2 Physical Methods and Techniques Part (ii) Mass Spectrometry By M. JARMAN Drug Metabolism Team Section of Drug Development Cancer Research Campaign Laboratory Institute of Cancer Research Clifton Avenue Sutton Surrey SM2 5PX 1 Introduction The period since the previous review' has been noteworthy for the explosive growth in the number of papers dealing with the new soft ionization technique of fast atom bombardment. This has not however been at the expense of other areas of mass spectrometry with the possible exception of field desorption. The Proceedings of the 9th International Mass Spectrometry Conference held in Vienna in 1982,2 continue to reflect the diversity of the subject as do the Proceedings of another regular series of meetings the 4th International Symposium on Quantitative Mass Spe~trometry.~ Subjects of substantial reviews not cited elsewhere have included fundamental aspects of electron impact i~nization,~ gas-phase chemistry of collision- ally activated ions,5 and analytical capabilities and 2 Ion Structure and Fragmentation Molecular orbital calculations predict and experiment confirms that stable ions exist which are unusual in being low-energy species without stable neutral counter- parts.' Thus the methyleneoxonium radical cation CH2-OH2t was found to be 45 kJ mol-' lower in energy than CH3-OHt and was subsequently generated from ethylene glycoL8 Other ions of the type CH,XH+ (X = halogen NH2 or SH) have been generated.' These ions have been referred to as 'radical ion dipole complexes' since CH2XH* can formally be represented as a tightly bound complex between ionized methylene and HX.Thus and typically the structure HF-+ CH2t was assigned" to the ion CH2FHt generated'.10 by loss of COz from CH2FC02H since collision activation afforded the ions HFt and abundantly CH2t but unlike the isomeric species CH,F$ no CH,'. Analogous more complex structures have been ' M. Jarman Annu. Rep. Prog. Chem. Secr. B 1982 78 3. * In?. J. Mass Spectrom. Ion Phys. 1982 Vol. 45; 1983 Vols. 4648. Biomed. Muss Specrrom. 1983 10 113-235. T. D. Maerk In?. J. Muss Spectrom. Ion Phys. 1982 45 125. K. Levsen and H. Schwarz Mass Spectrom. Rev. 1983 2 77. ' R. G. Cooks K. L. Busch and G. L. Glish Science 1983 282 273. ' W. J. Bouma J. K. Macleod R.H. Nobes and L. Radom Int. J. Mass Spectrom. Ion phys. 1983,46,235. ' W. J. Bouma J. K. Macleod and L. Radom J. Am. Chem. SOC. 1982 104 2930. J. L. Holmes F. P. Lossing J. K. Terlouw and P. C. Burgers Can. J. Chem. 1983 61 2305. lo H. Halim B. Ciommer and H. Schwarz Angew. Chem. Int. Ed. EngL 1982 21 528. 19 M. Jarman described such as ionized oxycarbenes‘ I (e.g. CH,COHt and CH,OCOH’) and halonium radical ions’’ (e.g. CH,XCH,t). The latter are noteworthy as containing a divalent halogen atom bonded to two carbons. Ion-molecule complexes as intermediates in gas-phase reactions are exten- sively discussed as part of a review’ of gas-phase analogues of solvolysis reactions which emphasizes the connection between chemistry in the mass spectrometer and reactions in solution.For example in solution alkyl 4-pyridyl ethers readily elimi- nated olefins following N-methy1ati0n.l~ In the gas-phase analogy the olefin was produced as an ion-molecule complex with C4H5NOt following proton transfer from the alkyl residue to the pyridyloxy moiety to form e.g. ion (1). If the olefin contained allylic hydrogen as in the isopropyl13 but not in the ethylI5 ether a second hydrogen-transfer yielded the ion (2) and m/z 96 as well as m/z95 was abundant in the mass spectrum. The propensity for pyridine nitrogen to abstract hydrogen intramolecularly has a practical application in determining the positions of branching or unsaturation in long-chain fatty acids by mass spectrometry of their picolinyl esters.16 Interest in C6H6 radical-cations continues and an interesting example is benzvalene’’ which appeared to retain the structure (3) when ionized as evidenced for example by the decompositions of its gas-phase adduct with buta-173-diene which are most readily interpreted in terms of the structure (4).Another recurrent theme is the comparative behaviour of odd- and even-electron ions and the McLafferty rearrangement has been studied from this viewpoint.’8 Using even-electron ions generated from aromatic diesters and diketones McLaff erty rearrangement of ketones [(5) -* (7) X = CH,] but not of analogous esters (X = 0) was established on the criteria of specific y-deuterium transfer (Y = ’H) and by the presence of an appropriate metastable peak but was judged less common than for radical cations.” J. K. Terlouw J. Wezenberg P. C. Burgers and J. L. Holmes J. Chem. SOC.,Chem. Commun. 1983 1121. ’’ Y. Apeloig B. Ciommer G. Frenking M. Karni A. Mandelbaum H. Schwarz and A. Weisz J. Am. Chem. SOC.,1983 105 2186. l3 T. H. Morton Tetrahedron 1982 38 3195. l4 G. Schmid and A. W. Wolkoff Can. J. Chem. 1972 50 1181. Is A. Maquestiau Y. van Haverbeke C. de Meyer A. R. Katritzky M. J. Cook and A. D. Page Can. J. Chem. 1975 53 490. l6 D. J. Harvey Biomed. Mass Spectrom. 1982 9 33. l7 D. L. Miller and M. L. Gross J. Am. Chem. SOC.,1983 105 4239. M. Zollinger and J. Seibl Inf. J. Mass Specrrom. Ion Phys. 1983. 47 363. Physical Methods and Techniques -Part (ii) Mass Spectrometry 3 Chemical Ionization The alkanols were among the earliest compounds studied by CI using methane” and isobutane,20 and their isobutane spectra were recently studied in detail.2’ Formation of the Lewis adduct [M + C4H9]+ requires more energy than protonation but becomes relatively more abundant for the higher members within a homologous series since these larger molecules are more efficient at distributing this energy.The ratio [M + H -H,O]+/[ M + H]+ was 3-4 fold greater in the spectra of the axial 3-and 4-methylcyclohexanols than in their equatorial counterparts since more steric strain is released in eliminating water from the axial position. For dihydroxy derivatives [A4 + HI’ was more abundant for isomers (8) and (9) where it is stabilized by hydrogen bonding than for Owing to the low internal energy content of [M -HI- anions negative ion CI is particularly effective for revealing such sterochemical differences and a recent example of its use is the assignment of ring D stereochemistry to 17[-alkyl-5a 14P-androstane- 14,17(-diol~.~~ (8) (9) (10) New re%gent gases have been introduced.Dimethyl ether produces reactive CH,CH=OH ions and among other discriminating gas-phase reactions was able to distinguish polycyclic hydrocarbons which would form Diels-Alder adducts giving intense [M + C,H,O]’ ions from isomers which would not.24 Reagents for locating olefinic bonds continue to be sought and dimethyl disulphide is a recent example.25 However the use of a selective reagent gas can be misleading should it react at other positions of unsaturation.In a different approach designed to circum- vent this problem epoxidation of the olefinic linkage and ring opening with ammonia or an amine to form the corresponding amino-alcohol introduced a heteroatom having a higher proton affinity than any already present and hence directing sub- sequent fragment at ion.26 Dimeric ions have received little attention as gas-phase products of chemical ionization. Although their formation obviously requires sufficient sample concentra- tion for intermolecular reactions to occur other factors influence their formation. ”) F. H. Field Acc. Chem. Rex 1968 1 42. 20 F. H. FieM J. Am. Chem. Soc. 1970 92 2672. ” F. J. Winkler F. 0. Gulacar F. Mermoud and A. Buchs Helu. Chim. Acta 1983 66 929.22 F. J. Winkler F. 0. Gulacar F. Mermoud D. Stahl T. Gauman and A. Buchs Inr. J. Mass Specrrom. Ion Phys. 1983 46 321. 23 J. C. Beloeil M. Bertranne D. Stahl and J. C. Tabet J. Am. Chem. Soc. 1983 105 1355. 24 T. Keough Anal. Chem. 1982 54 2540. 25 H. R. Buser H. Arn P. Guerin and S. Rauscher Anal. Chem. 1983 55 818. 26 M. Cervilla and G. Puzo,Anal. Chem.. 1983 55 2100. M. Jarman Thus at a given total ion current methyl a-D-glucopyranoside gave [M2 + H]+ more abundantly than did the p-anomer." Evidence points to hydrogen bonding as a factor stabilizing dimers as in the hydroxyazadamantanes (1 I)** and it is therefore likely that dimer formation will depend upon stereochemistry and prove a useful additional means of distinguishing stereoisomers.Gas-phase analogies with solution chemistry have been numerous in CI and the reaction between benzene and alkyl cations is a particularly well studied example. The large entropy loss for the reaction t-C,H,' + C6H6-t-C,H,.C,H,+ attributed to the large mass and moments of inertia of the reactants and to the restricted motions of the t-butyl group in the product ion contributed significantly to its thermal in~tability.'~ However the stability of benzenium ions increases with the number of alkyl substituents on the benzene ring owing to greater stabilization of the charge by the multiple alkyl substituents and o-xylene for example forms a stable adduct with t-C4H9+.30 Attention has been given to the experimental conditions needed to detect and analyse adduct ions in gas-phase aromatic sub~titution.~' They were collisionally stabilized in the ion source ion-selected using a double-focusing mass spectrometer and their spectra after collision-induced decomposition (CID) taken by scanning a final analyser.Wheland-type adducts from allyl (12; R = CH2CH=CH2) and isopropyl [R = CH(CH,),] cations were detected and their structures validated by comparison with the spectra of protonated allyl and isopropyl benzenes. Conversely the structures of the C3H5+ ions produced from the corres- ponding isomeric halides were determined by reacting them with benzene and examining the CID spectra of the ad duct^.^^ Although the generally accepted transition state for electrophilic aromatic substitu- tion in the liquid phase is a cT-bonded complex (12) n-complexes have been favoured in certain cases.In a study of gas-phase analogues of these processes,33 proton transfer from CH5+ to toluene was markedly disfavoured by an o-or p-fluorine substituent but only slightly in rn-fluorotoluene where alone the mesomeric effect of fluorine as in (13) and (14) opposed its electron-withdrawing inductive effect. In the T-complex with NO+ only the inductive effect operated and the reduction in affinity for NO+ on fluorine substitution was independent of the orientation of the substituent. 27 P. Finch R. A. Hancock M. C. Matulewicz H. Weigel and M. Jarman Curbohydr. Res. 1982 104 C9. 28 M. D. Bezoari P. Kovacic and A. R. Gagneux Org. Muss Spectrom. 1982 17 493.29 D. K. Sen Sharma S. Ikuta and P. Kebarle Can. J. Chem. 1982 60,2325. 30 F. Cacace G. Ciranni and P. Giacomello J. Chem. Soc. Perkin Trans. 2 1982 1373. 3' D. L. Miller J. 0. Lay Jr. and M. L. Gross J. Chem. SOC. Chem. Commun. 1982 970. 32 J. 0. Lay Jr. and M. L. Gross J. Am. Chem. SOC.,1983 105 3445. 33 J. A. Stone D. E. Splinter and S. Y. Kong Can. J. Chrrn. 1982. 60. 910. Physical Methods and Techniques -Part (ii) Mass Spectrometry (12) (13) (14) (15) Reactions other than aromatic substitution have been studied as gas-phase analogues. The carbanion produced from dimethyl adipate in the gas phase yielded the anion (15),34 a proposed intermediate in the analogous Dieckmann ester con- densation in solution. In contrast the acid-catalysed acyl-transfer reaction between methanol and vinyl acetate proceeded as with other esters in the gas phase via acyI -cation trans fe r3 -+ HY + AcXH' [HY--Ac+--XH]-+ AcYH+ + HX and not as previously thought possible via tetrahedral addition-complexes invoked for the corresponding reaction in solution.4 Fast Atom Bombardment A review36 of this technique provides a useful background against which recent progress can be assessed. Among the issues raised are the choice of bombarding gas matrix effects comparisons with other soft ionization methods particularly field desorption and the key question of how large a molecule can be lifted from the matrix. Regarding yields of ions in FAB spectra xenon is generally reckoned superior to the more common inert gases argon and neon on account of the higher momentum of the heavier gas at constant accelerating voltage.Thus in a study of peptides ion yields from the three gases were extrapolated to equivalent momentum and found to be identical.37 Other factors appear to operate for less polar molecules. Thus the yield of ions from vitamin B,z using Xe Ar and Ne closely reflected the ratio between their atomic weights (1 0.3 0.15) but for protoporphyrin IX dimethyl ester a chloroform-soluble material the ratio was 1 0.8 0.6 a much less steep decline than predicted.38 On the basis of volatility and atomic weight other less expensive elements should be suitable for FAB. Thus mercury and caesium have given ion yields similar to or greater than ~enon.~~,~' Glycerol is the most widely used matrix being relatively involatile but having good solvent properties.Not all compounds dissolve in glycerol and other media such as triethyl citrate diethanolamine and polyethylene glycol have advantages in certain cases.4' The characteristic [ M + HIf ion seen in positive-ion FAB spectra using glycerol or other hydroxylated solvents is a function of the matrix since the use of a non-hydroxylated medium such as Fomblin oil3' results in Mt instead. .3 4 D. J. Burinsky and R. G. Cooks J. Org. Chern. 1982 47 4864. 3 -5 J. K. Kim and M. C. Caserio J. Am. Chern. Soc. 1982 104 4624. 36 K. L. Rinehart Jr. Science 1982 218 254. 37 S.A. Martin C. E. Costello and K. Biemann Anal. Chern. 1982 54 2362.1x H. R. Morris M. Panico and N. J. Haskins Inr. J. Mass Spectrom. Ion Phys. 1983 46 363. 39 R. Stoll U. Schade F. W. Roellgen U. Giessman and D. F. Barofsky Inr. J. Mass Specfrorn. Ion Pbys. 1982 43 227. s. S. Wong R. Stoll and F. W. Roellgen Z. Naturforsch. Ted A 1982 37 718. " J. Meili and J. Seibl. In!. J. Mass Spectrom. [on Phys. 1983 46 367. 24 M. Jarman Also the nitrophenols studied by gas-phase FAB gave Mt and fragmentation patterns qualitatively like their EI though the negative-ion spestra differed in that EI gave predominantly [M -HI- whereas FAB afforded MY base peak. In glycerol solution FAB afforded the conventional [M + H]+ and [M -HI- ions. Interference from glycerol clusters can be a nuisance where these coincide with peaks in the spectrum of the sample.This can be overcome by adding acids when [M + HI+ is enhanced.37 Conversely the molecular radical ion Mt can be enhanced at the expense of [M + HIt by charge transfer complexation particularly if glycerol is replaced by the aprotic solvent dimethyl s~lphoxide.~~ Turning to specific applications FAB has been most extensively used in the analysis of peptides. Both molecular weights in excess of 5000 and amino-acid sequences have been determined. Studies of bovine insulin4u6 exemplify the high molecular weights (5729) accessible using FAB and high-field magnets. Careful examination of the molecular ion region has revealed three sets of overlapping isotope clusters corresponding to reduction of one two or all three of the disulphide bonds showing that the two component peptide-chains remain together even after reduction of all S-S linkages4' The spectra afford sequence information.Thus the positive-ion spectra of angiotensins exhibited systematic fragmentation between CH-CO bonds and between CON-C bonds in the peptide chain the N-terminal fragment retaining the charge.47 Negative-ion spectra afforded less fragmentation but were complementary in giving a series of fragments by NH-CH cleavage the C-terminal fragment retaining the charge. Complex proteins can be sequenced if the base sequence of the gene coding for that protein is Conventional GC-EI-MS sequencing of small peptide fragments and molecular weight determina- tion of larger fragments from trypsin digests using FAB can afford information as to how the base sequence of the gene should be read and help correct any errors in this sequence.Another exciting innovation is the study of enzymatic reactions by FAB. Hydrolysis of arginine methyl ester by trypsin and of angiotensin by carboxypeptidase Y proved compatible with the glycerol matrix. Moreover intermit- tent exposure to the fast-atom beam did not reduce the activity of the enzymes although continuous exposure for 20 minutes virtually abolished it.49 Other types of macromolecule give sequence information. Oligodeoxyribonucleo- tides were most successfully examined in the negative-ion Sequence-defining fragments in the negative-ion FAB spectrum derived from P-0 cleavage both at the 3' and the 5' end of the internucleotide linkage and were exemplified for d(A-C-T-C-G-A-T-G) ([M -HI- 2407 daltons) enabling controlled synthesis of such compounds to be monitored using FAB.52 42 B.Kralj V. Kramer and V. Vrscaj lnr. 3. Mass Spectrom. Ion fhys. 1983 46 399. 43 E. De Pauw Anal. Chem. 1983 55 2195. 44 M. Barber R. S. Bordoli G. J. Elliott R. D. Sedgwick A. N. Tyler and B. N. Green J. Chem. SOC. Chem. Commun. 1982 936. 45 A. Dell and H. R. Morris Blochem. Brophys. Res. Commun. 1982 106 1452. 46 A. M. Buko L. R. Phillips and B. A. Blair Biomed. Mass Spectrom. 1983 10 408. 47 M. Barber R. S. Bordoli D. R. Sedgwick and A. N. Tyler Biomed. Mass Specfrom. 1982 9 208. 48 K. Biemann Inr. J. Mass Specfrom. Ion fhys. 1982 45 183. 49 L. A. Smith and R.M. Caprioli Biomed. Mass Specfrom.,1983 10 98. 50 G. Sindona N. Uccella and K. Weclawek J. Chem. Res. (S) 1982 184. M. Panico G. Sindona and N. Uccella J. Am. Chem. Soc. 1983 105 5607. 52 G. Lutz. R. Frank and H. Bloecker. Nuckic Acids Res.. 1982. 10 4671. Physical Methods and Techniques -Part (ii) Mass Spectrometry h Carbohydrates have been studied using FAB. The negative ion spectra of simple monosaccharides contained prominent [M -HI- ions and the major fragmentations appeared to be unimolecular decompositions of this ion and were diagnostic of structure.53 For example the prominent ion at m/z 89 from [M -13-in the FAB spectrum of ribose could be accounted for by the sequence (16) --+ (18). Oligosac-charides gave sequence-defining fragments by cleavage at glycosidic linkage^.'^ Another application to carbohydrate derivatives was the analysis of intact in gluc~ronides,~~ particular a new class of drug conjugates quaternary glucuronides in which tertiary nitrogen is quaternized by the glucuronyl residue and which are therefore intractable to analysis by EI and CI.56 Sulphates are a class of conjugate which usually decompose on attempted derivatization but can be analysed intact using FAB as exemplified by dehydroepian- drosterone sulphate" and other steroid sulphate~.~~"~ Other classes of natural product which have been examined using FAB have included corrins,6' prostaglan- dins,62 and penicillin^.^^ Finally an application having relevance to solution chemistry is the use of FAB to study the complexes formed between crown ethers and metal ions and their salts.64 Metallic salts yielded complexes of the type [crown + M"++ For example the complex ion from PbCI2 contained the anion and had the structure [crown + PbCI]' whereas univalent cations e.g.the alkali metals gave complexes containing the cation only. The well known high affinity of 18-crown-6 for K+ is reflected in the increase in ion yielded with increasing [K'] which becomes asymptotic as the ratio [18-crown-6]:[K+] approaches unity. 5 Other Soft Ionization Procedures Desorption-ionization mass spectrometry has been used as a general term to describe those methods in which ions are sampled from the condensed phase.6s The term '' M. Barber R. S. Bordoli R. D.Sedgwick and J. C. Vickerman J. Chem. SOC.,Faraday Trans. I 1982 78 1291. 54 J. P. Johannis W. Heerma J. F. G. Vliegenthart B. N. Green A. S. Ivor G. Strecker and G. Spik Biomed. Mass Spectrom. 1983 10 420. 55 C. Fenselau L. Yelle M. Stogniew D. Liberato J. Lehman and P. Feng Int. J. Mass Spectrom. Ion Phys. 1983 46 41 I. 56 J. P. Lehman and C. Fenselau Drug Merab. Dispos. 1982 10 446. 57 S. J. Gaskell B. G. Brownsey P. W. Brooks and B. N. Green Biomed. Mass Specrrom. 1983 10 215. 58 J. G. Lier C. F. Beckner A. M. Ballatore and R. M. Caprioli Steroids 1982 39,599. 59 C. H. L. Shackleton V. R. Mattox and J. W. Honour J. Steroid Biochem. 1983 19 209. 60 M. C. Dumasia E. Houghton C. V. Bradley and D. H. Williams Biomed. MassSpectrorn.1983 10,434. " H. Schwarz K. Eckart and L. C. E. Taylor Org. Mass Specrrom. 1982 17 458. 62 R. C. Murphy W. R. Mathews J. Rokach and C. Fenselau Prostaglandins 1982 23 201. 63 M. Barber R. S. Bordoli R. D. Sedgwick A. N. Tyler B. N. Green V. C. Parr and J. L. Cower Biomed. Mass Spectrom. 1982 9 I I. 64 R. A. W. Johnstone and I. A. S. Lewis Int. J. Mass Spectrom. Ion Phvs..1983 46 451. 65 K. L. Busch S. E. Unger A. Vincze R. G. Cooks and T. Keough J. Am. Chem. SOC.,1982 104 1507. 26 M. Jarman encompasses FAB and other methods described in this section. Secondary-ion mass spectrometry (SIMS) is closely allied mechanistically to FAB. A major difference is that cationized ions are formed in SIMS by interaction between the sample and the metal surface on which it is conventionally coated.Thus a silver support gave [M + Ag]’ ions for example from peptides66 and from oligo~accharides,~~ which were useful in defining molecular weight. Molecular ions were predictably more abundant6’ from Xe’ bombardment than from Ar’. An interesting deveiopment of this principle is the use of heavy organic ions exemplified by [A4 -CHJ’ from trimethylpentaphenyltrisiloxane(m/z 53 l).40 Despite the growth of FAB as a relatively routine method field desorption (FD) continues to be a useful complementary technique particularly in the hands of investigators skilled in its use and has been recently reviewed.69 Alone among desorption methods FD commonly affords odd-electron molecular ions (Mt) although molecules lacking functional groups of low ionization potential such as carbohydrates or aliphatic amino-acids tend to yield protonated or cationized molecular ions.However the low tendency of cobyrinic esters to form this type of ion may be the reason why FAB afforded no molecular ion species from these corrins whereas FD of cobyrinic acid heptamethyl ester could afford Mt exclus-ive]~.~’ Another advantage over FAB exemplified in a comparative study of bis- pyridinium salts7’ is the often abundant formation of doubly charged ions in FD. It was proposed that the ions induced an ‘image’ charge of equal intensity and opposite sign which resists desorption of the ions but which is easily counteracted by the field in FD allowing desorption even of doubly charged ions.Plasma desorption (PD) continues to lead other desorption methods in its capabil- ity for analysing compounds of very high molecular weight. With oligonucleotides positive-ion PD afforded molecular weights and negative-ion spectra gave sequence determining fragments formed by C(3’)-0 and C(5’)-0 fission.72 The method has now been applied to peptides and sequence determining fragments have been observed.73 Insulin was studied using ‘271 ions which are more energetic than the conventional 252~f fission fragment^.'^ 6 Other Studies In parallel with the foregoing developments in desorption methods investigations continue into techniques for obtaining mass spectra on refractory compounds using conventional EI and CI sources and specially designed probes.Formerly the emphasis was on the use of CI and the term desorption chemical ionization (DCI) has been used to describe the technique. Recently reports of similar investigations using the EI mode have been numerous. It is likely however that CI mechanisms bb H. Kambara S. Hishida and H. Naganawa Org. Ma55 Spectrom. 1982 17 67. 67 K. Harada M. Suzuki and H. Kambara Tetrahedron Lett. 1982 23 2481. 68 H. Kambara Org. Mass Spectrom. 1982 17 29. 69 G. W. Wood Tetrahedron 1982 38 1125. 70 H. M. Schiebel and H. K.Schulten Biomed. Mass Spectrom. 1982 9 354. ” D. N. Heller J. Yergey and R. J. Cotter Anal. Chem. 1983 55 1310. 72 C. J. McNeal K. K. Ogilvie N. Y. Theriault and M. J. Nemer J. Am. Chem. Soc. 1982 104 972. 71 B.T. Chait B. F. Gisin and F. H. Field J. Am. C‘hem. SOC.,1982 104 5157. 74 P. Haakansson I. Kamensky B. Sundqvist J. Fohlman P. Peterson and C. J. McNeal J. Am. Chem. SOC.,1982. 104 2948. Physical Methods and Techniques -Part (ii) Mass Spectrometry often contribute to the formation of the ions observed in 'direct electron impact' as it has been termed.75 Thus amino-acids were reported as affording M' in one such but [M + HI' ions in another.76 Penicillins mainly gave Mt but did give [M + HI' in one example.77 The addition of ammonium chloride to salts of penicil- lins and bile acids afforded molecular ions of the free acids but above 300 "C sodium salts of bile acids also gave RCO,Na+ ions.7' Alkali attachment ions [M + Na]+ could also be produced at similar temperatures from sucrose and glucose in an EI source.79 Protonated molecular ions were also produced under DCI conditions in a CI source when only N2 was present implying that adjacent molecules on the probe served as the proton source.'o In this study FAB SIMS and DCI gave similar spectra for ecdysterone suggesting common features in the desorption ionization processes.However the 'in beam' techniques were thought unlikely to be competitive in studying really large polar molecules and an upper mass limit of ca. 1500 daltons was suggested. Finally the potential of Fourier transform mass spectrometry (FTMS) has been further explored.81 Collision-induced decompositions can be studied without the need for a tandem instrument and proton-bound dimers and metal-bound ions have been investigated,'* as has high-resolution mass measurement in the separation of isobaric product ions.'3 The range of ion lifetimes (lop3+-> Is) in FTMS was exploited in studying methyl nitrite as a CI reagent gas whose ion products change with time.'4 Short ion trapping times generated NO' which reacted mainly by hydride and hydroxide abstraction.Longer times afforded CH,O(NO)NO' which transferred NO' to yield [M + NO]+. 75 P. Traldi Org. Mass Spectrorn. 1982 17 245. 76 D. Dessort A. Van Dorsselaer S. J. Tian and G. Vincendon Tetrahedron Lett. 1982 23 1395. 77 M. Ohashi R. P. Barron and W. R. Benson J. Pharrn. Sci. 1983 72 508. 7X A. K. Bose B. N. Pramanik and P. L. Bartner J. Org. Chern. 1982,47 4008.79 E. Constantin Org. Mass Specrrorn. 1982 17 346. 80 R. N. Stillwell D. I. Carroll J. G. Nowlin and E. C. Homing Anal. Chern. 1983 55 1313. '' C. L. Wilkins and M. L. Gross Anal. Chern. 1981 53 1661A. X? R. C. Burnier K.B. Cody and B. S. Freiser J. Am. Chern. SOC.,1982 104 7436. H3 R. B. Cody and B. S. Freiser Anal. Chern. 1982 54 1431. x4 W. D. Reents Jr. R. C. Burnier R. B. Cody and B. S. Freiser Anal. Chern. 1982 54 1245.