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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