Mass Spectrometry of Thermally Unstable Molecules. Evaluation of Ionization Techniques Using Glutamine as zyx a Reference Compound William M. Lagna and Patrick S. Callery University of Maryland School of Pharmacy, 20 N. Pine St., Baltimore, Maryland 21201, USA A limiting factor in the application of mass spectrometry to biochemically important molecules has been the inability to analyze compounds without inducing thermal decomposition. Glutamine, which readily cyclizes to 2-pyrrolidone-5- carboxylic acid with zyxwvutsrqp loss of ammonia, is one of the more difficult of these non-volatile, thermolabile biomolecules to determine. Using a heated, direct probe, no molecular ion for glutamine was observed with electron impact ionization. A protonated molecular ion was detected with direct probe chemical ionization and thermospray. In both cases, the major ion formed was derived from the thermolysis product, 2-pyrrolidone-5-carboxylic acid. Analysis by fast atom bombardment and 252Cf plasma desorption mass spectrometry yielded a molecular ion as base peak with a small ion from the zyxwvuts 2-pyrrolidone-5-carboxylic acid. Ion evaporation produced only the molecular cations, and collisionally activated dissociation demonstrated that glutamine does not cyclize appreciably once protonated. These results suggest that glutamine can be used as a reference compound when analysis requires the optimization of conditions to produce a molecular ion. Furthermore, the relative intensity of the pyrolytic products, 2-pyrrolidone-5- carboxylic acid derived molecules, divided by the relative intensity of the molecular ions of glutamine provides a numerical evaluation of ionization conditions. INTRODUCTION Glutamine, consistent with its key role as nitrogen carrier in biological systems, is the object of a growing number of biochemical studies. Recent articles have dealt with the synthesis and determination of stable isotopes of glutamine to be used in studies delineating metabolic pathways.',' However, only the amide nitrogen in these studies was labeled. In an earlier paper, we described the synthesis and characterization of ~~-glutamine-2,5- 15N,,3 in which both the amide and amine nitrogens are labeled. Although several methods have been proposed for the aqueous solution hydrolyzes slowly at room temperature method has been established in the literature for the analysis of the doubly labeled compound, glutamine-2,5- NZ. The lability of the amide nitrogen, which in aqueous solution hydrolyzes slowly at room temperature to form glutamic acid-"N and amrn~nia-'~N, makes this a particularly difficult analysis. In previous methods, only the labeled amide nitrogen was enzymatically liber- ated, then assayed either as free ammonia or as a deriva- tive. These methods do not allow for the analysis of amine-labeled glutamine. Gas chromatography/mass spectrometry (GC/MS) methods are not readily employed because either the amide nitrogen is hydro- lyzed during the procedure, or is lost as ammonia when glutamine (1) thermally cyclizes to 2-pyrrolidone-5- carboxylic acid (2). In this communication we describe several mass spectrometric methods for the direct deter- 15 O=O-COOH zyxwvutsrqpo --+ HZN NHZ H mination of glutamine. We also describe a broader appli- cation of glutamine as a reference compound for charac- terization and comparison of the thermal nature of mass spectrometric ionization sources. EXPERIMENTAL Electron impact and chemical ionization mass spec- trometry Positive ion electron impact (EI) and chemical ioniz- ation (CI) of m-glutamine and ~~-ghtamine-2,5-~~N, were performed on an Extranuclear Simulscan quad- rupole mass spectrometer equipped with an Extranu- clear 1000 data system. A direct probe inlet was used for sample introduction. The probe was temperature programmed from 30 to 280 "C at 250 "C min-'. Source temperature was 160 "C, and electron energy was 70 eV in the EI mode. Methane or isobutane at 130 Pascal (1 torr) was the reagent gas in the CI mode. Chemical ionization was performed with an isothermally heated Extranuclear Laboratories CI probe using isobutane as the reagent gas. Thermospray liquid chromatography/mass spectrometry (LC/MS) Thermospray (TS) LC/MS was performed at the National Institutes of Health, Bethesda, Maryland, on a Biospec mass spectrometer interfaced to a Gilson liquid chromatograph. All positive ion analyses were repeated on a Finnigan MAT 4500 interfaced with a Gilson liquid chromatograph. The Vestec thermospray @ Wiley Heyden Ltd, 1985 CCC-0306-042X/85/ 120699-05 $02.50 BIOMEDICAL MASS SPECTROMETRY, VOL. zyxw 12, NO. 12,1985 699