Thomas D. McClure

The mass spectrometry of taxol.

The antitumor agent taxol has been examined by electron ionization, chemical ionization, and fast atom bombardment mass spectrometry. Three ion series are observed: (1) the M-series, characteristic of the intact molecule; (2) the T-series, with fragments derived from the taxane ring; and (3) the S-series representing the C-13 side chain. Neutral losses dominate each series of ions and serve to verify the presence and number of functionalities in each portion of the molecule. Fragmentation pathways and mechanisms of ion formation are proposed on the basis of product ion analysis and accurate mass measurements.

Analysis of Urinary Nucleosides

Conventional diagnosis, disease staging, and evaluation of therapy for cancer and acquired immune deficiency syndrome (AIDS) rely heavily on clinically observable measurements rather than biochemical tests. In the case of cancer, these measurements include tumor imaging and tissue biopsy and are accurate only after many cancer cells have been produced as a consequence of disease progression. A diagnosis of AIDS is established by detection of host-produced antibodies to the human immunodeficiency virus (HIV), whereas staging, measurement of response to therapy, and determining the type and severity of opportunistic infections (OI) suffered by AIDS patients is determined by measurement of the T- and CD-4 cell populations (1). For both cancer and AIDS, these methods can be unreliable; also, in some instances, they are painful and increase risk to the patient. For example, detection of mesothelioma (cancer of the pleural covering of the lung, most often the result of exposure to asbestos fiber) is an invasive procedure requiring rib resection to obtain tissue biopsy for cytological analysis. A dangerous level of unreliability in detecting HIV infection has been observed and results in a long lead time between HIV infection and production of antibodies. In a recent study (2), patients were shown to be infected with HIV for as long as 35 months before antibodies were detected. As a result of these deficiencies in clinical test methods, considerable research effort has been expended toward finding alternative indicators or “biomarkers” of cancer and AIDS.

Pharmacokinetic studies of the herbicide and antitumor compound oryzalin in mice

Oryzalin [3,5-dinitro-N,N-di(n-propyl)benzensulfanilamide] is a widely used sulfonamide herbicide that selectively inhibits microtubule formation in algae and higher plants. Oryzalin has also been found to be an inhibitor of intracellular free Ca2+ signaling in mammalian cells and to have antitumor activity in animals. Despite its widespread use there have been no reports of the pharmacokinetics of oryzalin in animals or man. A reversed-phase high-performance liquid chromatographic (HPLC) method was developed to measure oryzalin in biological fluids. Following repeated daily administration of oryzalin to mice by the i.p. route at 200 mg/kg, or the p.o. route at 300 mg/kg, peak plasma concentrations of up to 25 micrograms/ml were achieved. The half life for oryzalin in plasma of mice given i.p. oryzalin was 14.3 h with a clearance of 0.07 l/h. A major metabolite of oryzalin, N-depropyloryzalin, was identified in plasma and its structure confirmed by mass spectral analysis (M+H+ = 305). This metabolite was cleared more rapidly than oryzalin with a half life of 1.15 h and a clearance of 0.17 l/h. N-Depropyloryzalin caused similar inhibition of colony formation by HT-29 colon cancer cells as oryzalin with IC50 = 8 micrograms/ml. The results suggest that oryzalin and its N-depropyl metabolite can inhibit tumor colony formation at pharmacologically achievable levels.

Identification and Synthesis Of 5′-Deoxyxanthosine, a Novel Nucleoside in Normal Human Urine1

Abstract Methods used in the identification and synthesis of 5′-deoxyxanthosine (5′-dX), a novel nucleoside found in normal human urine, are described. The urine sample was separated into nucleoside components by HPLC. Low and high resolution GC/MS techniques were then used to assign a tentative structure of the new nucleoside as 5′-dX. Finally, after biochemical synthesis, a comparison of the reference material with the urinary sample using mass spectrometry confirmed the structure as 5′-dX.

3-Methyluridine in Normal Human Urine

Abstract 3-Methyluridine has been identified in normal human urine using gas chromatography mass spectrometry, confirming an earlier report of the presence of this modified nucleoside as a natural component of human biological fluids. Variable recoveries and the lack of quantitative data concerning normal or disease state levels of this compound make selection of 3-methyluridine a poor choice as an internal standard for the quantitation of other nucleosides in biological matrices.

Computerized Interpretation of the Electron Ionization Mass Spectra of Nucleoside TMS Derivatives

Abstract A computer program has been developed for the automated interpretation of mass spectra of TMS derivatives of nucleosides found in human urine. The m/z values in the unknown spectrum are compared to m/z values of 3 different ion series commonly observed in the mass spectra of nucleoside TMS derivatives.1 If a correlation exists, the unknown spectra are marked with color according to the scheme: 1) blue—molecular ion series, 2) red—base ion series and 3) yellow—sugar ion series. The program suggests a structural assignment for each of the marked ions and calculates a series related ion current. The calculated ion current is used to assign the of sugar contained in the unknown nucleoside.

Carotenoid Antioxidant Chemistry

β-Carotene and other carotenoids are thought to exert a protective effect on important biomolecules in vivo by trapping reactive radical species. The mechanisms by which carotenoids trap free radicals are not well understood, in part due to the unstable nature of intermediates formed in the radical-trapping reactions. We have developed mass spectrometry methods to characterize and measure relative concentrations of products formed by the reaction of β-carotene with free radicals. These methods require a minimum of sample handling and workup. We have used them to monitor the formation of β-carotene oxides and chain cleavage products in model systems. Application of new liquid chromatography-mass spectrometry (LC-MS) methods facilitated the identification of two previously undiscovered classes of β-carotene radical adducts. These products provide evidence that carotenoids trap radicals by both electron transfer and radical addition reactions.