MS and NMR

Journal Pre-proof Characterization of two unknown impurities in roxithromycin by 2D LC–QTOF/MS/MS and NMR Jian Wang (Conceptualization) (Resources) (Supervision) (Methodology) (Project administration), Jinjin Zhou (Validation) (Investigation) (Writing - original draft) (Methodology) (Project administration), Yu Xu (Validation) (Investigation) (Methodology) (Project administration), Bingqi Zhu (Writing - review and editing) (Investigation), Yong Jin (Investigation) (Supervision)

PII:

S0731-7085(20)30006-6

DOI:

https://doi.org/10.1016/j.jpba.2020.113196

Reference:

PBA 113196

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received Date:

2 January 2020

Revised Date:

18 February 2020

Accepted Date:

19 February 2020

Please cite this article as: Wang J, Zhou J, Xu Y, Zhu B, Jin Y, Characterization of two unknown impurities in roxithromycin by 2D LC–QTOF/MS/MS and NMR, Journal of Pharmaceutical and Biomedical Analysis (2020), doi: https://doi.org/10.1016/j.jpba.2020.113196

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Characterization of two unknown impurities in roxithromycin by 2D LC–QTOF/MS/MS and NMR

Jian Wanga,b, Jinjin Zhoua, Yu Xua, Bingqi Zhuc, Yong Jind,*

a

Zhejiang University of Technology, Hangzhou 310014, China Key Laboratory for Core Technology of Generic Drug Evaluation National Medical Product Administration, Zhejiang Institute for Food and Drug Control, Hangzhou 310052, China c d

Zhejiang Chinese Medical University, Hangzhou 310053, China Zhejiang Guobang Pharmaceutical Co, Ltd.

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b

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* Yong Jin: Tel.: +86 057187180358. E-mail address: [email protected]

Highlights

Two unknown impurities in roxithromycin were separated and characterized by 2D LC–QTOF MS/MS.

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The structures of the impurities were confirmed by NMR after purifying by preparative HPLC.

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The mechanisms for formation of the impurities were proposed.

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ABSTRACT

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

Two unknown impurities in roxithromycin were discovered and preliminarily characterized by two-

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dimensional liquid chromatography coupled with QTOF mass analyzer (2D LC–QTOF MS/MS). The column-switching technique of 2D LC made the chromatographic conditions in official standard of roxithromycin compatible with mass spectrometric detector. The complete MS/MS fragmentation patterns of the impurities were studied to obtain structural information of these impurities. Furthermore, these two impurities were separated and purified by preparative HPLC,

and their structures were confirmed by 1D and 2D nuclear magnetic resonance (NMR). Structural elucidation of two impurities by 1H NMR, 13C NMR, the 1H–1H COSY, HSQC and HMBC NMR spectra has been discussed. Based on high resolution MS/MS and NMR data, the structures of these two impurities were elucidated respectively as 11-O-[(2-Methoxyethoxy) methyl] roxithromycin and de(N-methyl)-N-formyl roxithromycin. In addition, the mechanisms for formation of the impurities were also proposed.

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Key words: roxithromycin, unknown impurity, 2D LC-QTOF MS, NMR, characterization 1. Introduction

Roxithromycin is classified as a second generation macrolide antibiotic and is currently widely

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used in clinical practice for its high bioavailability, strong tissue penetration, wide antibacterial

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spectrum and long half-life [1].

The impurity profile is always an important issue during the drug quality control. Even a very

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small amount of impurity can cause remarkably unwanted side effects[2]. To ensure the safety,

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impurities that exceed 0.1% must be identified according to ICH Guideline Q3A[3-9], therefore, it is necessary to characterize and control the impurities in roxithromycin. Roxithromycin monograph is

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included in European pharmacopeia 9.0[10], in which ten impurities named from A-J were described in the pharmacopeias. Additionally, another nine impurities in roxithromycin have been identified

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by LC-MS and reported in a previously published article [11]. In this study, two novel impurities, which have not been reported or identified till date to the

best of our knowledge, were discovered and detected in a batch of roxithromycin sample by HPLC. Two-dimensional liquid chromatography combined with QTOF mass analyzer was applied to preliminarily characterize these impurities. To confirm the proposed structures,

these two pure impurities were isolated by preparative HPLC. The structures of two impurities were confirmed by 1D and 2D nuclear magnetic resonance (NMR), and their structures were elucidated based on high resolution MS/MS and NMR data. The forming mechanisms of these impurities in roxithromycin were also studied.

2. Experimental 2.1 Materials

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Roxithromycin was synthesized by Zhejiang Guobang Pharmaceutical Co. Ltd (Shaoxing,

China). Impurity Ⅰand Ⅱwere isolated by preparative HPLC by Zhejiang Guobang Pharmaceutical Co. Ltd. Acetonitrile and methanol of HPLC grade was obtained from Merck Co. (Darmstadt,

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Germany), and water was purified by a Millipore Milli‐ Q‐ Gradient purification system.

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Ammonium dihydric phosphate, potassium dihydrogen phosphate and sodium hydrate were

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analytical grade and purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Ammonium formate was obtained from Sigma‐ Aldrich (St Louis, MO, USA).

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2.2 Two-dimensional LC system

Two-dimensional Agilent 1290 liquid chromatograph (Palo Alto, CA, USA), a system equipped

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with two binary pumps, was used in the study. The first dimension included a binary pump (G1312B), an auto-sampler (G4226B), a column thermostat (G1316C) and a diode array

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detector (G412A). Chromatographic separation in the first dimension was carried out at 20 ℃ using a Waters Xbridge C18 analytical column (150 mm × 4.6 mm, 3.5μm). The mobile phase of the first dimension included (A) ammonium dihydrogen phosphate solution at pH 4.3, which was prepared by dissolving 59.7 g of ammonium dihydrogen phosphate in 1000 mL water and adjusting to pH 4.3±0.1 with 5mol/L sodium hydroxide solution, and (B) water-acetonitrile (30:70, v/v). The

gradient elution conditions were: 0 min 34% B; 70 min, 48% B (hold for 5 min); 75.1 min, 34% B (hold for 15min). The flow rate of mobile phase was 1.30 mL·min−1 and injection volume was 20 μL. The detection wavelengths for roxithromycin was 205 nm. The second dimension was consisted of a binary pump (G4220A), a column thermostat (G1316C) and a diode array detector (G4212A). Chromatographic separation in the second dimension was carried out at 30 ℃ using a Shimadzu Shim-pack GISS C18 analytical column (50 mm × 2.1 mm, 1.9 μm). The

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mobile phase were (A) ammonium formate solution (10 mM) and (B) methanol. By applying

gradient elution conditions: 0 min 5% B; 5 min, 95% B; 5.5 min, 5% B; 9 min, 5% B in second dimension, appropriate retention times of impurities were obtained at the flow rate of 0.30 mL·min-

and the phosphate was eluted. The first and second dimensional columns were connected by means

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of two high pressure six-position and six-port switching valves and two 17 μL stainless

2.3 MS spectrometry

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steel quantitative loops.

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Two-dimensional liquid chromatography was coupled to a Agilent 6538 hybrid quadrupole time-of-flight mass spectrometer (Q-TOF) manufactured by Technologies Inc. (Palo Alto, CA,

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USA). The mass spectrometry detector (MSD) was equipped with an electrospray ionization (ESI) source. The ion source temperature was 300 °C and the needle voltage was always set at 4000 V.

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Nitrogen was used as drying gas at a flow rate of 12 L/min. The collision energy was varied from 5 V to 25 V to maximize the ion current in the spectra. ESI was performed in negative ion mode.

2.4 Semi-preparative HPLC The isolation of impurity Ⅰwas carried out on a C18 column filling with DACSP-100-8-ODSP (50 mm×250 mm, 8 μm). A mixed solution of 0.067mol/L ammonium dihydrogen phosphate

solution–acetonitrile (65:35, v/v) was used for the isocratic elution of the LC system. The flow rate was set at 20 mL/min and ultraviolet detection was carried out at 205 nm. 0.5 g drug substance was dissolved in a mixed solvent consisting of 6.0 mL methanol and 4.0 mL purified water. 7 mL sample solution was loaded after filtration. The target fraction was collected at 23.16 min and its pH was adjusted to 8.0 - 9.0 using sodium hydrate solution. After vacuum distillation, the fraction was purified again with this chromatographic system and the target fraction was collected at 25 min

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(Figure S1).

The isolation of impurity Ⅱwas carried out on a Pursuit XRs C18 column (21.2 mm×250 mm,10

μm). An isocratic LC method was applied using a mixed solution of 0.067 mol/L glacial acetic acid–

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acetonitrile (55:45, v/v) as the mobile phase. The flow rate was set at 50 mL/min and ultraviolet

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detection was carried out at 205 nm. 0.5 g drug substance was dissolved in a mixed solution, which

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consisted of 5.0 mL methanol and 2.0 mL purified water. After filtration, 7 mL sample was injected into the HPLC system. The target fraction was collected at 22-26 min (Figure S2).

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After collection by semi-preparative HPLC, the pH of target fraction was adjusted to 8.0-9.0 with sodium hydrate solution. A colorless oil was obtained after a series of procedures including

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vacuum distillation, ethyl acetate extraction, drying and vacuum distillation. After dissolving the colorless oil with a small amount of methanol, a proper amount of water was added. The pure

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impurities was obtained after solvent removing.

2.5 Software Instrument control and data acquisition were performed with Mass Hunter B.08.00 software from Agilent.

2.6 NMR spectroscopy

The 1H,

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C, DEPT 135°, HSQC, HMBC, COSY NMR (proton decoupled) spectra were

recorded on a Bruker 500M spectrometer using CDCl3 as the solvent. Protonand carbon chemical shifts were reported on δ scale in ppm, relative to tetramethyl silane (TMS) (δ= 0.00 ppm)

2.7 Sample preparation The drug substance was dissolved in potassium dihydrogen phosphate solution (0.1 M, pH 7.5)/methanol (70:30, v/v) and the concentration was 2.0 mg•mL−1.

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3. Results and discussion

3.1 LC separation and mass spectra of impurities

The mobile phase in the first dimensional system was selected according to the roxithromycin

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monograph in European Pharmacopeia. The HPLC chromatogram of roxithromycin in the first

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dimensional system were shown in fig.2 and three impurities, two of which were unknown

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impurities, were investigated in our study. Because the MS responses of the fragment ions from impurities were very weak in the positive mode, the negative mode was used. Based on the MS

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spectra in the negative mode, the adduct ions of the impurity Ⅰ, Ⅱand Ⅲwere [M+HCOO]- and at m/z 969.5763, 895.5063 and 969.5789, respectively. Table 1 shows the ESI-MS exact mass data and

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theoretical mass data on [M+HCOO] - of the impurities. The deviation values were all less than 5 ppm. ESI-MS/MS exact mass data of major product ions of impurities are also shown in Table 1.

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3.2 Identification of impurities by HPLC-MS/MS 3.2.1 Identification of impurity Ⅰand impurity Ⅲ In Figure S3, impurity Ⅰ and impurity Ⅲ exhibited a same adduct ion peak ([M+HCOO]-) at m/z 969.58 in negative ion mode by TOF high‐ resolution mass, indicating a same molecular formula of C45H84N2O17. It was suggested that they were isomers. The structure and fragmentation

pathways of impurity Ⅲwere reported in a previous paper [11]. The literature proposed that impurity Ⅲ was formed by the addition of (2-methoxyethoxy) methylium group in mycarose part of roxithromycin. Figure S4 presents the proposed structure and fragmentation patterns of impurity Ⅲ based on the LC-MS analysis in negative ion mode. The fragment ion at m/z 659.4142 showed a loss of 265 Da from the adduct ion at 969.5789, which was attributed to the cleavage of the bond between the 14-membered ring and mycarose as well as the (2-methoxyethoxy) methylium group.

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Fig. 3A presents the proposed structure and fragmentation patterns of impurity Ⅰ. The removal of mycarose from impurity Ⅰ led to the fragment ion at m/z 747.4660. The further loss of

mycaminose formed the fragment ion at m/z 572.3449. The fragment ions at m/z 641.4037 and

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466.2813 were formed by a loss of (2-methoxyethoxy) methylium group from the fragment ions at

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m/z 747.4660 and 572.3449 respectively. The above information indicated that the addition of (2methoxyethoxy) methylium group occurred in the 14-membered ring of the impurity Ⅰ. The exact

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position was confirmed by NMR in 3.2.2. Impurity Ⅰ and impurity Ⅲ have the same (2methoxyethoxy) methylium chain, but in different positions.

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3.2.2 Identification of impurity Ⅱ

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The adduct ion peak [M+HCOO]- of impurity Ⅱwas m/z 895.5063 based on the data obtained from TOF high‐ resolution mass (Fig.S3), indicating molecular formula of C41H74N2O16. And the

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adduct ion molecular weight of impurity Ⅱ is 59 Da more than the molecular weight of roxithromycin, indicating an adduct ion of HCOO- plus a difference of 14 Da. Fig.3B shows the proposed structure and fragmentation pattern of impurity Ⅱ. The fragment ion at m/z 673.3930 showed a loss of 222 Da from the adduct ion of impurity Ⅱ, which might be attributed to the cleavage of the bond between 14-membered ring and the mycarose. The fragment ion at m/z

484.2917 was formed by a further loss of 189 Da, which was 14 Da more than that of mycaminose. It was suggested that impurity Ⅱ formed through N-demethyl, N-formylation at the mycaminose part of roxithromycin.

3.3 Identification of impurities by NMR analysis In order to unequivocally elucidate the structure of two unknown impurities, NMR experiments were conducted after semi-preparative isolation of impurities. The assignment of NMR signals was

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performed for two impurities by means of 1H, 13C, DEPT 135° and 2D [correlated spectroscopy (COSY), heteronuclear single quantum coherence spectroscopy (HSQC), and Hheteronuclear multibond coherence spectroscopy (HMBC)] spectra in Table 2.

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3.3.1 Identification of impurity Ⅰby NMR spectra

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As shown in table 2, the 1H NMR spectrum exhibited thirty one groups of proton signals, whose area-to-area ratios were 1:1:1:1:1:1:1:2:5:3:4:7:3:1:1:2:1:2:1:6:1:1:1:1:7:3:10:6:3:3:3, and this

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indicated that impurity Ⅰcontained eighty four hydrogen atoms. Seven groups of hydrogen signals

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were observed in the low field. There were twenty four groups of proton signal peaks in the middle and high fields, including ten groups of overlapping proton signals, four groups of methine signals,

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one group hydroxyl hydrogen signal, three groups of methylene signals coupled with homocarbon, two groups of methyl hydrogen signals linked to heteroatoms and four groups of methyl signals

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linked to carbon atoms.

The 13C NMR and DEPT 135° spectra of impurity Ⅰindicated forty five carbon signals (Table

2). Six carbon signals were observed in the low field part, including one carbonyl quarter carbon signal, one oxime carbon signal, two tertiary carbon signals linked to two oxygen atoms and two secondary carbon signals. Thirty nine carbon signals were observed in the middle and high fields,

including nine tertiary carbon signals, three quarter carbon signal, four methylene signals, five methyl signals linked to heteroatoms, and four tertiary carbon signals, four methylene signals, ten primary carbon signals linked to carbon.

The combination of 13C-NMR and 1H-NMR spectra

revealed that impurity Ⅰwas oxime compounds containing carbonyl groups. Based on HSQC spectrum, other hydrogen proton signals except δH 3.34, δH 2.84, δH 2.44 and δH 2.21 , which were known as active hydrogen, could be assigned to the corresponding carbon.

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From the correlations of the 1H-1H COSY spectra in Fig 4, H-39 was correlated with H-38

and H-40, H-41 was correlated with H-40 and H-42, and H-42 was correlated with H-45, therefore, a linked fragment of O-C38-C39-C40-C41-C42(-O)-C45 could be revealed. Since H-34 showed

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correlations with H-37 and H-33, and H-33 showed correlations with H-48, the only possible

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linking fragment is C37-C34-C33-OH. H-2 showed correlations with H-14 and H-3, H-8 showed

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correlations with H-7 and H-17, H-28 showed correlations with H-13 and H-29, and H-4 showed correlations with H-5 and H-15, indicating the linking fragments of C3-C2-C14, C7-C8-C17, C13-

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C28-C19, and C5-C4-C15. H-19 showed correlations with H-20, H-10 showed correlations with H22, H-30 showed correlations with H-31, H-24 showed correlations with H-25, indicating the

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linked fragments of C19-C20, C10-C22, C30-C31 and C24-C25. Based on the combination analysis of HMBC spectrum and 1H-1H COSY spectrum, H-14

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was correlated with carbonyl C-1, indicating the linked fragment of C1-C2 as shown in Fig.4A. H15 was correlated with C-3, indicating the linked fragment of C3-C4. H-4 was correlated with C-6, indicating the linked fragment of C5-C6. H-16 was correlated with C-5, C-6 and C-7, and quaternary carbon C-6 was connected with hydroxyl indicating the linked fragment of C7-C6-C16. H-17 was correlated with C-9, indicating the linking fragment of C8-C9. H-22 was correlated with

C-9, indicating the linked fragment of C9-C10. H-11 was correlated with C-9 and C-10, indicating the linked fragment of C10-C11. H-27 was correlated with C-11, C-12, and C-13, and active H-47 was correlated with C-27, indicating the linked fragment of C11-C12 (-OH)-C13. H-13 was correlated with C-1, indicating the linking fragment of C1-O-C13. Based on the information above, the macrolide structure in the molecule was revealed. H-35 was correlation with C-31, C-32 and C-33, indicating the linked fragment of C31-C32(-C35)-C33. As H-36 was correlated with C-32, C-28

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was linked with methoxy. Since H-30 was correlated with C-3 and C-34, pyranoside was linked to

C-3. As H-43/44 was correlated with C-40, dimethylamino substituent position was in C-40. As H38 was correlated with C-5 and C-42, pyranoside was linked to C-5, and tertiary carbon C-39 was

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linked with hydroxyl. As H-21 was correlated with C-20 and H-18 was correlated with C-19, (2-

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methoxyethoxy) methyl group was connected with oximino group. As H-26 was correlated with C-25 and H-23 was correlated with C-24, (2- methoxyethoxy) methyl was connected to C-11 via a

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peroxy atom. By the analysis of the 1H–1H COSY, HSQC, HMBC NMR spectra, all protons and

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carbon signals were assigned and the planar carbon chain structure of the whole molecule was obtained. As a result, the chemical structure of impurity Ⅰwas 11-O-[(2-Methoxyethoxy)methyl]

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roxithromycin (Fig. 1B).

3.3.2. Identification of impurity Ⅱby NMR spectra

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As shown in table S1, the 1H NMR spectrum exhibited thirty four groups of proton signals,

whose area-to-area ratio were 1: 2: 1: 1: 1: 1: 1: 1: 1: 3: 1: 2: 1: 1: 3: 3: 1: 3: 1: 2: 1: 1: 1: 1: 2: 2: 3: 1: 3: 6: 6: 3: 6: 3, and this indicated that impurity Ⅱ contained eighty four hydrogen atoms. Four groups of hydrogen signals were observed in the low field. There were twenty nine groups of proton signal peaks in the middle and high fields, including six groups of overlapping proton signals, ten

groups of methine signals, two group methylene signal, four groups of methylene signals coupled with homocarbon, three groups of methyl hydrogen signals linked to heteroatoms and four groups of methyl signals linked to carbon atoms. The13C NMR and DEPT 135° spectra of impurity Ⅱ indicated forty one carbon signals. Six carbon signals were observed in the low field part, including one carbonyl quarter carbon signal, one oxime carbon signal, one an aldehyde group quarter carbon signal, two tertiary carbon signals

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linked to two oxygen atoms and one secondary carbon signals. Thirty five carbon signals were observed in the middle and high fields, including nine tertiary carbon signals, three quarter carbon

signals, two methylene signals, three methyl signals linked to heteroatoms, and four tertiary carbon

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signals, four methylene signals, ten primary carbon signals linked to carbon.

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Based on HSQC spectrum, hydrogen proton signals could be assigned to the corresponding

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carbon except δH 2.65, which was known as active hydrogen. From The correlations of the 1H-1H COSY spectra in Figure S7, H-35 was correlated with H-34 and H-36, H-37 was correlated with

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H-36 and H-38, and H-38 was correlated with H-41, therefore, the linked fragment of O-C34-C35C36-C37-C38-C41 was revealed. As H-30 showed correlations with H-33 and H-29, H-2 showed

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correlations with H-14 and H-3, H-8 showed correlations with H-7 and H-17, H-24 was correlation with H-13 and H-25, H-4 was correlation with H-5 and H-15, the only possible linking

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fragments were C33-C30-C29, C3-C2-C14, C7-C8-C17, C13-C24-C15,C5-C4-C15. H-19 showed correlations with H-20, H-10 showed correlations with H-22, H-26 showed correlations with H27, indicating the linking fragments of C19-C20, C10-C22 and C26-C27. Based on the HMBC spectrum combined with 1H-1H COSY spectrum in Figure S7, H-14 was correlated with carbonyl C-1, indicating the linked fragment of C1-C2-C14. H-15 was correlated

with C-3, indicating the linked fragment of C3-C4. H-4 was correlated with C-6, indicating the linked fragment of C5-C6. H-16 was correlated with C-5,C-6 and C-7, indicating the linked fragment of C7-C6-C16. H-17 was correlated with C-9, indicating the linked fragment of C8-C9. H22 was correlated with C-9 and C-11 indicating the linked fragment of C9-C10(-C22)-C11. H-23 was correlated with C-11, C-12 and C-13, indicating the linked fragment of C11-C12-C13. H-13 was correlated with C-,1 indicating the linked fragment of C1-O-C13. Therefore, the macrolide structure

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in the molecule was revealed. H-31 was correlation with C-27, C-29, C-30 indicating the linked

fragment of C27-C28(-C31)-C29. As H-32 was correlated with C-28, C-28 was linked with methoxy. As H-26 was correlated with C-3 and C-30, pyranoside was linked to C-3. As H-39/40 was

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correlated with C-36, N- methylformamide substituted in C-36. H-41 was correlated with C-37,

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indicating the linked fragment of C37-C38. As H-34 was correlated with C-5 and C-38, pyranoside

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was linked to C-5. As H-21 was correlated with C-20 and H-18 was correlation with C-19, (2methoxyethoxy) methyl group was connected to oximino group. In conclusion, the planar carbon

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chain structure of the whole molecule was obtained. By analysis of the1H–1H COSY, HSQC, HMBC NMR spectra, we assigned all proton and carbon signals. Therefore, the chemical structure

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of impurity Ⅱwas de(N-methyl)-N-formyl roxithromycin (Fig. 1C).

3.4 Formation of impurity Ⅰand impurity Ⅱ

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During the synthesis of roxithromycin, methoxyethoxychloromethyl ether (MEM chloride)

was added to react with erythromycin oxime in the final step[12] (Figure S11). In order to react completely with erythromycin oxime, excess MEM chloride was added, which led to the substitution of at multi-positions. Impurity Ⅰwas produced as a by-product in this reaction (Figure S11(B)). Impurity Ⅱ was formed via N-demethyl and N-formylation at the mycaminose part of

roxithromycin under heating condition (Figure S11(A)).

4. Conclusion Two novel impurities in roxithromycin were discovered and identified by two‐ dimensional liquid chromatography coupled with QTOF mass spectrometer and 1D and 2D nuclear magnetic resonance. Based on high resolution MS/MS and NMR data, the structures of two known impurity were characterized respectively as 11-O-[(2-Methoxyethoxy)methyl]Roxithromycin and de(N-

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methyl)-N-formyl roxithromycin. The two impurities were the by-products formed during the synthetic procedure.

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CRediT author statement Jian Wang: Conceptualization, Resources, Supervision, Methodology, Project administration. Jinjin Zhou: Validation, Investigation, Writing - Original Draft, Methodology, Project administration. Yu Xu: Validation, Investigation, Methodology, Project administration Bingqi Zhu: Writing - Review & Editing, Investigation. Yong Jin: Investigation, Supervision.

Declaration of interests

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The authors declare that they have no known competing financial interests or personal

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relationships that could have appeared to influence the work reported in this paper.

References [1] W.D. Li, C.G. Wang, J.G. Wang, Evaluation of relativity between impurity profile and synthesis technology of domestic roxithromycin, Chin J Pharm Anal. 35(2015) 320-327. [2] T. Zhuang, W. Zhang, L.J. Cao, H. Kai, Isolation, identification and characterization of two novel process-related impurities in olanzapine, J.Pharm. Biomed. Anal. 152 (2018) 188-196.

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[3] International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Q3B (R2), Impurities in New Drug Products, 2006, 1–16.

[4]P.X.Zhu, W.F.Yang, L.Y.Hong, Characterization of a novel process-related impurity in

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commercial bendazac lysine eye drops by LC-ESI-QTOF/MS/MS and NMR, J.Pharm. Biomed.

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Anal. 107 (2015) 437-443.

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[5]P.X.Zhu,J.X.Lu, L.Y.Hong, Characterization of an unknown impurity in doxofylline using LCMS and NMR, J.Pharm. Biomed. Anal. 140 (2017) 31-37.

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[6]J.Wang, Y.Xu, C.M.Wen, B.Q.Zhu, Application of a trap-free two-dimensional liquid chromatography combined with ion trap/time-of-flight mass spectrometry for separation and

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characterization of impurities and isomers in cefpiramide, Anal. Chim. Acta. 992 (2017) 42-54. [7] Y.Xu, F.Wang, J.N.Li, W.G.Shan, Separation and characterization of unknown impurities and

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isomers in flomoxef sodium by LC-IT-TOF MS and study of their negative-ion fragmentation regularities, J.Pharm. Biomed. Anal. 140 (2017) 81-90. [8] Y.Xu, D.D.Wang, L.Tang, J.Wang, Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-HPSEC × LC-IT-TOF MS, J.Pharm. Biomed. Anal. 145 (2017) 742-750.

[9]J.Wang, Y.Xu, Y.F.Zhang, H.Wang, Separation and characterization of unknown impurities in cefonicid sodium by trap‐ free two‐ dimensional liquid chromatography combined with ion trap time‐ of‐ flight mass spectrometry, Rapid Commun. Mass Spectrom. 31(2017)1541-1550. [10]European Pharmacopoeia, Edition 9.2017,pp.3513-3516. [11]F. Wang, H.X. Zeng, J.Wang, Characterization of Nineteen Impurities in Roxithromycin by HPLC/TOF and Ion Trap Mass Spectrometry, Chromatographia. 76 (2013)1683-1695.

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[12]C.D.Ma, H.Xu, X.Z. Li, S.Q.Xie, Synthesis technology of roxithromycin, China Prac Med.

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6(2011)145-146.

Figures Figure captions: Figure.1. Chemical structures of roxithromycin (A), impurity Ⅰ (B), impurity Ⅱ (C), and impurity Ⅲ (D). Numbering has been assigned only for NMR characterization of impurity Ⅰand Ⅱ. Figure.2. (A) UV (205 nm) chromatogram of roxithromycin, (B) UV (205 nm)

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chromatogram of blank.

Figure.3. (A) Proposed fragmentation pathways of impurity Ⅰin the negative ion mode; (B) Proposed fragmentation pathways of impurity Ⅱin the negative ion mode.

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Figure.4. The correlations of the 1H-1H COSY and HMBC（H→C）spectra of impurity

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Ⅰ.

Fig. 1. Chemical structures of roxithromycin (A), impurity Ⅰ (B), impurity Ⅱ(C), and

impurity Ⅲ (D). Numbering has been assigned only for NMR characterization of

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impurity Ⅰand Ⅱ.

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Fig. 2. (A) UV (205 nm) chromatogram of roxithromycin, (B) UV (205 nm)

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chromatogram of blank.

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Fig. 3. (A) Proposed fragmentation pathways of impurity Ⅰ in the negative ion mode;

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(B) Proposed fragmentation pathways of impurity Ⅱ in the negative ion mode.

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Fig. 4. The correlations of the 1H-1H COSY and HMBC（H→C）spectra of impurity Ⅰ.

Table Table Captions: Table 1 Summary of accurate mass measurements of impurities in negative-ion mode Table 2 1H, 13C, DEPT135°, HSQC and HMBC NMR data of impurity Ⅰ.

Table. 1. Summary of accurate mass measurements of impurities in negative-ion modea Deviation

[M+HCOO]-

(ppm)

C46H85N2O19-

969.5752

969.5763

1.13

Ⅱ

C42H75N2O18-

895.5020

895.5063

4.80

Ⅲ

C46H85N2O19-

969.5752

969.5789

3.82

Ⅰ

MS/MS fragmentation ions (m/z)

ro of

Experimental

[M+HCOO]-

[M+HCOO]-

747.4660、641.4037、572.3449、466.2813、174.1137 673.3930、484.2917 659.4142、484.2923

The data of theoretical [M+HCOO]- was calculated by the Mass Hunter software

-p

a

Theoretical

Imp.

DEPT 135°

re

Table. 2. 1H, 13C, DEPT135°, HSQC and HMBC NMR data of impurity Ⅰ. δH (ppm)

multiplicity J(Hz)

Position

δC (ppm)

5.21 5.10 5.06 5.00 4.82 4.76

d(7.4) dd(10.0,1.5) d(5.4) d(7.4) d(4.7) d(5.4)

18a 13 23a 18b 30 23b

/ / / / 175.90 169.62

/ / / / s s

O-47 O-49 O-46 O-48 C-1 C-9

4.42

d(7.3)

38

102.96

3.99

overlap

3 34

98.96 96.75

Key HMBC（H→C）

3.34 2.84 2.44 2.21 / /

17.59 / / / / /

C-27 / / / / /

d

C-38

4.42

83.88, 68.91,65.6

t t

C-23 C-18

5.06,4.76 5.21,5.00

78.48, 68.59 67.51

11

96.11

d

C-30

4.82

79.53, 72.84,65.61

20a

83.88

d

C-5

3.49

79.53, 74.93,39.65,37.42,26.30

C-3, C-6, C-4, C7,C-16

24

79.53

d

C-3

3.99

175.90, 96.11, 83.88, 44.68,39.65, 9.31

C-1, C-30, C-5, C2,C-4, C-15

na

lP

Position

ur

HSQC-H

δC (ppm）

Jo 3.82

Position

overlap

C-5, C-42, C-40 C-11, C-24 C-19 C-3, C-32, C-34

d

C-11

3.82

169.62, 98.96,77.70, 33.60

C-9, C-23, C-13, C10

19

78.07

d

C-33

2.99

65.61, 21.60,18.42

C-34, C-35,C-37

20b

77.70

d

C-13

5.10

175.90, 75.57,17.59, 10.98,15.46

C-1, C-12, C-27, C29,C-22

5 25 42 26 21 47 36 39 33

75.57 74.93 72.84 72.33 72.06 71.03 68.91 68.59 67.51

s s s t t d d t t

C-12 C-6 C-32 C-20 C-25 C-39 C-42 C-24 C-19

/ / / 3.82,3.58 3.49 3.22 3.49 3.82 3.58

/ / / / 67.51 102.96, 65.60 102.96 98.96 /

/ / / / C-19 C-38, C-40 C-38 C-23 /

2

65.61

d

C-34

3.99

96.11, 78.07, 72.84

C-30, C-33, C-32

49 10 40 46

65.60 59.09 58.79 49.56

d q q q

C-40 C-21 C-26 C-36

2.44 3.34 3.34 3.30

71.03 72.33 72.06 72.84

C-39 C-20 C-25 C-32

d

2.84

175.90, 79.53,39.65, 15.54

C-1, C-3,C-4, C-14

overlap

3.49

overlap

3.34

overlap

3.30 3.22 2.99

s dd(10.2,7.3) m

2.84

overlap

ro of

78.48

-p

3.58

8

q(6.7)

2.44

overlap

2.34

d(15.2)

31a

44.68

2.28

s

43,44

40.41

q

C-43,44

2.28

65.60

C-40

2.21

d(8.2)

48

39.65

d

C-4

2.02

83.88, 74.93,44.68, 9.31

C-5, C-6,C-2, C-15

2.02

m

t

C-7

1.49

169.62, 83.88,74.93, 27.15,26.30, 19.21

C-9, C-5,C-6, C-8,C16, C-17

34.98

t

C-31

2.34,1.49

96.11, 78.07,72.84, 21.60

C-30, C-33,C-32, C35

33.60

d

C-10

2.64

169.62, 78.48,75.57, 27.15,15.46

C-9, C-11,C-12, C8,C-22

41a

28.94

t

C-41

1.65,1.21

7

27.15

d

C-8

3.82

71.03, 68.91, 65.60, 21.48 169.62, 33.60

C-39, C-42, C-40, C45 C-9, C-10

31b

26.30

q

C-16

1.49

83.88, 74.93, 37.42

C-5, C-6, C-7

16

21.94

t

C-28

1.92,1.49

77.70, 10.98

C-13, C-29

28b

21.60

q

C-35

1.21

78.07, 72.84,34.98

C-33, C-32,C-31

dqd(14.7,7.3,2.4)

1.65

Jo m

overlap

C-2

lP

na

1.92

1.49

37.42

ur

4

re

2.64

28a

1.21

68.91, 28.94

C-42, C-41

37

19.21

q

C-17

1.00

169.62, 37.42,27.15

C-9, C-7,C-8

41b

18.42

q

C-37

1.25

78.07, 65.61

C-33, C-34

35

17.59

q

C-27

1.14

78.48, 77.70, 75.57

C-11, C-13, C-12

45

15.54

q

C-14

1.14

175.90, 79.53,44.68

C-1, C-3,C-2

22

15.46

q

C-22

1.21

169.62, 78.48,33.6

C-9, C-11,C-10

27

10.98

q

C-29

0.82

77.70, 21.94,75.57

C-13, C-28,C-15

14

9.31

q

C-15

1.06

83.88, 79.53,39.65

C-5, C-3,C-4

overlap

ro of

15 17 29

re

-p

d(7.4) d(6.9) t(7.4)

lP

1.06 1 0.82

C-45

na

1.14

overlap

q

ur

1.21

d(6.2)

Jo

1.25

21.48