Accurate determination of the presence, identity, concentration, and/or quantity of an analyte in a sample is critically important in many fields. Many techniques used in such analyses involve ionization of species in a fluid sample prior to introduction into the analytical equipment employed. The choice of ionization method will depend on the nature of the sample and the analytical technique used, and many ionization methods are available. Mass spectrometry is a well-established analytical technique in which sample molecules are ionized and the resulting ions then sorted by mass-to-charge ratio. In one technique, the degree of deflection of the ionized particles in a magnetic field is measured, from which the relative masses of ions can be calculated. In another technique, referred to as “time of flight” (TOF) mass spectrometry, ions are accelerated in an electric field, and their velocity determined using a time measurement. The mass-to-charge ratio can be readily determined, as it is proportional to an ion's velocity. Mass spectrometry has a number of significant applications, enabling the analysis of a wide range of molecular species, from drug-like small molecules to large proteins and cellular metabolites. The ability to couple mass spectrometric analysis, particularly electrospray mass spectrometric analysis, to separation techniques, such as liquid chromatography (LC), including high performance liquid chromatography (HPLC), capillary electrophoresis, or capillary electrochromatography, has meant that complex mixtures can be separated and characterized in a single process. The technology has been applied across a wide range of disciplines and has enabled advances in areas such as drug metabolism, proteomics, metabolomics, lipidomics, and imaging.
While the application areas for mass spectrometry have increased, its basic throughput has not changed significantly over the same time frame. This is predominantly due to the requirement that samples be subjected to a separation technique prior to introduction to the mass spectrometer to isolate analytes of interest from signal-suppressing matrix components. Improvements in HPLC system design, such as reductions in dead volumes and an increase in pumping pressure, have enabled the benefits of smaller columns containing smaller particles, improved separation, and faster run time to be realized. Despite these improvements, the time required for sample separation is still around one minute. Even if real separation is not required, the mechanics of loading samples into the mass spectrometer still limit sample loading time to about ten seconds per sample.
There has been some success in improving throughput performance. Simplifying sample processing by using solid-phase extraction, rather than traditional chromatography, to remove salts can reduce pre-injection times to under ten seconds per sample from the minutes per sample required for HPLC. However, the increase in sampling speed comes at the cost of sensitivity. Furthermore, the time saved by the increase in sampling speed is offset by the need for cleanup between samples. Processing samples in parallel using multiplexed chromatography systems can increase throughput, but the complexity of this approach can negatively impact system reliability and often preclude its use in high throughput screening (HTS) environments.
Techniques that rely on a laser to deliver ionization energy and free analytes from the sample matrix also offer some improvements in speed. In matrix-assisted laser desorption ionization (MALDI), ionization of the sample is a secondary process where laser energy is absorbed by either nanostructures in the plate surface topography or a matrix molecule. These excited molecules in turn ionize the target molecule via charge transfer. MALDI works well for peptides, small proteins, lipids, and oligonucleotides and can be performed at speeds of one second per sample but underperforms for a wide range of small molecules. A related technique, laser diode thermal desorption (LDTD), desorbs sample directly into the gas phase via a thermal pathway. However, application of LDTD in the literature has been mainly aimed at small drug-like molecules, as the thermal nature of this technique and the use of an ambient pressure chemical ionization (APCI) system make it unsuitable for both modified peptides and cellular metabolites in biochemical screening. Additionally, LDTD is slower than MALDI, requiring around ten seconds per sample.
Another limitation of current mass spectrometer loading processes is the problem of carryover between samples, which necessitates a cleaning step after each sample is loaded to avoid contamination of a subsequent sample with a residual amount of analyte in the prior sample. This requires time and adds a step to the process, complicating rather than streamlining the analysis.
Additional limitations of current mass spectrometers when used to process complex samples, such as biological fluids, are unwanted “matrix effects,” phenomena that result from the presence of matrix components (e.g., natural matrix components such as cellular matrix components, or contaminants inherent in some materials such as plastics) and adversely affect detection capability, precision, and/or accuracy for the analyte of interest. One such phenomenon is matrix ionization suppression, in which the presence of matrix components reduces the extent of analyte ionization and is observed as a loss in response. See, e.g., Volmer et al. (2006) LCGC North America 24(5):498-510, and Bruins et al. (1999) J. Chromatogr., A 863:115-122.
Several of the aforementioned limitations have been addressed by using acoustic droplet ejection (ADE) to deliver small amounts of a fluid sample from individual microtiter plate wells to a mass spectrometer or other analytical device. See Sinclair et al. (2016) Journal of Laboratory Automation 21(1):19-26 and U.S. Pat. No. 7,405,395 to Ellson et al. (Labcyte Inc., San Jose, Calif.), both of which are incorporated by reference in their entireties. Sinclair describes the application of an ultrasonic pulse to a well containing a sample of interest, generating a mist of tiny droplets that are ionized via application of an electric field and transported via a heated transport tube into a mass spectrometer. Use of the ADE process and equipment (Labcyte's Echo® 555, modified to couple to the input end of a mass spectrometer) was established to generate a signal from as little as three nanoliters of sample and enable the acquisition of over 10,000 data points per hour. While the sensitivity and speed of the ADE-based process were significant, and capable of supporting high-throughput screening, IC50 determination, and kinetic studies, some limitations remained. In particular, as noted by Sinclair et al., potential matrix effects can still be problematic. Additionally, for applications in which a consistent droplet size is necessary or desirable, the acoustic mist approach is less than ideal, insofar as droplets with different sizes are generated by a single acoustic burst.
An ideal system for loading a mass spectrometer or other analytical device would provide a process that is even faster than that described in Ellson et al. '395, and which would completely eliminate matrix ion suppression as well as the need for a cleaning step between sample loading events.