Mass Spectral Profile for Rapid Differentiating Beta-Lactams from Their Ring-Opened Impurities

High performance liquid chromatography tandem mass spectrometry (HPLC MS) has been widely used for β-lactam antibiotics determination. However, its application to identify impurities of these frequently used drugs is not sufficient at present. In this job, characteristic profiles of the collision induced dissociation (CID) spectra of both β-lactams and ring-opened β-lactams were extracted from the MS data of six β-lactam antibiotics and their forty-five impurities, and were confirmed by the MS data reported in the literature. These characteristics have been successfully applied to rapid differentiation of β-lactam and ring-opened β-lactam impurities in cefixime, cefdinir, and cefaclor. However, these characteristic profiles can only be obtained under low activating voltage. They did not display in the high energy activated CID spectra. Diagnostic fragmentations for determining the localization of double bond and substituents on the thiazine ring and the side chain were also observed. In addition, several characteristic fragmentations are hopeful to be used to differentiate the configurations of C-2 on the thiazine ring of ring-opened impurities, which is generally disadvantageous of mass spectrometry. Taken together, forty-five impurities were identified from the capsules of cefixime, cefdinir, and cefaclor.

Scheme S-7: Proposed pathway of fragment at m/z 337 formation from ring-opened cefdinir under low activating energy.

Identification of impurities in cefdinir
The representative chromatograms of cefdinir and its impurities are shown in figure 7. Apart from the main constituent (Compound 0), there are four peaks observed in extracted ion chromatogram at m/z 396 (Figure 7-B).
All display similar CID spectra as that of cefdinir. Compound 1`was proposed to be impurity E, cefdinir lactone product, according to the relative retention time and abundance (Scheme S-8). Compound 3 was identified similarly to be impurity G, cefdinir 7-epimer (Scheme S-8) . Compound 2 and 4 was suggested as unknown isomers.
Scheme S-8: Structures of cefdinir and impurities with β-lactam ring.
The pseudo-molecular ion at m/z 384 indicates that the molecular weight of compound 5 was 383, which is the same as impurity C of cefdinir. Its CID spectrum is observed to be almost the same as which of cefdinir (Figure S-5). Compound 5 was identified as impurity C of cefdinir (Scheme S-8) based on these data.
Protonated molecules at m/z 370 suggested that compound 10 and 11 were decarboxylation products of ring-opened cefdinir (compound 6 or 8). Their CID spectra were also observed to be in a ring-opened β-lactam profile ( Figure S-7). Different yields of fragments formed from loss of hydroxylamine were also observed in CID spectra of these compounds. Compound 10 and 11 were suggested to be impurity H of cefdinir (Scheme S-9) based on these data.
The pseudo-molecular ion of compound 12 and 13 were observed at m/z 428, which is 14 amu more than those of compound 6. The observation of fragment at m/z 395 (80% -90%), as well as the fragments at m/z 126, suggested that the side chains of compound 12 and 13 were the same as that of compound 6, and different substituent group on the furanone. Based on these observation, compound 12 and 13 were tentatively identified as

Identification of other impurities in cefaclor
Scheme S-10: Structure of impurities identified in cefaclor capsule. A, compound 1 and 2; B, compound 14 and 15; C, compound 7 and 13

Identification of compound 11 and 12
Pseudo-molecular ion of both compound 11 and 12 were observed at m/z 400, which is 14 amu more than indicated a primary amine located on the side chain, and their configurations of C-2 were the same. The neutral loss of 32 (m/z 315 [347-32] + , 100% for compound 11, 50% for compound 12) suggested that there was a methoxyl or a hydroxymethyl group. Compound 11 and compound 12 were identified to be methyl ester of

Identification of compound 14 and 15
Pseudo-molecular ions at m/z 501 suggested that the molecular weights of compound 14 and 15 were both 500 amu ( Figure S-9), which is the same as N-phenylglycyl cefaclor, the impurity H of cefaclor . High yield of fragment at 368 formed in the ion source indicated both compounds were unstable, and there was an N-phenylglycyl substituent group localize on the molecule. Extensive fragmentation of the daughter ion m/z 368 leads to a spectrum which is the same as that of compound 1 and 2. Compound 14 and 15 were identified as N-phenylglycyl delta-3-cefaclor based on these observations (Scheme S-10 B).

Identification of compound 7 and 13
CID spectra of both compound 7 and 13 display protonated molecule at m/z 364, which is 36 amu less than
Scheme S-12: Representative structure of β-lactam antibiotics and their impurities.
Most β-lactam antibiotic consist a core of thiazole lactam (or thiazine lactam) and an amide side chain localized on the lactam ring (Scheme S-12). Generally, R1 is an aromatic group and R3 is a carboxyl group. Much diversity has been observed of R3 and R4. The diversity of the substituent groups on the thiazole (or thiazine ring) and the side chain are related to the antimicrobial spectra and stabilities. Impurities of a β-lactam antibiotic include process impurities, degradants, and possible isomers. R5 is a carboxyl produced from the hydrolysis of the β-lactam. However, it is eliminated in decarboxylated impurities. Degradants with furone ring usually formed when R4 is methene or a vinyl group. Diagnostic fragmentations of β-lactams and their impurities have been obtained under low energy activated CID experiment. These characteristics disappeared when a CID spectrum was obtained under high energy activation.
Generally, mass spectrometry is not good at differentiating isomers. However, several diagnostic fragmentations are observed to be useful to determine the localization of a substituent group, even the configuration of chiral carbon in this job. The localization of double bond on the thiazine ring, which is generally difficult to be determined using MS data, has been successfully determined based on the yields of elimination of hydrogen chloride of cefaclor impurities. For the impurities with β-lactam ring, the fragments originate from the cleavage of lactam ring is the best diagnostic characteristic to determine the localization of the substituents.
Elimination of methanol, hydrogen chloride, as well as H 2 O, was used to determine the localization of the double bond on the thiazine ring. A double bond at delta-3 is proved to be advantageous to the elimination of R4 when it is a group linked with a heteroatom, e.g. methoxyl, hydroxyl or chloride. The effect of the localization of the double bond on the thiazine ring was also observed for the ring-opened impurities. Cleavage of R3 is observed in some ring-opened impurities. Its yield is supposed to be related to the configuration of C-2 of the thiazine ring without the fused β-lactam. Unfortunately, we failed to find any reference compound to determine what configuration it represents.
It is unimaginable to interpret the MS data obtained with a single compound, because there should be several