Chapter 6 Mass spectrometry: Isotope ratio mass spectrometry

119

CHAPTER 6

MASS SPECTROMETRY: ISOTOPE RATIO MASS SPECTROMETRY

THOMAS R. BROWNE ~, GEORGE K. SZABO ~ and ALFRED AJAMI 2 1Departments of Neurology and Pharmacology, Boston University School of Medicine; Neurology Service, Boston Department of Veterans Affairs Medical Center; 2Tracer Technologies, Inc.

Isotope ratio mass spectrometry (IRMS) determines the ratio of isotopes of a given element (e.g. H: 2H, 12C: 13C, ~4N: ~SN) present in a specimen. Typically, the total amount of each isotope also is determined. The amount of element derived from labeled drug in a specimen can be computed from the excess (over normal abundance) in labeled element isotope ratio and the total amount of element. The instruments that are traditionally associated with IRMS use multi-collector mass spectrometers that have the precision necessary to measure very small differences in isotope ratios that arise in nature. Thus, IRMS offers the potential of detecting and quantitating added label in any whole biological matrix. In pharmacologic studies, this procedure offers simplicity and flexibility similar to radioactive tracer procedures without the administrative and safely concerns of radioactive studies. Historically, whole biologcal matrix samples were combusted to elemental gases off-line, and manually collected and transfered to a dual inlet IRMS system. Two variant types of IRMS have been applied to pharmacologic research: continuous flow-isotope ratio mass spectrometry (CF-IRMS) and chemical reaction interface mass spectrometry (CRIMS). CF-IRMS uses a multi-collector mass spectrometer to measure isotope ratios of combusted gas species in a continuous flow of inert gas, while CRIMS uses traditional selected ion monitoring mass spectrometry (SIM-MS) measuring isotope ratios of small microwaveinduced plasma ion species. Neither of these techneques are classic IRMS, however, both are based on the concept of isotope ratio monitoring of tracer excess above natural abundance in the biomatrix. Both methods have been applied to mass balance/metabolite identification studies (see Chapter 11).

120 Both methods report promising preliminary results, but neither method is fully validated (see Chapter 11).

1. CONTINUOUS FLOW-ISOTOPE RATIO MASS SPECTROMETRY (CF-IRMS) 1.1. Technique

In CF-IRMS (Figure 1) organic sample is introduced into the high temperature combustion chamber of an elemental analyzer and flash combusted with a pulse of oxygen. The oxidized combustion gases (C02, N2, NOx and H20) are carried by a continuous stream of helium (hence, "continuous flow") through clean-up phases to a gas chromatography-isotope ratio mass spectrometry (GC-IRMS) device (eliminating the problematic step of transferring by hand combustion gas species to IRMS). NOx is reduced to N2 in a reduction tube (Cu); H20 is removed in an H20 trap; and C02 is removed (for N2 determina-

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121 tions) in a C02 trap. N2 and C02 are separated on a GC column and carried to an ion source where an ion beam is generated for each combustion gas. In the magnetic sector of the MS, the beam is separated into multiple beams depending on the mass-to-charge ratio of the various isotopically labeled ions. The beam for N2 is separated into three ion beams in the magnetic sector, and ion currents are measured at preset masses (m/z 28, 29, 30) in Faraday cup collectors. The intensity of ions at these m/z values are used to calculate 14N/14N, 15N/14N and 1SN/15N ratios. The mass spectrometer data system also calculates total nitrogen. In a biological sample containing a 15N tracer, an increase in masses 29 and 30 above natural abundance (atom percent) will correlate with the concentration of enriched tracer as atom percent excess (APE). Similar considerations apply for 13C (1) except that the mass spectrometer is tuned to measure m/z 44([12C02]), 45([13C 02] + [12C 170 160]) and 46([12C 180 160]), 1.2. H i s t o r y

IRMS with 15N and 13C tracers has been used extensively in the past for environmental, metabolic and nutritional studies but has been used little for pharmacokinetic studies (1-8). Hachey et al. (1) provided an excellent review of technical considerations of IRMS for nutrition and biomedical research. Benedetti and Pataky (4) measured the elimination of lSN-labeled urea in rats using an early spectroscopic analytical method. In 1974, Von Unruh et al. (9) and in 1976 Sano et al. (10) each demonstrated mass balance and metabolite identification techniques with 13C, labeled aspirin in human urine using combustion/mass spectrometric isotope ratio measurements. In 1985, Nakagawa et al. (11) performed a successful IRMS mass balance study of antipyrine in rats using 15N and 13C labeling. These studies did not lead immediately to routine animal or human applications, presumably because of the problems with early IRMS methods discussed below. Goodman and Brenna (12) described a high precision (at natural abundance) gas chromatographic combustion-isotope ratio mass spectrometric (GCC-IRMS) system capable of performing metabolite identification and mass balance for pharmaceuticals. Similar to the GC-pyrolizer-MS system used earlier by Sano et al. (10), the GCCIRMS system relies on isotope ratio measurements from combusted GC peaks. However, this is a problem in metabolite studies where thermolabile polar conjugates are present. Inadequate derivitization of nonvolatile drugs and metabolites would also be a problem with methods that used GC as the preliminary separation technique. These issues would also prove to be limitations for early GC-CRIMS methods. In 1993, Browne and coworkers (3,

122 13-16) were the first to provide evidence that stable isotope labeling and newly developed, commercially available CF-IRMS instruments could be used for whole matrix human mass balance studies and detection of labeled liquid chromatographic (LC) peaks for metabolite identification studies. However, it should be noted that LC peaks were individually collected and processed offline as discrete samples for CF-IRMS analysis. The optimal instrument would directly interface an HPLC to a continuous flow combustion isotope ratio mass analyser. Classic IRMS instrumentation was problematic because: (1) each instrument was unique and "made by hand"; (2) complete liberation of all atoms of a given molecule by manual oxidation techniques was difficult; (3) transfer by hand of N2 and CO2 gases from Dumas combustion techniques to IRMS was problematic; (4) each specimen was run manually (lack of automation); and (5) factors 1-4 made IRMS difficult and inconsistent. Recently, refined commercially-available instruments using a helium carrier gas to carry combustion products to the IRMS have become available from three sources (Europa Scientific, Ltd.; Finnegan MAT; and Micromass, Inc.). The authors purchased a Europa (Europa Scientific, Inc., Franklin, Ohio, USA) ANCL-SL (elemental analyzer) 20/20 (mass analyzer) CF-IRMS and found the instrument performed up to specifications and with very high precision as delivered (see Chapter 11 ).

1.3. Assumptions, Validation, Applications, Advantages and Disadvantages These topics are covered for pharmacologic applications of CF-IRMS in Chapter 11.

2. CHEMICAL REACTION INTERFACE MASS SPECTROMETRY (CRIMS)

2.1. Technique In CRIMS (Figure 2), the effluent molecules from a gas chromatograph (GC) or HPLC column (after solvent removal) are converted to low molecular weight ionic species, using a reactant gas in a microwave induced helium plasma with sufficient energy to break all chemical bonds (17-26). In the presence of an abundance of the reactant gas, the atoms of each analyte recombine into a common set of simple molecular species. The labeled and unlabeled species of small molecules are then detected and quantitated by selected ion monitoring (SIM) with either a magnetic sector or quadrupole mass spectrometer or

123

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an isotope ratio mass spectrometer (19). For example, in deuterated drug studies, H2 is used as a reductive reactant gas. The resulting CRIMS products are: HD (from deuterium labeling), CH4, HCN, H2S, H20 and C2H2. However, an MS with sufficient resolving power is required to measure HD at m/z 3.022 (18, 20). Strongly oxidizing reactant gases such as S02 are more useful for ~3C and ~5N isotopic detection and SIM can be performed at m/z 45 (~3C02) and m/z 31 (15N0) on a conventional quadrapole MS. Element selective detection (e.g. CL, Br, S, P, Se) is also possible with CRIMS methods for drug and metabolite molecules that contain these elements. Because of the inability of GC to analyse underivatized nonvolatile/thermolabile compounds (e.g. drug conjugates), only HPLC-CRIMS is satisfactory as a general method for mass balance/metabolite identification studies of new drugs with unknown metabolites.

2.2. History Recently, Abramson (20) provided an extensive review of the developments in CRIMS technology. CRIMS was introduced in 1982 (21) for use in conjunction with GC. In the following years a number of studies were performed using GC-CRIMS and deuterium, 13C and 15N labeling (22-24). The first attempt to combine HPLC with CRIMS utilized a moving belt interface (25). Later, a particle beam LC-MS interface (the Vestec Universal Interface) was used to join HPLC with CRIMS (26). This is the current state-of-the-art HPLC-CRIMS instrument which has been successfully applied to separation and detection

124 of deuterium, 13C and 15N labeled drugs (17-18, 26). More recently, postcolumn modifications to the HPLC interface have improved sensitivity such that LC-CRIMS had superior metabolite identification over on-line radiometric ~4C detection of a doubly labeled (~4C ~5N) anxiolytic drug (buspirone) in bile and urine (27-29). The principle need for post-column modifications of the particle beam LC-MS interface arises from the fact that HPLC gradient elution techniques are necessary to resolve polar metabolites. Unfortunately, aqueous mobile phase containing polar metabolites tends not to form uniform finely nebulized particles that readily desolvate in the universal interface. Post-column addition of an organic modifier (e.g. methanol) to the LC eluent stream reduces the variability of CRIMS response and improves instrument sensitivity for quantitation (27). This modification is critical for LC-CRIMS mass balance studies since accurate mass balance quantitation by this technique requires the measurement of the sum of all the possible labled peaks in a given sample. This is not a trivial point since each sample analyzed by LCCRIMS will have labeled peaks that cover a wide dynamic range of isotope ratio measurments. Thus, a drug that forms many minor metabolites may give total mass balance quantitation errors simply due to a lack of sensitivity of individual isotope ratio measurments for minor metabolites. As these technical details are obviated, LC-CRIMS promises to be an exciting and powerful technique for pharmacologic tracer studies.

2.3. Assumptions, Validation, Applications, Advantages and Disadvantages These topics are covered for pharmacologic applications of CRIMS in Chapter 11.

ACKNOWLEDGMENTS Supported by the United States Department of Veterans Affairs. We wish to thank Fred P. Abramson, Ph.D. for a critical review of this paper.

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