Characterization of a new component and impurities in josamycin by trap-free two-dimensional liquid chromatography coupled to ion trap time-of-flight mass spectrometry

Article abstract of DOI:10.1002/rcm.8439

Rationale: The toxicities of the impurities of a drug will affect the clinical effects and cause potential health risk; therefore, it is essential to study profiles of the impurities. In this study, a new structural type of component and two acid degradation impurities in josamycin were discovered and characterized for the further improvement of official monographs in pharmacopoeias. Methods: The component and acid degradation impurities in josamycin were separated and preliminary characterized by trap-free two-dimensional liquid chromatography coupled to high-resolution ion trap time-of-flight mass spectrometry (2D LC/IT-TOF MS) in both positive and negative electrospray ionization mode. The eluent of each peak from the first dimensional chromatographic system was trapped by a switching valve and subsequently transferred to the second dimensional chromatographic system, which was connected to the mass spectrometer. Full scan MS was firstly conducted to obtain the exact m/z values of the molecules. Then LC/MS/MS and LC/MS/MS/MS experiments were performed on the compounds of interest. Results: A new structural type of component, which was named as josamycin A, and two acid degradation impuritiess, which were identified as impurity I and impurity II, were discovered in josamycin. Their structures and fragmentation pattern were deduced according to MSⁿ data. Furthermore, josamycin A was synthesized and impurity I was separated by preparative HPLC. The structures of josamycin A and the impurities were confirmed by ¹H NMR and ¹³C NMR data. Conclusions: Josamycin A was produced when the hydroxyl group on the macrolide of josamycin was oxidized into a carbonyl group. Impurity I and impurity II were produced by the loss of one molecule of acetyl mycaminose from josamycin and josamycin A, respectively. Compared with josamycin, the experimental results showed that josamycin A had a higher antibacterial activity with similar cytotoxicity, while impurity I had no antibacterial activity but a higher cytotoxicity. As a result, the control of impurity I is significant.

Full text of DOI:10.1002/rcm.8439

wang jian (Orcid ID: 0000-0001-5870-851X)
Characterization of a new component and impurities in josamycin by trap-free
two-dimensional liquid chromatography coupled to ion trap time-of-flight mass
spectrometry
Guijun Liu1, Yu Xu1, Jing Sang2，Bingqi Zhu2, Jian Wang1,2,*
1 Zhejiang University of Technology, Hangzhou 310014, China
2 Zhejiang Institute for Food and Drug Control, Hangzhou 310052, China
*Jian Wang: Tel.: +86 057187180358. E-mail address: wangjianhw2000@aliyun.com.
Rationale: Impurities’ toxicities of drug will affect the clinical effect and cause potential
health risk, therefore, it is essential to study impurities profiles. In this study a new structural
type of component and two acid degradation impurities in josamycin were discovered and
characterized for the further improvement of official monographs in pharmacopoeias.
Methods: The component and acid degradation impurities in josamycin were separated and
preliminary characterized by a trap-free two-dimensional liquid chromatography coupled to
high resolution ion trap time-of-flight mass spectrometry (2D LC/IT-TOF MS) with both
positive and negative electrospray ionization modes. Eluent of each peak from the first
dimensional chromatographic system was trapped by a switching valve and subsequently
transferred to the second dimensional chromatographic system, which was connected to MS.
Full scan MS was firstly conducted to obtain the exact m/z values of the molecules. Then
LC-MS/MS and LC MS/MS/MS experiments were performed on the compounds of interest.
Results: A new structural type of component, which was named as josamycin A, and two
acid degradation impuritiess, which were identified as impurity Ⅰ and impurity Ⅱ, were
discovered in josamycin. Their structures and fragmentation pattern were deduced according
to MSn data. Furthermore, the josamycin A was synthesized and impurity Ⅰwas separated by
preparative HPLC. The structures of josamycin A and impurities were confirmed by 1H NMR
and 13C NMR data.
Conclusions: Formation mechanism: Josamycin A was produced when the hydroxyl group
on the macrolide of josamycin was oxidized into carbonyl. Impurity Ⅰand impurity Ⅱwere
produced by the loss of one molecule of acetyl mycaminose from josamycin and josamycin A,
respectively. Compared with josamycin, the experiment results showed that josamycin A had
a higher antibacterial activity with similar cytotoxicity, while impurity Ⅰhad no antibacterial
activity but a higher cytotoxicity. As a result, the control of impurity Ⅰis significant.
Keywords: josamycin, new component, impurity, trap-free 2D LC/IT-TOF MS
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/rcm.8439
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1 INTRODUCTION
Many 16-membered macrolides and their derivatives had relatively good gastric tolerance
and low tendency to produce allergy and induce resistance1. Josamycin and its derivatives are
typical examples2-6. Josamycin (leucomycin A3) produced by fermentation of streptomyces
narbonensis var. Josamyceticus7, is consist of 16-membered ring aglycone and disaccharide
(Figure 1).Similar to erythromycin8, josamycin exhibited a broad-spectrum antibacterial
activities, and was used in the clinical treatment of respiratory tract infections and sexually
transmitted diseases, caused by Gram-positive bacteria, which includes mycoplasma
pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, and bordetella pertussis9-10. In
particular, josamycin is an alternative medicine for patients exhibiting penicillin allergy and
developing resistances to penicillin11-12 and 14- and 15-membered macrolides13. The
immunomodulatory properties independent of its antibacterial actions of josamycin may have
clinical relevance to inflammation, such as periodontal diseases14. In addition, the current
reports on synthesized leucomycin analogues have revealed high antibacterial activity and
anticancer activity, which indicated that these types of antibiotics can be good starting
material for antibacterial and anticancer drug candidates15.
However, the components and impurities of macrolide antibiotics are generally
complicated because commercial josamycin is produced through fermentation and extracted
from a mixture of numerous structurally related compounds. The compounds possessing
antibacterial activities were called as components, and had potential to develop into new
drugs. The compounds which have no antibacterial activity but toxicity were called as
impurities, and their control is critical during the manufacturing of drugs. Guidelines on the
identification and qualification of impurities above a certain threshold have been issued by
the International Conference on Harmonization of Requirements for Registration
Pharmaceuticals for Human Use (ICH)16. Therefore, the structures of these components and
impurities should be characterized to ensure the effectivity and safety.
The two-dimensional liquid chromatography coupled to high resolution ion
trap/time-of-flight mass spectrometry (2D LC/ IT-TOF MS) had great advantage in
establishments of impurity profiles17-19. The 2D LC/MS overcomes the drawbacks of
incompatibility between non-volatile salt mobile phase and ionization process in mass
spectrometry. The TOF MS is capable to characterize minor complex structural components
at trace levels due to its high sensitivity, efficiency and selectivity20-22. In this article, trap-free
2D LC coupled to IT-TOF MS has applied to be separated and identified the components and
impurities in josamycin. Each peak eluted from the first dimensional chromatography was
trapped by a switching valve and sent to the second dimensional chromatography for
desalting. This 2D chromatography successfully convert the non-volatile mobile phase in the
first dimension to volatile mobile phase in the second dimension before entering IT-TOF MS
for analysis.
The known component and impurities in josamycin were determined by HPLC at
detection wavelength of 231 nm according to Chinese Pharmacopoeia. However, a new
structural type of component and acid degradation impurities in josamycin were discovered at
a detection wavelength of 280 nm. Although some researchers have characterized some
impurities in josamycin using LC/MS approach23-27, the components and acid degradation
impurities of josamycin discovered in this study have not been reported. As presented in
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Figure 1, the structures of a new component which was named as josamycin A, and two acid
degradation impurities in josamycin were characterized preliminarily by 2D LC/IT-TOF MS
with positive and negative electrospray ionization modes. Based on MSn data, the structure
and fragmentation pathways of new component and acid degradation impurities of josamycin
were elucidated by referring to published article on structural analogues28. Subsequently,
josamycin A was synthesized and degradation impurity was separated by preparative HPLC
for structural confirmation, and the structures of josamycin A and impurities were confirmed
by NMR. The forming mechanisms of josamycin A and degradation impurities in josamycin
were also studied. Furthermore, antibacterial activity and cytotoxicity of these component
and acid degradation impurities in josamycin were studied.
2 EXPERIMENTAL
2.1 Materials
Josamycin (batch number: 13359-201302) used in this study was purchased from National
Institutes for Food and Drug Control (Beijing, China). HPLC grade methanol was purchased
from Merck (Darmstadt, Germany), ammonium acetate and phosphoric acid were purchased
from Sigma-Aldrich (St. Louis, USA). Analytical grade hydrochloric acid was purchased
from Lanxi Yongli Chemical Industry Co. Ltd. (Jinhua, China), Analytical grade Sodium
hydroxide was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) and
de-ionized water used in this study was producedby a Millipore Milli-Q-Gradient purification
system.
2.2 Trap-free two-dimensional LC apparatus
A trap-free two-dimensional Nexera-XR liquid chromatography system (Shimadzu, Kyoto,
Japan) equipped with four binary pumps was used. The first dimensional liquid
chromatography system included two binary pumps (LC-30AD), an auto-sampler
(SIL-30AC), a column thermostat (CTO-20A) and a diode array detector (SPD-M20A).
Chromatographic separation in the first dimension was carried out at 60℃ using a ZORBAX
SB-C18 analytical column (150 mm × 4.6 mm, 3.5μm). The mobile phase of the first
dimension were (A) 0.1 M ammonium acetate solution, whose pH was adjusted to 4.5 with
phosphoric acid, and (B) methanol-acetonitrile (55:5, v/v). The elution program were as
follows: 0 min, 40% B (hold for 10 min); 30 min, 60% B; 45 min, 60% B; 46 min 40% B
(hold for 9 min). The mobile phase flow rate was 1.0 mL min−1 and injection volume was 10
μL. The second dimensional chromatography system was consisted of two binary pumps
(LC-20AD), a column thermostat (CTO-20A) and a photo diode array detector (SPD-20A).
Chromatographic separation in the second dimension was carried out at 60 ℃ using a
Shimadzu Shim-pack GISS C18 analytical column (50 mm × 2.1 mm, 1.9 μm). The mobile
phase of the second dimension were (A) 20 mM ammonium acetate solution and (B)
methanol with gradient conditions: 0 min, 5% B; 5 min, 95% B; 5.5 min, 5% B; 9 min, 5% B.
The mobile phase flow rate was 0.3 mL min-1. And the detection wavelengths of UV detector
were set at 231 nm and 280 nm simultaneously. The first and second dimensional columns
systems were connected by means of two high pressure six-position and six-port switching
valves, which were equipped with six 20 μL stainless steel quantitative loops. Loop 1~5 were
used to retain target component, loop 6 was used as a drain loop to exchange the first and
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second dimension mobile phase The first dimension column effluent was detected by diode
array detector, and then target peak was fractioned in sample loops based on their retention
time. The first dimension column effluent was sent to sample loop just before the target peak
appeared, the flow line was switched to waste when the target peak reached the loop center.
The second dimension mobile phase directly flowed to the MS (or drain) without passing
through any loops in the initial state. During LC-MS analysis, the second dimension mobile
phase flowed and pushed the sample within loop to second dimension column, then to MS.
2.3 MS spectrometry
Trap-free two-dimensional liquid chromatography coupled to 2D LC/IT-TOF MS from
Shimadzu Corp. (Kyoto, Japan). The mass spectrometry detector (MSD) was equipped with
an electrospray ionization (ESI) source. The ionization mode was positive and negative
alternatingly. 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 desolvation line (CDL) and
heat block: 200 ℃; detector voltage: 1.56 V; scan range: 100-1000 m/z; the mode of MSn:
manual to MSn. The mode of ESI was in both positive and negative ion mode.
2.4 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.
2.5 NMR spectroscopy
All NMR spectra measurements were performed on a Bruker AVIII 600MHz spectrometer
13
1
13
(1H=600 MHz, C=101 or 151MHz) at 25℃ using CDCl3 as the solvent. H NMR and C
NMR chemical shift values were reported on the δ scale in ppm, relative to trimethylsilane
(TMS) (=0.00).
2.6 Sample preparation
Preparation of josamycin solution: The bulk drug of josamycin was dissolved in ammonium
acetate solution (0.1 M, pH 4.5)-methanol-acetonitrile (40:55:5, v/v) solution and the
concentration was 2.0 mg ml-1.
Acid degradation: 50 mg of josamycin was dissolved by 1 mL hydrochloric acid at the
concentration of 0.1 M and kept for 2 hours in water bath at the temperature of 37℃. After 2
hours, the solution was neutralized by 0.1 M sodium hydroxide and diluted with ammonium
acetate solution (0.1 M, pH 4.5)-methanol-acetonitrile (40:55:5, v/v) to 10 mL.
3 RESULTS AND DISCUSSION
3.1 Structural elucidation of josamycin A
The first dimensional chromatogram of josamycin were shown in Figure 2. Figure 2A showed
chromatogram of josamycin detected at the wavelength of 231 nm, and Figure 2B showed
chromatogram of josamycin detected at the wavelength of 280 nm. Josamycin A was
discovered and detected at the wavelength of 280 nm. ESI-MS exact mass data and
theoretical mass data on [M+H]+ and [M-H]- of the new component in josamycin were shown
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in Table 1, the values of deviation in both positive ion and negative ion modes were basically
less than 5 ppm. ESI-MSn exact mass data on major product ions of the new component in
josamycin in positive ion modes were shown in Table 2.
The formula of josamycin A was C42H67NO15 based on the data obtained from TOF MS
as shown in Figure 3. Figure 4 presented the proposed structure and fragmentation pattern of
josamycin A. Josamycin A exhibited [M+H]+ at m/z 826.4592 and [M-H]- at m/z 824.4439
indicated that the molecular weight of josamycin A was 825, which was 2 Da less than that of
josamycin. The protonated molecule at m/z 826.4592 of josamycin A fragmented into the
product ion at m/z 598.3113 by a loss of 228 Da, which corresponded to the glycosidic
cleavage between the mycaminose and the mycarose. In the MS3 spectrum of the fragment
ion at m/z 598.3113 resulted in the ion at m/z 407.2010 and m/z 192.1227, which
corresponded to the cleavage of the ether bond between 16- membered ring and mycaminose.
The fragmentation pathways of losingone mycarose and one mycaminose was in agreement
with the published paper28. Furthermore, the fragment ion at m/z 407.2010 lost one H2O,
one CH3COOH and one CH3OH successively resulted in the product ion at m/z 389.1929, m/z
329.1714 and m/z 297.1460, respectively. Another fragmentation pathway of the product ion
at m/z 407.2010 was losing one CH3COOH, one CH3OH and one H2O successively and
resulted in the ion at m/z 347.1788, m/z 315.1564 and m/z 297.1460. The fragment ion at m/z
297.1460 further neutral loss of one CO resulted in the ion at m/z 269.1526. Besides, the
ultraviolet absorption wavelength of the josamycin A shifted toward longer wavelength
compared with josamycin, which indicated the increased conjugated structure. From the
above information, josamycin A was an oxidation product of josamycin by oxidizing the
hydroxyl group on the macrolide to carbonyl.
3.2 Synthesis of josamycin A
100 mL dichloromethane solution containing 1.2 g Dess-Martin periodinane was added to a
100 mL dichloromethane solution with 1.0 g josamycin as solute constant stirring were
maintained at room temperature until the disappearance of josamycin, which was monitored
by thin-layer chromatography. Solvent was removed under reduced pressure. The residue was
purified by column chromatography to give the product as white powder. The synthetic route
of josamycin A was shown in Figure 5.
3.3 Structural confirmation of synthetic josamycin A by 1H NMR and 13C NMR
All NMR spectra measurements were performed on a Bruker AVIII 600MHz spectrometer
1
(1H=600 MHz, 13C=101 MHz) at 25℃ using CDCl3 as the solvent. The H NMR and 13C
NMR spectra of josamycin A and the correspondence between structural units and peaks in
their NMR spectra were analyzed, the molecular structure of josamycin A together with the
atom numbering and NMR data were shown in Figure 1 and Table S1. The changes of
josamycin A in 13C NMR chemical shift compared with josamycin and C-9 hydroxyl
substituted josamycin derivatives3-6 were clear. The presence of carbonyl group on position
C-9 in 16-membered ring was proved by the analysis of the 13C NMR spectra of josamycin A
in which an additional signal at 202.8 ppm(C=O) was detected. The proton hydrogen signal
on position C-9 disappeared, while proton and carbon signals assigned to the mycarose and
mycaminose parts in josamycin A were not changed.
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The data from the mass spectrum and LC retention time of josamycin A agree with the
data obtained from synthetic josamycin A, which confirmed that josamycin A corresponded
the structure of josamycin A.
3.4 Structural elucidation of acid degradation impurities
Figure 6 showed the first dimensional chromatogram of josamycin after acid degradation,
impurity Ⅰwas detected in josamycin after acid degradation at the wavelength of 231nm as
presented in figure 6A and impurity Ⅱ at 280 nm as presented in figure 6B. The ESI-MS
exact mass data and theoretical mass data on [M+H]+ and [M-H]- of the two acid degradation
impurities in josamycin in both positive and negative ionization modes were shown in Table
1 and the values of deviation were basically less than 5 ppm. ESI-MSn exact mass data of
major product ions of the impurities in josamycin in positive ionization mode were shown in
Table 2.
The formula of impurity Ⅰ was C30H49NO11 based on the data from TOF high resolution
mass spectrometer. Figure 7 presented the proposed structure and fragmentation pattern of
impurity Ⅰ. The fragmentation pathway of the protonated molecule at m/z 600.3400
concerned the loss of one H2O and led to the ion at m/z 582.3244. Subsequently, the cleavage
of ether bond occurred and the loss of C8H17NO4 from the ion at m/z 582.3244, which
produced the ion at m/z 391.2130. Further loss of one CH3COOH and one CH3OH from the
ion at m/z 391.2130 conducted to the ions at m/z 331.1892 and m/z 299.1646, respectively.
The fragmentation pathway of the product ion at m/z 299.1646 concerned the loss of 18 Da,
which was identified as H2O, and led to the ion at m/z 281.1532. From the above information,
it was concluded that the impurity Ⅰwas an acid degradation impurity.
The formula of impurity Ⅱ was C30H47NO11 based on the data from TOF high resolution
mass spectrometer. Impurity Ⅱ exhibited [M+H]+ at m/z 598.3239 and [M-H]- at m/z
596.3094, which indicated that the molecular weight of impurity Ⅱwas 597 and 2 Da less
than impurity Ⅰ. Figure 8 presented the proposed structure and fragmentation pattern of
Impurity Ⅱ. The deprotonated molecule at m/z 596.3094 initially gave rise to the ion at m/z
536.2922 due to the loss of one CH3COOH. Moreover, the ultraviolet absorption wavelength
of impurity Ⅱshifted toward longer wavelength compared with that of impurity Ⅰ. Therefore,
the structure of impurity Ⅱwas that the hydroxyl group on the macrolide of impurity Ⅰwas
oxidized to carbonyl, resulting in impurity Ⅱ conjugated chains lengthened and absorbing
wavelength redshift.
3.5 Preparation of impurity Ⅰ
1.0 g of josamycin was dissolved by 100 mL 0.1 M hydrochloric acid and the solution was
kept for 2 hours in water bath at 37℃. After 2 hours, the solution was neutralized by 0.1 M
sodium hydroxide, which was furtherly extracted with dichloromethane three times with 50
mL each time. The extracted dichloromethane solution was combined organic layer and dried
using anhydrous Na2SO4. The white powder was obtained by evaporating solvent under
reduced pressure. Then, the impurity Ⅰ was isolated using a Shimadzu semi-preparative
HPLC.
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3.6 Structural confirmation of impurity Ⅰby 1H NMR and 13C NMR
All NMR spectra measurements were performed on a Bruker AVIII 600MHz
spectrometer (1H=600 MHz, 13C=151 MHz) at 25℃using CDCl3 as the solvent. The 1H NMR
13
and C NMR spectra of impurity Ⅰand correspondence between structural units and peaks
in their NMR spectra were analyzed. The molecular structure of impurity Ⅰtogether with the
atom numbering and NMR data were shown in Figure 1 and Table S2. Comparison of the
NMR data between josamycin5 and impurity Ⅰshowed that signals assigned 16-membered
ring and mycaminose were similar. The proton and carbon signals assigned to the mycarose
were not detected in impurity Ⅰ, which means the loss of mycarose
3.7 Forming mechanism, antibacterial activity and cytotoxicity of josamycin A and
degradation impurities in josamycin
Josamycin A was an oxidative product of josamycin as the hydroxyl group on the macrolide
was oxidized to carbonyl. Impurity Ⅰ was produced by the loss of one molecule of acetyl
mycaminose from josamycin, and impurity Ⅱ produced by the loss of one molecule of
mycaminose from josamycin A under acidic condition. The Impurity Ⅰand impurity Ⅱwere
existed and could be detected in commercial josamycin. Because of the increased conjugated
structure, the ultraviolet absorption wavelengths of the josamycin A and impurity Ⅱshifted
toward longer wavelength compared with josamycin and impurity Ⅰ. Josamycin and impurity
Ⅰexhibited the maximum absorption at the wavelength of 231 nm, whereas josamycin A and
impurity Ⅱ showed the maximum absorption at the wavelength of 280 nm. The formation
mechanisms of josamycin A and acid degradation impurities were shown in Figure 1.
The antibacterial activity was determined by cup-plate method (two-dose assay)
reference to the statistical method of biological assay in the Chinese Pharmacopoeia 2015.
The result showed that the activity of josamycin A was 1.5 times high josamycin and the
impurity Ⅰ had no antibacterial activity. Whereas, the experimental result of cytotoxicity
showed that there was no significant difference between the josamycin A and josamycin, and
the survival ratio of cell L929 was identical under the five concentrations of josamycin A and
josamycin. Impurity Ⅰshowed higher cytotoxicity than josamycin, and the survival ratio of
cell L929 was lower with the application of impurity Ⅰ. The relationship of drug
concentration and bacteriostatic rate was shown in Figure 9. This result indicated that content
of impurity Ⅰshould be control in josamycin.
4 CONCLUSIONS
The structures of a new component and two acid degradation impurities in josamycin were
discovered and identified by 2D LC/IT-TOF MS. Josamycin A (new component) was
synthesized and impurity Ⅰ was separated by preparative HPLC, and their structures was
1
confirmed by H NMR and 13C NMR data. Josamycin A was an oxidative product of
josamycin, whose the hydroxyl group on the macrolide were turned into carbonyl during the
oxidation process. Impurity Ⅰ was produced by the loss of one molecule of acetyl
mycaminose from josamycin, and impurity Ⅱ was produced when josamycin A lost one
molecule of mycaminose. Compared with josamycin, experiment results showed that
josamycin A presented a higher antibacterial activity and little difference in cytotoxicity, and
impurity Ⅰ had no antibacterial activity but a higher cytotoxicity. Therefore, impurity Ⅰ
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should be controlled. Based on characterization of impurities, this study revealed the
mechanism of impurity production and can provide guidance for pharmaceutical companies
to improve the manufacturing process and reduce impurity content. The result from this
research also established scientific basis for quality control of josamycin.
ACKNOWLEDGMENT
This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.
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TABLE 1 ESI-MS exact mass data and theoretical mass data on [M+H]+ and [M-H]- of
josamycin A and impurities in josamycin in positive ion (a) and negative ion (b) modes.
Theoretic
[M+H]+
Theoretical
[M-H]-
(m/z)
Com
p.
al
Deviati
onb
Deviati
onb
formula
formula
(m/z)
[M+H+](m/z)
[M-H]-(m/
z)
(ppm)
(ppm)
A
I
826.4592
600.3389
598.3204
C42H68NO15
C30H50NO11
C30H48NO11
826.4582
600.3378
598.3222
1.21
1.83
-3.01
824.4439
598.3217
596.3094
C42H66NO15
C30H48NO11
C30H46NO11
824.4438
598.3233
596.3094
0.12
-2.67
3.02
Ⅱ
a: the data of theoretical [M+H]+ and [M-H]- was calculated by software of Shimadzu ccurate
Mass calculator.
b: the deviation was calculated by the following formula: (M-M0)/m*106, where M was the
measured value of the ion mass; M0 was the theoretical value of the ion mass; m was the
mass of the ion.
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TABLE 2 ESI-MSn exact mass data on major product ions of the josamycin A and impurities
in joamycin in positive ion (a) and negative ion (b) modes.
(a) positive
MS/MS
Precursor ion (m/z)
[M+H]+
MS/MS/MS fragmentation
Name
fragmentation
ions (m/z)
in MS/MS/MS
(m/z)
ions (m/z)
826.4592
598.3113，407.2010 598.31
407.2007，389.1929，375.2253，
347.1788，329.1714，315.1564，
297.1460
Josamycin A
600.3389
582.3203, 391.2114, 582.32
331.1930, 299.1653,
391.2148
Impurity Ⅰ
281.1541, 192.1267
(b) negative
Name
MS/MS
Precursor ion (m/z)
in MS/MS/MS
[M-H]-
MS/MS/MS fragmentation
fragmentation
ions (m/z)
536.2922
(m/z)
ions (m/z)
596.3094
Impurity Ⅱ
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FIGURE 1 The forming mechanisms and chemical structures of josamycin A and acid
degradation impurities. The dotted lines marked their main differences in 16-membered ring.
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FIGURE 2 The first dimensional chromatogram of josamycin, (A) detected at the wavelength
of 231 nm, (B) detected at the wavelength of 280 nm.
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FIGURE 3 MS full scan ESI+ (A) and ESI- (D), the MS2 (B) and MS3 (C) spectra in
both positive ionization of josamycin A.
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FIGURE 4 Proposed fragmentation pathways of josamycin A in the positive ion mode.
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FIGURE 5 Structure and synthetic route of josamycin A.
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FIGURE 6 First dimensional chromatogram of josamycin after acid degradation, (A) detected
at the wavelength of 231 nm, (B) detected at the wavelength of 280 nm.
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FIGURE 7 Proposed fragmentation pathways of impurity Ⅰin the positive ion mode.
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FIGURE 8 Proposed fragmentation pathways of impurity Ⅱin the negative ion mode.
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FIGURE 9 Relationship of drug concentration and bacteriostatic rate by MTT assay. The
absorbance was measured at 490 nm, relative growth ratio (RGR) was calculated according to
following formula: RGR% =Average absorbance of test group/ Average absorbance of control
group×100%.
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