See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/239593279

Structure elucidation of degradation products of the antibiotic drug amoxicillin

Part I: Examination of the degraded drug products by fragmentation with ion
trap MS n

Article

CITATIONS READS
0 836

2 authors:

Edgar Nägele Ralf Moritz

Agilent Sandoz
57 PUBLICATIONS   1,082 CITATIONS    9 PUBLICATIONS   132 CITATIONS   

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Edgar Nägele on 18 December 2013.

The user has requested enhancement of the downloaded file.

 Structure elucidation of degradation products
of the antibiotic drug amoxicillin
Part I: Examination of the degraded drug products by
fragmentation with ion trap MSn

Application Note Edgar Nägele

Ralf Moritz

O
OH
O
N CH3
O
HO CH3
S
NH

NH 2

Abstract

Today, it is necessary to identify and confirm the identity of all com-

pounds appearing in the process of the discovery and the development
of a new drug substance in the pharmaceutical industry.
In this Application Note the identification and confirmation of degrada-
tion products from a final dosage of the antibiotic drug amoxicillin
obtained under stress conditions will be demonstrated by structure
elucidation with LC ion trap MS/MS and MS3. The identity confirmation
of the degradation products by accurate mass measurement of the
molecular ions and of the CID fragments by LC/ESI oaTOF will be
discussed in Part II of this work1.
Introduction
In modern pharmaceutical drug
discovery and development it is of
crucial importance to identify an
unknown compound with the high-
est possible confidence because
of its potential toxic effects on
humans. Such a compound could
be, for instance, the pharmaceuti-
cal active substance itself, a minor
byproduct out of the production
process, a secondary substance
in a drug isolated from a natural
source, a metabolite created in
the human body or a degradation
product of the pharmaceutical
agent created under harsh storage
conditions.
Widely used mass spectrometric
tools for the structure elucidation
are ion trap instruments with
MS/MS and MSn capabilities. With
these instruments it is possible to
break the molecular ion of the
investigated substance in frag-
ments, which are useful for the
structure elucidation. It is possible
to do this under full control by a
repeatable isolation and fragmen-
tation of the fragments derived
from the original substance.
Recently, the use of an ion trap for
MSn fragmentation and structure
elucidation together with an ESI
oaTOF instrument to measure
accurate molecular mass and
calculate of the empirical formula,
and consequently the identity
confirmation of an unknown com-
pound was impressively demon-
strated in several published appli-
cations. For instance, MS/MS and
MS3 experiments were performed
to gain structural information for
the identification of a photooxy-
genation product of a broadspec-
trum antibiotic used for live stock2
and for the identification of highly
2
complex polyene macrolides iso- vent A: Water, 10 mM ammoni-
lated from Streptomyces noursei um formate, pH 4.1; Solvent B:
by means of an ion trap moni- ACN. Column flow: 8 µL/min,
tored purification process3. Primary flow: 500-800 µL/min.
In this Application Note the iden- Gradient: 0 min 0 % B, 1 min 0 %
tification of degradation products B, 13 min 25 % B, 23 min 25 %
from the antibiotic drug amoxicillin B. Stop time: 23 min. Post time:
obtained under harsh conditions 15 min.
will be demonstrated by means of • The Agilent 1100 Series
structure elucidation with an LC autosampler was used to make
ion trap MS/MS and MS3. The injections of 1 µL. The sample
obtained and interpreted data are loop was switched to bypass
used to build up a degradation after 1 minute to reduce delay
pathway of amoxicillin. volume.
• The mass spectrometers were
operated under the following
Experimental conditions:
Equipment Part I – Ion Trap MS:
• The ESI ion trap (Part I of this Source: ESI in positive
work) and the ESI TOF MS mode
analysis (Part II of this work) Dry gas: 5.0 L/min
was performed with the Agilent Dry temp.: 300 º
1100 Series LC/MSD ion trap Nebulizer: 15psi
XCT plus and with the Agilent Target: 150,000
LC/MSD TOF equipped with a Max. accum. time: 50 ms
dual sprayer source for the Scan: 200-60
simultaneous infusion of the Averages: 2
reference mass solution. Automated MS/MS and MS3
• The LC system used was an
Agilent 1100 Series capillary LC Part II – ESI TOF MS:
system containing a capillary Source: ESI in positive
pump with a micro vacuum mode with dual
degasser, a micro well-plate spray for refer-
autosampler with a thermostat ence mass
and a column compartment. Dry gas: 7.0 L/min
• The column used was a Dry temp.: 300 ºC
ZORBAX SB-Aq, 0.3 mm x 150 Nebulizer: 15 psi
mm, 3.5 µm. Scan: 50-1000
• The software used for instru- Fragmentor: 150 V or 300 V
ment control was ChemStation for CID
A10.02, ion trap software 4.2, Skimmer: 60 V
TOF software A.01 and for data Capillary: 5000 V
analysis the ion trap data analy- • Sample preparation: The antibi-
sis software 3.2 and Analyst QS otic amoxicillin was stressed
software. under acidic conditions.
Approximately 1 mL of amoxi-
Methods cillin solution (25 mg/mL in
• The Agilent 1100 Series capil- DMSO) was added to 1 mL
lary pump was operated under 0.1 M HCl solution. The sample
the following conditions: Sol- was stirred for 1 hour at room
temperature (RT = 25 ºC) and
 then diluted (1:10 with DMSO). ions and the CID fragments in together with the peak for the iso-
Results and discussion Part II of this work1. The mass of meric compound (4) at 10.5 min
the protonated amoxicillin (1) (figure 2A). The amoxicillin peak
The degradation of amoxicillin (1) at m/z 366.1 was extracted at a also contains a potassium adduct
was induced by subjecting the retention time of 4.0 minutes from at m/z 404.0 and a product gener-
pure drug substance to harsh con- the base peak chromatogram ated under the conditions of mass
ditions, as described in the experi-
mental section. Aliquots of this
solution were collected at various Intens. BPC 50-600
1. Amoxicillin
timeframes and subjected to capil- x107
2. Amoxicillin penicilloic acid
lary chromatography to separate the 8 3 3. Amoxicillin penilloic acid I and II
accumulated degradation products. 4 4. Diketopiperazine amoxicillin
The base peak chromatogram 6 5. 4-Hydroxyphenylglyl amoxicillin
(BPC) obtained clearly shows the
degradation of amoxicillin into 5
4 1
various products (figure 1). The
chromatogram also assigns the
2
degradation products, which were 2
identified by structure elucidation
with ion trap MS/MS and MS3 0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 Time [min]
experiments and confirmed by
accurate mass determination with
ESI TOF MS and empirical formu- Figure 1
la calculation for the molecular BPC of amoxicillin (1) and its degradation products

Intens. Intens. +MS, 4.7 min

x10 7
2.0
A Amoxicillin EIC 366 +All MS
x106 B 366.1
6 404.0
1.5 349.0
4
1.0

0.5 2

0.0 0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 Time [min] 100 200 300 400 500 m/z
Intens.
+MS, 4.7-5.2min
x105 366.0
6 349.0
4 C MW (C16H19 N3O5S): 365,10 D
2
0 [M+H] += 366.11
+MS2(366.0), 4.7-5.2min
x105 349.1 [M+H] + - NH3 = 349.08
4 160.0
2 H COOH
160.0 207.0 O
0 H NH 2 N C H 3114.0
+MS3(366.0->349.1), 4.7-5.2min 349.0
x104 NH
S CH3
4 207.0 H
114.0 H
233.9 O
2 305.1 HO (1)
160.0
0
100 150 200 250 300 350 m/z

Figure 2
Amoxicillin (1) (C16H19N3O5S), [M+H+]+ = 366.11 m/z
A) EIC of amoxicillin. B) MS of amoxicillin at 4.7 min.
C) MS, MS/MS and MS3 between 4.7 and 5.2 min of amoxicillin. D) Fragmentation of amoxicillin according to the MS/MS and MS3.
3
spectrometry analysis by the loss
of ammonium at m/z 349.0 (figure
2B). The product ion of ammoni-
um loss is also generated under
MS/MS conditions in the ion trap
as a major product at this stage
(figure 2C). The MS3 analysis
starting with this ion results in
two new ions at m/z at 114.0 and
at m/z 160.0, which could be easi-
ly assigned to the corresponding
fragmentation (figures 2C and 2D).
The degradation of amoxicillin (1)
in an acidic medium starts with
the opening of the four membered
beta lactam ring and yields the
product amoxicillin penicilloic
acid (2) containing a free car-
boxylic acid group, which gives a
higher polarity to this molecule.
This leads to a shift towards an
earlier retention time in the RP LC
separation, which is shown in the
EIC of protonated amoxicillin
penicilloic acid (2) at m/z 384.0 at
2.6 minutes (figure 3A). The mass
spectrum shows the protonated
molecular ion of (2) and also its
potassium adduct at m/z 422.0
(figure 3B). The fragmentation of
the molecular ion of (2) resulted
in two product ions. The ion
resulting from a loss of ammonia
at m/z 376.1 and the product ion
from additional decarboxylation
resulting from the loss of the free
carboxylic acid group at m/z 323.1
(figure 3C). This ion is the precur-
sor of the following MS3 fragmen-
tation leading to the fragment ions
with m/z 189.0 and the fragment
ion of the thiazolidine ring at

Intens. Intens.
EIC 384 +All MS +MS, 2.6min
x10 6 A x106 B 384.0
1.5 2.5
2.0
1.0 1.5
1.0
0.5
0.5 422.0

0.0 0.0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 Time [min] 150 200 250 300 350 400 450 500 m/z
Intens.
+MS, 2.5-2.6min
x105
4
C 310.8 MW (C16H21N3O6S): 383,12 1
D
174.9 212.8 384.0 [M+H]+= 384.12
2
[M+H]+ - NH3 = 367.09
0 [M+H]+- NH3 - CO2 = 323.41
x105 323.1
+MS2(384.0), 2.5-2.6min
O OH
H COOH
1.0
367.1 NH 2 NH C H3
0.5 H
367.1 323.1
0.0 NH
S CH3
x10 4 +MS3(384.0->323.1), 2.5 -2.7min H
H
3 189.0 O
2 HO 189.0
160.0
1 228.9 305.1 (2)
160.0
0
100 200 300 400 500 600 m/z

Figure 3
Amoxicillin penicilloic acid (2) (C16H21N3O6S), [M+H]+ = 384.12 m/z
A) EIC of amoxicillin penicilloic acid.
B) MS of amoxicillin penicilloic acid at 2.5 min.
C) MS, MS/MS and MS3 between 2.5 and 2.6 min of amoxicillin penicilloic acid.
D) Fragmentation of amoxicillin penicilloic acid according to the MS/MS and MS3.

4
m/z 160.0 (figure 3D).
Starting with compound (2) there
are two possible ways of further
degradation. The first one is based
on the decarboxylation of the free
carboxylic acid and leads to the
stereoisomeric compounds amoxi-
cillin penilloic acid I and II (3).
The second possible degradation
reaction of intermediate (2) is the
closure of a new stable six mem-
bered ring to Diketopiperazine
amoxicillin (4). Both reaction
products were identified in the
amoxicillin solution stored under
harsh conditions. The protonated
stereo isomeric amoxicillin penil-
loic acids I and II (3) both at
m/z 340.2 were extracted from the
BPC at 6.7-7.3 and 8.5-9.0 minutes
(figure 4A). They were found as
the protonated species as well as
in the form accompanied by potas-
sium at m/z 378.0 (figure 4B).
From the MS/MS analysis the
product of a loss of ammonium
(figure 4C) and from the MS3 frag-
mentation the molecule fragment
with m/z 188.9 were assigned
(figure 4D). Identical mass spectra
and fragmentation patters where
obtained for both stereo isomers

Intens. Intens. 340.2 +MS, 6.7min

EIC 340 +All MS
x107 A x10 6 B
2.0 4

1.5 3

1.0 2
0.5 1 323.1 378.0

0.0 0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 Time [min] 100 200 300 400 500 m/z
Intens.
+MS, 6.7 -7.3min
340.1
x10 6 MW (C15H21N3O4S): 339,12 D
1.0
0.5
C 323.1 [M+H] += 340.13
0.0
x106 +MS2(340.4), 6.7-7.3min [M+H] +- NH3 = 323.10
2 323.1
H COOH
1
188.9 229.0 H NH2 CH3
0 NH
323.1 NH
x10 5 +MS3(340.1->323.1), 6.7-7.3min CH3
4 188.9 S
229.0 H
2 305.1 O 188.9
HO (3)
0
100 150 200 250 300 m/z

Figure 4
Amoxicillin penilloic acid I and II (3) (C15H21N3O4S), [M+H]+ = 340.13 m/z
A) EIC of amoxicillin penilloic acid I and II .
B) MS of amoxicillin penilloic acid at 6.7 min.
C) MS, MS/MS and MS3 between 6.7 and 7.3 min of amoxicillin penilloic acid.
D) Fragmentation of amoxicillin penilloic acid according to the MS/MS and MS3.

5
under both peaks. the protonated form of the com- ring and the five membered thia-
The second reaction product pound is also accompanied by zolidine ring yielding a fragment
derived from compound (2) the potassium giving the positive at m/z 206.2 and a fragment at
protonated diketopiperazine charge as indicated at m/z 404.0 in m/z 160.0 (figure 5C). Further MS3
amoxicillin (4) at m/z 366.0 was the mass spectrum (figure 5B). In fragmentation yields the product
extracted from the BPC and the the MS/MS experiment the mole- ion at m/z 114.1 obtained by the
corresponding peak is shown in cule undergoes fragmentation by cleavage of a carboxylic acid
the EIC at a retention time of 10.4 cleavage of the bond between the group from the thiazolidine ring
minutes (figure 5A). In addiiton, six membered diketopiperazine

Intens . Intens.
x10 7 366.0 +MS, 10.4min
2.0
A EIC 366 +All MS
x10 6 B
3

1.5
2
1.0 Diketopiperazine amoxicillin
1
0.5
404.0
0.0 0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 Time [min] 100 200 300 400 500 m/z
Intens.
+MS, 10.5 -10.8min 366.0
x10 6
1.0
C
MW (C16 H19 N 3O5S): 365,10 D
160.0 [M+H] += 366.11
0.0 H COOH
x10 6 160.0 +MS2(366.2), 10.5-10.8min
HN CH3 114.1
0.5 H
O N
0.0 206.2 S C H3
6000 +MS3(366.0 ->160.0), 10.5 -10.8min
4000 114.1 N O 160.0
2000 H 206.2
0 HO (4)
100 150 200 250 300 350 m/z

Figure 5
Diketopiperazine amoxicillin (4) (C16H19N3O5S), [M+H]+ = 366.11 m/z
A) EIC of diketopiperazine amoxicillin.
B) MS of diketopiperazine amoxicillin 10.4 min.
C) MS, MS/MS and MS3 between 10.4 and 10.7 min of diketopiperazine amoxicillin.
D) Fragmentation of diketopiperazine amoxicillin according to the MS/MS and MS3.

6
moiety (figures 5C and 5D). m/z 515.0 was extracted from the zolidine ring. The MS3 step unrav-
In another reaction pathway BPC at 9.3 minutes (figure 6A) eled the structure with a loss of a
amoxicillin (1) reacts in a nucle- and found together with the carbonyl group and finally yielded
ophilic attack on itself whereas the potassium adduct (figure 6B). The a benzylic amino fragment at
benzylic carbonyl group is attacked MS/MS measurement reveals a m/z 122.1 (figure 6C and 6D).
by the free amino group and under- product at m/z 498.0 from a sepa- The final degradation pathway of
goes condensation to 4-Hydroxy- ration of ammonium and also a amoxicillin (1) with the identified
phenylglyl amoxicillin (5). The product at m/z 339.1 from degra-
protonated molecular ion at dation of the five membered thia-

Intens. Intens.
EIC 515 +All MS +MS, 9.3min
x10
7 A x10
6 515.0
3
1.25 B
1.00 552.9
2
0.75
0.50 1
0.25
0.00 0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 Time [min] 100 200 300 400 500 m/z
Intens.
+MS, 9.3-9.7min
x10 6 515.0 MW (C24H26N4O7S): 514,14
1.0 D
0.5 C 498.1 [M+H]+= 515.16

0.0 [M+H]+- NH3 = 498.13

x10 6 +MS2(515.0), 9.3-9.7min 339.1 H 2N H 339.1
498.1 O
0.50 311.1 H COOH
498.1 O
311.1 NH N C H3
0.00 HO
H
x10 5 +MS3(515.0->339.1), 9.3-9.8min NH
S CH3
2 311.1 H
H
1 122.1 O
HO
122.1 (5)
0
100 150 200 250 300 350 400 450 m/z

Figure 6
4-Hydroxyphenylglyl amoxcillin (5) (C24H26N4O7S) [M+H]+= 515.16 m/z
A) EIC of 4-Hydroxyphenylglyl amoxcillin. B) MS of 4-Hydroxyphenylglyl amoxcillin 10.4 min. C) MS, MS/MS and MS3 between 9.3 and 9.7 min of
4-Hydroxyphenylglyl amoxcillin. D) Fragmentation of 4-Hydroxyphenylglyl amoxcillin according to the MS/MS and MS3.

H COOH H COOH
H COOH O OH
O NH2 NH2 CH3
NH CH3 H NH
NH2 N CH3 H
H NH
NH CH3 S CH3
NH S
S CH3 H
H H H
H O O
HO
O HO
HO
1 2 3
H COOH
H2N H 1. Amoxicillin
O HN CH3
H COOH
H 2. Amoxicillin penicilloic acid
O O N CH3
NH S 3. Amoxicillin penilloic acid
N CH3
H
HO
NH
S CH3 N O 4. Diketopiperazine amoxicillin
H H
H 5. 4-Hydroxyphenylglyl amoxicillin
O HO
HO
5 4
Figure 7
Degradation pathway of amoxicillin.
7
 degradation products is summa-
rized in figure 7.
Conclusion
The presented work describes
the elucidation of the degradation
pathway of the pharmaceutical
substance amoxicillin, a commonly
used antibiotic drug. The creation
of degradation products, which
may possibly be formed under
harsh storage or production condi-
tions was artificially induced. The
degradation products created from
amoxicillin were separated by
capillary LC and analyzed by an
MS ion trap in MS/MS and MS3
mode. The fragments obtained
were used for the structure eluci-
dation of the possible degradation
products and for the creation of a
degradation pathway. Further re-
sults of this work will be described
in Part II1, which presents the
confirmation of the obtained ion
trap results by the measurement
of accurate mass and empirical
formula confirmation of the degra-
dation products by LC/ESI oaTOF.
In Part III4 the obtained knowl-
edge about the degradation of
amoxicillin will be applied for the
identification of minor byproducts
in a drug development formulation
trial by accurate mass determination
with LC/ESI oaTOF.
View publication stats
References 4.
Nägele, E., Moritz, R., “Structure
1. elucidation of degradation prod-
Naegele, E., Moritz, R., “Structure ucts of the antibiotic drug amoxi-
elucidation of degradation prod- cillin – Part III: Identification of
ucts of the antibiotic drug Amoxi- minor byproducts in a formulation
cillin – Part II: Identification and trial with accurate mass measure-
confirmation by accurate mass ment by ESI TOF and ion trap
measurement with ESI TOF of the MRM” Agilent Publication Num-
compound ions and the fragments ber 5989-2470EN, 2005.
after CID.” Agilent Publication
Number 5989-2348EN, 2005.
2.
Eichhorn P., Aga D.S., “Identifica-
tion of a photooxygenation prod-
uct of chlorotetracycline in hog
lagoons using LC/ESI-ion-trap-MS
and LC/ESI-time-of-flight-MS.”
Anal. Chem. 76: 6002-6011,
2004.
3.
Bruheim P., Borgos S.E.F., Tsan P.,
Sellta H., Ellingsen T.E., Lancelin
J.-M., Zotchev S.B., “Chemical
diversity of polyene macrolides
produced by Streptomyces nour-
sei ATCC11455 and recombinant
strain ERD44 with genetically
altered polyketide synthase
NysC.” Antimicrob. Agents
Chemother. 48: 4120-4129, 2004.
Edgar Nägele is Application
Chemist at Agilent Technologie,
Waldbronn, Germany.
Ralf Moritz is Analytical Chemist
at Sandoz GmbH, Kundl, Austria.
www.agilent.com/chem/1100
Copyright © 2005 Agilent Technologies
All Rights Reserved. Reproduction. adaptation
or translation without prior written permission
is prohibited. except as allowed under the
copyright laws.
Published June 1, 2005
Publication Number 5989-2347EN