Journal of Pharmaceutical and Biomedical Analysis

Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-HPSEC × LC-IT-TOF MS

Yu Xua, DanDan Wanga, Lan Tanga, Jian Wanga,b,∗
a Zhejiang University of Technology, Hangzhou 310014, China
b Zhejiang Institute for Food and Drug Control, Hangzhou 310052, China
a r t i c l e i n f o a b s t r a c t

Article history:
Received 5 June 2017
Received in revised form 30 July 2017 Accepted 31 July 2017
Available online 2 August 2017
Keywords: Polymerized impurity Cephalosporin
High performance size exclusion chromatography
2D-HPSEC × LC-IT-TOF MS

Eleven unknown allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and char- acterized by a trap-free two-dimensional high performance size exclusion chromatography (HPSEC) and reversed phase liquid chromatography (RP-HPLC) coupled to high resolution ion trap/time-of-flight mass spectrometry (2D-HPSEC × LC-IT-TOF MS) with positive and negative modes of electrospray ionization method. Separation and characterization the allergic polymerized impurities in β-lactam antibiotics were on the basis of column-switching technique which effectively combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In the first dimension HPSEC, the column was Xtimate SEC-120 analytical column (7.8 mm × 30 cm, 5 µm), and the gradient elution used pH 7.0 buffer-acetonitrile as mobile phase And the second dimension analytical column was ZORBAX SB-C18 (4.6 × 150 mm, 3.5 µm) with ammonium formate solution (10 mM) and ammonium formate (8 mM) in [acetonitrile-water (4:1, v/v)] solution as mobile phase. Structures of eleven unknown impurities were deduced based on the high resolution MSn data with both positive and negative modes, in which nine impurities were polymerized impurities. The forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied. The question on incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely by multidimensional heart-cutting approaches and online demineralization technique, which was worthy of widespread use and application for the advantages of stability and repeatability. 

1. Introduction

Cephalosporin, broad-spectrum β-lactam antibiotics, are widely used in human and animal therapy [1]. Polymerized impurities of cephalosporins are generated easily during produc- tion and storage of the material [2]. A large number of clinical trials and studies have confirmed that polymerized impurities in the β-lactam antibiotics can cause immediate hypersensitivity, so determination and control of polymerized impurities in the β-lactam antibiotics is extremely important [3].
Since the polymerized impurity of β-lactam antibiotics is very unstable, it is very difficult to prepare and standardize the impu- rity reference substance. The British Pharmacopoeia (BP 2009) uses RP-HPLC for identification of the polymerized impurities peaks of
∗ Corresponding author at: Zhejiang Institute for Food and Drug Control, Hangzhou 310052, China.
E-mail address: [email protected] (J. Wang).

amoxicillin, ampicillin, cefotaxime sodium by comparison of their relative retention times (RT) between drugs and polymers. How- ever, the practice proved that using RP-HPLC for identification of the polymerized impurities is problematic due to the diffi- culty to obtain polymerized impurities reference substance [4]. Polymerized impurities of cephalosporins including cefotaxime, ceftriaxone, cefoperazone and ceftazidime are controlled by a gel filtration chromatography (GFC) in Chinese Pharmacopoeia (ChP) [5]. At present, the most common method for determination the polymer in cephalosporin is Sephadex G-10 gel chromatography, which shows low column efficiency, poor separation and often need a longer time for analysis [6]. Compared with Sephadex G-10 gel chromatography, high performance gel chromatography has the advantages of high sensitivity, good separation and short analysis time. In recent years, high performance gel chromatography has been widely used in the detection of the polymerized impurities in cephalosporin because of its better separation ability, short analysis time and high sensitivity [7,8].

http://dx.doi.org/10.1016/j.jpba.2017.07.063 0731-7085/© 2017 Published by Elsevier B.V.

Based on the request of ICH, the structures of impurities whose content are over 0.1% need to be confirmed. Therefore, it is nec- essary to characterize the structures of polymerized impurities in cephalosporin that has a great significance in improving the quality of cephalosporin [9]. Liquid chromatography-mass spectrometry (LC–MS) has evolved as an identification tool for the characteriza- tion of drug impurities and degradation products [10]. IT-TOF-MS combines the multistage fragmentation function of ion trap full scan mode with high resolution and sensitivity and the accurate determination of molecular weight by TOF mass spectrometry, which greatly improves the ability of accurate tracing components in a complex matrix.
However, all the above LC–MS methods were based on volatile
mobile phase. In non-volatile systems, the selectivity and sensi- tivity were limited. A non-volatile system is used in the official analytical method of Chinese Pharmacopoeia for detection of the polymerized impurities in cephalosporin. Thus, the characteriza- tion of unknown peaks in a non-volatile system, based on data obtained from a volatile LC–MS method, is problematic [11]. Two- dimensional liquid chromatography-mass spectrometry, which has undergone a dramatic development over the last decade, can solve these problems. In the multidimensional heart-cutting approaches (LC-LC), the first chromatographic dimension (1D) can be applied to trapped the aimed impurities by valve-switching and stored in 20ul quantitative loop using the mobile phase with non-volatile salt, then the second chromatographic dimension (2D) removes the non-volatile salt using methanol and pure water as mobile phase [12,13], leading to TIC chromatogram of LC–MS consist with the LC chromatogram of the official analytical method in the peak sequence of impurities. Jian Wang et al. characterized the oxi- dation degradation products in tigecycline by a two-dimensional liquid chromatography combined with Q orbitrap mass spectrom- etry [11]. Because the mobile phase of non-volatile buffer solution did not need to be replaced by volatile buffer solution, the phar- macopeia methods using non-volatile buffer were applicable to LC–MS methods, which lead either the shorter researching time or the risk of missing some impurities due to the retention time variation was avoided. However, the efficiency of this method would be reduced due to the technique only had one loop. Jinlin Zhang et al. used trap-free two-dimensional liquid chromatography to identify the impurities in doxycyline hyclate [14]. Since trap- free two-dimensional liquid chromatography which means do not need a trap column and has six quantitative loops that can handle five impurities simultaneously, making the analysis method more rapid, convenient and time-saving than other analytical methods of two-dimensional liquid chromatography.
In this study, structures of eleven impurities in cefodizime,
cefmenoxime and cefonicid were characterized by trap-free 2D- HPSEC LC-IT-TOF MS with positive and negative electrospray ionisation modes. Each peak eluted from the non-volatile system (one-dimension gel analytical column) was trapped by valve- switching and stored in a 20 µl quantitative loop, then sent to the volatile mobile phase (two-dimension C18 analytical column), which is connected to MS. Structures and fragment pathways of eleven unknown impurities were investigated by complete frag- mentation patterns, in which nine impurities were polymerized impurities. And the forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied. This method had provided the basis of the separation and analysis of β-lactams polymerized impurities, and it could be used to improve the quality of product. To date, there is no report concerning characterization and forming mechanisms of polymerized impurities of cefodizime, cefmenoxime and cefonicid. Hence present research work is under- taken considering general interest.

2. Experimental

2.1. Materials

Cefodizime (batch number: 1609241) used in this study was obtained by Zhejiang Jingxin Pharmaceutical Co. Ltd. (Shaoxing, China). Cefonicid (batch number: 060401) used in this study was obtained by Zhejiang Huidisen Pharmaceutical Co. Ltd. (Hangzhou, China). Cefmenoxime (batch number: 20101018) used in this study was obtained by Zhejiang Huidisen Pharmaceutical Co. Ltd. (Hangzhou, China). Acetonitrile was purchased from Merck (Darmstadt, Germany), ammonium formate was purchased from Sigma-Aldrich (St. Louis, MO, USA), dibasic sodium phosphate (Ana- lytical reagent), sodium dihydrogen phosphate (Analytical reagent) and Sodium carbonate (Analytical reagent) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), and water was HPLC grade (generated by a Millipore Milli-Q-Gradient purifi- cation system).

2.2. Instrumentation

2.2.1. Trap-free two-dimension LC apparatus
A trap-free two-dimension Nexera-XR liquid chromatograph (Shimadzu, Kyoto, Japan), a system equipped with a binary pump 2 was connected to a Shimadzu SIL-30AC autosampler. The first dimension included a binary pump (LC-30AD), auto-sampler (SIL-30AC), column thermostat (CTO-20A) and diode array detec- tor (SPD-M20A). Chromatographic separation in the first dimension chromatography was carried out at 40 ◦C using a Xtimate SEC-120 analytical column (7.8 mm 30 cm, 5 µm). The mobile phase of 1D-LC: pH 7.0 buffer [0.005 mol/L dibasic sodium phosphate solu- tion – 0.005 mol/L sodium dihydrogen phosphate solution (61:39, v/v)]-acetonitrile with gradient conditions: 0 min 5% B (hold for 15 min); 18 min, 20% B (hold for 2 min); 23 min, 5% B (hold for 3 min). The mobile phase flow rate was 0.80 mL min−1 and injec- tion volume was 30 µL. The second dimension was consisted of a binary pump (LC-20AD), column thermostat (CTO-20A) and UV/VIS detector (SPD-20A). The mobile phase of 2D chromatog- raphy of method A (the chromatogram was shown in Fig. S1(A)):
(A) ammonium formate solution (10 mM) and (B) acetonitrile with gradient conditions: 0 min 5% B; 5 min, 95% B; 5.5 min, 5% B; 9 min, 5% B, using a Shimadzu Shim-pack GISS C18 analytical column (50 mm 2.1 mm, 1.9 µm). The mobile phase of 2D chromatog- raphy of method B (the chromatogram was shown in Fig. S1(B)):
(A) acetic acid solution (0.1%, v/v) and (B) acetonitrile with gra- dient conditions: 0 min 5% B (hold for 10 min); 12 min, 15% B (hold for 13 min); 45 min, 60% B (hold for 10 min); 55.1 min, 5% B (hold for 10 min), using a ZORBAX SB-C18 analytical column (4.6 150 mm, 3.5 µm). The mobile phase of 2D chromatography of method C (the chromatogram was shown in Fig. S1(C)): (A) ammonium formate solution (10 mM) and (B) ammonium formate (8 mM) in [acetonitrile-water (4:1, v/v)] solution with gradient con- ditions: 0 min 12% B; 9.3 min, 16% B; 15 min, 20% B; 18 min, 40% B; 19 min, 12% B(hold for 10 min), using a ZORBAX SB-C18 analytical column (4.6 150 mm, 3.5 µm). The mobile phase flow rate was
0.40 mL min−1. The column temperature was at 40 ◦C and the detec-
tion wavelength was 254 nm in the first and second dimension. The first and second dimension columns were connected by means of two high speed/high pressure six-position and six-port switch- ing valves, and equipped with six 20 µL stainless steel loops. Fig. 1 showed the schematic plot of instrument structure. In Fig. 1a, the switch valve 1 and valve 2 were set to the “sample collection”, and sample were directly transferred to the 20 µL quantitative loops with non-volatile mobile phase by control of valve 3 and valve 4. The system retained this configuration for 26 min. After, the switch valve 1 and valve 2 was turned to the “LCMS analysis”(Fig. 1b), thus

 

Fig. 1. A schematic of the instrumental set-up used for LC × LC–IT-TOF MS analyses, using two six-position, six-port valves equipped with six 20 µL sample loops.
connecting ion trap/time-of-flight mass spectrometer. The trapped analytes in quantitative loops were transferred to the second ana- lytical column with the volatile mobile phase. Whether substances drain to MS or drain to waste was controlled by the valve 5.

 

2.2.2. MS spectrometry
Trap-free two-dimensional liquid chromatography was cou- pled to ion trap/time-of-flight mass spectrometer(2D LC-IT-TOF MS)from Shimadzu Corp. (Kyoto, Japan). The mass spectrome- try detector (MSD) was equipped with an electrospray ionization (ESI) source. The ionization mode was positive and negative alter- natively. The interface and MS parameters were as the follows: nebulizer gas flow:1.5 L min−1 (nitrogen); Collision gas: argon; the temperature of curve desolution line (CDL) and heat block: 200 ◦C; detector voltage: 1.56 V; scan range: 100–2000 m/z; the mode of MSn: manual to MSn.
The structures of cefodizime, cefonicid and cefmenoxime pre- sented both amino and carboxylic groups, leading to the response in the mass spectrometry either in the positive ionization or in the negative ionization, so in this study we used the both modes to pre- dict and verify the structures of cefodizime, cefonicid, cefmenoxime and their impurities.

2.2.3. Software
All data acquired were processed by Shimadzu LC-solution software (Kyoto, Japan). Formula predictor from Shimadzu Corp. (Kyoto, Japan) was used to simulate and study the fragmentation behavior of the described compounds.

2.2.4. Sample preparation
10 mg of efodizime sodium was dissolved with water to make a solution of 10 mg mL−1. 10 mg of cefonicid sodium was dissolved with water to make a solution of 10 mg mL−1. And 10 mg of cef- menoxime hydrochloride was dissolved with 5 mg mL−1 sodium carbonate solution to make a solution of 10 mg mL−1.

3. Results and discussion

3.1. Selection of chromatographic conditions

The non-volatile mobile phase in the official analytical method of Chinese Pharmacopoeia for cefodizime was used as the mobile phase in the first dimension system. Cefodizime, cefonicid and cefmenoxime could be completely separated from various degra- dation products and impurities. To permit the use of liquid chromatography-mass spectrometry analysis, the mobile phase in the second dimension system must be a volatile, and concentra- tion of salt should be as low as possible. Fig. S1 showed the second

Fig. 2. First dimension gel chromatography of cefodizime sodium(A), cefonicid sodium(B) and cefmenoxime hydrochloride(C). The insert pictures were the second dimension RPLC-UV of six polymer peaks, a-Peak 1; b- Peak 2; c- Peak 3; d-Peak 1; e- Peak 2; f- Peak 3. h of detection = 254 nm.

Table 1
ESI–MS exact mass data and theoretical mass data on [M+H+] and [M H]− of eleven impurities of cefodizime, cefmenoxime and cefonicid in the positive and negative ion modes.a

Imp. formula [M+H]+
(m/z) [M−H+]
(m/z) Theoretical [M+H](m/z) Theoretical [M-H](m/z) Deviation (ppm)
I II C34 H35 N11 O13 S6 C28 H28 N8 O10 S5 998.0796
797.0620 996.0640
795.0454 998.0813
797.0605 996.0667
795.0459 0.80/−2.71
1.88/−0.62
III
IV C28 H27 N8 O10 S5 C34 H33 N11 O12 S6 731.0367
980.0700 729.0246
978.0586 731.0387
980.0707 729.0241
978.0562 −2.74/0.89
−0.71/2.45
V C34 H33 N9 O11 S7 968.0405 966.0271 968.0417 966.0272 −1.24/-0.10
VI C36 H39 N11 O13 S6 1026.1127 1024.0967 1026.1126 1024.0980 −0.10/-1.56
VII C18 H26 N8 O14 S4 707.0510 705.0368 707.0524 705.0379 −1.98/-1.56
VIII C18 H20 N6 O9 S3 561.0524 559.0363 561.0527 559.0381 −0.53/-3.22
IX C18 H24 N8 O13 S4 689.0446 687.0286 689.0436 687.0277 1.45/1.31
X C30 H32 N14 O11 S5 925.1018 923.0922 925.1013 923.0906 0.54/1.73
XI C14 H15 N5 O6 S2 414.0524 412.0410 414.0537 412.0391 −3.14/4.61
a The data of theoretical [M+H]+ and [M−H]− was calculated by software of Shimadzu accurate mass calculator.
dimension RPLC-UV chromatograms of peak 2 in Fig. 1 of different LC methods. Compared with the chromatograms of methods A and B, method C which used ammonium formate solution (10 mM) and ammonium formate (8 mM) in [acetonitrile-water (4:1, v/v)] solu- tion as the mobile phase in the second dimension system could achieve better separation.

3.2. 2D LC Separation and mass spectra results of impurities

Fig. 2 showed the gel chromatography of cefodizime sodium(A), cefonicid sodium(B) and cefmenoxime hydrochloride(C) in the first dimension, there were six polymerized peaks before the main peaks could be separated by the chromatographic condition in the first dimension system with the detection wavelength 254 nm (peaks 1–6). To check whether each of the impurity peaks was pure or not,

it was important to confirm that all the impurities eluting before the main peaks. The effluent of each peak (peaks 1–6) on the gel chromatographic system before the main peak was switched to the second dimension chromatographic system to obtain RPLC- UV chromatogram of six polymer peaks (seen the insert figure in Fig. 2) and analyzed by LC–MS. There were eleven impurities could be separated in the second dimension and the structures of eleven products were deduced based on the high-resolution MSn data, in which nine impurities were polymerized impurities. Table 1 showed eleven impurities of cefodizime, cefmenoxime and cefonicid in the positive and negative ion modes, and the values of deviation were basically less than 5 ppm. Table 2 showed ESI–MSn exact mass data of major product ions of eleven impurities in both positive and negative ion modes, and proposed chemical structures

Table 2
ESI–MSn exact mass data of major product ions of cefodizime, cefmenoxime and cefonicid and eleven high-molecule weight impurities in positive and negative ion modes.

Imp. [M+H]+(m/z) MS2 fragmentation ions (m/z) MS3 fragmentation ions (m/z)
I 998.0796 585.0261 396.0461
980.0520, 954.0783, 414.0532
II 797.0620 779.0353, 608.0648
III 731.0367 542.0529, 498.0583, 470.0583, 220.0908
IV 980.0700 791.0709, 730.0544, 636.0644
V 968.0405 779.0442, 718.0277, 396.0365
VI 1026.1127 585.0280 396.0451
422.0822
VII 707.0510 663.0490, 497.9773, 470.9738
705.0368 661.0480, 617.0567
VIII 561.0524 365.0750
559.0363 487.0454 423.0747,379.0866,
423.0842, 379.0938, 345.1034, 327.0921 345.1009
IX 689.0446 671.0256 581.0544
645.0511, 627.0299, 581.0595
687.0286 643.0463, 599.0551
X 925.1018 881.0969, 809.0832, 765.0880, 748.0833, 704.0694
XI
Imp. VII 414.0515 [M−H+](m/z)
705.0368 396.0429, 370.0633, 352.0544, 326.0746, 169.0486
MS2 fragmentation ions (m/z) 661.0480, 617.0567
MS3 fragmentation ions (m/z)
VIII 559.0363 487.0454 423.0747,379.0866,
423.0842, 379.0938, 345.1034, 327.0921 345.1009
IX 687.0286 643.0463, 599.0551

 

Fig. 3. Chemical structures of cefodizime, cefonicid and cefmenoxime and impurities 1–11.

Fig. 4. (a) Full scan spectrum of impurity 1 showing [M+H]+ ions at m/z 998.0796. (b) MS2 spectrum of m/z 998.0796 ion. (c) MS3 spectrum of m/z 585.0261 ion. (d) Full scan spectrum of impurity 1 showing [M−H+] ions at m/z 996.0640.
of cefodizime, cefmenoxime and cefonicid and eleven impurities were shown in Fig. 3.

3.3. Structure elucidation

3.3.1. Impurity I
TOF high resolution mass showed that the formula of impurity I was C34H35N11O13S6. Fig. 4 revealed that [M+H+] at m/z 998.0796 and [M+Na+] at m/z 1020.0680 appeared in the full scan of ESI+. The protonated molecule at m/z 998.0796 of impurity I fragmented into the product ion at m/z 954.0783 by means of the loss of 44 Da, corresponding to CO2, further fragmented into the product ion at m/z 585.0261 by the cleavage of amide bond between C-18 and N-29 and loss of C14H15N5O4S2 structure. Subsequent loss of one 2- mercapto-4-methyl-5-thiazoleacetic acid (MMTA) led to the ion at m/z 396.0461. And the ion at m/z 980.0520 was formed by elimina- tion of one H2O. The protonated molecule at m/z 998.0796 showed the cleavage of amide bond between C-18 and N-29 and loss of C20H20N6O7S4 structure conducted to the fragment ion at m/z 414.0532. From the above information, it was concluded that impu- rity I was resulting from intermolecular aminolysis of β-lactam ring by the amine of the second molecule with the hydroxyl sub- stituting MMTA [15]. Fig. 5 presented the proposed structure and fragmentation pattern of impurity I.

3.3.2. Impurity II
TOF high resolution mass showed that the formula of impurity II was C28H28N8O10S5. The protonated molecule at m/z 797.0620 of impurity II fragmented into the product ion at m/z 608.0648 by means of the loss of 189 Da, corresponding to MMTA. Another frag- mentation pathway of the protonated molecule at m/z 797.0620 concerned the loss of 18 Da, corresponding to one H2O, conducting to the fragment ion at m/z 779.0353. These experiments revealed that structure of impurity II was formed by intermolecular aminoly- sis at the position of C-29 by the amine of the second molecule with the hydroxyl substituting MMTA. Fig. S2 presented the proposed structure and fragmentation pattern of impurity II.

3.3.3. Impurity III
TOF high resolution mass showed that the formula of impurity III was C28H27N8O10S5. The protonated molecule at m/z 731.0367 of impurity III fragmented into the product ion at m/z 542.0592 by

loss of one MMTA, further fragmented into the ion at m/z 498.0583 by the loss of one CO2 and the ion at m/z 220.0908 by elimination of C7H6N2O3S2 structure and one H2S. Another fragmentation path- way of the ion at m/z 542.0529 concerned the loss of one CO2 and one CO, conducting to the fragment ion at m/z 470.0583. From the above information, the structure of impurity III was deduced that the carbon positive ion formed by the bond cleavage at the posi- tion of C-10 in one molecule of cefodizime, and the carbon positive ion could nucleophilic attack of the atom at the S-25 of another molecule with the cleavage of amide bond. Fig. S3 presented the proposed structure and fragmentation pattern of impurity III.

3.3.4. Impurity IV
The formula of impurity IV was C34H33N11O12S6 based on the data from TOF high resolution mass spectrometer. The frag- mentation pathway of the protonated molecule at m/z 980.0700 fragmented into the product ion at m/z 791.0709 by the loss of one MMTA, further fragmented into the ion at m/z 730.0544 by elim- ination of one NH3 and one CO2. Another fragmentation pathway of ion at m/z 791.0709 concerned the loss of C3H2N2S2 structure, leading to the ion at m/z 636.0644. From the above information, it was concluded that impurity IV was resulting from intermolecular aminolysis of β-lactam ring by the amine of the second molecule with losing one MMTA and forming a five-member lactone ring. Fig. S4 presented the proposed structure and fragmentation pattern of impurity IV.

3.3.5. Impurity V
TOF high resolution mass showed that the formula of impurity V was C34H33N9O11S7. The protonated molecule at m/z 968.0405 fragmented into the product ion at m/z 779.0442 by the loss of one MMTA, further fragmented into the ion at m/z 718.0277 by loss of one H2O and one NH3. The product ion at m/z 779.0442 showed the cleavage of the amide bond between N-15 and C-30 conducted to the ion at m/z 396.0365. Based on the information above, the structure of impurity V was that the carboxyl group at the position of C-30 of one molecule cefodizime reacted with the amino group at the position of N-15 of the second molecule to form an amide bond accompanied with amide bond cleavage at the position of N-29. Fig. S5 presented the proposed structure and fragmentation pattern of impurity V.

Fig. 5. proposed fragmentation pathways of impurity I in the positive ion mode.
3.3.6. Impurity VI
High resolution TOF mass scanning in both positive and negative modes proved that the formula of impurity VI was C36H39N11O13S6. The protonated molecule at m/z 1026.1127 of impurity VI frag- mented into the product ion at m/z 585.0280 and the ion at m/z 442.0822 by cleavage of amide bond between C-8 and N-29. The product ion at m/z 585.0280 fragmented into the ion at m/z 396.0451 by the loss of one MMTA. From the above information, the structure of impurity VI was that intermolecular aminolysis of β-lactam ring by the amine of the second molecule with the hydroxyl substituting MMTA. Subsequently, the carbon positive ion formed by the bond cleavage at the position of C-31, and the carbon positive ion could nucleophilic attack of the amine at the position of N-41 of the third molecule with the cleavage of amide bond and hydroxyl substituting MMTA. Fig. S6 showed the mass spectral fragmentation of impurity VI in both modes.
3.3.7. Impurity VII
Data obtained from TOF high resolution mass spectrometer in both positive and negative ion mode showed that the formula of impurity VII was C18H26N8O14S4. The protonated molecule at m/z 707.0510 fragmented into the product ion at m/z 663.0490 by losing one CO2 and the ion at m/z 497.9773, which was easy to produced, by the cleavage of the bond between N-1 and C-6 and elimination of C5H9NO4 structure. The fragment ion at m/z 470.9738, which originated from the loss of one molecule of NH3 from the ion at m/z 497.9773. In the negative ion mode, the product ion at m/z 661.0480, which originated from the loss of one molecule of CO2 from the precursor ion at m/z 705.0368, further fragmented into the product ion at m/z 617.0567 by the loss of one CO2. Based on the information above, the structure of impurity VII was that inter- molecular aminolysis of β-lactam ring by the amine of the second molecule, and one molecule in the structure of impurity VII was the cleavage of amide bond of cefonicid with oxygen addition occurred on the sulphurs, and the other one was the cleavage of amide bond of cefonicid accompanied with hydroxyl substituting 5-Mercapto- 1-methyltetrazole (3-TSA) and the cephem ring was hydrolyzed.

Figs. S7 and S8 presented the proposed structure and fragmentation pathway of impurity VII.

3.3.8. Impurity VIII
The structure of impurity VIII was C18H20N6O9S3 based on the data from TOF high resolution mass spectrometer. The fragmen- tation pathway from the protonated molecule at m/z 561.0524 concerned the loss of 3-TSA leading to the ion at m/z 365.0750. In the negative ion mode, the precursor ion at m/z 559.0363 frag- mented into the product ion at m/z 487.0454 by losing one CO and one CO2 and the ion at m/z 423.0842 by elimination of one SO2, further loss of one CO2 to form the ion at m/z 379.0938. The frag- ment ion at m/z 345.1034, which originated from the loss of one molecule of H2S from the ion at m/z 379.0938, further fragmented into the product ion at m/z 327.0921 by the loss of one H2O. The impurity VIII was a hydrolysis product of cefonicid, the structure of impurity VIII was deduced as the hydrolysis of β-lactam ring of cefonicid. Figs. S9 and S10 presented the proposed structure and fragmentation pattern of impurity VIII.

3.3.9. Impurity IX
The formula of impurity IX was C18H24N8O13S4 based on the data from TOF high resolution mass spectrometer. The protonated molecule at m/z 689.0446 of impurity IX fragmented into the prod- uct ion at m/z 671.0256 by means of the loss of one H2O. Subsequent loss of one CO and one H2O conducted to the fragment ion at m/z 581.0595. Another fragmentation pathway of the protonated molecule at m/z 689.0446 fragmented into the product ion at m/z 645.0511 by losing one CO2 and the ion at m/z 627.0299 by elimina- tion of one H2O. In the negative ion mode, the precursor ion at m/z 687.0286 fragmented into the product ion at m/z 643.0463 by the loss of one CO2 and the ion at m/z 599.0551 by elimination of one CO2. From the above information, the structure of impurity IX was that intermolecular aminolysis of β-lactam ring by the amine of the second molecule, and one molecule was the cleavage of amide bond of cefonicid with oxygen addition occurred on the sulphurs, and the other one was the cleavage of the amide bond of cefoni- cid accompanied with hydroxyl substituting 3-TSA. Figs. S11 and

S12 presented the proposed structure and fragmentation pattern of impurity IX.

3.3.10. Impurity X
The formula of impurity X was C30H32N14O11S5 based on the data from TOF high resolution mass spectrometer. The fragmen- tation pathway from the protonated molecule at m/z 925.1018 concerned the loss of one 5-mercapto-1-methyltetrazole leading to the ion at m/z 809.0832. Subsequent loss of one CO2 conducted to the ion at m/z 765.0880. The product ion at m/z 765.0880 frag- mented into the ion at m/z 748.0833 by the loss of one NH3 and the ion at m/z 704.0694 by the elimination of one CO2. Another frag- mentation pathway of the ion at m/z 925.1018 concerned the loss of one CO2, leading to the ion at m/z 881.0969. From the above infor- mation, the structure of impurity X was formed by intermolecular aminolysis of β-lactam ring by the amine of the second molecule with losing one 5-mercapto-1-methyltetrazole and forming a five- member lactone ring and the β-lactam ring was hydrolyzed. Fig. S13 presented the proposed structure and fragmentation pattern of impurity X.

3.3.11. Impurity XI
Data obtained from TOF high resolution mass spectrometer in both positive and negative ion mode shows that the formula of impurity XI was C14H15N5O6S2. The protonated molecule at m/z 414.0515 fragmented into the product ion at m/z 396.0429 by the loss of one H2O and the ion at m/z 352.0544 by elimination of one CO2, subsequent loss of one C6H5N2O2S structure leading to the ion at m/z 169.0486. Another fragmentation pathway of the protonated molecule at m/z 414.0515 concerned the loss of 44 Da, correspond- ing to one CO2, conducting to the fragment ion at m/z 370.0633, further loss of one CO2 conducted to the ion at m/z 326.0746. From the above information, impurity XI was formed by losing one 5- mercapto-1-methyltetrazole with forming a five-member lactone ring and the β-lactam ring of cefmenoxime was hydrolyzed. impu- rity XI was a hydrolysis product of cefpiramide. Fig. S14 presented the proposed structure and fragmentation pattern of impurity X.

3.4. Forming mechanism of ˇ-lactam antibiotic polymerization in cephalosporins

β-lactam antibiotic polymerization usually could be divided into two categories: 1) occurred in the nucleus of the N-type polymerization: First, the β-lactam ring was opened to form a secondary amino group with nucleophilic attack ability, then nucle- ophilic addition reaction happened to the carbonyl group of the other cephalosporin molecule, and the N-type polymerization was formed such as impurities I and VI; 2) side-chain involvement in the L-type polymerization: The active group in the 7-side chain directly attacked the carbonyl carbon atoms in the β-lactam ring to form a polymer such as impurity V [16].
Moreover, β-lactam antibiotics included ring-closed and ring- open polymer [17]. The polymerization process could be seen from the impurity I–X. It could be seen that the β-lactam ring-open was the structural basis of the polymerization. It was found that the energy of the β-lactam ring and amide conjugation can determine the molecular reactivity. The amide nitrogen atoms of the β-lactam ring generally extended out of a certain length of the plane com- posed of three adjacent carbon atoms to form the taper of the β-lactam ring. When the taper of the ring increased, the tension energy and the conjugate energy of the ring were decreased, the reaction activity of the molecule was increased, and the opening ring became easy [18].

4. Conclusions

Eleven allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and characterized by a trap-free 2D- HPSEC LC-IT-TOF MS with positive and negative modes of electrospray ionization method. Through the multidimensional heart-cutting approaches and online demineralization technique, the contradiction between non-volatile mobile phase and mass spectrometry was solved. The TIC chromatogram of LC–MS could be in conformity with the LC chromatogram of the official analytical method in the peak sequence of impurities. Moreover, an effective method to characterize the polymerized impurities in β-lactam antibiotics may be established on the basis of column-switching technique which effectively combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. Struc- tures of eleven impurities in cephalosporins were deduced based on the high resolution MSn data with both positive and negative modes, in which nine impurities were polymerized impurities. And the forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied.

Acknowledgment

This research is supported by the food and drug science and technology project of Zhejiang food and drug administration in 2016.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2017.07.063.

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