Direct Sample Analysis/Time-of-Flight Mass Spectrometry

There have been many developments in mass spectrometry ionization techniques in recent years, enabling the investigation of diverse sample types across the molecular weight range. Some of these techniques allow for the ionization and analysis of samples at atmospheric pressure and without the application of high voltages. These techniques are known collectively as ambient ionization techniques.

A number of ambient ionization methodologies are now commercially available. One of these is the Direct Sample Analysis™ (DSA™) system (PerkinElmer, Waltham, MA), which is integrated with a time-of-flight (TOF) mass spectrometer. The ionization region design is shown in Figure 1. A stream of nitrogen gas is heated and ionized via a corona discharge needle in a field-free region contained within the DSA assembly. The heated and ionized gas is passed across a sample, which is supported by using either metal gauze (lower inset) or glass capillary (upper inset). Sample components with sufficient volatility are vaporized and ionized by the action of the nitrogen beam, and the resulting ions are drawn into the mass spectrometer. The ion source is equipped with a liquid inlet at the head of the probe assembly. While not essential to DSA operation, the inlet allows the user to introduce solutions that modify ionization behavior, as described herein, or to introduce internal calibration solutions. When the liquid inlet is used, an associated stream of nitrogen gas is used to nebulize the flow.

Figure 1 – Diagram of DSA ion source design. Components are described in the text. Sample supports are shown in insets, with designs presented for glass capillary tubes (upper) and metal grids (lower).

The AxION™ TOF 2 (PerkinElmer) is a high-resolution mass spectrometer, meaning it can record ion masses to better than five parts-per-million (ppm) precision relative to their expected masses, and with very high accuracy for natural isotope peak abundance determinations. Mass spectra are recorded at a high rate (up to 70 spectra per second). This is important for accurate peak intensity recording across the complete mass range, especially for relatively volatile species whose rate of desorption and ionization can be quite fast, and tend to produce transient signals. In contrast to scanning devices, such as quadrupole mass filters, TOF instruments record “all the ions, all the time” and, combined with fast spectral acquisition rates, ensure the recording of these signals. This is particularly important for quantitative measurements, as described later, and for DSA-TOF data collection, which happens in the seconds per sample time scale.

The ambient nature of DSA means that there are very few obstacles to a cursory test of almost any solid sample or solution. Unlike many other ionization techniques, small quantities of sample submitted to DSA pose little risk of impairing system performance, such as may happen with a clogged HPLC injector from incompatible samples. Thus DSA-TOF can find a place in many laboratory work flows in which high sample throughput is required, particularly where measurements are qualitative and cursory. Examples include reaction chemistry assessment and compound identification or confirmation. With data collection time on the order of 30 sec per sample or less, the time-saving impact on appropriate work flows can be quite dramatic, with potential overall savings measured in hours.

Applications in forensics analysis

Potential applications can be found in many arenas. The example shown in Figure 2 is in the field of forensics analysis and demonstrates the application of DSA-TOF to gunshot residue analysis.

Figure 2 – Positive ion DSA mass spectrum of extracted gunshot residue. Signals for methyl and ethyl centralite are clearly observed.

In this study, samples were prepared by a simple solvent extraction. This spectrum was recorded in positive ion mode, and ions corresponding to molecules known as methyl centralite and ethyl centralite are easily identified by their high-resolution mass measurements. These molecules are commonly used as burn rate modifiers and stabilizers in the production of smokeless powder.

The same extracts may be analyzed by using negative ion detection, and this is achieved with a simple switch in the polarity of the TOF. The negative ion spectrum in Figure 3 was obtained with a solvent modifier, where a solution of 2% methylene chloride in methanol was introduced at a flow rate of 10 μL/min via the DSA source’s liquid inlet. This procedure allows the generation of chloride anion from the solvent, which can then attach to molecules vaporized by the nitrogen gas that can act as acceptors. A chloride ion adduct of nitroglycerin is observed; the presence of the chloride can be seen in the expanded inset figure, showing the distinctive 3:1 isotope ratio pattern.

Figure 3 – Negative ion DSA mass spectrum of extracted gunshot residue. Chloride anion adduct signals for nitroglycerin (inset) are generated using a diluted methylene chloride infusion, as described in the text.

Once the presence of chlorine has been established, the measured accurate mass of the adduct ion is sufficient to assign the signal as derived from nitroglycerin, with the recorded m/z value within 0.1 ppm of the calculated exact mass for the chloride ion adduct. Note that nitroglycerin does not give a distinctive signal in either positive or negative ion mode in the absence of the modifying solution.

The liquid inlet may be employed in a variety of ways. The authors attempted to use DSA in a study related to food adulteration, directed toward measuring a variety of fruit and nut oils. The major fraction of these oils is made up of triglycerides, which comprise a glycerol molecule esterified with three long-chain fatty acids. The variations in their carbon chain lengths and degrees of unsaturation result in a molecular profile that is fairly characteristic of the underlying origin of the unadulterated products, while even low levels of adulteration may result in a diagnostic shift in molecular profile.

When these oils are examined using DSA, spectra typically show little or no molecular diagnostic signal. Instead, the spectra are dominated by fragment ions resulting from thermal degradation of the sample components prior to ionization. However, when the technique is modified by introducing a flow of 100 mM ammonium acetate solution at 5 μL/min via the liquid inlet, intense signals in the molecular region corresponding to ammonium ion adducts [M+NH4]+ are produced.

Figure 4 shows the resulting DSA mass spectra of commercial grape seed and olive oils. Inspection of the data reveals the distinct profiles of these oils, and using this technique, it is straightforward to assess triglyceride molecular profiles in a 20-sec measurement and to detect even quite small differences in composition among multiple samples. This is in contrast to the traditional approaches, which involve lengthy chromatography or fatty acid methyl ester (FAME)-based methods.

Figure 4 – Ammonium adduct ion mass spectra of olive oil (lower panel) and grape seed oil (upper panel), generated as described in the text. Different mass profiles distinguish samples due to varying fatty acid contents of the composite triglycerides.

Potential of ambient ionization

The potential for ambient ionization techniques to deliver quantitative data in a high-throughput manner is an exciting but also a particularly challenging opportunity. It is well known that mass spectrometry, like most analytical techniques, lacks any inherent quantitative properties. Variations in fundamental ionization properties among molecules are akin to differences in spectroscopic properties due to differing functional groups, for example.

Additional challenges in quantitation arise when multiple molecular species are exposed to a mass spectrometer’s ionization source at the same time. The potential of one molecular species to interfere with the ionization of a different species is a phenomenon commonly known as ion suppression. This can be alleviated to some extent by using appropriate chromatographic techniques, thereby introducing a temporal separation among the species that may undergo ion suppression, but even here careful assessment of the presence and extent of ion suppression may be required.

In an ambient ionization method, chromatographic separation is not employed. This offers the advantage of analytical speed, but also elevates the likelihood that suppression will occur. This implies that for most “real-world” samples, drawn from a chemical or biological matrix, quantitation using ambient methodologies poses severe challenges and requires particular care and attention.

As with MS methods hyphenated with chromatography, quantitation using DSA is best approached if appropriate internal standard compounds are available. The best standards for this type of work are analogs of the species under investigation synthesized to contain stable isotope labels, such as deuterium or carbon-13. Standards may be spiked into each sample under analysis at a known concentration, with the data then processed by comparing the ratio between labeled and nonlabeled peaks for the compound(s) of interest.

Figure 5 shows a negative ion DSA spectrum of a commercial apple juice sample, diluted 200-fold and spiked with 50-ng/μL of a D3-labeled standard of malic acid. The deprotonated signal for the natural component is observed at m/z 133.0546, with the standard observed three masses higher. The assumption is made that any ion suppression effects apply equally to labeled and nonlabeled forms, and therefore an area ratio of these two peaks allows a rapid assessment of malic acid concentration.

Figure 5 – Quantitation DSA mass spectrum of malic acid in apple juice. Intensity abundance ratio between the ion at m/z 133 and m/z 136 (D3-labeled malic acid spiked into juice) allows single-point quantitation.

These examples show that the DSA ionization source coupled with the AxION 2 TOF mass spectrometer can provide rapid interrogation of many sample types under ambient conditions. Actual analysis time for each sample is in the 15- to 60-sec range, depending on the sample type. For some molecules, desorption profiles can be short, on the order of 1 or 2 sec. To accurately record these profiles requires an integrating detector, such as a TOF-MS device, recording all of the ions, all of the time. The technique is well suited to samples requiring a qualitative assessment, but quantitation is approachable under controlled circumstances.

Conclusion

The availability of isotope labeled standards is highly beneficial to quantitation design, and fortunately standards for many commonly studied species are commercially available, because the same standards are employed for quantitation using GC/MS and LC/MS techniques. The AxION DSA offers the advantage of rapid analysis on the order of seconds per sample, reduced experimental complexity, minimal solvent consumption, and elimination of the need for LC systems when DSA methods are able to substitute for LC/MS approaches.

Dr. Andrew Tyler, Ph.D., is Global Field Technical Lead for mass spectrometry, and Avinash Dalmia, Ph.D., is a Staff Scientist, PerkinElmer, Inc., 940 Winter St., Waltham, MA 02451, U.S.A.; tel.: 781-663-5512; e-mail: andrew.tyler@perkinelmer.com.

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