sensitivity. The aperture diameter - or in some instruments, the diameter of the tube through which ions are sampled - has increased from 0.125 mm in 1982 up to a diameter of greater than 0.5 mm on many commercial systems today. This provides more ions, but also requires more efficient vacuum system designs to handle the gas load.

Figure 3: Cross-sectional view of the IonDrive QJet showing the supersonic free jet (supersonic flow region in red) and the gas flow along the axis.

With as many ions as possible sampled through the aperture, the next challenge is to focus them into the mass spectrometer. A larger aperture results in higher gas flow and higher pressure in the first vacuum stage. The gas expands in a supersonic free jet through the aperture, forming a characteristic barrel-shock structure as shown in Figure 3.

The high gas flow and pressure provide a strong drag force on the ions, carrying the ions down the axis at high speeds making conventional focusing optics ineffective.

Radio-frequency (RF) dynamic focusing fields (so-called RF ion guides) have been developed over many years to provide ion focusing in this pressure regime. At higher pressure, there is a need for better ion guides that contain and focus ions. Some designs employ ion funnels or ring guides, which use a series of aperture lenses to converge the ion beam.

RF Quadrupole

In AB SCIEX systems, we have developed the QJet ion guide, a form of RF quadrupole that focuses the ions while allowing the gas to be pumped away. For the AB SCIEX QTRAP 6500 System, our new and highest-performance instrument, we have developed a two-section QJet that captures the ion beam from the first orifice and focuses it through the following aperture with an efficiency of approximately 50%, an impressive figure of merit under the circumstances.

As indicated by the growth curve in Figure 1, the current level of sensitivity achieved by the QTRAP 6500 System represents a significant improvement over the earlier generation AB SCIEX QTRAP 5500 system. This improvement is due to the increased efficiency provided by the heating and desolvation in the IonDrive Turbo V ion source combined with the larger orifice and two-section IonDrive QJet described above.

An important development in this product has also been to increase the dynamic range of the pulse-counting detection system so that count rates of greater than 50 million ions per second can be measured without saturation. This is a major improvement over the more conventional pulse-counting detection systems that were limited to an upper count rate of only about 5 million ions per second.

The combination of a high-dynamic range continuous-dynode multiplier and a new amplifier that provides low noise and allows operation of the multiplier at a relatively low gain has resulted in a dynamic range of greater than six orders of magnitude, which now better matches the dynamic range of the ion source.

As the analytical needs of the bioanalysis community continue to increase, more and better approaches to generating, sampling, and detecting ions by mass spectrometry will be required. However, the growth curve of sensitivity will become more and more difficult to maintain as we approach the limit of measuring and detecting nearly every ion injected.

The most significant challenges ahead are in developing mass spectrometry techniques that are more selective (to reduce noise from isobaric interferences), faster, and more cost efficient. The need for creative physicists, chemists and engineers to answer this challenge in instrument design will continue.