Note 51: Development and Characterization of a New Chemical Reaction Interface for the Detection of Nonradioisotopically Labeled Analytes Using Mass Spectrometry (CRIMS)

Jiuwei Teng, Yohannes Teffera, and Fred Abramson, Dept. of Pharmacology, The George Washington University, 2300 Eye Street, NW, Washington, DC 20037.

(presented at EAS '96)

INTRODUTION

We report the development of an improved microwave cavity for use in Chemical Reaction Interface Mass Spectrometry (CRIMS). CRIMS relies on mass ratio measurement for the identification of isotopically labeled species. The accurate measurement of isotope ratios eliminates the need for radioactive species as tracers. This approach has many advantages and applications particularly in the study of pharmocological agents.

Background

The CRIMS method was developed at George Washington University in the early 1980's [1-3]. The technique replaces the need for radioactive tracers by measuring the ratios of stable isotopes. In order to distinguish isotopes of different mass from molecular species, as (M+H)⁺ etc., the analytes are broken down and mixed with a reactant gas in a microwave cavity. This destroys chemical information about the original molecule but generates the same simple products for all precursors. For example, analytes containing enriched ¹³C may be detected by reaction with SO₂ to produce CO₂. The ratio of ¹²CO₂ (44 amu) and ¹³CO₂ (45 amu) may then be monitored to selectively identify ¹³C enriched analytes.

CRIMS

Chemistry

Abramson [3] and others [4-6] have developed a variety of CRIMS chemistries to distinguish between the isotopes of specific elements. These elements include: H, C, N, O, P, S, Cl, Se, and Br. The reactant gases employed include NF₃, SO₂, H₂, N₂, and HCl of which NF₃ and SO₂ appear to be the most versatile. Key to the selection of reactant gas is the generation of volatile and stable products whose masses do not overlap with other atomic or molecular species. It is preferable to generate products, as HF and DF, which have unique masses in comparison to products, as ¹H²H, which requires high mass resolution to be distinguished from ¹H₃⁺. Signals resulting from the natural abundance of the tracer isotope can easily be subtracted from the total signal to determine that part resulting solely from enrichment. For example, enriched ¹³CO₂ signal can be found from ¹³CO₂ = m/z 45 - 0.0119 * m/z 44. The factor 0.0119 accounts for both the natural ¹³C and ¹⁷O contributions.

Figure 1 - CRIMS Cavity

CRIMS Advantages

CRIMS is intended to eliminate the need for radioactive tracers by determining the ratios of stable isotopes. Radioactive tracers are currently used in a variety of industries and have the advantage that other than a small change in mass the tagged species is not chemically modified. Their primary disadvantages are the need to produce and handle radioactive material and the dangers of introducing radioactive isotopes into living organisms. This is a severe limitation in Pharmacology and Biochemistry. Other techniques that can distinguish stable isotopes include inductively coupled plasma mass spectrometry, inductively coupled and microwave-induced plasma spectroscopy, and isotope-ratio monitoring mass spectrometry. The last of these techniques is the closest analog of CRIMS. It involves the complete oxidation of the analyte in a CuO furnace and the mass spectrometric detection of CO₂ from which carbon isotopic composition can be determined. Matthews and Hayes [7] report performance for a CuO furnace similar to that of the chemical reaction interface. CRIMS has the advantages that it operates at lower pressure so that the volume of the reaction cavity has a much smaller effect on chromatographic band broadening. The CuO furnace also requires regular replacement or regeneration of the CuO. In contrast, the low pressure of the CRIMS cavity means that the consumption on reactant gas is minimal. An additional advantage of the CRIMS system is that ¹³C and ¹⁵N can be monitored simultaneously whereas the furnace chemistry requires that CO₂ be removed from the system to avoid interference between N₂ and CO. While radiochemical detection methods are normally considered highly sensitive, this is only true for off-line measurements where analysis time is not a limiting factor. In on-line measurements, as those made when analyzing chromatographic effluent, the detection time is limited to the width of the chromatographic peak and the detection limits are dramatically reduced. As a consequence, both GC/CRIMS and HPLC/CRIMS have been shown to produce substantially better signal to noise and/or resolution then on-line radiometric detection (for ¹⁴C) [3].

Figure 2 - Dissambled CRIMS cavity

Design of a CRIMS Cavity

A new CRIMS cavity is shown in Figure 1. This device is either placed inside a GC oven for GC/CRIMS or after a desolvation apparatus for HPLC/CRIMS. In either case, a transfer line (fused silica capillary) is used to transport the reaction products to a mass spectrometer. Microwave power is supplied through a gold plated sidearm (long enough to reach outside of a GC oven if needed.) The cavity is tuned via two finely threaded pole pieces located on opposite ends of the cavity. These parts are indicated in the disassembled cavity shown in Figure 2. Also shown are the reactant gas inlet and reaction tube. The (1/16 mm i.d.) reaction tube confines the chromatographic effluent and maintains a low pressure to sustain a plasma. The plasma occurs only inside of this tube since the remainder of the cavity is at atmospheric pressure. A number of improvements have been made in this version of the microwave cavity. These addressed problems that had been observed in earlier versions. First among these was poor electrical conductivity between the pole pieces and the bulk of the cavity. This contact is made along a 3/4-32 thread. When the pole piece was rotated, intermittent changes in impedance affected the tuning of the cavity. A second problem was the deterioration of the center conductor in the side arm. This piece (12 gauge Cu rod) become very hot when the cavity was in use and could be bent in the process of connecting a cable. Contact between the pole pieces and the CRIMS body was improved by the introduction of gold gaskets and a compression mechanism. This is shown in Figure 3A. It is now possible to tighten the end plates and assure good electrical contact between the body and poles. This is expected to dramatically improve the tuning process for the microwave plasma. Initial (DC) resistance measurements have been performed and these indicate that an improvement has been made. (See below.) Two changes have been made in the construction of the side arm in order to improve the lifetime of the center conductor. First, additional PTFE supports have been placed inside the arm to prevent bending of the rod when connectors are attached. This will prevent shorting between the center rod and outer tube. (An event that can damage the microwave power supply.) Second, the connection between the center rod and the induction loop has been made with a screw instead of a silver solder joint. The center rod can therefore be easily exchanged and the need to replace the entire sidearm is eliminated. A view of the side arm showing the induction loop and PTFE supports is presented in Figure 3B.

Figure 3A - CRIMS Gold Gasket Compression Mechanism

Measurements Measurements have been made of the resistance between the CRIMS body and pole pieces. This was accomplished with a Keithley 2010 multimeter and Kelvin (4 lead) probes. The results are shown in Figure 4. The relative standard deviation of the measurements improved from 90 to 44 % with the introduction of the gold gasket. The average resistance was also reduced from 15 to 0.36 milliohms. Between each measurement the pole piece was rotated and the gasket was tightened if present. While these measurements were made with DC potentials it is hoped that similar results will be observed at microwave frequencies.

Figure 3B - CRIMS Center Rod

Conclusions

A new microwave cavity for use in Chemical Reaction Interface Mass Spectrometry has been produced. This cavity includes several improvements over previous designs and is expected to be both more reliable and easier to use. The device is currently undergoing tests as part of a complete CRIMS system. It will be examined for plasma stability and component lifetime. Further results will be made available at: http://www.sisweb.com

References

1. Abramson F. P.; Markey, S. P. Proc ASMS Conf. 1982, 30, 866- 867.

2. Markey S. P.; Abramson F. P. Anal. Chem. 1982, 54, 2375-6.

3. Abramson F. P. Mass Spectrom. Rev. 1994, 13, 341-356.

4. Heppner, R. A. Anal. Chem. 1983, xx, 2170-2174.

5. Morre', J. T.; Moini, M. Biol. Mass Spectrom.1992, 21, 693-9.

6. Li, G.; Moini, M.; Rerez, F.; Ibarra, F. E.; Sandoval, D. Proc. ASMS Conf. 1994, 42, 293-4.

7. Matthews, D. E.; Hayes, J. M. Anal. Chem. 1978, 50, 1465-73.