Application of LC-NMR and LC-TOF-MS in the identification ...shodhganga.inflibnet.ac.in/bitstream/10603/8540/12/12_chapter 4.pdf · Rabeprazole Sodium tablets 4.1 Introduction Rabeprazole,

Chapter 4 95

Application of LC-NMR and LC-TOF-MS in the identification and

characterization of degradation products of

Rabeprazole Sodium tablets

4.1 Introduction

Rabeprazole, (±) Sodium-2-[4-(3-methoxypropoxy)-3-

methylpyridin-2-yl] methyl sulfinyl]-1H-benzimidazole is a proton

pump inhibitor which covalently binds and inactivates the gastric

parietal cell proton pump (H+/K+ ATPase). It is an important

alternative to H2 antagonists and an additional treatment option to

other proton pump inhibitors in the management of acid-related

disorders [1]. It has also demonstrated efficacy in healing and giving

symptomatic relief for gastric and duodenal ulcers, as well as a high-

eradication rate of the microorganism Helicobacter pylori when

associated with antimicrobial therapy [2]. The molecular structure of

Rabeprazole Sodium is shown in Scheme 4.1.

In general, solid APIs are formulated with excipients as tablets

or capsules. Since the active ingredient is interacting with the

excipients and the formulated product is stored at different

conditions, the study of stability of APIs is critical in the drug

development process. Many factors can affect the stability of a

pharmaceutical product, some of them includes the stability of the

active ingredient, the manufacturing process, the environmental

conditions (such as heat, light and moisture during storage), as well

as some chemical reactions like oxidation, reduction, hydrolysis and

Chapter 4 96

racemization that might occur [3&4]. Study of stability under stressed

conditions is very important, since it can cause many degradation

reactions.

The identification of process related Rabeprazole impurities in

the bulk substance by LC-MS and spectral data (IR and NMR) were

reported[5]. Rabeprazole Sodium photodegradation products were

recently published [6]. The instability of Rabeprazole under acidic

conditions is known; hence it is manufactured as enteric coated

tablets [7]. However, the stability of Rabeprazole Sodium tablets under

stressed conditions is not reported. Hence, the present manuscript

deals with the identification and characterization of three degradation

products, obtained by storage of the tablets at stressed conditions [40

°C/75% RH] for six months. Of the three degradation products, the

most polar degradation product was isolated by preparative HPLC.

But other remaining two degradation product could not be isolated

due to low resolution, hence HPLC hyphenated techniques (LC-NMR

and LC-MS) were utilized for the structure identification.

HPLC hyphenated techniques are now widely used for the

structure elucidation of trace amounts of the degradation products

without complicated isolation process. LC-MS has been one of the

powerful techniques for the identification of small quantities of drug

degradation products [8]. Recently LC-NMR has been increasingly

utilized to obtain detailed structural information of degradation

products [9&10].

Chapter 4 97

4.2 Experimental

4.2.1 Materials and reagents

Samples of pure Rabeprazole Sodium and degraded Rabeprazole

tablets were procured for DRL, Hyderabad, India.

4.2.2 Analytical HPLC

An In-house LC gradient method was developed for the

separation of all possible related substances of Rabeprazole Sodium.

This LC method was able to detect all the degradation products with

good resolution. The HPLC conditions are shown in Table 4.1.

Table 4.1 Analytical HPLC conditions

ColumnSymmetry Shield C18, 250mm × 4.6 mm, 5µm

particle size column (Waters, Ireland)

Buffer

3.4 gm of KH2PO4 in 1000 mL and 1.0mL of

trimethylamine, pH 6.7 with dilute phosphoric

acid

Solvent – A 90 : 10 (v/v) buffer : Acetonitrile

Solvent – B 10 : 90 (v/v) buffer : Acetonitrile

Gradient Program(T / %B)

0/5, 50/65, 60/65, 62/5, 70/5

Flow rate 1.0 mL/min

Detection 280 nm

Injection volume 10 μL

Diluent 1 : 1 (Water : CH3CN)

Chapter 4 98

4.2.3 Preparative HPLC conditions

An in-house gradient preparative method was developed for the

separation of the degradation products. The Preparative HPLC

conditions are shown in Table 4.2

Table: 4.2 Preparative HPLC conditions

Column 250mm × 20mm, 5 μm, RP C18 (ZodiacCompany)

Solvent – A Buffer (1.0 gm of Ammonium Acetate in 1000mLof water)

Solvent – B Acetonitrile


0/5, 10/5, 11/95, 30/95, 31/5, 40/5


Detection 280 nm

Injection volume 5.0 mL

Diluent 80 : 20 (Methanol : Water)

4.2.5 UPLC -TOF Mass Spectrometry

An in-house gradient UPLC method was developed. The UPLC

conditions are shown in Table 4.3.

4.2.6. NMR spectroscopy

The NMR experiments were performed on Varian spectrometer

operating at 500 MHz, Unity INOVA, in D2O at 25º C. The proton

chemical shifts were reported on scale in ppm, relative to HOD

(=4.76ppm) as internal standard.

Chapter 4 99

Table 4.3 UPLC conditions

Column C18 50x2.1mm 1.7µm particle size (Watercorporation, Manchester, UK)

Solvent – A Buffer (0.01 M Ammonium Acetate)

Solvent – B Acetonitrile


0/5, 0.23/5, 1.64/50, 2.42/85, 3.19/85,3.51/5, 4/5


Detection 280 nm

Injection volume 5.0 µL

Diluent 1 : 1 (Acetonitrile : Water)

4.2.7. LC-NMR spectroscopy

LC–NMR was performed on a Varian LC–NMR instrument

(Varian Associates, Inc., Palo Alto, CA) using a Pro Star pump system,

a Pro Star UV detector, an Unity INOVA 500 MHz NMR spectrometer

and a micro flow LC–NMR probe. The probe has 1H{13C} channels (1H

observed with 13C decoupling) with pulsed-field gradient along z axis.

The active sample volume of the probe was approximately 60 L and

the transfer time from the UV cell to the active volume was calibrated

to be 21 s at a flow rate of 1.0 mL/min. Proton NMR experiments

were performed in ‘stop-flow’ mode, where the HPLC flow was halted

after the sample elution fraction was transferred to the NMR probe

which was equilibrated at 25°C. Pulse sequence ‘lc1d’ was used.

Double solvent suppression was applied on the proton resonances of

Water and Acetonitrile. One-dimensional (1D) proton NMR spectra

Chapter 4 100

were recorded into 32K data points with a spectral width of 9500 Hz

and 1.7 sec of acquisition time. A total of 2000 transients were

collected in approximately 1.5 hr for each 1D proton spectrum. The

LC conditions are shown in Table 4.4.

Table 4.4 LC conditions for LC-NMR

Column Zorbax SB CN 250 mm×4.6 mm, 5.0mparticle size (Agilent Technologies, UK)

Solvent – A D2O (99.9%, Cambridge Isotope Laboratory,MA)

Solvent – B Chromasolv® Acetonitrile (Riedel-de Haen)


0/5, 1.0/5, 10/85, 15/85, 12/95, 20/95,25/5, 35/5

Flow rate 1.0 ml / min

Detection 280 nm

Injection volume 5.0 µL

Diluent 1 : 1 (Acetonitrile : Water)

Chapter 4 101

4.3 Results and discussions

4.3.1 LC-UV analysis of degradation products.

During the stability studies of Rabeprazole tablets, the tablets

were kept at 40 C/75% RH for six months. The HPLC analysis of the

tablets has shown ca. 0.03 to 0.3% of unknown degraded products

DP-I, DP-II and DP-III at RRT's ca. 0.17, 0.22 and 0.28, respectively.

The chromatograms of Rabeprazole tablets, before and after

degradation, are shown in Figure 4.1. The known impurities of

Rabeprazole are shown with their names in the chromatogram.

4.3.2 Enhancement of degradation products

Isolation of 0.3% degradation product is a tedious job. In order

to enhance the degradation products, approximately 20 gm of

Rabeprazole tablet blend was kept in an aultoclave at 105 °C for five

days. The purity of the blend before and after autoclave was

determined using HPLC. It was observed that these degraded

products increased to ca. 1.0%. The enriched sample of Rabeprazole

blend was subjected to preparative HPLC for the isolation of the

degradation products.

Chapter 4 102

Figure 4.1 Typical HPLC chromatograms : (a) Rabeprazole Sodiumtablets (initial) (b) Stressed Rabeprazole Sodium tablets (6months at 40 °C/75% RH)

4.3.3 Isolation of degradation products

Two grams of sample with enhanced levels of degradation

products was subjected to preparative HPLC under the conditions

describe in Section 4.2.3. The fractions, collected by preparative

HPLC, were analyzed by analytical HPLC conditions described in

Section 4.2.2. The DP-I fraction was isolated with ~ 95% purity,

where as the DP-II and DP-III were collected together because of low

resolution in the preparative method. These fractions were

Chapter 4 103

concentrated on rotaevaporator, to remove the solvents viz.,

Acetonitrile and water. The Ammonium Acetate present in the

concentrated fractions was removed by subjecting to preparative

HPLC by using only water and Acetonitrile (50:50) as mobile phase.

The isolated fractions were again concentrated using rotaevaporator.

The DP-I was obtained as a pale yellow solid with chromatographic

purity of ~ 98%. The DP-II and DP-III were obtained as white powder

with chromatographic purity of ~95% together. The DP-I was

characterized by using NMR and LC-TOF-MS. The combined material

of DP-II and DP-III was subjected for LC-NMR and LC-TOF-MS data.

4.3.4 Structural chemistry of degradation products

Generally it is easy to identify the impurities / degradation

products of any API, if the spectral data of the degradation products

were compare with those of API. So, the spectral data of the DP-I, DP-

II and DP-III was compared with those of Rabeprazole Sodium .

The numbering scheme followed for Rabeprazole in the

discussion is given in Scheme 4.1.

12

34

56

7

89 10

11 12

N

HN

S N

H3C O

O CH3

O

Chemical Formula: C18H21N3O3SExact Mass: 359.1304

Scheme 4.1 Structure of Rabeprazole with numbering

Chapter 4 104

The reported NMR assignments of Rabeprazole Sodium [5] were

used for the comparison purpose. The NMR assignments are given in

Table 4.5. The HR-MS data of Rabeprazole and the degradation

products are given in Table 4.6. These spectral data will be referred in

the discussion of structure elucidation of these degradation products

in Sec 4.3.5, 4.3.6 and 4.3.7.

Table 4.51H NMR assignments of Rabeprazoel Base, DP–I, DP-II and DP–III.No.1 1H Rabeprazole Na2 DP-I DP-II DP-III

1,4 2H7.30 (m)[117.39]

7.30(m)(126.47)

7.3 (m) -

2,3 2H7.65 (m)[118.40)

7.55(m)(117.50)

7.6 (m) -

5 Ha4.80 (d, 13.5)

[61.13)-

- -

Hb 4.70 (d, 13.5)(61.13)

- - -

6 1H8.21 (d, 6.0)

(148.17)7.90(d, 6.0)

(142.58)- 8.20 (d, 6.0)

7 1H6.95 (d, 6.0)

106.176.50(d, 6.0)

(117.47)- 7.1 (d, 6.0)

8 3H2.14 (s)(10.76)

1.95 (s)(14.11)

- 2.10 (s)

9 2H4.10 (t, 6.0)

(65.09)-

- 4.20 (t, 6.0)

10 2H1.97 (m)(28.74)

-- 1.95 (m)

11 2H3.48 (t, 6.0)

(68.39)-

- 3.60 (t, 6.0)

12 3H 3.21 (s) - - 3.20 (s)1 Refer the structural formula given above for numbering.2 This column gives the 1H chemical shift, multiplicity and coupling constant.

The 13C NMR chemical shifts are given in the parenthesiss-siglet, d-doublet, dd-doublet of doublet, ddd-doublet of doublet of a doublet,t-triplet and br-broad.

Chapter 4 105

Table 4.6 ES-MS & TOF-MS data

ES-MS+ve / -ve

TOF +ve / -ve Elementalcomposition

Rabeprazole - - C18H21N3O3S

DP-I 270.1/ 268.1 270.1093 / 268.0030 C14H12N3O3/C14H10N3O3

DP-II 199.0 / 197.1 199.0197 / 197.0019 C7H7N2O3S/C7H5N2O3S

DP-III 226.1 / 224.3 226.1092 / 224.0916 C11H16NO4/C11H14NO4

4.3.5 Identification of DP – I

In the positive and negative high resolution mass spectra, the

protonated and deprotonated, [M+H]+ and [M-H]-, molecular ions were

detected at m/z 270.1093 and 268.0939 respectively (Figures 4.2 and

Figure 4.3). The even m/z number of [M+H] + and [M+H] - ions suggest

that DP-I contains odd number of nitrogen atoms (nitrogen rule).

From these results the possible molecular formulae for positive and

negative data were found to be C14H12N3O3 and C14H10N3O3

respectively. The molecular formula of DP-I was found to be

C14H11N3O3. On comparing the molecular formulae of DP-I

(C14H11N3O3) with Rabeprazole (C18H21N3O3S), it is clear that DP-I is

less than Rabeprazole by C4H10S1. It is interesting to note the absence

of Sulphar atom in the DP-I. Further the missing component of C4H10

is indicative of the absence of the long aliphatic chain connected to

the pyridine ring of Rabeprazole.

Chapter 4 106

Figure 4.2 HR-MS spectrum of DP-I (+ve)

Figure 4.3 HR-MS spectra of -ve DP-I

The proton NMR spectra of Rabeprazole and DP-I were

compared. The proton NMR spectrum of DP-I (Figure 4.4) showed

signals corresponding to benzimidazole and methyl substituted

pyridine moieties. The signals of 3-methoxypropoxy group were not

observed in DP-I. The COSY spectrum (Figure 4.5) of DP-I showed two

set of correlation, (i) 7.90 (d, 6.0Hz) & 6.50 (d, 6.0Hz) and (ii) 7.30 (2H,

m) & 7.55 ppm (2H, m). The signals of the spin systems, viz., (i) and

(ii) correspond to pyridine and benzimidazole moieties respectively.

Chapter 4 107

The HSQC (Figure 4.6) correlations further confirmed the presence of

these moieties. The quaternary carbon chemical shifts were extracted

from HMBC spectrum (Figure 4.7). There is no significant difference

in the chemical shifts of benzimidazole ring, while the signals of

pyridine ring in DP-I is significantly shielded with respect to

Rabeprazole indicating possibilities of some substitution changes in

the pyridine moiety.

Till now all the aromatic 1H signals are explained. The absence

of 1H signals (in the aliphatic region) due to the 3-methoxy propoxy

group is in agreement with the hypothesis mentioned above. The only

signal in the aliphatic region is a methyl signal at 1.95 ppm which is

significantly shielded with respect to the methyl signal at 2.14 ppm

connected to pyridine moiety of Rabeprazole.

The HSQC and HMBC correlations indicate that there is no

change to the benzimidazole ring. The quaternary carbons, inferred

from HMBC data, at 183.96 and 170.1 ppm can be attributed to two

carbonyl groups. The expanded region of HMBC experiment is shown

in Figure 4.8. The quaternary carbon at 184.0 ppm showed long-

range proton correlations to a methine at 7.90 ppm and a methyl

group at 1.95 ppm (s). This places this quarternary carbon atom as a

ketone group para position to the nitrogen in the pyridine moiety. On

the other hand, the quaternary carbon signal at 170.1 ppm did not

show any long range HMBC correlations (Figure 4.8). The absence of

correlations to any proton showed that the acid group is farther to

Chapter 4 108

protons. This quarternary carbon atom is indicative of a carbonyl

carbon of carboxylic acid group as inferred from the molecular

formula determined by HR-MS studies. As the benzimidazole group is

intact, the carbon ortho to the nitrogen atom of pyridine ring is the

only possible place where the –COOH group can be connected. In the

HMBC experiment, the aromatic methine at 7.90 ppm showed

additional correlations to the quartenary carbon at 150.5 ppm. The

signal at 150.5ppm also shows correlation to the methyl signal at 1.95

ppm. These correlations indicate that 150.5 ppm signal can be

attributed to the quarternary carbon ortho to the nitrogen in the

pyridine moiety. Only one quartenary carbon signal at 148.3 ppm

remains unexplained. This signal could be assigned to the

quarternary carbon atom between the nitrogen atoms of benzimidazole

group. The correlation shown by this signal to the aromatic methine

at 7.90 ppm connects the pyridine ring to the benzimidazole ring

through its ring nitrogen atom.

Thus, from the NMR and the LC-TOF-MS data the structure of

the DP-I was identified as 1-[1H-benzo[d]imidazol-2-yl]-3-methyl-4-

oxo-1, 4-dihydro-2-pyridinecarboxylic acid.

Chapter 4 109

Scheme 4.2 Structures of Rabeprazole and DP-I

Figure 4.4 1H NMR spectrum of DP-I in D2O+CD3CN

Chapter 4 110

Figure 4.5 COSY NMR spectrum of DP-I

Figure 4.6. HSQC NMR spectrum of DP-I

Chapter 4 111

Figure 4.7. HMBC spectrum of DP-I

Figure 4.8. HMBC spectrum of DP-I expansion

4.3.6 Identification of DP – II


protonated and deprotonated, [M+H] + and [M-H]-, molecular ions were

Chapter 4 112


4.10). The odd m/z number of [M+H] + and [M+H] - ions suggest that

DP-II contains even or no nitrogen atoms (nitrogen rule). From these

results the possible molecular formulae for positive and negative data

were found to be C7H7N2O3S and C7H5N2O3S respectively. The

molecular formula of DP-II was found to be C7H6N2O3S. On

comparing the molecular formulae of DP-II with Rabeprazole

(C18H21N3O3S), there is a difference of C11H15N.

Figure 4.9 DP-II +ve HR-MS data and Elemental composition results

Chapter 4 112










Chapter 4 112










Chapter 4 113

Figure 4.10 DP-II -ve HR-MS data and Elemental composition

results

The proton NMR data was collected by LC-NMR. The proton

NMR spectra of Rabeprazole and DP-II were shown in Figure 4.11.

The DP-II NMR spectrum showed only two signals in the aromatic

region, at 7.30 (m) and 7.60 ppm (m). The splitting pattern and the

chemical shift showed that these two signals correspond to

benzimidazole moiety, four protons. The molecular formula showed

Chapter 4 113


results







Chapter 4 113


results







Chapter 4 114

that there are six protons in DP-II. The remaining two protons were

not observed. These can be attributed to exchangeable proton. One

exchangeable proton can be attributed to again benzimidazole moiety.

Then the molecular formula assigned to bezimidazole becomes

C7H5N2. The remaining molecular formula found to be SO3H. This

can be attributed to sulfonic acid moiety on benzimidazole moiety.

The structure of DP-II was characterized as 1H-benzo[d]

imidazole-2-sulfonic acid from LC-NMR and LC-TOF-MS data. The DP-

II was further confirmed by the comparison of authentic sample

purchased from Sigma Aldrich.

Scheme 4.3 Structures of Rabeprazole and DP-II

Chapter 4 115

Figure 4.11 1H NMR spectra of Rabeprazole Base and DP-II (LC-

NMR)

4.3.7 Structure Elucidation of DP – III



detected at m/z 226.1105 and 224.0916 respectively (Figures 4.12

and 4.13). The even mass number of [M+H] + and [M+H] - ions suggest

that DP-III contains odd number of nitrogen atoms (nitrogen rule).


negative data were found to be C11H16NO4 and C11H14NO4 respectively

(Figures 4.12 and 4.13). The molecular formula of DP-III was found to

Chapter 4 115


NMR)










Chapter 4 115


NMR)










Chapter 4 116

be C11H15NO4. On comparing the molecular formulae of DP-II with

Rabeprazole (C18H21N3O3S), there is a difference of C7H6 and there is

an addition of one oxygen atom.

The overlaid proton NMR data of Rabeprazole and DP-III are

shown in Figure 4.14. The DP-III proton NMR spectrum showed

signals at 2.00 (m, 2H), 2.10 (s, 3H), 3.20 (s, 3H), 3.60 (t, 2H), 4.20 (t,

2H), 7.10 (d, 1H) and 8.20ppm (d, 1H). On comparison it was

observed that these signals correspond to 4-(3-methoxypropoxy)-3-

methyl-2-pyridine moiety. The signals corresponding to benzimidazole

and the methylene signals were absent. The molecular formula of 4-

(3-methoxypropoxy)-3-methyl-2-pyridine moiety corresponds to

C10H14NO2. The remaining molecular formula corresponds to C1H1O2.

This can be easily attributed to the substitution of free acid group on

the pyridine at ortho-position.

Thus, from the 1D LC-NMR and the LC-TOF-MS data the

structure of DP-III was identified as 4-(3-methoxypropoxy)-3-

methylpicolinic acid.

Chapter 4 117

Figure 4.12 HR-MS spectra of +ve DP-III

Chapter 4 117


Chapter 4 117


Chapter 4 118

Figure 4.13 HR-MS spectra of -ve DP-III

Chapter 4 118


Chapter 4 118


Chapter 4 119

N

HN

S N

H3C O

O CH3

O1

2

34

56

7

8

9 1011 12

N

H3C O

O CH3

6

7

8

9 1011 12

O

HO

Chemical Formula: C11H15NO4Exact Mass: 225.1001


Scheme 4.4 Structures of Rabeprazole and DP-III

Figure 4.14 1H NMR spectra of Rabeprazole and DP-III (LC-NMR)

4.4 Formation of the degradation products

The formation of DP-I could be due to the rearrangement of

Rabeprazole structure. Detailed studies of similar rearranged

impurities were reported by Arne Brändström, et.al for Omeprazole

Chapter 4 119

N

HN

S N

H3C O

O CH3

O1

2

34

56

7

8

9 1011 12

N

H3C O

O CH3

6

7

8

9 1011 12

O

HO









Chapter 4 119

N

HN

S N

H3C O

O CH3

O1

2

34

56

7

8

9 1011 12

N

H3C O

O CH3

6

7

8

9 1011 12

O

HO









Chapter 4 120

[11-15]. The DP-II and DP-III could be formed due to the cleavage of the

bond adjacent to sulphur followed by oxidation.

4.5 Conclusions

To conclude, three unknown degradation products of

Rabeprazole Sodium tablets were observed during the accelerated

stressed conditions. The most polar DP-I was isolated by preparative

HPLC and characterized by NMR and MS. The low resolution

degradation product, DP-II and DP-III were characterized by the HPLC

hyphenated techniques, LC-NMR and LC-MS-TOF. The LC-MS

spectra provided the molecular formulae of DP-II and DP-III and the

molecular structures were elucidated by LC-NMR analysis.

Complementary use of these two hyphenated techniques facilitated in

the unambiguous structure identification of the degradation products.

Chapter 4 121

4.6 REFERENCES

1 C. I. Carswel, K. L. Goa, Drugs, 61, 2001, 2327-56.

2 J. Bart, W. Hahne, Aliment. Pharmacol. Ther. 16, 2002, 31-33.

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Buenos Aires, 1975.

4 B. Kommanaboyina, C. T. Rhodes, Drug Dev. Ind. Pharm., 25,

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5 G. M. Reddy, B. V. Bhaskar, P. P. Reddy, P. Sudhakar, J. Moses

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E. S. Schapoval, J. Pharm. Biomed. Anal. 46, 2008, 88-93.

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Alminger, Kjell Ankner, Ulf Junggren, Bo Lamm, Peter

Chapter 4 122

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Documents

Application of LC-NMR and LC-TOF-MS in the identification ...shodhganga.inflibnet.ac.in/bitstream/10603/8540/12/12_chapter 4.pdf · Rabeprazole Sodium tablets 4.1 Introduction Rabeprazole,