Anal. Chem. 1003, 65,2380-2388
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Structurally Diagnostic Ion-Molecule Reactions and Collisionally Activated Dissociation of 1,4=Benzodiazepines in a Quadrupole Ion Trap Mass Spectrometer Tracy Donovan McCarley and Jennifer Brodbelt’ Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712-1167
The ion-molecule reactions of various l,4-benzodiazepines and dimethyl ether ions were studied witha quadrupoleion trap mass spectrometer.The methoxymethylene ions of dimethyl ether selectively react with 3-hydroxy-l,4-benzodiazepines (temazepam,oxazepam) to form (M + 13)+adducts by methylene substitution, and they react with 1,4-benzodiazepines that do not have hydroxyl substituents (diazepam, nordiazepam, nitrazepam) to form (M + 15)+adductby a simple methyl cation transfer. These adducts are formed by elimination of methanol or formaldehyde, respectively, from (M + CHzOCH# precursor ions. Ion-molecule reactions of model compoundswith dimethyl ether ions suggest that the reactive site in the formation of (M + 15)+adducts is the imine functional group of the l,4-benzodiazepines,while the reactive site for formation of (M + 13)+ adducts involves a functional group interaction between the hydroxyl and carbonyl functional groups. Fragmentation induced by chemical ionization and collisionally activated dissociation provides further structural information for the differentiation of l,4-benzodiazepines. Also, the gas-phase basicities of diazepam and temazepam have been estimated by bracketing techniques to be between 220.7 and 222.2 kcal/mol. INTRODUCTION The 1,4-benzodiazepines are the most widely prescribed drugs in the world, with diazepam (Valium) being the most well-known. They are a class of sedatives and tranquilizers that are used commonly to treat anxiety, insomnia, muscle spasms, and seizures.’ The analysis of these drugs is important for pharmacological studies as well as in clinical, forensic, and law enforcement applications due to their potential for addiction, abuse, and overdose.293 A variety of instrumental methods are used in the analysis of 1,4benzodiazepines including gas c h r o m a t ~ g r a p h y ,high~~ performance liquid chromatography (HPLC)? thin-layer chromatography? immunoassays,gfluorine nuclear magnetic (1)Priest, R. G. In Benzodiazepines Today and Tomorrow; Priest, R. G.,Filho, U. V., Armein, M., Skreta, M., Eds.;UniversityPark Baltimore, MD, 1980;pp 4-5. (2)Finkle, B. S.;McCloskey,K. L.;Goodman, L. S. J.Am. Med. Assoc. 1979,242,429-434. (3)Parker, K. M.; White, B. N.; Beattie, D. J.; Altmiller, D. H. Clin. Chem. 1988,34,748-750. (4)Ahuja, S. In Ultratrace Analysis of Pharmaceuticals and Other Compounds of Interest;Ahuja, S.,Ed.; Chemical Analysis Series 85;John Wiley and Sons: New York; 1986;pp 50-51. (5)Maurer, H.; Pfleger, K. J. Chromatogr. 1987,422, 85-101. (6)Japp, M.; Garthwaite, K.; Geeson, A. V.; Osselton, M. D. J. Chromatogr. 1988,439,317-339. (7)Drouet-Coassolo, C.; Aubert, C.; Coassolo, P.; Cano, J.-P. J. Chromatogr. 1989,487,295-311. 0003-2700/93/0365-2380$04.00/0
resonance,”Jelectrochemical methods,11J2 Fourier transform Raman and infrared spectros~opies,~~ ion mobility spectrometry,l4 and mass spectrometry.1”20 The primary focus of mass spectrometric studies has been correlating fragmentation patterns to structural features of 1,4-benzodiazepines and their metabolites.1619 In a study of the electron ionization-induced fragmentation of 1,Cbenzodiazepines, the molecular ions fragmented mainly by loss of H’,HCN, CO, and HCO to form smaller ring systems. The 5-phenyl substituent was detected by intense peaks at mlz 77 and 51, and the precursor ion or major fragments lost C1 or NO2 from position 7. All spectra showed characteristic peaks a t mlz 151, 165, 177, 193, and 205. Substituents in positions 3 and 4 of the diazepine ring influenced the fragmentation patterns significantly and thus allowed differentiation of three classes of 1,4-benzodiazepines: 3,4unsubstituted l,4-benzodiazepines, 3-hydroxy-l,4-benzodiazepines,and N4-0~ides.l~ However, in the electron ionization studies, the molecular ion intensity of some 1,4-benzodiazepines was particularly low, so softer fast atom bombardment (FAB) ionization was utilized for molecular weight determination.20 Abundant protonated molecules were formed, as well as fragment ions a t mlz 151,165, 194, and 205 that were also characteristic upon electron ionization. High-energy collisional activation (mass-analyzed ion kinetic energy spectrometry) of the FAB-generated protonated molecules produced additional fragment ions that were structurally diagnostic. The use of chemical ionization reagents in the mass spectrometry of l,4-benzodiazepines has been limited to ammonia positive ion chemical ionization for proton transfer21s22 and methane electron capture negative ion chemical ioni~ation.~39~~ The combination of site-selective ion-molecule reactions with collisionallyactivated dissociation (CAD) offers greater promise for differentiation of related 1,4-benzodiazepinesdue to the enhanced specificity and sensitivity of this method. One primary objective of the present study was to examine (8)Valk, 0. K.; Olajos, S.; Cserhati, T. J. Chromatogr. 1990,499,361371. (9)Jones, C. E.; Wians, F. H., Jr.; Martinez, L. A.; Merritt, G. J. Clin. Chem. 1989,35,1394-1398. (10)Bhattacharyya, P. K.; Grant, A. Anal. Chim.Acta 1982,142,249257. (11)Vire, J. C.; Hermosa, B. G.;Patriarche, G. J. A n d L e t t . 1986,19, 1839-1851. (12)Ribes, A. F.; Osteryoung, J. Anal. Chem. 1990,62,2632-2636. (13)Neville, G. A.; Shurvell, H. F. J. Raman Spectrosc. 1990,21,9-19. (14)Lawrence, A. H. Anal. Chem. 1989,61,343-349. (15)Grostic, M. F. InBiochemical Applicationsof Mass Spectrometry; Waller, G. R., Ed.; Wiley-Interscience: New York, 1972;pp 575-588. (16)Sadee, W. J. Med. Chem. 1970,13,475-479. (17)Rendic, S.;Klasinc, L.; Sunjic, F. K.; Kajfez, F.; Kramer, V.; Mildner, P. Biomed. Mass Spectrom. 1975,2, 97-106. (18)Budzikiewicz, H.;Mohr, N. Org. Mass Spectrom. 1981,16,329330. (19)Gioia, B.; Arlandini, E.; Giacconi,P.; Rossi, E.; Stradi, R. Biomed. Mass Spectrom. 1984,11,408-414. (20)Ghezzo, E.;Traldi, P.; Minghetti, G.; Cinellu, M. A.; Bandini, A. L.; Banditelli, G.; Zecca, L. Rapid Commun.Mass Spectrom. 1990,4, 314-317. 63 1993 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
1,4-benzodiazepine
RI -
R3 R7 -
Diazepam
CH,
H
Nordiazepam
H
H
Ternazepam oxazepam
CH, H
OH OH
Nitrazeuam
H
H
c1
c1 C1 C1
NO2
Flgure 1. 1+Benzodlazeplne structures.
the chemical ionization of 1,4-benzodiazepines by selective ion-molecule reactions with dimethyl ether and ethylene oxide ions in a quadrupole ion trap mass spectrometer in order to evaluate the differences in reaction products and to determine whether the reactive trends of these larger molecules mimic the trends observed for smaller model subunits of the benzodiazepines. The 1,4-benzodiazepines chosen for this study include diazepam, nordiazepam, temazepam, oxazepam, and nitrazepam. As can be seen from Figure 1,they only differ by subtle substituent variations a t positions 1,3, and 7 and afford a useful set of models for systematic evaluation of site-selective bimolecular reactions. Dimethyl ether ions were chosen as the reactive species because it had been shown previously that ion-molecule reactions of organic substrates and dimethyl ether ions result in structurally diagnostic adducts, allowing the differentiation of isomers and compounds with slight structural variations, while still forming abundant protonated molecules needed for molecular weight determination.2"27 Low-energy collisional activation of the ion-molecule reaction products along with comparisons of the reactions and dissociation of subunits of the benzodiazepines has been used to characterize the ions and their fragmentation patterns. In addition, the gas-phase basicities of diazepam and temazepam have been determined using the bracketing technique.28s29
EXPERIMENTAL SECTION Ion trap experimentswere performed in a Finnigan quadrupole ion trap mass spectrometer (ITMS)mv31heated to a temperature of 100 O C . Samples were introduced into the vacuum chamber through a direct-inlet probe to a nominal pressure of (0.8-2.0) x 10-6 Torr. The probe temperature was raised to 120-180 "C to promote desorptionof the l,4-benzodiazepines. Dimethyl ether or ethylene oxide was used as a chemical ionization reagent gas at a pressure of 1 X 106 Torr. The reactive ions of dimethyl ether are methoxymethylene, CH30CH2+,at m/z 45, and protonated dimethyl ether, (CH&OH+, at mlz 47. Protonated
ethylene oxide at mlz 45 and methylated ethylene oxide at mlz 57 are the reactive ions formed by electron ionization of ethylene oxide. For deuterium labeling experiments, acetone-4 and dimethyl-de ether were used as reagent gases at 1 X 1od Torr. Acetoned6 was used as a deuteronating agent to form (M + D)+ ions, whereas dimethylde ether was used to elucidate the mechanisms of formationof the (M + 13)+and (M + 15)+product ions. Helium was admitted as a buffer gas at 1mTorr (corrected gauge pressure measured by an ionization gauge attached to the vacuum chamber). The reagent gas was ionized by a 0.1-1.0-ms electronionization pulse. After an ion-molecule reaction period of 50-100 ms, the product ion spectrum was recorded using the mass-selective instability mode to eject ions from the trap to an electron m~ltiplier.3~ To enhance the peak resolution in cases where several isotopic distributions overlap, the apex isolation modes was used to selectivelyisolate regionsof particularinterest in the mass spectrum prior to the use of the mass-selective instability mode. Alternatively, a protonated benzodiazepine or adduct of interest was isolated by apex isolations consisting of a -200 to -350 V dc potentialcombinedwithan appropriateradio frequency (rf) voltage applied to the ring electrode. Isolated ions were then collisionallyactivated by a supplementaryac tickle voltage applied across the end caps at the axial frequency of motion of the ion of interest, typically 1-5 V, for 5 ms at a qr value of 0.4, before the spectrum was recorded. For reagent isolation experiments, reagent ions of interest were isolated after a sufficient ionmolecule reaction period and then allowed to react with the 1,4benzodiazepines. Benzodiazepine samples of approximately 150 pg were sufficient to produce steady signals for about 30 min. Samples of diazepam, nordiazepam, oxazepam, and nitrazepam were generously donoted by Hoffmann-LaRoche (Nutley, NJ). Temazepam was purchased from Sigma Chemical Co. (St. Louis, MO), and N-benzylidenemethylamine,3-amino-1-propanol,4-amino1-butanol,N&-diethylmethylamine, 3-hydroxy-3-methyl-2-butanone, and 1,4-diaminobutane were purchased from Aldrich Chemical Co.,Inc. (Milwaukee,WI). Cyclohexanolwas purchased from Fluka Chemika-Biochemika (Ronkonkoma, NY), cyclohexanone from J. T. Baker, Inc. (Phillipsburg,NJ), nitrobenzene from Mallinckrodt (Paris,KY), and chlorobenzenefrom Spectrum Chemical Mfg. Corp. (Gardena, CA). All samples were used without further purification. Dimethyl ether was purchased from Matheson Gas Products, Inc., ethylene oxide was purchased from Linde Specialty Gases (Danbury, CT), and dimethyl-de ether was purchased from Isotec, Inc. (Miamisburg, OH).
RESULTS AND DISCUSSION Ion-Molecule Reactions of lY4-Benzodiazepinesand Dimethyl Ether Ions: Formation of (M + 13)+,(M + 15)+, and (M + 45)+ Adducts. The ion-molecule reaction products of the 1,4-benzodiazepines and dimethyl ether ions are listed in Table I, and a representative ion-molecule reaction spectrum is presented in Figure 2a. The 3'Cl isotope ions as well as fragment ions in the low-mass range have been omitted from Table I for simplicity. Upon reaction with the dimethyl ether ions, all of the 1,4-benzodiazepines were protonated, all compounds formed either (M + 13)+ or (M + 15)+products, and all except temazepam formed (M 45)+ products. In order to determine which dimethyl ether ion is responsible for the formation of the product ions, the methoxymethylene cation at mlz 45, CH30CH2+, and protonated dimethyl ether a t mlz 47, (CH&OH+, were individually isolated and allowed to react with the 1,Cbenzodiazepines. Protonated dimethyl ether protonated the benzodiazepines, while the methoxymethylene cation produced the other products. The selective formation of (M 13)+ and (M 15)+ products is analytically useful since it allows differentiation
+
(21) Min, B. H.; Garland, W. A. J. Chromtogr. 1977,139, 121-133. (22) Joyce, J. R.; Bal, T. S.; Ardrey, R. E.; Stevens, H. M.; Moffat, A. C. Biomed. Mass Spectrom. 1984, 11, 2&1-289. (23) Miwa, B. J.; Garland, W. A.; Blumenthal, P. Anal. Chem. 1981, 53,193-191. (24) Graland, W. A.; Min, B. H. J. Chromtogr. 1979, 172, 219-286. (25) Brodbelt, J.; Liou-C.-C.; Donovan,T. Anal. Chem. 1991,63,12051209. (26) Donovan, T.; Brodbelt, J. J. Am. SOC.Mass Spectrom., in press. (27) McCarley,T. D.; Brodbelt, J.,SubmittedtoBioZ.Mass Spectrom. (28) Dzidic, I. J. Am. Chem. SOC.1972,94, 8333-8335. (29) Long, J.; Munson, B. J. Am. Chem. SOC.1973,95,2421-2432. (32) Stafford, G. C., Jr.; Kelley, P. E.; Syka, J. E. P.; Reynolds, W. E.; (30) Louris, J. N.; Cooks, R. G.; Syka, J. E. P.; Kelley, P. E.; Stafford, Todd, J. F. J. Int. J. Mass Spectrom Ion Processes 1984, 60, 86-98. G. C.; Todd, J. F. J. Anal. Chem. 1987,59, 1677. (31) Louris,J.N.;Brodbelt,J.S.;Cooks,R.G.;Glish,G.L.;VanBerkel, (33) Weber-Grabau, M.; Kelley, P. E.; Syka, J. E. P.; Bradshaw, S. C.; Brodbelt, J. S. Proceedingsof the 35thAnnualConferenceoftheAmerican G. J.; McLuckey, S. A. Int. J . Mass Spectrom. Ion Processes 1990, 96, Society for Mass Spectrometry; 1987; p 1114. 117.
+
+
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
Table I. Ion-Molecule Reaction Products of Benzodiazepines and Dimethyl Ether Ions. banzediampine,
(MH- 74)+ ( M H- 63)+ (MH- 46)+ (MH- 30)+ (MH- 28)+ (MH- 18)+ ( M+ H)+ (M+ 13)+ (M+ 15)+ ( M+ 27)+ (M+ 45)+
Mw
diazepam,
-1,
284
-
nordiazepam,
270
10 (0)
-
-
5 (0)
-
50 (0)
-
0 (5)
