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141
4.1 INTRODUCTION
Sultamicillin, chemically known as Methylene(2S,5R,6R)-6-[[(2R)-
aminophenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylate(2S,5R)-3,3-dimethyl-4,4,7-trioxo-
4λ6-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, which is a mutual
(joint) prodrug of ampicillin and sulbactam compounds attached together
with ester connection. The chemical structure of sultamicillin is shown in
Fig. 4.1
H
N
S
CH3
CH3
O
H
H
O
N
NH2
H
H
O
O
N
S
OO
H3C
H3C
O
H
OO
Fig. 4.1 Chemical structure of Sultamicillin
This mutual prodrug is one of the antibiotics with plenty
antimicrobial spectrum for the treatment of childhood pneumonia. The
irretrievable β-lactamase inhibitor sulbactam has been combined
chemically via ester linkages with ampicillin to form sultamicillin [1]. It
was composed of double esters of formaldehyde hydrate in which one of
the hydroxyl groups is esterified with ampicillin and sulbactam. It is
hydrolyzed quickly in neutral or faintly alkaline conditions, while
hydrolyzed; it forms ampicillin and hydroxylmethyl sulbactam or
sulbactam and hydroxylmethyl ampicillin by different routes [2]. It is
142
obtainable in both oral and parenteral preparations for child
(pediatric)use. Sultamicillin is also a valuable treatment option for a
multiplicity of pediatric infections, bacterial infections in children
including those due to β-lactamase-producing organisms [3, 4]. The use
of β-lactam and β-lactamase inhibitor mixtures, particularly ampicillin
and sulbactam, as empiric treatment or prophylaxis for number of
pediatric infections are healthy established, and have been extensively
reviewed over number of years [3-7]. The antimicrobial action of
Sultamicillin had been established in vitro against extensive range of
gram-positive and negative organisms and as well as anaerobes [3, 8].
Literature
This mutual pro-drug antibacterial activity was explained in 1985
by Kawasaki and etal [9]. They explicates that, sultamicillin have greater
bactericidal activity against both B. fragilis and S. aureus than
bacampicillin and cefadroxil drugs. A number of synthetic procedures
were available as a patents and publications in the literature for
preparation of sultamicillin, its drug forms like tosilate, napsilate,
amoxicillin analogues, and intermediate compounds during the decades
[10-14]. Furthermore, so many microbial [15-18] and bioequivalence
studies have demonstrated for sultamicillin. The oral suspension is
highly palatable and can be administered without regard to meal times.
This regimen is likely to be more convenient than others for the
143
treatment of children, as it fits more easily into the normal daily routine,
and may lead to greater compliance [19]. Superficial skin and soft tissue
infections are common pediatric problems treated in an ambulatory
setting with oral antibiotics [20] and sultamicillin is exploiting in the
treatment of superficial skin and soft tissue infectivity’s in children [21].
It was first developed in 1987 by Pfizer Inc and marketed under the
trade name Unasyn [22] and saltum. The degradation impurity formed
under stability storage conditions have been studied in this research
work and discussed in this chapter. In this study, sultamicillin was
subjected to stress/forced and formal/proper stability storage conditions
(accelerated and real/long time storage) as per ICH [23]. An unknown
impurity was observed when sultamicillin stability samples under
analysis, which is other than the known impurities [24-27]. Stability
studies of sultamicillin and its degradation behavior have been reported
in literature [28-29]. In general, the impurity profile of any drug
substance/product study has to be conceded as ICH to recognize and to
distinguish all the unknown impurities [30]. To the best of our
familiarity, this impurity has not been reported in literature till date.
Present scientific research work transaction with detection of impurity,
isolation, structural elucidation and formation of this degradation
product.
144
4.2 EXPERIMENTAL
4.2.1 Samples, chemical and reagents
Sultamicillin and its known related substances were used for this
work gifted from Aurobindo Pharma Ltd, Research Centre Laboratories.
Sodium dihydrogen orthophosphate (dihydrate), acetonitrile, methanol,
acetic acid, formic acid solvents, ammonium acetate, orthophosphoric
acid and potassium bromide (spectroscopy (IR) grade) were purchase
from Merck (India), formaldehyde was procured from Sigma aldrich and
water (pure milli-Q) was used with the assist of millipore purification
system.
4.2.2 High performance liquid chromatography
a) Preparation of mobile phase
Mobile phase A was phosphate buffer pH 3.0 (it was prepared by
dissolving 4.69 g of sodium dihydrogen orthophosphate in 1 liter water,
and pH was adjusted to 3.0(±0.05) by using diluted phosphoric acid).
Acetonitrile solvent was used as Mobile phase B. For the sample
preparations, diluent was prepared by addition of solution A with
solution B in the ratio of 7:3 vol/vol (solution A: methanol and
acetonitrile in the ratio of 2:8 vol/vol and solution B: Dissolved 1.56 g of
sodium dihydrogen orthophosphate (dihydrate) in 900 ml of water and
added 7.0 ml of orthophosphoric acid and diluted to 1000 ml with water).
145
b) Preparation of sample solution
About 50 mg of sultamicillin sample accurately weighed and
transferred into a 100 ml clean and dry flask added 70 ml of Solution A
and sonicated for about 1 minute. Added 26 ml of Solution B, mix and
sonicated for about 1 minute and diluted to volume with Solution B.
This solution was filter through 0.45 µ or finer porosity membrane filter.
c) Methodology
The analysis was carried out on branded column, Kromasil C18,
100 mm long, 4.6 mm i.d., 3.5µm particle size. Injection volume
(quantity) was 10µl, the flow rate was reserved as 1.0 ml/minute and
column oven was maintained at 25°C temperature. For the detection, UV
was selected as 215 nm depending upon its chromophore activity, and
analysis data acquisition time was 20 min. The HPLC pump was in
gradient mode and gradient plan was as follows:
Time (min) Mobile Phase [A] (% vol/vol)
Mobile Phase [B] (% vol/vol)
0.01 95 05
15.0 30 70
20.0 30 70
22.0 95 05
30.0 95 05
146
4.2.3 LC/MS/MS analysis
a) Preparation of mobile phase
1 ml of formic acid was taken in a 1 liter of water and filtered
through a nylon:66 membrane filter of 0.45µ pore size using Millipore
vacuum pump (0.1% v/v formic acid solution) and acetonitrile was used
as mobile phase B.
b) Preparation of sample solution
About 100 mg of sultamicillin sample accurately weighed and
transferred into a 100 ml fresh and dry volumetric flask, added 70 ml of
Solution A (as prepared in HPLC methodology) and sonicated for about 1
minute. Added 26 ml of Solution B (as prepared in HPLC methodology),
mix and sonicated for about one minute and diluted to volume with
Solution B. This solution was filtered all the way through 0.45µ or
higher quality porosity membrane filter.
c) Methodology
The turbo ion spray/scatter voltage was maintained at 5500 mV
and temperature was set at 375°C. Pure nitrogen gas was used as
auxiliary gas and curtain gas. Zero air was used as nebulizer gas. LC-MS
spectra were obtained from m/z 100-1000 in 0.1 amu steps with 2.0 s
dwell time. The analysis of the samples were carried out by using YMC
PACK ODS-AQ , 250 mm long, 4.6 mm i.d., 5µm particle size column.
For detector response was carried out at 215 nm, flow rate was kept as
147
1.0 ml/min and data acquisition time was 30 min. The gradient (pump)
series was as follows,
Time (min)
Mobile Phase [A] (% vol/vol)
Mobile Phase [B] (% vol/vol)
0.01 95 05
20.0 30 70
30.0 20 80
32.0 95 05
40.0 95 05
4.2.4 Preparative liquid chromatography
a) Preparation of mobile phase
The mobile phase A was 0.2% ammonium acetate solution (2g of
ammonium acetate was dissolved in 1000 ml water), this was adjusted to
pH adjusted to 5.0(±0.1) with glacial acetic acid and acetonitrile solvent
was used as mobile phase B for this experiment.
b) Methodology
Hyperprep HS C18 500 mm long, 30 mm i.d., (make: Thermo
Scientific) filled with 10 µm particle size was employed for isolation of the
impurity. Flow rate was 30 ml/min and UV detection was carried out at
215 nm. The gradient program is,
148
Time (min) Mobile Phase [A] (% vol/vol)
Mobile Phase [B] (% vol/vol)
0.01 100 0
10.0 90 10
25.0 80 20
40.0 70 30
55.0 60 40
65.0 55 45
80.0 50 50
100.0 40 60
120.0 20 80
4.2.5 MS/TOF (Time-o-flight mass) analysis
The mass parameters for MS/TOF analysis were hexapole Rf,
capillary voltage 3000 v, cone voltage 25 v, source temperature 120°C,
desolvation temperature 300°C, ion energy 1.0 v and collision energy 8v.
The samples were directly infused using a syringe at a concentration of 2
mg/ml in methanol.
4.2.6 Nuclear magnetic resonance (NMR)
Experiments using deuterated dimethylsulfoxide (DMSO-d6) as
solvent and tetramethylsilane (TMS) as internal standard at 25°C
temperature. For 1H NMR and 13C NMR spectrometer, the operating
frequencies were 300.1315 MHz and 75.4748 MHz; number of scans was
149
32 and 1062 respectively. LB 0.3 Hz and SF 300 MHz parameters were
used for data processing.
4.2.7 Fourier transform infrared spectroscopy (FT-IR)
Triturate about 2 mg of sample in about 400 mg of potassium
bromide (previously dried at 80°C). Pellet was prepared by using pellet
maker by applying 8-10 tons of pressure and IR spectrum was recorded
between 650 and 4000 cm-1 by doing the blank correction by using KBr
pellet.
4.2.8 Stability studies
Stress stability testing is to be carried out to recognize the
potential degradation products otherwise to elucidate the inherent
stability individuality of the drug substance [23].
4.2.8a Stress stability studies
Sultamicillin drug substance was subjected to dry heat thermal
exposure at 105°C for 120 hrs. The drug substance was subjected to
60°(±2°)C at different time periods viz., 12 hrs, 1 day, 2, 5, 10 and 15
days respectively.
4.2.8b Formal stability studies
Three different batches of sultamicillin drug substance were
subjected to evaluate the degradation under formal temperature and
humidity conditions i.e. 40°C(±2°) /75%RH(±5%), 25°C(±2°)/60%RH(±5%)
at various time intervals from 1 to 3 months. These samples were also
150
kept at refrigerator condition (5°C (±3°) for 2, 3 and 6 months. All these
samples were packed in a lowdensity polyethylene bag; this bag was
congested with a twirl tie with a plastic tie. This bag was placed in into a
second lowdensity polyethylene bag, and this was warm sealed and
further located in a triple laminate (20µ
thickness)(polyethyleneterephthalate - aluminium foil - linear lowdensity
polyethylne) bag, this was as well heat sealed. Lastly, this pack was
located in a well closed high density polyethylene (HDPE) container. All
samples were withdrawn periodically from particular environmental
conditions. The sample solutions were prepared and injected into HPLC
as per section 4.2.2
4.3 RESULTS AND DISCUSSION
4.3.1 Detection and identification of impurity
After the evaluation of stability data, EP impurity A (sulbactam) is
increased up to 0.4% for 3 months at accelerated conditions, 0.4% for 6
months at real time storage when compared with initial samples.
However, interestingly EP impurity G (sultamicillin dimer) was observed
as decreasing trend when compared with initial samples and stability
sample chromatograms of sultamicillin shows, in thermal stress and
formal stability conditions, an unknown impurity was originated along
with recognized impurities, these known related substances have been
reported in European pharmacopoeia official monograph [24]. This
151
unknown impurity was eluted after the elution of sultamicillin EP
impurity G at a relative retention time around 1.44 with respect to
sultamicillin peak. The European pharmacopoeial impurities were shown
in Fig. 4.2 and the stability assessment of sultamicillin with respect to
related substances test were tabulated in Table 4.1 to Table 4.3.
Impurity Chemical Structure RRT
Ph.Eur. Impurity A [Sulbactam Acid] N
S
OO
CH3
CH3
O
H
CO2HH
0.40
Ph.Eur. Impurity B [p-Toluenesulfonic acid]
SO3H
H3C 0.50
Ph.Eur. Impurity C [Ampicillin]
H
N
S
CH3
CH3
O
H H
N
O
H COOH
NH2H
0.55
Ph.Eur. Impurity D [Penicilloic acids of Sultamicillin]
H
HN
S
O
CH3
CH3
O O
O
NS
O
H
CH3
CH3HH
O
O
O
N
CO2H
NH2H
**
0.94
Ph.Eur. Impurity E H
N
S
O
NH
H H
O
O
CH3
CH3
O O
O
NO
O
H
CH3
CH3HH
N
H3CO
CH3
1.09
Ph.Eur. Impurity F [Ampicillin Sultamicillin amide]
H
N
S
O
NH
H H
O
O
CH3
CH3
O O
O
NS
O
H
CH3
CH3HH
NH
O
O
N
S
O
N
HH
O
HH2N
O
H3C
H3C
H
H
1.27
Ph.Eur. Impurity G [Sultamicillin dimer]
H
N
S
O
NH
H H
O
O
CH3
CH3
O O
O
NS
O
H
CH3
CH3HH
HN
O
O
O
N
NH2HHN
S
O
CH3
CH3
O O
O
NS
O
H
CH3
CH3HH
O
O
O
H*
*
1.42
Fig. 4.2 Pharmacopoeial impurities of sultamicillin
152
Table 4.1 Stability comparison data at accelerated storage
Storage Condition: 40°C ( ± 2o) / 75% RH( ± 5%)
Name of the Related Substance
Sample-1 Sample-2 Sample-3
Initial 1M 2M 3M 6M Initial 1M 2M 3M 6M Initial 1M 2M 3M 6M
EP impurity A 0.12 0.41 0.46 0.45 0.40 0.12 0.36 0.37 0.36 0.35 0.12 0.55 0.50 0.45 0.31
EP impurity B 0.04 0.04 0.03 0.04 0.04 0.13 0.10 0.12 0.13 0.14 0.28 0.31 0.34 0.37 0.40
EP impurity C 0.22 0.36 0.35 0.33 0.30 0.16 0.31 0.31 0.29 0.28 0.20 0.36 0.36 0.32 0.26
EP impurity D 0.11 0.17 0.22 0.26 0.28 0.11 0.17 0.19 0.22 0.27 0.11 0.23 0.29 0.30 0.36
EP impurity F 0.17 0.16 0.13 0.12 0.10 0.23 0.17 0.15 0.12 0.12 0.21 0.13 0.14 0.13 0.14
EP impurity G 0.75 0.53 0.55 0.49 0.22 0.57 0.41 0.44 0.42 0.20 0.50 0.37 0.41 0.37 0.25
153
Table 4.2 Stability comparison data at real time storage
Storage Condition: 25°C (± 2o) / 60% RH (± 5%)
Name of the Related Substance
Sample-1 Sample-2 Sample-3
Initial 1M 2M 3M 6M Initial 1M 2M 3M 6M Initial 1M 2M 3M 6M
EP impurity A 0.12 0.28 0.35 0.33 0.39 0.12 0.27 0.28 0.31 0.34 0.12 0.38 0.40 0.41 0.43
EP impurity B 0.04 0.02 0.03 0.03 0.04 0.13 0.09 0.12 0.12 0.11 0.28 0.29 0.34 0.36 0.31
EP impurity C 0.22 0.33 0.34 0.33 0.34 0.16 0.21 0.30 0.29 0.31 0.20 0.33 0.36 0.42 0.36
EP impurity D 0.11 0.12 0.14 0.14 0.16 0.11 0.10 0.14 0.14 0.16 0.11 0.16 0.17 0.16 0.20
EP impurity F 0.17 0.17 0.13 0.14 0.15 0.23 0.15 0.16 0.17 0.19 0.21 0.11 0.14 0.15 0.16
EP impurity G 0.75 0.59 0.64 0.64 0.42 0.57 0.45 0.48 0.48 0.32 0.50 0.39 0.41 0.39 0.26
154
Table 4.3 Stability comparison data at real time storage (fridge conditions)
Storage Condition: 5°C(±3°)
Name of the Related Substance
Sample-1 Sample-2 Sample-3
Initial 2M 3M 6M Initial 2M 3M 6M Initial 2M 3M 6M
EP impurity A 0.12 0.18 0.17 0.22 0.12 0.13 0.13 0.19 0.12 0.18 0.19 0.26
EP impurity B 0.04 0.03 0.04 0.04 0.13 0.11 0.11 0.12 0.28 0.33 0.36 0.30
EP impurity C 0.22 0.26 0.28 0.30 0.16 0.19 0.18 0.24 0.20 0.25 0.26 0.31
EP impurity D 0.11 0.10 0.11 0.11 0.11 0.11 0.12 0.11 0.11 0.11 0.12 0.13
EP impurity F 0.17 0.13 0.13 0.14 0.23 0.21 0.20 0.21 0.21 0.18 0.16 0.17
EP impurity G 0.75 0.68 0.63 0.55 0.57 0.52 0.45 0.42 0.50 0.46 0.44 0.35
155
The detection level of the unknown degradation impurity
throughout the stability storage studies at different conditions is given in
Table 4.4. The experimental results revealed that the level of impurity
formed in sultamicillin samples stored at 60°C(±2°) as stress storage was
more than that of samples stored at 40°C(±2°)/75%RH(±5%),
25°C(±2°)/60%RH(±5%) and 5°C±3°C as regular stability storage. In
60%RH(±5%) storage, up to ~1% level is observed. However, in
remaining conditions it is detected below 1% level. The unknown
impurity formation of chromatograms under this degradation studies
was shown in the Fig. 4.3
156
Storage & Time Period
Sulbactam Ampicillin Impurity at RRT ~ 1.44
Sum of RS
105°C(±2°)/120 Hrs 0.30 0.65 0.87 5.14
60°C(±2°)
Initial ND 0.19 0.11 1.79
12 Hours 0.20 0.41 0.41 3.27
1 Day 0.46 0.54 1.26 6.27
2 Days 0.40 0.56 1.16 5.87
5 Days 0.36 0.54 1.02 4.96
10 Days 0.34 0.59 1.12 6.29
15 Days 0.34 0.70 1.11 7.46
40°C(±2°) / 75% RH(±5%)
Initial 0.12 – 0.12 0.16 – 0.22 ND – 0.11 1.32 – 1.62
1 Month 0.36 – 0.55 0.31 – 0.36 0.58 – 0.77 2.56 – 3.30
2 Months 0.37 – 0.50 0.31 – 0.36 0.66 – 0.92 3.06 – 3.80
3 Months 0.36 – 0.45 0.28 – 0.32 0.64 – 0.87 2.92 – 3.73
25°C(±2°) / 60% RH(±5%)
1 Month 0.27 – 0.38 0.21 – 0.33 0.27 – 0.37 1.77 – 2.36
2 Months 0.28 – 0.40 0.30 – 0.36 0.36 – 0.52 2.19 – 2.82
3 Months 0.31 – 0.41 0.29 – 0.42 0.38 – 0.48 2.23 – 2.99
5°C±3°C
2 Month 0.13 – 0.18 0.19 – 0.26 0.11 – 0.19 1.57 – 1.89
3 Months 0.13 – 0.19 0.18 – 0.26 0.11 – 0.15 1.39 – 1.76
6 Months 0.19 – 0.26 0.24 – 0.31 0.12 – 0.20 1.64 – 1.93
Table 4.4 Study of formation of impurity at different stability storage conditions
157
Fig. 4.3 HPLC chromatograms of sultamicillin stress stability thermal degradations
105°C/24hrs
60°C/24hrs
40°C/75%RH-3M
158
4.3.2 Isolation of impurity by Prep. HPLC
The electrospray ionization mass spectrum of this unknown
impurity shows m/z value 607 [(M+H)+] in positive ion mode by LC-MS
analysis. To enrich this impurity in Sultamicillin, about 1 to 2 g of drug
substance was taken in a glass Petri dish; small quantity of water was
spilled out on this sample, and was kept at 80°C temperature for about
16 to 18 hours in a vacuum oven. The as such (initial) and enriched
samples were subjected to chromatographic analysis. The requisite
impurity was increased to a level of around 2.50% when measuring up to
with as such sample. The enriched sample was taken in a glass beaker,
and added about 10 ml of combination of water and acetonitrile in the
ratio of 4:6 v/v, and was sonicated to dissolve. This solution was loaded
into the preparative column using the conditions stated in section 2.3.
Fractions of greater than 96% were combined together, concentrated on
rotavapour to eliminate acetonitrile solvent. These fractions were loaded
into a preparative column and eluent was treated with water for
exclusion of ammonium acetate used for isolation. Finally, column was
washed with water and acetonitrile mixture in the ratio of 2:8 vol/vol,
one more time eluent was concerted using rotavapour to eradicate
acetonitrile. The aqueous solution was lyophilized using freeze dryer
(Virtis advantage 2XL).The impurity was obtained as almost white
powder, with purity greater than 95.0%.
159
4.3.3 Structural elucidation of impurity
Further this impurity was co-injected with Sultamicillin into HPLC
to verify/confirm the retention and relative retention times. The HPLC
chromatograms of sultamicillin spiked with pharmacopoeial known
impurities along with new impurity and Sultamicillin spiked with new
impurity were shown in the Fig. 4.4.
Fig: 4.4 HPLC chromatograms of sultamicillin spiked with european pharmacopoeial impurities including new impurity (a) and sultamicillin spiked with new impurity (b)
(a)
(b)
160
The ESI mass spectrum of this impurity displayed a protonated
molecular ion at m/z 607 amu [(M+H)+] in +ve ion mode [mass spectrum
shown in the Fig. 4.5] which is 12 units more than that of sultamicillin
protonated molecular ion.
This impurity was further subjected to TOF, analyzed by MS/TOF
as per analysis conditions mentioned in section 4.2.5 and shows a
molecular ion peak at m/z 629.1368 amu [(M+Na)+] as sodium adduct
against the theoretical mass of m/z 629.1352 amu [(M+Na)+]. The mass
error between calculated and observed masses is 2.5 ppm. The MS/TOF
spectrum of impurity was shown in the Fig. 4.6.
The 1H NMR and 13C NMR spectra of this impurity show, chemical
shift (\) values are comparable with Sultamicillin except at phenyl glycine
and lactam moieties of ampicillin linkage. Moreover, the close inspection
of sultamicillin structure and proposed structure of unknown
degradation impurity with their NMR interpretation data. The chemical
atom positions of sultamicillin and proposed structure of unknown
impurity were represented in Fig. 4.7 as its structures, the 1H NMR and
DEPT NMR spectra of both the molecules have been showed in Fig. 4.8 to
4.11. The comparative NMR data of these two structures was presented
in Table 4.5.
161
Fig. 4.5 Mass spectrum of impurity
162
Fig. 4.6 MS/TOF spectrum of impurity
163
21'
22'
23
22
21
20
19
1817
16
15
1413
12
11
10'
109
87
6 5
43
2
1'
1N
S
CH3
CH3
O
H
H
O
N
NH2
H
H
O
O
N
S
OO
H3C
H3C
O
H
O
O
H
H
(a)
21'
22'
23
22
21
20
2419
1817
15
14
13
12
11
10'
109 8 7
65
43
2
1'
1N
S
CH3
CH3
O
H
H
O
N
H
O
O
N
S
OO
H3C
H3C
O
H
OO
HN
(b)
Fig. 4.7 Chemical structure of sultamicillin (a) and chemical structure of new impurity (b)
164
Fig. 4.8 1H NMR Spectrum of Sultamicillin
165
Fig. 4.9 1H NMR Spectrum of Impurity
166
Fig. 4.10 DEPT NMR Spectrum of Sultamicillin
167
Fig. 4.11 DEPT NMR Spectrum of Impurity
168
s, singlet; d, doublet; dd, doublet of doublet; m, multiplet; brs, broad singlet; q,quartet; t, triplet, ABq, AB quartet, ♠, assumed from HMBC. a, refer structures for numbering. J, spin coupling constant; *, assumed from 1H-1H-COSY and #, assumed from HSQC.
Table 4.5 1H, 13C NMR, DEPT, HSQC & HMBC assignments for sultamicillin and impurity
Number Positiona
Sultamicillin Impurity
1H, δ (ppm), multiplicity 13C, δ (ppm) Multiplicity
[DEPT, HMQC & HMBC]
1H, δ (ppm), multiplicity 13C, δ (ppm) Multiplicity
[DEPT, HMQC & HMBC]
1 & 1’ δ1.36 & δ1.42 (2s-6H) δ18.4 , δ 20.3 2 X CH3# δ1.36 & δ1.45 (2s-6H) δ17.5, δ19.4 2 X CH3#
2 - δ63.0 -C♠ - δ70.1 -C♠
3 δ5.20 (dd-1H, J=5.21, 1.65
Hz) δ61.3 -CH*# δ5.20 (dd-1H, J=5.21, 1.65 Hz) δ60.43 -CH*#
4 δ3.26 & 3.70 (ABq, 2H) δ38.2 -CH2*# δ3.26 & δ3.70 (ABq-2H) δ38.2 -CH2*#
5 - δ167.2 -C♠ - δ167.1 -C♠
6 δ4.57 (s-1H) δ70.5 -CH# δ4.64 (s-1H) δ69.50 -CH#
7 - δ174.8 -C♠ - δ174.7 -C♠
8 δ5.92 & 5.95 (ABq- 2H) δ81.9 -CH2# δ5.92 & δ5.95 (ABq-2H) δ81.9 -CH2#
9 - δ173.9 -C♠ - δ172.9 -C♠
10 & 10’ δ1.47 & 1.61 (2s-6H) δ27.2, δ30.8 2 X CH3# δ1.47 & δ1.65 (2s-6H) δ26.4, 29.6 2 X CH3#
11 - δ64.9 -C♠ - δ70.4 -C♠
12 δ4.50 (s-1H) δ58.8 -CH# δ4.56(s-1H) δ61.92 -CH#
13 δ5.50 (d-1H) δ68.2 -CH# δ5.61 (m-1H) δ60.17 -CH#
14 δ5.59 (d-1H) δ58.8 -CH# δ5.61 (m-1H) δ66.25 -CH#
15 - δ166.7 -C♠ - δ166.7 -C♠
16 δ8.75 (brs-1H) - - - - -
17 - δ172.9 -C♠ - δ172.2 -C♠
18 δ4.51 (s-1H) δ62.8 -CH# δ4.54 (s-1H) δ60.90 -CH#
19 - - - δ4.00 (m-H) - -
20 - δ140.0 -C♠ - δ138.0 -C♠
21 & 21’ δ7.39 (d-2H) δ128.9 2 X CH# δ7.38 (d-2H) δ129.1 2 X CH#
22 & 22’ δ7.25 (dd-2H) δ127.6 2 X CH# δ7.26 (dd-2H) δ128.0 2 X CH#
23 δ7.28 (t-1H) δ128.0 1 X CH# δ7.28 (t-1H) δ128.7 1 X CH#
24 - - - δ4.52 & 4.79 (2m-2H) δ82.1 CH2*#
169
A significant 13C chemical shift has been observed at 13, 14 and 18
positions. At 14th signal position, down field shift from \ 58.8 to \ 66.25,
at signal position 13, an up field shift from \ 68.2 to \ 60.17 and at
signal position 18, an up field shift from \ 62.8 to \ 60.90 have been
observed, representing that the possibility of cyclization in the phenyl
glycine moiety. At position 24, additional signals of \ 4.52 and \ 4.79 in
1H NMR and \ 82.1 in DEPT and 13C NMR represents the methylene
signal of imidazolone group of this impurity. In comparison with
Sultamicillin, amide -NH signal is establish to be absent at position 16th
and an extra chemical shift value \4.00 corresponds to -NH signal of
imidazolone moiety at 19th position, which is an exchangeable proton
with deuterium oxide (D2O). This observable fact has further been
definite from the connection observed between cross peaks of key
protons at 19 and 24 positions in 1H-1H COSY NMR spectrum. All the
signal assignments / correlations have further been confirmed by using
2D-NMR testing like 1H-1H COSY, NOESY, HSQC and HMBC and spectra
of impurity was shown in Fig. 4.12 to 4.15 respectively.
170
During the NMR interpretation of HMBC spectrum of impurity, long
range correlation between \ 4.52 & \ 4.79 at position 24 and \ 172.2 at
position 17 together with their chemical shifts reveals that 14, 17 and 24
position carbons are connected to nitrogen atom. Further all quaternary
carbons were interpreted based on the long range correlations observed
in the HMBC spectral cross peaks.
171
Fig. 4.12 2D-NMR 1H-1H COSY Spectrum of impurity
172
Fig. 4.13 2D-NMR 1H-1H COSY Spectrum of impurity
173
Fig. 4.14 2D-NMR HSQC Spectrum of impurity
174
Fig. 4.15 2D-NMR HMBC Spectrum of impurity
175
The structure of this new impurity was proposed for the ion at m/z
607 is supported by the presence of main fragments at m/z 577, 405 and
361 obtained from the mass spectra and major fragmentation was shown
in Fig. 4.16.
N
S
CH3
CH3
O
H
H
O
N
H
O
O
N
S
OO
H3C
H3C
O
H
OO
HN
405
361
577
Fig. 4.16 Probable mass fragmentation of impurity
In addition to that, this impurity was further established by FT-IR
spectral data. As of the FTIR impurity spectrum, the absence of a
characteristic strong absorption band at ~1641 cm-1 in contrast with
sultamicillin to amide -C=O stretch supports the existence of
imidazolone group moiety. A comparative FT-IR data of the unknown
impurity with sultamicillin was summarized in Table 4.6 and IR spectra
of sultamicillin and impurity was publicized in Fig. 4.17 and 4.18.
176
Fig. 4.17 FT-IR spectrum of Sultamicillin
177
Fig. 4.18 FT-IR spectrum of Impurity
178
IR absorption bands (cm-1)/KBr
Sultamicillin Impurity
3396 ,3299 (m) -NH stretch
2977, 2926 (w) -Aliphatic stretch
1790 (s) -C=O (lactam) stretch
1682 (s) -C=O (ester) stretch
1641 (s) -C=O (amide) stretch
1602, 1514 (m) -C=C (aryl) stretch
1319 (s) -SO2 asymmetric stretch
1179 (s) -SO2 symmetric stretch
1014 (m) -C-O-C symmetric stretch
709 (m) -Aryl -CH out of plane bend
3428 (s) -NH stretch
2973, 2931(w) -Aliphatic stretch
1788 (s) -C=O (lactam) stretch
1705 (s) -C=O (ester) stretch
-- --
1633, 1495 (m) -C=C (aryl) stretch
1321 (s) -SO2 asymmetric stretch
1176 (s) -SO2 symmetric stretch
1019 (s) -C-O-C symmetric stretch
709 (m) -Aryl -CH out of plane bend
Table 4.6 FT-IR spectral data for sultamicillin and impurity
Based on the elemental analysis, theoretical values C: 51.47; H:
4.98; N: 9.24; S: 10.57 and experiential values C: 51.38; H: 4.91; N: 9.18
S: 10.50 proposed that the elemental composition of this impurity is
C26H30N4O9S2 with m/z 606 and LC-MS/TOF analysis is also justifying
the obtained formula and mass. Based on the obtained spectral data,
name of this impurity has been proposed as Methylene(2S,5R,6R)-6-(5-
oxo-4-phenylimidazolidine-1-yl)-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylate-(2S,5R)-3,3-dimethyl-4,4,7-
trioxo-4λ6-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate.
179
Formation of impurity
Based on the data obtained from stability studies, the formation of
impurity is depends on temperature. To justify this, carried out the
experiments at a temperature 60°C (±2°) up to 15 days in the similar
packaging conditions and the samples were subjected to HPLC analysis.
The results exposed that the level of impurity formed in samples stored
at 60°C(±2°) was more than that of samples stored at
40°C(±2°)/75%RH(±5%), 25°C(±2°)/60%RH(±5%) and 5°C(±3°), which are
already discussed/tabulated in Table 4.1 to 4.3.
Sultamicillin in thermal stress conditions degrades to ampicillin,
sulbactam compounds and formaldehyde is released as a byproduct [2].
Formaldehyde further reacts with amino group in sultamicillin leading to
formation of this impurity. The chemical pathway of the formation of
impurity shown in Fig. 4.19. The probable mechanism for the formation
of this impurity is given in Fig. 4.20.
180
H
N
S
CH3
CH3
O
H
H
O
N
NH2
H
H
O
O
N
S
OO
H3C
H3C
O
H
OO
H
SULTAMICILLIN
Heating
H
N
S
CH3
CH3
O
H
COOHH
O
N
NH2
H
H
+N
S
OO
CH3
CH3
O
H
COOHH
+ HCHO
STEP-I
H
N
S
CH3
CH3
O
H
H
O
N
NH2
H
H
O
O
N
S
OO
H3C
H3C
O
H
OO
H
HCHO+
SULTAMICILLIN FORMALDEHYDE
Heating
N
S
CH3
CH3
O
H
H
O
N
H
O
O
N
S
OO
H3C
H3C
O
H
OO
HN
IMPURITY
STEP-II
Fig. 4.19 The chemical pathway of formation of impurity
181
H H
O
H2ON
S
CH3
CH3
O
H
H
O
N
H
O
O
N
S
OO
H3C
H3C
O
H
OO
HN
SULTAMICILLIN
IMPURITY
H
N
S
CH3
CH3
O
H
H
O
N
NH2H
H
O
O
N
S
OO
H3C
H3C
O
H
OO
H
H
N
S
CH3
CH3
O
H
H
O
N
HNH
H
O
O
N
S
OO
H3C
H3C
O
H
OO
H
OH
H
H
H
N
S
CH3
CH3
O
H
H
O
N
HNH
H
O
O
N
S
OO
H3C
H3C
O
H
OO
H
OH
H
H
Fig. 4.20 The mechanism for the formation of impurity
The formation of impurity was further verified by carrying out the
experiments during this research work. Sultamicillin was mixed with
Para formaldehyde (~10%w/w) and packed in the similar packaging
conditions as mentioned in stability studies section and stored at
60°C(±2°) up to one day (24 hours), the exposed samples were subjected
to HPLC analysis. The results showed that, this impurity was found at
about 10% level in 12 hours sample, and further it increased with time.
The experimental results are reported in Table 4.7.
182
Time Period 60°C(±2°)
Sulbactam Ampicillin Impurity at RRT ~ 1.44
Sum of RS
Initial - 0.06 0.12 ND 1.01
Sultamicillin as such
6 Hours 0.06 0.14 0.3 1.2
12 Hours 0.08 0.18 0.9 1.7
1 Day 0.34 0.67 0.8 4.5
Sultamicillin mixed with
formaldehyde (10%w/w)
1 Hour 0.11 0.36 1.1 2.7
12 Hours 0.11 0.25 11.6 13.7
1 Day 0.44 0.47 21.2 29.7
Table 4.7 Evaluation of formation of impurity – experiment results
183
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