Electrospray mass spectrometry in the structural characterization of cephalosporins

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 34, 268È275 (1999)

Electrospray Mass Spectrometry in the StructuralCharacterization of Cephalosporins¤

Simone Tenconi,1 Laura De Filippo,1 Marco Da Col,1 Anna Maria Gioacchini2 and Pietro Traldi2*1 Ribbon Srl, Via Mascheroni 25, Milan, Italy2 CNR, Area di Ricerca, Corso Stati Uniti 4, I-35020 Padova, Italy

Six cephalosporins of pharmacological interest, cephalexin, cephuroxime, cephazolin, cephoperazone sodium salt,cephatrizin free acid and cephonicid disodium salt, were analysed by electrospray mass spectrometry. [M Ô Na ]—anions were produced in high yield in the case of cephalexin, cephuroxime, cephazolin and cephoperazone, leadingto signals at least two orders of magnitude more intense than those related to [M + Na ]‘ cations observed in thepositive ion mode. In cephatrizin, [M Ô H ]— represented the most abundant species, whereas in cephonicid the[M Ô 2Na + H ]— anions were easily produced. No fragment ions were detectable in the electrospray spectra ofany of the compounds, and MSn turned out to be essential to draw the fragmentation patterns. Most of thesepatterns were related to the substituent of the 7-aminocephalosporin nucleus, suggesting that the nucleus itself ishighly stable. Copyright 1999 John Wiley & Sons, Ltd.(

KEYWORDS: electrospray ionization ; negative ions ; cephalosporins ; multiple mass spectrometry ; low-energy collisions

INTRODUCTION

Cephalosporins, Ðrst isolated and identiÐed by Brotzenin 1948, are still an interesting class of b-lactam anti-biotics because of their power against a large number ofboth Gram-positive and Gram-negative organisms.Cephalosporin structures are based on the 7-aminocephalosporinic acid nucleus (with a condenseddihydrothiazole ring in its skeleton) and are generallystable in acid media and in the presence of penicillase.1Many e†orts have been made to synthesize cephalo-sporins with various physico-chemical properties(mainly liposolubility) by varying the substituents.2

Cephalosporins have been the object of mass spectro-metric investigations since 1964.3 The ionizationmethod employed at that time was electron ionization(EI), which required derivatization of the polar groupscontained in the molecule. With this approach, clearcharacterization of the cephalosporins under study wasachieved and the fragmentation pattern was found to beclosely related to the original structure. As observed inthe case of the penicillins, cleavage of the b-lactam ringwas the main, common, primary fragmentation

* Correspondence to : P. Traldi, CNR, Area di Ricerca, Corso StatiUniti 4, I-35020 Padova, ItalyE-Mail : Favretto=padr1.pd.cnr.it.

¤ This paper is dedicated to the memory of Professor Dr Wilhelm J.Richter

pathway, together with the side-chain amide bond andC(3)ÈR bond.

The further development of desorption/ionizationmethods, such as Ðeld desorption (FD)4 and fast atombombardment (FAB),5 led to the development of newapproaches for the mass spectrometric analysis ofcephalosporins, without derivatization being essential.For example, the Ðeld desorption of derivatized andunderivatized cephalosporins led to clear identiÐcationof their molecular mass. Fragment ions due to b-lactamring cleavage, like those observed in EI conditions, werethought to be caused by pyrolysis on the hot emitter.6

Thermospray high-performance liquidchromatography/mass spectrometry (HPLC/MS) hasbeen successfully used to analyse non-volatile and ther-mally labile compounds,7h9 including several b-lactams.10h14 Structurally di†erent underivatizedcephalosprins have been studied in detail by laser-induced evaporation, desorption chemical ionizationand fast atom bombardment mass spectrometry.15Another e†ective technique is atmospheric pressureionization, used for investigating drug residues inbiological matrices.16

Recently, electrospray ionization (ESI)17 linked withmultiple tandem mass spectrometry (MSn)18 has beenemployed in the structural characterization of a series ofpharmaceutically important penicillin sodium salts.19 Inthese conditions, it was observed that [M[ Na]~anions were the sole peaks present in the spectra, withan abundance two or three orders of magnitude higherthan that of the [M ] Na]` positive ion. MSn experi-

CCC 1076È5174/99/040268È08 $17.50 Received 29 July 1998Copyright ( 1999 John Wiley & Sons, Ltd. Accepted 22 October 1998

ESI-MS OF CEPHALOSPORINS 269

Table 1. Commercial names structural and elemental formulae and molecular masses of the cephalosporins under investigation(Mr)(compounds 1–6)

Copyright ( 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 268È275 (1999)

270 S. TENCONI ET AL .

Scheme 1

ments also produced a fragmentation pathway relatedto b-lactam ring cleavage which was di†erent to thatusually obtained from positive precursor ions. Thenegative fragmentation pathway gave better structuralinformation.

These results led us to undertake an analogousstudy on a series of pharmacologically important cep-halosporins, widely employed as antibacterial drugs,i.e. cephalexin sodium, sodium (6R,7R)-7-[(R)-2-amino -2 -phenylacetamido] -3 -methyl - 8 -oxo -5 - thia -1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (1) ; cephur-oxime sodium, sodium (7)-(6R,7R)-3-(carbamoyloxy-methyl)-7-[2-(2-furyl)-2-(methoxyimino)acetamido]-8 - oxo - 5 - thiaazabicyclo [ 4.2.0 ] oct - 2 - ene - 2 - car-boxylate (2) ; cephazolin sodium, monosodium (6R,7R)-3 - [ ( 5 - methyl - 1 , 3 , 4 - thiadiazol - 2 - yl ) thiomethyl ] -8 - oxo - 7 - [ 2 - ( 1H - tetrazol - 1 - yl ) acetamido ] - 5 - thia -1 - azabicyclo[4.2.0]oct - 2 - ene - 2 - carboxylate (3 ) ;cephatrizine propylene glycol, 7-M[amino-(4-hydroxy-phenyl )acetyl]-aminoN -8-oxo-3-[ (1H -1 ,2 ,3- thiazol-4 - ylthio ) methyl ] - 5 - thia - 1 - azabicyclooct - 2 - ene -2-carboxylic acid solvatate with propane-1,2-diol(4) ; cephonicid disodium (6R,7R)-7-[(R)-mandel-amido]-8-oxo-3-M[1- ( sulphomethyl ) -1H - tetrazol -5 -yl ] methyl N - 5 - thia - 1 - azabicyclo [ 4.2.0 ] oct - 2 - ene -2-carboxylate (5) ; and cephoperazone sodium, sodium(6R , 7R ) -7 -[ (R ) -2 - (4 -ethyl -2 ,3 -dioxo-1-piperazine-carboxamido ) - 2 - ( p - hydroxyphenyl ) acetamido ] - 3 -M[(1-methyl-11-tetrazol-5-yl ) thio]methylN -8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (6). Thestructural and elemental formulae and molecularmasses of these compounds are listed in Table 1.

EXPERIMENTAL

Chemicals

Water and methanol were of HPLC grade (Sigma, StLouis, MO, USA). The cephalosporins (compounds 1È6)were obtained from Ribbon (Milan, Italy).

Mass spectrometric measurements

Samples were dissolved in methanolÈwater (50 : 50, v/v)at a concentration of 10 lM. ESI-MSn experiments wereperformed using a Finnigan (San Jose, CA, USA) LCQion trap mass spectrometer. Cephalosporin solutionswere introduced into the electrospray source at 8 llmin~1 using the syringe pump of the instrument. TheESI source was operated at 3.8 kV and the capillaryheater was set to 170 ¡C. Experiments were performedin the negative ion mode. The ions of interest were iso-lated monoisotopically in the ion trap and collisionallyactivated with 35% ejection r.f. amplitude at standardHe pressure.

RESULTS AND DISCUSSION

In a previous investigation, the behaviour of a series ofpenicillins in ESI conditions was studied.19 For all thecompounds examined, it was found that, in ESI, theproduction of negative ions was more abundant thanthat of positive ions. MSn experiments produced a frag-mentation pathway relating to the cleavage of theC(6)ÈC(7) bond of the b-lactam ring, which was di†er-ent to that described for positive ions. MSn experimentsallowed the deÐnition of a general fragmentationpattern, valid for all the penicillins studied.

Similarly, the ESI of compounds 1È6 in the presentstudy led to [M[ Na]~ anions for 1È3 and 6,[M[ H]~ for 4 and [M[ 2Na ] H]~ for 5, whichwere all at least two orders of magnitude more abun-dant than the corresponding [M ] Na]` or [M] H]`cations. This behaviour may be ascribed to the pre-existence of [M[ Na]~ ions in the methanol solutionsinjected into the ESI source ; in other words, being salts,these compounds can exist in neutral solution inanionic form together with their metal cations, andhence their primary ESI negative ion spectra are whollypredictable. In the case of 5, in which an groupSO3Na



Table 2. Negative ion ESI mass spectra (MS) and sequential MSn-traces obtained for compounds 1–6

is present, the only ion detectable in negative ion ESIconditions was the [M[ 2Na ] H]~ anion, suggestingthe presence of species in the molecular structure.SO3HAccording to these ESI data, although there is a closeanalogy between the behaviour of penicillins andcephalosporins, it is limited to the ease with which theyboth produce [M [ Na]~ ions. In fact, although thepenicillins produced a common fragmentation pattern,this was not the case for the cephalosporins, whichrevealed speciÐc collisionally activated fragmentationroutes, strongly dependent on the R1 and R2 substit-uents. Hence the loss from [M[ Na]~,CO2[M[ H]~ or [M[ 2Na ] H]~ ions (the only col-lisionally generated process observed for [M[ Na]~ions from penicillin sodium salts) was absent for 2 and 3and accompanied by other decomposition routes for 1and 6 (see Table 2 and Scheme 1). This behaviour maybe related to the intrinsic difficulty of the dihydrothia-zole ring in managing the negative charge once hasCO2been lost. This difficulty, which does not arise in thecase of the thiazolidine ring of penicillins, may lie in the

unfavourable location of the negative charge on the C(2)carbon atom, sp2 hybridized. Consequently, Hrearrangement processes may take place, in order toyield more stable products, such as those shown inScheme 1. Another explanation for the di†erent behav-iour among compounds 1È6 may lie in the e†ect(s) ofthe R2 substituent, which, although it may a†ect nega-tive charge stabilization after the loss of (favouringCO2or inhibiting H rearrangement process(es)), may alsopromote di†erent and more energetically favoureddecomposition routes.

Multiple MS/MS experiments were found to beessential to obtain the fragmentation patterns of com-pounds 1È6. The related data are summarized in Table2 and, as examples, the MS3 traces are reported in Figs1È3.

Compound 1 shows only the formation, from theion (a), of an ion species at m/z 189[M[ Na[ CO2]~(see Table 2, MS3 experiments). These fragments orig-

inate from the cleavage of N(1)ÈC(8) and C(6)ÈC(7)bonds, i.e. through the process always occurring in the



Figure 1. MS3 traces of (a) 1 and (b) 2 obtained by collision ofcollisionally generated ions at m /z 302 and 362, respectively.

case of positively charged molecular species (either M`Õof derivatized compounds or MH` generated by di†er-ent desorption/ionization methods) of cephalosporinsand penicillins,15 but not detected in the fragmentationof [M[ Na]~ anions of penicillin sodium salts.19 Thisfragmentation pattern was only observed with 1 andmust be related to thermodynamic aspects (in particularto the stability of the products). It may originate fromthe a@ ion through the mechanism shown in Scheme 2.

Compounds 2, 3 and 6 show highly favoured primaryloss of R2H, responsible for the most abundant peak inthe collisional spectra of the related [M[ Na]~ ions(see Table 1). The HÕ engaged in the R2H loss is prob-ably one of those in position C(4), and the fragmenta-tion process implies the formation of a diradical anion,as shown in Scheme 3. This species may easily rearrangeto tricyclic species b, highly stable from the thermody-namic points of view; b ions are the precursors of aseries of further decomposition pathways, speciÐc forthe examined compounds. Thus, loss from b ionsCO2occurs for 2 and 3, leading to the species at m/z 318 and277, respectively. The former shows a further loss of

leading to the ion at m/z 284, whereas the latterH2S,decomposes through loss of R1H. In the case of 6 (m/z528), b ions pass through a series of decompositionpathways, summarized in Scheme 4. The ion at m/z 386originates by cleavage 1 and loss of the R1 substituent,with H rearrangement. Cleavage of the b-lactam ringalso occur (2 and 3 in Scheme 4), giving rise to the frag-


ments at m/z 343 and 371. The latter, through loss ofthe R1 substituent, leads to the most abundant ion inthe collisional MS3 trace (m/z 229). All these fragmenta-tion pathways may easily be explained by the b ion inan anion form originating after the loss of R2H, due toits high stability.

For 5, a di†erent behaviour of ion a was observed. Inthis case, two product ions were observed, at m/z 306and 262, which cannot be explained by fragmentationpathways such as those already described, but by cleav-age of the dihydrothiazole ring (Scheme 5).

Lastly, the [M- ion of 4 (m/z 417) showed aCO2Na]~loss of leading to a fragment at m/z 400, and theNH3 ,cleavage of C(7)ÈC(8), N(1)ÈC(6) and S(5)ÈC(4) bondsled to the fragment at m/z 223 containing the R1moiety. MS4 experiments showed that the ion at m/z400 led to fragments at m/z 299, due to a further R2H2loss, whereas the ion at m/z 223 underwent further NH3loss, giving rise to the anion at m/z 206.

In conclusion, the mass spectrometric behaviour inESI conditions of these cephalosporins is mainly due toR1 and R2 substituents. Like the penicillins, in ESI con-ditions, compounds 1È6 give rise to abundant [M-Na]~,but the cleavage of the C(7)ÈC(8) bond, which is theÐrst step of most fragmentation processes in penicillins,is completely suppressed. The data obtained suggestthat, in negative ion conditions, the cephalosporinskeleton has a higher bond strength than that of thepenicillins.



Scheme 2


Scheme 3



Scheme 4



Scheme 5

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Electrospray mass spectrometry in the structural characterization of cephalosporins