Characterization of the components of meleumycin by liquid chromatography with photo-diode array detection and electrospray ionization tandem mass spectrometry

Journal of Pharmaceutical and Biomedical Analysis 84 (2013) 69–76

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Characterization of the components of meleumycin by liquid chromatography with photo-diode array detection and electrospray ionization tandem mass spectrometry Ming-juan Wang a , Ya-Ping Li a , Yan Wang a , Jin Li a , Chang-qin Hu a,∗ , Jos Hoogmartens b , Ann Van Schepdael b , Erwin Adams b a b

Department of Antibiotics, National Institutes for Food and Drug Control, Beijing 100050, PR China KU Leuven, Faculteit Farmaceutische Wetenschappen, Laboratorium voor Farmaceutische Analyse, O&N2, PB 923, Herestraat 49, B-3000 Leuven, Belgium

a r t i c l e

i n f o

Article history: Received 11 March 2013 Received in revised form 26 May 2013 Accepted 27 May 2013 Available online xxx Keywords: Liquid chromatography–mass spectrometry Photo-diode array detection Meleumycin Component identification Macrolide antibiotic

a b s t r a c t Reversed-phase liquid chromatography coupled with photo-diode array (PDA) detection and electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to characterize the components of meleumycin, a 16-membered macrolide antibiotic produced by fermentation. In total 31 components were characterized in commercial samples, including 12 impurities that had never been reported before and 12 others that were partially characterized. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of known components. Their ultraviolet spectra and chromatographic behavior were used to confirm the proposed structures: e.g. max shift from 232 nm to 282 nm would indicate the presence of an ␣-, ␤-, ␥-, ␦-unsaturated ketone instead of a normal ␣-, ␤-, ␥-, ␦-unsaturated alcohol in the 16-membered ring of the examined components. Compared to other methods, this LC/MSn method is particularly advantageous to characterize minor components at trace levels in multi-components antibiotics, in terms of sensitivity and efficiency. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Meleumycin is a mixture of 16-membered macrolide antibiotics containing about 50% of midecamycin A1 and about 50% of other components with similar structures, such as midecamycin A2, leucomycin A8, A6 and A4 (Fig. 1) [1,2]. When the in vitro and in vivo activities of midecamycin A1 and meleumycin were compared by microbiological assay, MIC (minimum inhibitory concentration) test and their pharmacokinetics curves in rabbits, no significant differences were found between midecamycin A1 and meleumycin. Midecamycin A2 showed similar activity against Micrococcus luteus and Bacillus subtilis to that of midecamycin A1 while the activity of leucomycin A6 was about 20% lower [3]. The ICH guidelines for new drug substances prescribe that impurities should be identified above the 0.1% level. Though antibiotics from fermentation origin are not subject to this ICH guideline, it is necessary to characterize as much as possible related substances to ensure the safety and batch-to-batch reproducibility of commercial meleumycin. In the current Chinese Pharmacopeia (ChP), the amounts of midecamycin A1, leucomycin A6 as well as the total amount of

∗ Corresponding author. Tel.: +86 10 67095308; fax: +86 10 65115148. E-mail address: [email protected] (C.-q. Hu). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.05.043

midecamycin A1 + midecamycin A2 + leucomycin A4 + leucomycin A6 + leucomycin A8 are controlled [4]. However, many minor components are still unknown and require characterization. There are some literature reports on the identification of active components of this kind of macrolide antibiotics [1–3,5–16], but only a few refer to midecamycin or meleumycin and most of them focus on separation and quantitation [2,3,17]. The aim of this work is to identify the unknown compounds in commercial meleumycin samples by liquid chromatography with photo-diode array (PDA) detection and electrospray ionization tandem mass spectrometry (LC–PDA–ESIMS/MS). The UV spectrum of the compound obtained by a PDA detector is determined to get information about the chromophore. If its maximum absorbance wavelength (max ) is at 232 nm, it will indicate that the compound has the normal structure of an ␣, ␤-, ␥-, ␦- unsaturated alcohol in the macrocyclic lactone. If its max shifts to 282 nm, it will reveal the presence of an aberrant ␣, ␤-, ␥-, ␦- unsaturated ketone instead. Further details about the structures of unknown compounds are deduced by comparison of their fragmentation patterns with those of midecamycin A1 and some other known components. The chromatographic properties of known components in meleumycin are also reported to illustrate the retention behavior of macrolide antibiotics in reversed-phase high-performance liquid chromatography (RP-HPLC), which will be helpful in confirming the proposed structures of unknown components.

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M.-j. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 84 (2013) 69–76

R3 CH3 α 9 γ δ

6 H3CO

5

O

O

4

HO

3 H3C

H 3C

CH2R4

β

CH 3

N

3'

O

O

OH

CH3

3"

mycaminose

O

OR1

O

CH 3 OR2 4" CH3

acetylated mycarose Components

R1

R2

R3

R4

[M+H]+

Peak No.a

Comment major component 4

midecamycin A1

COCH2CH3

COCH2CH3

OH

CHO

814.5

20

midecamycin A2

COCH2CH3

COCH2CH2CH3

OH

CHO

828.5

25

leucomycin A8

COCH3

COCH3

OH

CHO

786.5

11

specified minor

leucomycin A6

COCH3

COCH2CH3

OH

CHO

800.5

17

component 4

leucomycin A4

COCH3

COCH2CH2CH3

OH

CHO

814.5

22

meleumycin D

COCH2CH3

H

OH

CHO

758.5

7

reported

meleumycin B2

COCH2CH3

COCH3

OH

CHO

800.5

15

meleumycin

X4

COCH2CH2CH3

COCH2CH3

OH

CHO

828.5

23

components 1-2

X3

COCH3

COCH2CH3

=O

CHO

798.4

21

midecamycin A3

COCH2CH3

COCH2CH3

=O

CHO

812.5

25b

4 -depropionyl-4 -butyryl X3

COCH3

COCH2CH2CH3

=O

CHO

812.5

26

3-deacetyl-3-butyryl X3

COCH2CH2CH3

COCH2CH3

=O

CHO

826.5

8

midecamycin A4

COCH2CH3

COCH2CH2CH3

=O

CHO

826.5

c

midecamycin A4 (isoform)

COCH2CH3

COCH(CH3)2

=O

CHO

826.5

d

6-hydroxyethyl leucomycin A6

COCH3

COCH2CH3

OH

CH2OH

802.5

12

6-hydroxyethyl midecamycin A1

COCH2CH3

COCH2CH3

OH

CH2OH

816.5

14/16

6-carboxymethyl X4

COCH2CH2CH3

COCH2CH3

OH

COOH

826.4

1

leucomycin V

H

H

OH

CHO

702.5

2

newly identified components

leucomycin U

COCH3

H

OH

CHO

744.5

3

4 -depropionyl X4

COCH2CH2CH3

H

OH

CHO

772.4

10

leucomycin A7

H

COCH2CH3

OH

CHO

758.6

13

a

Refer to Fig. 2 for the peak number.

b

In the front part of peak 25;

c, d

Only found in midecamycin RS. Peaks are eluted between peaks 14-15 and 17-18, respectively. Their 4’’-substituents may be butyryl (COCH2CH2CH3) or isobutyryl (COCH(CH3)2).

Fig. 1. Chemical structures of midecamycin A1 (major component) and specified, reported and newly identified components of meleumycin.

M.-j. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 84 (2013) 69–76

2. Experimental 2.1. Reagents and samples HPLC-grade acetonitrile was obtained from Fisher Scientific (Fairlawn, NJ, USA). Ammonium formate (99%) was supplied by Acros Organics (NJ, USA) and analytical grade aqueous ammonia was obtained from Beijing Chemical Works (Beijing, China). A milliQ water purification system (Millipore, USA) was used to further purify glass-distilled water. Midecamycin RS (batch no. 0337-9803, containing 95.9% of midecamycin A1) and meleumycin RS (batch no. 0341-9402, containing 49.0% of midecamycin A1) were from National Institutes for Food and Drug Control, PR China. Meleumycin samples examined were obtained from Nanyang Pukang Pharmaceutical Company (Henan, China) (Company 1), Jiangsu Ping-guang Pharmaceutical Factory (Jiang, China) (Company 2) and Shanghai Quanyu Biotechnology Neixiang Pharmaceutical Company (Shanghai, China) (Company 3), respectively. 2.2. Instrumentation The liquid chromatography/mass spectrometry (LC/MS) system consisted of a 3201 S1-2 binary pump, a 3202 S1-2 vacuum degasser, a 3014 S1-2 column heater, a 3012 S1-2 column switch system, a 3133 S1-2 sampler from SHISEIDO (Tokyo, Japan), an Accela PDA detector (Thermo Fisher Scientific Inc., Massachusetts, USA) and a 3200Q TRAP mass detector (Applied Biosystems Inc., California, USA), controlled by Analyst® software (version 1.4.2). 2.3. Chromatographic conditions A mobile phase containing acetonitrile – 100 mmol L−1 ammonium formate (pH 7.6, adjusted with aqueous ammonia) (45:55, v/v) was used at a flow rate of 0.5 mL min−1 . The Poroshell 120 SBC18 column (150 mm × 4.6 mm i.d., 2.7 ␮m) (Agilent Technologies, Santa Clara, California, USA) was maintained at 35 ◦ C. Samples were prepared in the mobile phase at a concentration of 2.0 mg mL−1 . The injection volume was 5 ␮L. Ultraviolet detection was performed from 200 to 400 nm (extraction: 232 nm). 2.4. Mass spectrometry Tuning and MS/MS investigation of midecamycin A1 was carried out according to the manufacturer’s instruction. The optimized mass spectrometric conditions were as follows: electrospray ionization (ESI) positive ionization mode, Declustering Potential (DP) 50, Entrance Potential (EP) 10, Collision Energy (CE) 40, Curtain gas: 20.0, Ion Source gas 1: 65.0, Ion Source gas 2: 60.0, IonSpray Voltage (IS): 5500.0, Temperature (TEM): 500.0, Interface Heater: on. The acquisition of the first-order mass spectra (Q1 scan) was from 200 to 1000 u. The ions of interest were isolated in the ion trap and activated at a CE of 40 to get Enhanced Product Ion (EPI) spectra in the range of 200–1000 u. Each protonated molecule [M+H]+ was confirmed based on our previous work [1,2,17]. The presence of minor adduct ions of [M+Na]+ and [M+K]+ will further confirm each [M+H]+ . In the case of peaks containing multiple components, we used the “precursor Ion (Prec)” method of Analyst® software to confirm an individual protonated molecule. 3. Results and discussion 3.1. Development of the LC–PDA–ESI-MS/MS method Based on the ChP method [4], the system using 100 mmol L−1 ammonium formate (pH 7.6) – acetonitrile was selected for

71

separation of the meleumycin complex. To be compatible with LC/MS, a minor modification was made to the above system: the pH was adjusted using aqueous ammonia instead of triethylamine. First, a Venusil XBP C18 column (size: 250 mm × 4.6 mm i.d., 5 ␮m) (Agela Technologies, China) was selected based on our antibioticscolumn selection recommendation database [18]. As shown in Fig. 2, this column gave good separation of meleumycin at a flow rate of 1.0 mL min−1 . Using the same mobile phase, a Poroshell 120 SB-C18 column (150 mm × 4.6 mm, 2.7 ␮m) (Agilent, USA) was also evaluated. This new shell-type particle (1.7 ␮m nonporous core surrounded by 0.5 ␮m porous shell) column should have a great chance to improve separation of complex compounds [19]. As shown in Fig. 2, the Poroshell 120 SB-C18 column gave better results at a flow rate of 0.5 mL min−1 in terms of peak shape and separation. So, the Poroshell 120 SB-C18 column was used further in our characterization experiments. 3.2. UV spectra, fragmentation pathway and chromatographic behavior of known components of meleumycin 3.2.1. UV spectra of midecamycin A1 and other known components of meleumycin As expected, midecamycin A1 showed a maximum UV absorption (max ) at 232 nm which corresponded to the presence of the structure of ␣-, ␤-, ␥-, ␦- unsaturated alcohol in the macrocyclic lactone [1,6]. Other known components of meleumycin, such as leucomycin A8, A6, A4 and midecamycin A2 with the structure of ␣-, ␤-, ␥-, ␦- unsaturated alcohol (R3 = OH, see Fig. 1 for their structures) all showed max at 232 nm. Thus, the max information obtained by the PDA detector can be used to identify the chromophore of unknown components in meleumycin. 3.2.2. Fragmentation pathway of known components of meleumycin In accordance with other macrolide antibiotics [1,5–16], most product ions of midecamycin A1 ([M+H]+ m/z 814) were formed by cleavage of glycosidic bonds or the hydroxyl group: the neutral losses of 200 u and 191 u correspond to the successive cleavage of 4 -propionyl mycarose and mycaminose at C-5, respectively, while the loss of 18 u corresponds to the cleavage of the hydroxyl group at C-9. Different cleavage order could result in various product ions, as shown in Fig. 3. The presence of the characteristic disaccharide ion (F4) with m/z 374 confirmed that the 4 -acylated substituent group was propionyl. Further fragmentation of the aglycone ion (F5) with m/z 405 was the same as in literature [5–8,14]. Similar fragmentation pathways were observed in the MS/MS spectra of other known components of meleumycin: in case of leucomycin A4 and midecamycin A2, as their 4 - substituents were butyryl (14 u more than propionyl), their neutral losses of the acylated mycarose were 214 u instead of 200 u, and their corresponding disaccharide ions were at m/z 388 instead of 374. Meanwhile, the presence of characteristic aglycone (AG) ions at m/z 405, 331, 299, 281 revealed that the 3-O-substituent (R1 group, see Fig. 1) of midecamycin A2 was also propionyl, while the characteristic AG ions at m/z 391, 331, 299, 281 verified that the 3-O-substituents of leucomycin A4, A6 and A8 were all acetyl. The fragmentation patterns as summarized in Fig. 3 will be used to reveal more structure details about the unknown compounds in the meleumycin complex. 3.2.3. Chromatographic behavior of known components of meleumycin The retention behavior of the components of meleumycin complex was linked to their hydrophobic properties (see Figs. 1 and 2): the more hydrophobic the R1 or R2 substituent, the longer the retention time (RT), e.g.

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M.-j. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 84 (2013) 69–76

Fig. 2. Comparison of LC-UV chromatograms of meleumycin RS obtained by Venusil XBP C18 (250 mm × 4.6 mm i.d., 5 ␮m) (A) and Poroshell 120 SB-C18 (150 mm × 4.6 mm i.d., 2.7 ␮m) (B). Mobile phase: acetonitrile – 100 mmol L−1 ammonium formate (pH7.6, adjusted with aqueous ammonia) (45:55, v/v). Flow rate: 1.0 mL min−1 (Venusil XBP C18 )/0.5 mL min−1 (Poroshell 120 SB-C18 ). Column temperature: 35 ◦ C. UV detection: 232 nm. Meleumycin RS solution: 2.0 mg mL−1 . Injection volume: 20 ␮L.

RT(meleumycin A4) > RT(leucomycin A6) > RT(leucomycin A8) , and RT(midecamycin A1) > RT (leucomycin A6) . Meanwhile, it could be noticed that the influence of R2 in the acylated mycarose on the chromatographic behavior of the meleumycin complex was larger than that of R1 in the macrocyclic lactone: e.g. RT(leucomycin A4) > RT(midecamycin A1) . Similar retention behavior was also observed in the bitespiramycin complex and acetylspiramycin components [1,5–7]. The chromatographic behavior of known components would be useful to verify the rationality of the proposed structures of unknown related substances.

3.3. Investigation of unknown components in commercial meleumycin samples Five commercial samples from three different origins were screened for unknown components. The major components of meleumycin in the investigated samples were similar, but their relative amounts varied slightly, as shown in Table 1. The similarity

of different batches from company 2 indicated that its production process was repeatable. As the meleumycin complex is produced by fermentation, its related substances may arise from the fermentation process or degradation during storage. Identification of unknown components in commercial samples was discussed based on their structural differences compared to midecamycin A1. The characterization results are summarized in Table 2. In total 31 components were characterized in commercial samples, including 12 impurities that had never been reported before (see newly identified components in Fig. 1, except midecamycin A4 (isoform)) and 12 others that were partially characterized (isomers or with aberrant mycarose sugar). 3.3.1. Related substances with aberrant macrocyclic lactone 3.3.1.1. Related substances with the structure of ˛-, ˇ-, -, ı- unsaturated ketone. Unlike midecamycin A1, the compounds in peaks 21, 26 and 25* (25* : the front part of peak 25) showed max at 282 nm, indicating that their chromophores in the 16-membered macrocyclic lactone were aberrant. Furthermore, no neutral loss of water

M.-j. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 84 (2013) 69–76

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Table 1 Relative amounts of components in meleumycin samples from different origins. Component

Peak No.a

RS

Company 1

Meleumycin D Leucomycin A8 Meleumycin B2 Leucomycin A6 Midecamycin A1 Leucomycin A4 X4 Midecamycin A2

7 11 15 17 20 22 23 25

2.2% 2.6% 5.0% 15.8% 45.3% 2.9% 2.8% 5.7%

2.0% 3.0% 3.5% 11.4% 52.8% 2.0% 1.9% 4.4%

Company 2 2.6% 3.9% 4.0% 11.3% 50.3% 2.0% 2.0% 4.6%

2.6% 3.6% 4.0% 11.4% 50.7% 2.3% 2.4% 5.0%

Company 3 2.3% 3.2% 4.0% 11.6% 50.9% 2.2% 2.3% 4.9%

3.2% 4.8% 3.3% 10.5% 53.0% 2.0% 1.1% 3.0%

RS = meleumycin reference substance (batch no. 0341-9402, National Institutes for Food and Drug Control, P.R. China). a Refer to Fig. 2 for peak numbers.

corresponding to the hydroxyl at C-9 was observed in their MS/MS spectra, and their characteristic AG ions groups were at m/z 407, 347, 315, 297 (peaks 21 and 26) and at m/z 421, 347, 315, 297 (peak 25*), all 16 u higher than those of leucomycin A8/A6/A4 (m/z 391, 331, 299, 281) and midecamycin A1/A2 (m/z 405, 331, 299, 281), respectively. According to literature, 1 the structure of an ␣-, ␤-, ␥-,

␦- unsaturated ketone in which a carbonyl has replaced the normal hydroxyl at C-9 of the macrocyclic lactone, would make the max shift to 282 nm. Based on the above results and literature, the aberrant chromophores of these compounds were characterized as ␣-, ␤-, ␥-, ␦- unsaturated ketone. The neutral losses of peaks 21/25* and 26 corresponding to acetylated mycarose were 200 u

Fig. 3. Fragmentation pathway of midecamycin A1.

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M.-j. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 84 (2013) 69–76

Table 2 List of meleumycin major components and its related substances in commercial samples. Peak No.a

[M+H]+

Identification

1 2 3 4 5 6 7 8 9

826.4 702.5 744.5 816.5 758.4 + 748.5 758.5 758.5 826.5 842.5

6-Carboxymethyl X4 Leucomycin V Leucomycin U Unknown Unknown Meleumycin D (isoform) Meleumycin D 3-Deacetyl-3-butyryl X3 X4 with aberrant mycarose (Mr 214)

772.4 844.4 786.5 802.5

4 -Depropionyl X4 compound with aberrant mycarose (Mr 230) Leucomycin A8 6-Hydroxyethyl leucomycin A6

758.6 814.5

Leucomycin A7 Midecamycin A1 (isoform)

816.5 800.5 800.5 826.5

6-Hydroxyethyl midecamycin A1 Leucomycin A6 (isoform) Meleumycin B2 (isoform) Midecamycin A4 (isoform)

800.5 858.5 816.5 800.5 826.5 814.5 814.5 814.5 798.4 814.5 828.5 828.5

Meleumycin B2 Compound with aberrant mycarose (Mr 244) 6-Hydroxyethyl midecamycin A1 Leucomycin A6 Midecamycin A4 (isoform) Midecamycin A1 (isoform) Midecamycin A1 (isoform) Midecamycin A1 X3 Leucomycin A4 X4 X4 (isoform)

812.5c 828.5 812.5

Midecamycin A3 Midecamycin A2 4 -Depropionyl-4 -butyryl X3

10 11 12 13

14 b 15 16 17 b

18 19 20 21 22 23 24 25 26 a b c

Comment

Reported component

See Section 3.3.2. Specified component

Reported component

Specified component

Major component Specified component Reported component

Specified component

Refer to Fig. 2B for the peak numbers. Only found in midecamycin RS, which are between peaks 14 and 15, 17 and 18, respectively. In the front part of peak 25.

and 214 u, which revealed that their 4 -substitutes were propionyl and butyryl, respectively. Their neutral losses corresponding to mycaminose were all 191 u, indicating that their mycaminose was normal. Thus, the compounds in peaks 21 ([M+H]+ m/z 798), 26 ([M+H]+ m/z 812) and 25* ([M+H]+ m/z 812) were identified as X3, 4 -depropionyl-4 -butyryl X3 and midecamycin A3, respectively (see Fig. 1). Their retention behavior was in accordance with that observed for known components of meleumycin (see Section 3.2.3.), which confirmed the rationality of the proposed structures. The UV spectrum (max at 282 nm) and fragmentation behavior (AG ions at m/z 435, 347, 315, 297, all 16 u higher than those of 3O-butyryl derivatives) of peak 8 ([M+H]+ m/z 826) were similar to those of the above compounds, which indicated that it also has the structure of an ␣-, ␤-, ␥-, ␦- unsaturated ketone. Its neutral losses of 200 u and 191 u and corresponding characteristic disaccharide at m/z 374 indicated that its 4 -substituent was propionyl and that the mycaminose was normal. Based on the above information, the structure of the compound was proposed as 3-deacetyl-3-butyryl X3 (Fig. 1). In midecamycin RS, two other compounds ([M+H]+ m/z 826, isomers) with the structure of an ␣-, ␤-, ␥-, ␦- unsaturated ketone were eluted between peaks 14–15 and 17–18, respectively. Their AG ions were both at m/z 421, 347, 315, 297, all 16 u higher than those of 3-O-propionyl derivatives, meanwhile their max were both at 282 nm. The neutral losses of 214 u and 191 u revealed that their 4 -substituent was (iso)butyryl and that their mycaminose was normal, which was further confirmed by the disaccharide ion at m/z 388. Thus, the two compounds ([M+H]+ m/z 826) were identified as

midecamycin A4 or its isomer (see Fig. 1). Other techniques, such as nuclear magnetic resonance (NMR), are required to determine the difference between the two compounds. 3.3.1.2. Related substances with a different C-6 substituent. The fragmentation behaviors of the compounds in peaks 12 ([M+H]+ m/z 802), 14 and 16 ([M+H]+ m/z 816) were similar to those of leucomycin A6 and midecamycin A1, except that their characteristic AG ions at m/z 393, 333, 301, 283 (peak 12) and m/z 407, 333, 301, 283 (peaks 14 and 16) were all 2 u higher than those of 3-O-acetyl/3O-propionyl derivatives. Their UV spectra showing max at 232 nm indicated the presence of an ␣-, ␤-, ␥-, ␦- unsaturated butadiene structure in the macrocyclic lactone. Compounds with similar fragmentation behaviors were also observed in many other macrolide antibiotics and their positions of modification were proposed at C6 [5,6,8,13,14]. So, the compounds in peaks 12 ([M+H]+ m/z 802), 14 and 16 ([M+H]+ m/z 816) were identified as 6-hydroxyethyl leucomycin A6 and 6-hydroxyethyl midecamycin A1 (or its isomer), respectively (see Fig. 1). The max of the compound in peak 1 ([M+H]+ m/z 826) was at 232 nm, which indicated that it has a normal ␣-, ␤-, ␥-, ␦- unsaturated butadiene structure. Its AG ions were at m/z 435, 347, 315 and 297, all 16 u higher than those of 3-O-butyryl derivatives. Its neutral losses were 200 u and 191 u, respectively. A weak loss of 18 u corresponding to water could also be observed. Based on the above information and literature report [5,8], the structure of the compound in peak 1 ([M+H]+ m/z 826) was characterized as 6carboxymethyl X4, as shown in Fig. 1.

M.-j. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 84 (2013) 69–76

3.3.1.3. Related substances with a different C-3 substituent. The compounds in peaks 2 ([M+H]+ m/z 702), 3 ([M+H]+ m/z 744), 7 ([M+H]+ m/z 758) and 10 ([M+H]+ m/z 772) were identified as leucomycin V, leucomycin U, meleumycin D and 4 -depropionyl X4, respectively (see Fig. 1). Their neutral losses were all 144 u (unacetylated mycarose), 191 u and 18 u, respectively. Their aglycone ions F5 (refer to Fig. 3) were at m/z 349, 391, 405 and 419, respectively, while their successive ions F6–F8 were all at m/z 331, 299 and 281, in accordance with those of 3-hydroxyl, 3-O-acetyl, 3-O-propionyl and 3-O-butyryl derivatives, respectively [5–8,14]. Their UV spectra (max at 232 nm) and chromatographic behavior (RT(4 -depropionyl X4) > RT(meleumycin D) > RT(leucomycin U) > RT(leucomycin V) ) confirmed the proposed structures. Similarly, the compounds in peaks 13 ([M+H]+ m/z 758), 15 ([M+H]+ m/z 800) and 23 ([M+H]+ m/z 828) were characterized as leucomycin A7, meleumycin B2 and X4, respectively. Their F5 ions were at m/z 349 (peak 13), 405 (peak 15) and 419 (peak 23) and their neutral losses corresponding to acetylated mycarose were 186 u (peak 15) and 200 u (peaks 13 and 23), respectively, meanwhile their other neutral losses (−191 u, −18 u) were normal. The structures of these compounds are proposed in Fig. 1. Their chromatographic behaviors were in accordance with those observed for known meleumycin components: RT(leucomycin A6) > RT(peak 13, leucomycin A7) > RT(leucomycin V) , RT(peak 23, X4) > RT(midecamycin A1) , confirming that their retention was correlated to their hydrophobic properties. The fact that RT(leucomycin A6) > RT(peak 15, meleumycin B2) and RT(midecamycin A2) > RT(peak 23, X4) verified that the influence of the 4 -substituent on the retention behavior was bigger than that of the C3-substituent. Their max at 232 nm further confirmed the proposed structures. 3.3.2. Unknown compounds with aberrant mycarose Compounds with an aberrant mycarose were found in peaks 9 ([M+H]+ m/z 842), 10 ([M+H]+ m/z 844) and 15 ([M+H]+ m/z 858). Their neutral losses were 214 u (peak 9), 230 u (peak 10), and 244 u (peak 15), all followed by losses of 18 u and 191 u. Their corresponding characteristic disaccharide ions were at m/z 388 (peak 9), 404 (peak 10) and 418 (peak 15), indicating that their aberrant mycarose had a neutral loss of 214 u, 230 u and 244 u, respectively. Their AG ions at m/z 419, 331, 299, 281 (peak 9) or 405, 331, 299, 281 (peaks 10 and 15) revealed that they were 3-O- butyryl and 3-O- propionyl derivatives, respectively. Their max at 232 nm confirmed that they had a normal ␣-, ␤-, ␥-, ␦- unsaturated butadiene structure in the macrocyclic lactone. Though the aberrant mycarose of peak 9 may be 4 -(iso)butyryl mycarose, the retention behavior of the proposed structure was not consistent with that of known meleumycin components, e.g. its RT was shorter than that of leucomycin A8. Thus, it was concluded that it may have another form of mycarose (Mr 214). Further NMR experiments are necessary to reveal the exact structures of these aberrant mycarose moieties, but due to their low amount and the fact that the impurities can not be easily isolated, NMR experiments are not straightforward. 3.3.3. Isomers of known meleumycin components As observed in other macrolide antibiotics [1,5–9,13–16], isomers of midecamycin A1 and some other components with the same m/z values and similar product ions were also found in meleumycin, e.g. isomers of midecamycin A1 in peaks 13, 18 and 19, isomer of leucomycin A6 in peak 14, isomer of meleumycin D in peak 6, isomer of meleumycin B2 in peak 14, isomer of X4 in peak 24, etc. However, positional isomers and even spatial isomers cannot be distinguished by LC/MS only and as mentioned before already, NMR experiments are not straightforward.

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4. Conclusion A new shell-type particle (1.7 ␮m nonporous core surrounded by 0.5 ␮m porous shell) column (Poroshell 120 SB-C18 , 150 mm × 4.6 mm i.d., 2.7 ␮m) was used for the separation of the meleumycin complex. It gave slightly better results than a traditional C18 column for basic compounds in terms of peak shape and separation. The components in commercial meleumycin samples were characterized based on the combined information of their UV spectrum, MS/MS fragmentation pathway and chromatographic behavior. In total 31 components were found in commercial samples in this work, including 12 new related substances that had never been reported before and 12 others that were only partially characterized. All meleumycin components identified arise from fermentation. Six meleumycin/midecamycin components were found to have the structure of an ␣-, ␤-, ␥-, ␦- unsaturated ketone, which demonstrated that it was also necessary to detect such related substances at 282 nm to control well the quality of commercial meleumycin products. It was also found that leucomycin A6 was the second major component in meleumycin and its normalized peak area percentage was within 10–16% by LC-UV (232 nm) (see Table 1). Due to its relative lower in vivo and in vitro activity compared to midecamycin A1 [3], it is suggested to control properly its amount in meleumycin. Acknowledgements Financial support by the Twelfth Five-year National Science and Technology Support Program ‘The research and development of new material for separation and integration demonstration’ (Fund No. 2012BAK25B02) (PR China) is gratefully acknowledged. 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.2013.05.043. References [1] M. Hu, C.Q. Hu, Identification of the components of 16-membered macrolide antibiotics by LC/MS, Anal. Chim. Acta. 535 (2005) 89–99. [2] Y.L. Yang, Y.P. Li, Y.H. Wen, C.Q. Hu, Analysis of the components of meleumycin and the quality control, Chin. J. Pharm. Anal. 28 (2008) 341–344. [3] W.L. Xue, J.R. Zhang, Q. Wu, R. Zhang, L.X. Wang, Comparison of in vitro and in vivo bioactivity of meleumycin, midecamycin and its major components, Chin. J. Antibiot. 18 (1993) 42–46. [4] Chinese Pharmacopoeia Commission. Chinese Pharmacopoeia, vol. II, China Medicine Science and Technology Press, Beijing, 2010, pp. 303. [5] M.J. Wang, M. Pendela, C.Q. Hu, S.H. Jin, J. Hoogmartens, A. Van Schepdael, E. Adams, Impurity profiling of acetylspiramycin by liquid chromatography-ion trap mass spectrometry, J. Chromatogr. A 1217 (2010) 6531–6544. [6] M.J. Wang, J. Xue, W.B. Zou, Y. Wang, C.Q. Hu, J. Hoogmartens, E. Adams, Identification of the components of bitespiramycin by liquid chromatography–mass spectrometry, J. Pharm. Biomed. Anal. 2012 (66) (2012) 402–410. [7] X.G. Shi, S.Q. Zhang, J.P. Fawcett, D.F. Zhong, Acid catalysed degradation of some spiramycin derivatives found in the antibiotic bitespiramycin, J. Pharm. Biomed. Anal. 36 (2004) 593–600. [8] M. Pendela, C. Govaerts, J. Diana, J. Hoogmartens, A. Van Schepdael, E. Adams, Characterization of impurities in spiramycin by liquid chromatography/ion trap mass spectrometry, Rapid Commun. Mass. Spectrom 21 (2007) 599–613. [9] M. Pendela, S. Beni, E. Haghedooren, L. Van den Bossche, B. Noszal, A. Van Schepdael, J. Hoogmartens, E. Adams, Combined use of liquid chromatography with mass spectrometry and nuclear magnetic resonance for the identification of degradation compounds in an erythromycin formulation, Anal. Bioanal. Chem. 402 (2012) 781–790. [10] J.Y. Li, C.Y. Ma, H.Y. Wang, Y.H. Wang, L.Z. Wu, Y.G. Wang, On-line identification of 4 -isovalerylspiramycin I in the genetic engineered strain of S. spramyceticus F21 by liquid chromatography with electrospray ionization tandem mass spectrometry, ultraviolet absorbance detection and nuclear resonance spectrometry, J. Chromatogr. A 1217 (2010) 1419–1424. [11] D.F. Zhong, X.G. Shi, L. Sun, X.Y. Chen, Determination of three major components of bitespiramycin and their major active metabolites in rat plasma

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