CHAPTER CHAPTER CHAPTER CHAPTER ––––IIIIIIII

STABILITY INDICATING

IMPURITIES METHOD FOR

RABEPRAZOLE DRUG PRODUCT

AND

CHARACTERISATION OF

THREE POTENTIAL DEGRADATION

PRODUCTS

Section (i): Brief account of Rabeprazole

Rabeprazole sodium [RAB], chemically known as 2-[[(4-(3-methoxypropoxy)-3-methyl-

2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole [1], is a proton pump inhibitor, and used to treat

gastroesophageal reflux disease (GERD), a condition in which backward flow of acid from the

stomach causes heartburn and possible injury to the esophagus (the tube that connects the throat

and stomach); it heals esophagus, and prevents further damage to the esophagus. RAB is also

used to treat Zollinger-Ellison syndrome and ulcers (sores in the lining of the stomach or

intestine), and is used in combination with other medications to eliminate Helicobacter pylori, a

bacterium that causes ulcers [2].

In general, solid active pharmaceutical ingredients (APIs) are formulated with excipients as tablets or

capsules. Since the active ingredient interacts 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 which include the stability of the active ingredient, the manufacturing

process, environmental conditions (such as heat, light and moisture during storage), as well as some chemical

reactions such as oxidation, reduction and hydrolysis that might occur [3, 4]. RAB has been reported in the

literature as thermal, acidic and photo sensitive [5], As RAB is sensitive to acid, pharmaceutical dosage form is

manufactured as enteric coated tablets [6-10]. RAB is a white to light yellow crystalline, hygroscopic material with a

molecular weight of 359.44. It is soluble in water. RAB drug substance and drug product is official in IP. The

Chemical structure of RAB is as follows:

Rabeprazole :

2-[[(4-(3-methoxypropoxy)-3-methyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole

In literature, limited LC methods (LC- MS, HPLC, ELSD, NMR, Preparative HPLC, TLC and ESI-

MS/MS) [11-19] are reported for the determination of RAB in pharmaceutical preparations. A few of the

degradation and other impurities of RAB are reported in literature [20, 21]. Currently, the quantification of

impurities is one of the most difficult tasks in pharmaceutical analysis, especially if number of impurities required to

be determined is more and, preferably in a single chromatographic run. Ultra-performance liquid chromatography

(UPLC) is one of the tools which uses a column packed with sub-2-µm particles. These particles operate at elevated

linear velocities of mobile phase to drastically increase the resolution, sensitivity and speed of analysis. Because of

its speed and sensitivity, this technique has gained considerable attention in recent years for pharmaceutical and

biomedical analyses.

This chapter deals with a reproducible stability-indicating RP UPLC method for the quantitative

determination of RAB along with identification and characterization of three unknown degradation impurities

formed during the storage of the drug product at stressed conditions 40°C/75% RH (Relative Humidity) for 6

months. These impurities are isolated by preparative HPLC and their structures are elucidated using LC-MS, High

Resolution Mass Spectroscopy (HRMS) and NMR spectroscopy. UPLC method is able to accurately detect and

quantify three impurities along with six other impurities. This method is successfully validated according to

International Conference of Harmonization (ICH) guidelines [22].

Section (ii): Isolation and characterization of three unknown impurities, Development and

Validation of nine impurities in Rabeprazole Tablets

This section describes the isolation and characterization of three unknown degradation products found in

Rabeprazole (RAB) tablets during the stability studies. Though some of the impurities and degradation products are

reported in literature, these three impurities are not reported to the best of our knowledge. This section also reports

various aspects relating to the development and validation of stability indicating UPLC method for nine impurities

including three degradants in RAB tablet dosage form.

1. Experimental

1.1. Chemicals

RAB tablets (delayed release) are formulated at Dr Reddy’s laboratories Ltd, Hyderabad, India. RAB drug

substance, impurities, working standards and RAB tablets are obtained from Dr.Reddy’s laboratories Ltd,

Hyderabad, India. The HPLC grade acetonitrile, methanol and analytical grade KH2PO4, NaOH, HCl, triethyl amine

and ortho phosphoric acid are purchased from Merck, Germany. High purity water is prepared by using Milli Q Plus

water purification system. Chemical name, structures of RAB and its impurities are described in Table 2.2.1

Table 2.2.1 Chemical name and structure of RAB and its 9 impurities

Name Structure IUPAC Name

Rabeprazole

N

S

N

HN

O

CH3

OOCH3

2-[[(4-(3-methoxypropoxy)-3-

methyl-2-

pyridinyl)methyl]sulfinyl]-1H-

benzimidazole

Impurity A

1-(1H-benzo[d]imidazol-2-yl)-

3-methyl-4-oxo-1,4-

dihydropyridine-2-carboxylic

acid

Impurity B

2-[[[4-(3-Methoxypropoxy)-3-

methyl-2-pyridinyl-1-

oxide]methyl]sulphinyl]-1H-

benzimidazole

Impurity C

2-[[[4-Methoxy-3-methyl-2-

pyridinyl]methyl]-sulphinyl-1H-

benzimidazole

Impurity D

N

HN

S

O

N

Cl

2-[[[4-Chloro-3-methyl-2-

pyridinyl]methyl]sulphinyl]-1H-

benzimidazole

Impurity E

2-[[[4-(3-Methoxypropoxy)-3-

methyl-2-

pyridinyl]methyl]sulphonyl]-

1H-benzimidazole

Impurity F

2-[[[4-(3-Methoxy propoxy)-3-

methyl-2-

pyridinyl]methyl]thio]-1H-

benzimidazole

Impurity I

2-Amino-1H-benzimidazole

Impurity II

1H-Benzimidazol-2-ol

Impurity III

2-Benzimidazolethiol

1.2. Determination of appropriate UV wavelength

A suitable wavelength for the determination of RAB and its impurities are identified by taking the

overlay spectra from 200–400 nm of all impurities and RAB from PDA detector.

1.3. Instrumentation and chromatographic / spectrometric conditions

The Waters UPLC system is used for method development and method validation with a diode array

detector. The output signal is monitored and processed using M/s waters empower software. Mettler XS 205 Dual

Range balance is used for weighing of samples and standard. Photo stability studies are carried out in a Sun Text

XLS+ photo stability chamber. Thermal stability studies are performed in a Merck hot air oven.

The ESI MS spectrum is recorded using Aapplied bio systems API 4000 Q-trap connected to Agilent 1100

HPLC with photodiode array detector. The HRMS spectrum is recorded using Waters Quadrupole time-of-flight

MS. NMR spectra are recorded in DMSO-d6 using Unity INOVA Varian 500 MHz spectrometer.

1.3.1 Liquid Chromatographic method

Acquity BEH Shield, RP18, 100 x 2.1 mm i.d., 1.7 µm column is used for method development and

validation. Phosphate buffer is prepared by using potassium dihydrogen orthophosphate, pH adjusted to 6.0 with

diluted orthophosphoric acid. Mobile phase A consists of a phosphate buffer and acetonitrile in the ratio of 90:10

(v/v). Mobile phase B consists of a phosphate buffer and acetonitrile in the ratio of 45:55 (v/v). Column temperature

is maintained at 25°C, injection volume is 3 µL and detection wavelength at 280 nm. The UPLC system is operated

with a gradient mode at a flow rate of 0.4 mL/min and data acquisition time is 18 min. The gradient program is set

as Time (min)/%B; T0.01,/10, T1.1/00, T1.5/00, T5.0/20, T7.0/30, T10.0/30, T12.0/50, T14.0/70, T16.0/80, T16.5/50, T17.0/10

and T18.0/10.

1.3.2 Liquid chromatography mass spectrometry (LC/HR/MS)

Mass spectrometry compatible chromatographic method is developed for the analysis of RAB and its

impurities on a Water Symmetry Shield RP18, 250 x 4.6 mm, 5 µm column. Mobile phase A consisting of

Ammonium acetate buffer and Ammonia (30%) solution of pH 6.4 and acetonitrile in the ratio of 90:10 (v/v).

Mobile phase B consists of water and acetonitrile in the ratio of 10:90 (v/v). Diluent is prepared by mixing

acetonitrile, water and ammonia solution (30%) in the ratio of 80:20:1 (v/v/v). Injection volume is 10 µl, flow rate

1.0 mL/min, UV detection is carried out at 280 nm and data acquisition time is 70 min. The gradient program is set

as Time (min)/%B; T0.01,/05, T50/65, T60/65, T62/05 and T70/05. The mass spectra of impurities are recorded on AB-

4000 Q-trap LC-MS/MS - Applied Biosciences (Agilent) mass spectrometer.

1.3.3 1H NMR

The 1H NMR data of Impurity I, Impurity II and Impurity III are recorded in DMSO-d6 at 400 MHz on

Varian Mercury plus 400 MHz spectrometer. The chemical shift values are reported on δ scale in ppm with respect

to TMS (0.00ppm) as internal standard.

1.4. Diluent

Diluent is prepared by mixing methanol, water and diethylamine in the ratio of 80:20:1 (v/v/v).

1.5. Preparation of standard solution

A Stock Solution of RAB (500 µg/mL) is prepared by dissolving an appropriate amount of the drug in

diluent. Working solution of 1 µg/mL is prepared from the stock solution for the determination of related

substances. The overlay chromatogram of RAB standard and blank is shown in Fig 2.2.1.

Fig 2.2.1 Overlay chromatogram of blank and diluted standard

1.6. Preparation for Test solution

20 tablets are crushed to a fine powder and tablet powder equivalent to 100 mg of RAB is

transferred to 200 mL volumetric flask. About 150 mL of diluent is added and sonicated for 30

minutes with intermediate shaking (Sonicator temperature is maintained between 10°C - 15°C).

Sample solutions are allowed to come to room temperature and then diluted to volume with

diluent (500 µg/mL). Part portion of solution is centrifuged at 3000 rpm for 10 min. The

supernatant test solution is used for analysis. Placebo sample is prepared in the same way by

taking the placebo equivalent weight present in a test preparation.

1.7. Impurity stock preparations

Impurity stock solutions are prepared individually by weighing accurately about 10 mg each into 100 mL

volumetric flasks. 25 mL of diluent is added and dissolved with aid of sonication and made upto volume with

diluent to obtain stock solution of 100 µg/mL each of impurity.

1.8. Preparation of Test solution with spiking of Impurities

20 tablets are crushed to a fine powder and tablet powder equivalent to 100 mg of RAB and 2.5 mL of

RAB impurity stock solution is transferred to 200 mL volumetric flask. About 150 mL of diluent is added and

sonicated for 30 minutes with intermediate shaking (Sonicator temperature is maintained between 10°C - 15°C).

Sample solutions are allowed to come to room temperature and then diluted to volume with diluent (500 µg/mL).

Part portion of solution is centrifuged at 3000 rpm for 10 min. The supernatant test solution is used for analysis. The

overlay chromatogram of test spiked with impurities and placebo are shown in Fig 2.2.2.

Fig 2.2.2 Overlay chromatogram of placebo and RAB test spiked with impurities

1.9. Specificity

As per ICH guidelines development and validation of stability indicating methods is required for all

pharmaceutical dosage forms. The current ICH guidelines do not describe degradation conditions for stress study.

The forced degradation conditions, stress agent concentration and time of stress, are found to be effective based on

% degradation. Preferably between 20% to 30 % of degradation is recommended for active material to make the

right assessment of stability indicating nature of the chromatographic methods. The optimization of such stress

conditions which can yield desired % degradation is based on experimental conditions. Chromatographic run times

are decided for placebo and samples subjected to force degradation in order to provide an indication of the stability

indicating properties and specificity of the method. The stress conditions employed are acid, base, neutral, peroxide,

heat, humidity and light. After the degradation treatments are completed, the samples are allowed to equilibrate to

room temperature, neutralized with acid or base (as necessary) and made up with diluent to get a concentration of

500 µg/mL of RAB. Peak purity test is carried out for RAB peak by using PDA detector in stress samples. Specific

conditions are described below:

1.9.1. Placebo (excipients) interference

Samples are prepared in duplicate by taking the weight of placebo approximately

equivalent to its weight in the test sample as described in the test preparation.

1.9.2. Effect of acid hydrolysis

RAB tablet powder equivalent to 100 mg of RAB is transferred into 100 mL round bottom flask, treated

with 5 mL of 0.1N HCl for 15 minutes at 60°C. The sample is allowed to equilibrate to room temperature,

neutralized with base and resulting solution is prepared as per test procedure to obtain final concentration 500

µg/mL of RAB. Part portion of solution is centrifuged at 3000 rpm for 10 min and supernatant solution is used for

analysis.

1.9.3. Effect of base hydrolysis

RAB tablet powder equivalent to 100 mg of RAB is transferred into 100 mL round bottom flask, treated

with 5 mL of 0.1N NaOH for 30 minutes at 60°C. The sample is allowed to equilibrate to room temperature,

neutralized with acid and resulting solution is prepared as per test procedure to obtain final concentration 500 µg/mL

of RAB. Part portion of solution is centrifuged at 3000 rpm for 10 min and supernatant solution is used for

analysis.

1.9.4. Effect of neutral hydrolysis

RAB tablet powder equivalent to 100 mg of RAB is transferred into 100 mL round bottom flask, treated

with 5 mL of water for 30 minutes at 60°C. The sample is allowed to equilibrate to room temperature and resulting

solution is prepared as per test procedure to obtain final concentration 500 µg/mL of RAB. Part portion of solution is

centrifuged at 3000 rpm for 10 min and supernatant solution is used for analysis.

1.9.5. Effect of oxidation

RAB tablet powder equivalent to 100 mg of RAB is transferred into 100mL round bottom flask, treated

with 5 mL of 3% H2O2 for 5 minutes at 60°C. The sample is allowed to equilibrate to room temperature and

resulting solution is prepared as per test procedure to obtain final concentration 500 µg/mL of RAB. Part portion of

solution is centrifuged at 3000 rpm for 10 min and supernatant solution is used for analysis.

1.9.6. Effect of humidity and heat

To evaluate the effect of moisture and heat, thin layers of tablets powder are distributed over a glass plate.

The plate is stored at 25ºC/90% RH (Relative Humidity) for about 7 days. A similar sample is kept in an oven at

105°C for 6 hrs. Then the samples are prepared as described in the test preparation.

1.9.7. Effect of UV and visible light

To study the photochemical stability of the drug product the powder from the tablet is exposed to 1200 K Lux

of visible light and 200 W h/ m2 of UV light by using photo stability chamber. After exposure the samples are

prepared as described in test preparation.

2. Isolation and purification of three unknown impurities

2.1. Impurity-I (RRT 0.10)

2.1.1. Impurity enrichment

RAB tablet powder equivalent to 1000 mg of RAB is refluxed with 10 % Hydrogen peroxide solution at

60°C for 30 minutes and centrifuged at 4000 RPM for 30 minutes. The solution is analyzed by analytical LC method

and about 14.75% targeted impurity is observed in stress sample.

2.1.2. Isolation by preparative HPLC

The enriched sample is purified by injecting into the below given preparative HPLC conditions and the

fractions are collected using automatic fraction collectors attached with preparative HPLC. Fraction collection is

based on target retention times. Inertsil C18 column (250 × 20 mm i.d., 5 µm) is used for preparative analysis.

Mobile phase-A consists of water and acetonitrile in the ratio of 90:10 (v/v). Mobile phase-B consists of acetonitrile

and water in the ratio of 90:10 (v/v). Preparative LC is carried out at a flow rate of 16 mL/min, Gradient (T/%B, 0/5,

10/5, 15/90, 30/90, 32/5, 40/5) at ambient temperature, detection wavelength is 285 nm. Enriched sample solution is

injected into the preparative HPLC system using a rheodyne injector. Peak cut criteria for the isolated impurity is

selected based on the peak retention time. The impurity fractions are collected from about 40 injections and the

fractions are pooled. Acetonitrile present in the pooled fraction is evaporated using rotavapour at room temperature.

After evalopration of the total solvent, aqueous layer is kept for lyophilization. The chromatographic purity of the

lyophilized compound is found to be 96.7%. The identity of the impurity is confirmed by ESI MS. In a +ve mode

the protonated molecular ion found at m/z 134. The impurity isolated and purified is subjected to further

characterization studies. The chromatogram of pure compound 0.1RRT impurity is given in Fig 2.2.3.

Fig 2.2.3 Chromatogram of pure compound 0.1 RRT impurity

2.2. Impurity-II (RRT 0.18)

2.2.1. Enrichment of impurity

RAB tablet powder equivalent to 1000 mg of RAB is refluxed with 50 mL of 0.2 N HCl at 60°C for 20

minutes and centrifuged at 4000 RPM for 30 minutes. The solution is analyzed by analytical LC method and about

6.77% of targeted impurity is observed in stress sample

2.2.2. Purification by Preparative HPLC

The enriched sample is purified by injecting into preparative HPLC and the fractions are collected using

automatic fraction collectors attached with preparative HPLC. Fraction collection is based on target retention times.

Zodiac C18 column (250 × 20 mm i.d., 5 µm) is used for preparative analysis. Mobile phase-A consists of water and

acetonitrile in the ratio of 90:10 (v/v). Mobile phase-B consists of acetonitrile and water in the ratio of 90:10 (v/v).

Preparative LC is carried out at a flow rate of 16 mL/min, Gradient (T/%B, 0/5, 10/5, 35/20, 40/80, 55/80, 60/5,

65/5) at ambient temperature and detection wavelength is 285 nm. The enriched sample solution is injected into the

preparative LC system using a rheodyne injector. Peak cut criteria for the isolated impurity is selected based on the

peak retention time. The impurity fractions are collected from about 40 injections and the fractions are pooled.

Acetonitrile present in the pooled fraction is evaporated using rotavapour at room temperature. After evalopration of

the total solvent, aqueous layer is kept for lyophilization. The chromatographic purity of the lyophilized compound

is found to be 98.4%. The identity of the impurity is also confirmed by ESI MS. In a +ve ionization mode the mass

spectrum displayed protonated molecular ion at m/z 135. The impurity isolated and purified is subjected for further

characterization studies. The chromatogram of pure compound 0.18RRT impurity is given in Fig 2.2.4.

Fig 2.2.4 Chromatogram of pure compound 0.18 RRT impurity

2.3. Impurity-III (RRT 0.31)

2.3.1. Impurity enrichment

RAB tablet powder equivalent to 1000 mg of RAB is refluxed with 50 mL of 0.2 N HCl at 60°C for 30

minutes and centrifuged at 4000 RPM for 30 minutes. The solution is analyzed by analytical HPLC method and

about 5.3% of targeted impurity is observed in stress sample.

2.3.3. Purification by Preparative HPLC

The enriched sample is purified by injecting into preparative HPLC and the fractions are collected using

automatic fraction collectors attached with preparative HPLC. Fraction collection is based on target retention times.

Zodiac C18 column (250 × 20 mm i.d., 5 µm) column is used for preparative analysis. Mobile phase-A consists of

water and acetonitrile in the ratio of 90:10 (v/v). Mobile phase-B consists of acetonitrile and water in the ratio of

90:10 (v/v). Preparative LC is carried out at a flow rate of 16 mL/min, Gradient (T/%B, 0/5, 10/5, 35/20, 40/80,

55/80, 60/5, 65/5) at ambient temperature and detection wavelength is 285 nm. An Enriched sample solution is

injected into the preparative LC system using a rheodyne injector. Peak cut criteria for the isolated impurity is

selected based on the peak retention time. The impurity fractions are collected from about 40 injections and the

fractions are pooled. Acetonitrile present in the pooled fraction is evaporated using rotavapour at room temperature.

After evalopration of the total solvent, aqueous layer is kept for lyophilization. The chromatographic purity of the

lyophilized compound is found to be 97.5%. The identity of the impurity is confirmed by ESI MS. In +ve ionization

mode the mass spectrum displayed protonated molecular ion at m/z 151. The impurity isolated and purified is

subjected for further characterization studies. The chromatogram of pure compound 0.31 RRT impurity is given in

Fig 2.2.5.

Fig 2.2.5 Chromatogram of pure compound 0.31 RRT impurity

3. Method validation

3.1. Relative retention times and relative response factors

Relative retention times (RRT) and Relative response factors (RRF) are established for all the known impurities

of RAB by injecting all the impurity standards. Relative response factors are established for all the known

impurities of RAB against RAB. RRFs are established based on ratio of slope of impurities to the slope of RAB.

Slope value obtained with linearity calibration plots. The RRT and RRF values of RAB impurities are shown in

Table 2.2.2.

Table 2.2.2 RRT and RRF values of impurities of RAB

S.No. Name RRT RRF

1 Impurity A 0.09 1.28

2 Impurity B 0.68 1.55

3 Impurity C 0.76 1.21

4 Impurity D 0.98 1.44

5 Impurity E 1.13 1.00

6 Impurity F 1.36 1.13

7 Impurity I 0.10 1.57

8 Impurity II 0.18 1.43

9 Impurity III 0.31 2.09

3.2. Precision

The precision of test method is evaluated by analyzing six individual test preparations of RAB samples by

spiking test preparation with RAB impurities solution at 0.5% concentration of each impurity with respect to test

concentration. The Relative standard deviation is calculated for the response of each impurity. The Intermediate

precision of test method is evaluated on different UPLC systems by different analyst on different columns in different

days.

3.3 Limits of Detection (LOD) and Quantification (LOQ)

The LOD and LOQ for impurities of RAB are estimated at a signal-to-noise ratio of about 3:1 and 10:1

respectively by injecting a series of diluted solutions with known concentration. Precision study is carried at the

LOQ level by injecting six individual preparations of all impurities. % RSD is calculated for the content of each

impurity. Accuracy at LOQ level is evaluated in triplicate for the impurities by spiking at the estimated LOQ level.

3.4. Linearity

Linearity solutions for the impurities are prepared by diluting impurity stock solution to get the solutions of

impurities having different concentrations. The solutions are prepared at different concentration levels from LOQ to

200 % of the normal limit concentration for RAB impurities. The peak area versus concentration data is treated by

least-squares linear regression analysis.

3.5. Accuracy

Recovery study is conducted to determine accuracy of impurities method for the quantification of all

impurities in RAB tablets. The study is carried out in triplicate at LOQ, 25%, 50%, 100%, 150% and 200% of the

target concentration (0.5% ) of each impurity. The percentages of recoveries for RAB impurities are calculated

against spiked amounts.

3.6. Robustness

To determine the robustness of the test method, experimental conditions are purposely altered one after the

other to establish their effect, RRTs of all RAB impurities are measured. The effect of flow rate is studied at 0.4 ±

0.05 ml/ min. The effect of column temperature is studied at 25ºC ± 5 ºC (at 20 and 30ºC). The effect of percent

organic strength is studied by varying acetonitrile percentage from -10% to +10% while the other mobile phase

components are held constant. To study the effect of buffer pH, 0.2 units changed from 5.8 to 6.2, while the other

mobile phase components are held constant.

3.7. Solution stability and mobile phase stability

The solution stability of RAB standards and test preparation spiked with impurities is established by

allowing solutions on bench top at controlled room temperature for 24 hours. The solutions are stored in volumetric

flasks by tightly capping. The amounts of RAB and its impurities in the above solutions are measured. The stability

of mobile phase is also determined by analyzing freshly prepared solution of RAB and its impurities at 24 hours

intervals for 48 hours using same lot of mobile phase.

4. Results and discussion

4.1. Determination of suitable wavelength

Based on the UV spectra (Fig 4.3.3), 280 nm is selected for quantification all the impurities of RAB. UV

spectra of RAB and its impurities are shown in Fig 2.2.6

Fig 2.2.6: UV spectra of RAB & its impurities.

4.2. Optimization of chromatographic conditions

RAB and its related substances have ionizable functional groups such as carboxyl, amino groups etc. the

reverse phase LC method development is more appropriate for determination of impurities in RAB tablets. Water

symmetry shield RPl8, 250 mm x 4.6 mm, 5.0 µm particle size column is selected for development. The

dissociation constant of RAB is 4 to 5, hence various experiments are conducted using phosphate, citrate and acetate

buffers by maintaining its pH (between 4 to 6) close to RAB pKa value along with different organic modifiers in

mobile phase. Good separation among all impurities is observed with phosphate buffer and 0.1 % TEA at pH 6.4.

Acetonitrile is selected over methanol as organic modifier owing to its suitability for improved resolution between

RAB and its related compounds. Separation is achieved in the below mentioned conditions: Mobile phase A

consists of 25mM potassium phosphate buffer and 0.1% triethylamine of pH 6.4 (pH adjusted to 6.4 ± 0.05 using

orthophosphoric acid or potassium hydroxide solution) and acetonitrile in the ratio of 90:10 (v/v). Mobile phase B

consists of water and acetonitrile in the ratio of 10:90 (v/v). Injection volume is 20 µl, flow rate 1.0 mL/min and

column oven temperature maintained at 25°C. UV detection is carried out at 280 nm and data acquisition time is 70

minutes. The gradient program is as follows: Time (min)/%B; T0.01,/5, T50/65, T60/65, T62/5 and T70/5. This LC

method is able to detect all the impurities with longer run time (about 70 minutes). The stability sample analysis is

performed in these conditions. Impurity at 0.27 RRT is found to be observed more than identification threshold and

also adequate separation is not observed as one of the other impurities is closely eluting. Hence decided to fine tune

the method for better separation of closely eluting peaks and also with an objective of shorter runtime. To reduce

run time further it is decided to conduct the experiments on UPLC system using an Acquity BEH C18, 100 x 2.1

mm, 1.7 µ column with mobile phase flow rate of 0.4 mL/min. Lower particle size column is used to achieve the

resolution of individual impurities and degradents from RAB. Mobile phase A consists of pH 6.4 phosphate buffer

& acetonitrile in the ratio of 90:10 (v/v) and Mobile phase B consists of pH 6.4 phosphate buffer & acetonitrile in

the ratio of 30:70 (v/v) respectively. All impurities are well separated but Impurity I, II & III are eluted very closely.

Different gradient programs are tried with same mobile phase combination, separation is achieved in 20 minutes but

long eluting peaks and more blank peaks are observed. Analytical column is replaced with a fully endcapped

Acquity BEH Shield, RP18, 100 x 2.1 mm, 1.7 µ column due to its high efficiency and suitability for polar moieties

compared with other commercially available octadecyl silanized silica packed columns for getting proper baseline.

Number of blank peaks is significantly controlled but impurity A and impurity 1 are not resolved properly. Gradient

program and pH of the buffer in mobile phase are modified and separation is achieved within 18 min compared to

run time of 70 min as per HPLC method. The separation is achieved with following chromatographic conditions:

Acquity UPLC with BEH Shield, RP18 column (100 x 2.1 mm i.d., 1.7 µm particle size), Mobile phase A consists

of phosphate buffer and acetonitrile in the ratio of 90:10 (v/v), Mobile phase B consists of phosphate buffer and

acetonitrile in the ratio of 45:55 (v/v). The phosphate buffer is prepared by using Potassium dihydrogen

orthophosphate and pH adjusted to 6.0 with dilute orthophosphoric acid. Column temperature is maintained at 25°C.

The injection volume is 3 µL. Detection wavelength is 280 nm. The UPLC system is operated with a gradient mode

at a flow rate of 0.4 mL/min and data acquisition time is 18 min. The gradient program is as follows, Time

(min)/%B; T0.01,/10, T1.1/00, T1.5/00, T5.0/20, T7.0/30, T10.0/30, T12.0/50, T14.0/70, T16.0/80, T16.5/50, T17.0/10 and

T18.0/10.

4.3. Structural characterization of three unknown impurities

4.3.1. Impurity –I (RRT 0.10)

The isolated and purified impurity (0.10 RRT) is subjected to LC-MS studies. Electro spray Ionization

mass spectrum in +ve mode of RAB 0.10 RRT impurity is presented in Fig 2.2.7. The compound mass spectrum

shows that the mass number is 133, as it displayed a protonated molecular ion at 134. This indicates that the

molecular weight of the impurity is 226 mass units less than the RAB molecular weight.

The isolated and purified impurity (0.10 RRT) is subjected to high resolution mass (HRMS) spectral

studies. HRMS spectrum in +ve mode of 0.10 RRT impurity is presented in Fig 2.2.8. The HRMS spectrum showed

that the impurity is having molecular formula of C7H7N3 with exact mass of 133.0715 daltons. The molecular

formula shows the absence of sulphur, 3 oxygenn less, 11 carbons and 14 protons less when compared to RAB. This

clearly indicates the possibility of cleavage of RAB.

The isolated and purified impurity (0.10 RRT) is subjected to NMR spectral studies. 1H NMR spectrum is

recorded in DMSO-d6. The 1H NMR spectrum has showed peaks at 10.7, 7.09, 6.84 and 6.12 ppm corresponding to

seven protons. The peaks at 10.7 and 6.12 ppm found to be from exchangeable protons. The chemical shift values

are reported on δ scale in ppm with respect to TMS (0.00ppm) as internal standard. The spectra are presented in Fig

2.2.9. The COSY spectrum showed correlation between 7.09 and 6.84 ppm peaks which shows that these two are

coupled (Fig 2.2.10). Based on the above discussion Imp-I structure is proposed as mentioned below (Table 2.2.3).

Table 2.2.3 NMR assignments of Rabeprazole Impurity-I

N

H

N

NH2

2

1

6

5

4

3

8

7 9

2-Amino-1H-Benzimidazole

Position

1H δ (ppm ) Multiplicity

##

1 NH 10.70 Br

3, 6 2H 7.09 M

4, 5 2H 6.84 dd, 3.0,5.5

9 NH2 6.12 S

## This column gives the

1H-

1H multiplicity, s-singlet, dd-doublet of doublet, br-broad, m-multiplet.

Fig 2.2.7 LC-MS (ESI+ve) Spectrum of 0.10 RRT impurity of RAB

Fig 2.2.8 HRMS Mass spectrum of 0.10 RRT impurity of RAB

Fig 2.2.9 1H NMR spectrum of 0.10 RRT impurity of RAB in DMSO-d6

Fig 2.2.10 gDQ-COSY spectrum of 0.10 RRT impurity of RAB in DMSO-d6

4.3.2. Impurity II at 0.18 RRT

The isolated and purified impurity (0.18 RRT) is subjected to LC-MS studies. The ESI mass spectrum in

+ve mode of 0.18 RRT impurity is presented in Fig 2.2.11. The compound mass spectrum shows that the mass

number is 134, as it displayed a protonated molecular ion ion at m/z=135. This indicates that the molecular weight

of the impurity is 225 mass units less than the RAB, which clearly indicates that the one moiety cleaving from RAB.

The isolated and purified impurity (0.18 RRT) is subjected to high resolution mass (HRMS) spectral

studies. HRMS spectrum in +ve mode of RAB 0.18 RRT impurity is presented in Fig 2.2.12. The HRMS spectrum

showed that the impurity is having molecular formula of C7H6N2O with exact mass of 134.05 daltons. The

molecular formula shows the absence of sulphur and one nitrogen, 2 oxygens less, 11 carbons and 15 protons less

when compared to RAB. This clearly indicates the possibility of cleavage of RAB.

The isolated and purified impurity (0.18 RRT) is subjected to NMR spectral studies. 1H NMR spectrum is

recorded in DMSO-d6. The Spectra is presented in Fig 2.2.13. The 1H NMR spectrum showed two sets of signals at

6.95 and 10.58 ppm. The 6.95 ppm protons are attached to two carbon signals coming at 108 ppm and 120 ppm from

HSQC data. The 10.58 ppm signal found to be an exchangeable proton. HSQC data shows that the signal at 6.95

ppm correspond to four protons where as the peak at 10.58 ppm correspond to two protons (Fig 2.2.14)

Based on above discussion Imp-II structure is proposed as mentioned below

(Table 2.2.4)

Table 2.2.4 NMR assignments of Rabeprazole Impurity-II

N

H

N

OH2

1

6

5

4

3

8

7 9

1H-Benzimidazol-2-ol

Position

1H δ (ppm ) Multiplicity

##

1 NH 10.58 br

3, 6 2H 6.95 s

4, 5 2H 6.95 s

9 OH 10.58 br

##This column gives the

1H-

1H multiplicity, s-singlet, br-broad

Fig 2.2.11 LC-MS (ESI+ve) Mass spectrum of 0.18 RRT impurity of RAB

Fig 2.2.12 HRMS Mass spectrum of 0.18 RRT impurity of RAB

Fig 2.2.13 1NMR spectrum of 0.18 RRT impurity of RAB in DMSO-d6

Fig 2.2.14 gHSQC spectrum of 0.18 RRT impurity of RAB in DMSO-d6

4.3.3. Impurity-III at 0.31 RRT

The isolated and purified impurity (0.31 RRT) is subjected to LC-MS studies. Electro spray mass spectrum

in +ve mode of 0.31 RRT impurity is presented in Fig 2.2.15. The compound mass spectrum shows that the mass

number is 150, as it displayed a protonated molecular ion ion at m/z=151. This indicates that the molecular weight

of the impurity is 209 mass units less than the RAB molecular weight, which clearly indicates that the one moiety

cleaving from RAB.

The isolated and purified impurity (0.31 RRT) is subjected to high resolution mass (HRMS) spectral

studies. HRMS spectrum in +ve mode of RAB 0.31 RRT impurity is presented in Fig 2.2.16. The HRMS spectrum

showed that the impurity is having molecular formula of C7H6N2S with exact mass of 150.03 daltons. The molecular

formula shows the absence of one nitrogen, 3 oxygen’s, 11 carbons and 15 protons when compared to RAB. This

clearly indicates the possibility of cleavage of RAB.

The isolated and purified impurity (0.31 RRT) is subjected to NMR spectral studies. 1H NMR spectrum is

recorded in DMSO-d6. The Spectra is presented in Fig 2.2.17. The 1H NMR spectrum has showed two signals at

7.14 and 12.53 ppm. The 12.53 ppm signal found to be an exchangeable proton. The 13

C NMR spectrum showed

carbon signals at 168.27, 133.16, 121.89 and 109.55 ppm (Fig.2.2.18).

Based on above discussion Imp-III structure is proposed as mentioned below

(Table 2.2.5)

Table 2.2.5 NMR assignments of Rabeprazole Impurity- III

N

H

N

SH2

1

6

5

4

3

8

7 9

2-Benzimidazolethiol

Position

1H δ (ppm ) Multiplicity

##

1 NH 12.53 Br

3, 6 2H 7.14 M

4, 5 2H 7.14 M

9 SH 12.53 br

##

This column gives the 1H-

1H multiplicity, m-multiplet, br-broad.

Fig 2.2.15 ESI +ve MS spectrum of 0.31 RRT impurity of RAB

Fig 2.2.16 HRMS spectrum of 0.31 RRT impurity of RAB

Fig 2.2.17 1H NMR spectrum of 0.31 RRT impurity of RAB in DMSO-d6

Fig 2.2.18 13

C NMR spectrum of 0.31 RRT impurity of RAB

4.4. Method validation

4.4.1. Precision

The % RSD of replicate test preparations spiked with impurities (Intra-day and inter day

precision study) is found to be less than 4.6, conforming good precision of the method. All

values are well within the acceptance criteria i.e. % RSD not more than 15.0 %. The data is

presented in Table 2.2.6 to 2.2.7.

Table 2.2.6 Results of precision of test method for RAB impurities

Preparatio

n

Impurit

y A

Impurit

y B

Impurit

y C

Impurit

y D

Impurit

y E

Impurit

y F

Impurit

y I

Impurit

y II

Impurity

III

Prep-1 0.497 0.464 0.510 0.502 0.449 0.509 0.503 0.473 0.493

Prep-2 0.493 0.470 0.510 0.509 0.450 0.509 0.505 0.475 0.489

Prep-3 0.493 0.467 0.513 0.513 0.448 0.512 0.506 0.477 0.493

Prep-4 0.486 0.470 0.508 0.518 0.449 0.508 0.503 0.472 0.485

Prep-5 0.479 0.449 0.470 0.523 0.436 0.493 0.489 0.462 0.486

Prep-6 0.488 0.467 0.510 0.516 0.449 0.505 0.504 0.474 0.489

Avg 0.489 0.465 0.508 0.514 0.447 0.506 0.502 0.472 0.489

%RSD 1.3 1.7 1.1 1.4 1.2 1.3 1.3 1.1 0.7

Table 2.2.7 Results of intermediate precision of test method for RAB impurities

Preparat

ion

Impurit

y A

Impurit

y B

Impurit

y C

Impurit

y D

Impurit

y E

Impurit

y F

Impurit

y I

Impurity

II

Impurity

III

Prep-1 0.470 0.438 0.505 0.446 0.435 0.506 0.499 0.465 0.512

Prep-2 0.467 0.447 0.508 0.41 0.452 0.511 0.498 0.466 0.518

Prep-3 0.466 0.449 0.502 0.479 0.443 0.514 0.500 0.466 0.504

Prep-4 0.469 0.443 0.503 0.493 0.447 0.512 0.498 0.463 0.517

Prep-5 0.470 0.449 0.507 0.465 0.454 0.510 0.501 0.465 0.516

Prep-6 0.472 0.444 0.503 0.434 0.437 0.508 0.499 0.467 0.512

Avg 0.469 0.445 0.505 0.463 0.445 0.510 0.499 0.465 0.513

%RSD 0.5 1.0 0.5 4.6 1.7 0.6 0.2 0.3 1.0

4.4.2. LOQ and LOD

The determined limit of detection (LOD), limit of quantification (LOQ) and precision at LOQ values for

RAB impurities are reported in Table 2.2.8.

Table 2.2.8 LOD, LOQ and precision at LOQ for RAB and its impurities

Name of the Impurity

LOD LOQ

Concentration in

‘%’ S/N ratio

Concentration in

‘%’

S/N

ratio

% RSD

LOQ

RAB

impurities

Impurity A 0.0279 2.702 0.0846 10.326

0.0

Impurity B 0.0287 2.734 0.0871 10.957 0.0

Impurity C 0.0400 2.295 0.1214 9.182 1.7

Impurity D 0.0663 2.423 0.2009 10.058 1.0

Impurity E 0.0384 2.528 0.1164 9.944 1.6

Impurity F 0.0534 2.126 0.1618 9.055 2.3

Impurity I 0.0232 2.291 0.0705 9.099 3.0

Impurity II 0.0384 2.753 0.1165 11.259 1.8

Impurity III 0.0419 2.445 0.1269 9.859 0.0

4.4.3. Linearity

A linear calibration plot for RAB

and the correlation co-efficient is found to be about 0.999. The result shown in

correlation exists between the peak area and concentration of the analyte

RAB impurities is obtained over the calibration range LOQ (~0.07)

efficient is found to be about 0.999. The result shown in Fig 2.2.19 indicates that an excellent

correlation exists between the peak area and concentration of the analyte for all impurities.

LOQ (~0.07) to 5.1 µg/mL

2.2.19 indicates that an excellent

Fig 2.2.19 Linearity graphs of RAB impurities

4.4.4. Accuracy

The percentage recoveries of all impurities in

RAB impurities. The % recovery values for

4.4.5. Robustness

To determine the robustness of the developed method, experimental conditions

the elution pattern, separation between

deliberate varied chromatographic conditions (flow rate, column temperature, pH of

composition of organic solvent), all analytes are adequately resolved and elution orders remained unchanged. RRT

impurities

The percentage recoveries of all impurities in RAB samples are found to be between 85.0 to 115.0 for

The % recovery values for RAB impurities are presented in Table 2.2.9

To determine the robustness of the developed method, experimental conditions are deliberately altered and

the elution pattern, separation between RAB and its impurities and tailing factor for RAB are recorded.

deliberate varied chromatographic conditions (flow rate, column temperature, pH of buffer in

composition of organic solvent), all analytes are adequately resolved and elution orders remained unchanged. RRT

samples are found to be between 85.0 to 115.0 for all

e deliberately altered and

are recorded. In all the

buffer in mobile phase and

composition of organic solvent), all analytes are adequately resolved and elution orders remained unchanged. RRT

of all the known impurities for all deliberately varied conditions along with original conditions are summarized in

Table 2.2.10.

4.4.6. Solution stability and mobile phase stability

The stability of diluted standard solution is estimated against freshly prepared standard and found that

solution is stable up to 2 days, as the difference in assay is found to be less than 1.0% up to 2 days. The results from

test solution stability confirmed that the solution has to be injected freshly for impurities quantification as it is not

stable at bench top even for one day. The variability in the estimation of RAB impurities is within ± 0.04% during

mobile phase stability experiments. The results from mobile phase stability experiments confirmed that mobile

phases are stable up to 48 hours for impurities quantification analysis. The results are summarized in Table 2.2.11.

Table 2.2.9 Recovery results of RAB impurities in RAB tablets

Amount

spikeda

%Recoveryb

Imp. A Imp. B Imp. C Imp. D Imp. E Imp. F Imp. I Imp. II Imp. III

LOQ 97.5±0.7 92.1±0.9 102.1±1.1 95.2±3.1 101.2±1.2 91.3±0.4 103.9±0.8 99.1±1.6 94.6±1.3

25% 98.6±0.0 94.8±0.5 101.3±0.4 99.5±1.2 99.7±1.7 104.7±0.0 106.1±0.4 100.6±0.5 100.5±0.5

50% 106.2±0.6 90.3±1.3 93.7±0.5 99.0±0.2 97.9±0.2 97.9±0.8 103.3±0.2 103.7±0.2 91.5±0.7

100% 101.5±0.4 95.0±0.2 101.3±0.2 92.2±0.5 104.9±0.1 90.7±0.1 104.5±0.1 102.3±0.4 92.4±0.2

a All impurities of RAB individually spiked on test preparation at 0.5 % level

b Mean ± % RSD for three determinations

150% 99.6±0.1 93.8±0.5 101.8±0.3 94.8±0.4 100.9±0.2 88.3±2.7 105.0±0.3 101.0±0.5 92.8±0.8

200% 92.1±0.1 94.2±0.6 101.0±0.2 94.9±0.8 99.1±0.4 88.2±1.8 104.5±0.6 100.5±0.9 95.3±0.8

Impurity

Name

RRT’s of the impurities

As per

the

method

condition

s

Flow rate Column

temperature

pH of the

buffer

Acetonitrile composition

Mobile Phase A Mobile Phase B

0.35

mL/min

0.45

mL/min 20°C 30°C 5.8 6.2 90% 110% 90% 110%

Imp-A 0.09 0.09 0.08 0.09 0.08 0.08 0.08 0.09 0.09 0.08 0.09

Imp-B 0.68 0.68 0.71 0.68 0.69 0.68 0.68 0.67 0.69 0.66 0.68

Imp-C 0.98 0.98 0.97 0.97 0.97 0.98 0.97 0.98 0.98 0.98 0.98

Imp-D 0.76 0.75 0.78 0.76 0.76 0.75 0.75 0.75 0.77 0.74 0.76

Table 2.2.10 Results of Robustness study

Table 2.2.11 Results of mobile phase stability

4.4.7. Results of specificity studies

All the placebo and stressed sample solutions are injected into the UPLC system with

photodiode array detector as per the described chromatographic conditions. Chromatograms of

placebo solutions have shown no peaks at the retention time of RAB and its impurities. This

indicates that the excipients used in the formulation do not interfere in estimation of impurities in

RAB tablets.

Significant degradation is observed in acid hydrolysis, thermal stress, water hydrolysis and oxidative

conditions. Degradation is minimal in base hydrolysis, light stress and humidity stress conditions. All degradant

peaks are well resolved from RAB peak in the chromatograms of all stressed samples. The chromatograms of the

stressed samples are evaluated for peak purity of RAB using Empower software. For all forced degradation samples,

purity angle for RAB peak is found to be less than purity threshold. This indicates that there are no hidden peaks in

Imp-E 1.13 1.11 1.15 1.12 1.12 1.12 1.12 1.09 1.15 1.09 1.12

Imp-F 1.36 1.32 1.45 1.34 1.36 1.33 1.35 1.28 1.43 1.29 1.34

Imp-I 0.10 0.11 0.09 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10

Imp-II 0.18 0.18 0.16 0.17 0.17 0.17 0.17 0.18 0.17 0.17 0.18

Imp-III 0.31 0.32 0.26 0.30 0.29 0.30 0.30 0.31 0.30 0.29

0.31

% of impurities

Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F Imp-1 Imp-2 Imp-3

Initial 0.5064

0.4709 0.5264 0.5024 0.5185 0.5188 0.5034 0.4730 0.4928

After 48 h 0.5113

0.4954 0.5354 0.4781 0.4907 0.5228 0.5053 0.4763 0.5197

Difference

from Initial 0.005 0.025 0.009 0.024 0.028 0.004 0.002 0.003 0.027

main analyte peaks of RAB. The % of degradation observed during stress study is summarised in Table 2.2.12. The

chromatogram and purity plots of all stress samples are shown in Fig 2.2.20 to 2.2. 27.

Table 2.2.12 Summary of forced degradation studies

Stress condition % degradation Purity

Angle

Purity

Threshold

Treated with 0.1N HCl for 15 min at 60°C 6.9 0.021 1.020

Treated with 0.1N NaOH for 30min at 60°C 1.4 0.052 1.012

Treated with 3%H2O2 for 5min at 60°C 10.5 0.026 1.048

Exposed to Heat for 105°C for 6 hrs 8.6 0.030 1.009

Treated with purified water for 30min at 60°C 5.0 0.032 0.264

Exposed to UV stress (200 W h/ m2 of UV light) 1.0 0.043 1.017

Exposed to Visible stress (1200 K Lux of visible light) 1.3 0.038 1.024

Exposed to humidity at 25 °C, 90% RH for 7 days 1.3 0.044 1.010

Fig 2.2.20 Chromatogram and purity plots of acid stress sample

Fig 2.2.21 Chromatogram and purity plots of base stress sample

Fig 2.2.22 Chromatogram and purity plot of peroxide stress sample

Fig 2.2.23 Chromatogram and purity plot of water stress sample

Fig 2.2.24 Chromatogram and purity plot of UV stress sample

Fig 2.2.25 Chromatogram and purity plot of visible stress sample

Fig 2.2.26 Chromatogram and purity plot of heat stress sample

Fig 2.2.27 Chromatogram and purity plot of humidity stress sample

5.0. Conclusions

The isolation, purification and characterization of the three unknown impurities (RRT

0.10, 0.18 and 0.31) that are observed to be increasing during the accelerated stability studies

in RAB tablets are identified. The chemical names are 2-Amino-1H-benzimidazole for 0.10 RRT

impurity, 1H-Benzimidazol-2-ol for 0.18 RRT impurity and 2-Benzimidazolethiol for 0.31 RRT

impurity.

The RP-LC method developed for impurity determination in RAB tablets is precise, accurate, sensitive,

specific, robust and rugged. The test method is validated as per International Conference on Harmonization. The

behavior of RAB under various stress conditions is studied. All degradation products and process impurities are well

separated from each other and from RAB. This demonstrates the stability- indicating power of the method. The

information presented in this study is very useful for quality monitoring of RAB tablets and can be used to check

drug product quality during stability studies.

References

1. Shigeru S, Norihiro U, Shuhei M, Katsuya T, Seiichiro N, Makoto O, Naoyuki S, Toshihiko K, Masatoshi F,

Preparation of 2-[(4-alkoxypyrid-2-

yl)methylthio]benzimidazoles, -benzothiazolels, and -benzoxazoles, Eur. Pat.

Appl. EP 268,956 [CA 110:23889a].

2. Bart J, Hahne W, rabeprazole based therapy in Helicobacter pylori eradication, Aliment. Pharmacol. Ther.

2002, 16, 31–33.

3. Nudelman NS, Estabilidad de Medicamentos, El Ateneo, Buenos Aires.

1975.

4. Kommanaboyina B, Rhodes CT, Trends in stability testing, with emphasis on stability during distribution

and storage, Drug Dev. Ind. Pharm. 1999, 25, 857–868

5. Cassia V Garcia, Norma S Nudelman, Martin Steppe, Elfrides E S Schapoval, Structural elucidation of

rabeprazole sodium photodegradation products, J. Pharm. Biomed. Anal. 2008, 46, 88 - 93.

6. Janssen-Cilag, Pariet® –Rabeprazole. 1999.

7. Mathew M, Das Gupta V, Bailey R, Stability of omeprazole solutions at various pH values as determined

by high-performance liquid chromatography, Drug Dev. Ind. Pharm. 1995, 21, 965–971.

8. Radi A, Adsorptive stripping square-wave voltammetric study of the degradation of lansoprazole in

aqueous solutions, Microchem. J. 2002, 73, 349–354.

9. Qaisi A, Tutunji M, Tutunji L, Acid Decomposition of Omeprazole in the Absence of Thiol: A Differential

Pulse Polarographic Study at the Static Mercury Drop Electrode (SMDE), J. Pharm. Sci. 2006, 95, 384–

391.

10. Vadas EB, in: Genaro AR (Ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott

Williams &Wilkins, Philadelphia. 2000, 980–985.

11. Garcia CV, Nudelman NS, Steppe M, Schapoval EES, Structural elucidation of rabeprazole sodium photo

degradation products, J. Pharm. Biomed. Anal. 2008, 46, 88–93.

12. Reddy GM, Bhaskar BV, Reddy PP, Sudhakar P, Babu JM, Vyas K, Reddy PR, Mukkanti K, Identification and

characterization of potential impurities of rabeprazole sodium, J. Pharm. Biomed. Anal. 2007, 43, 1262–

1269.

13. Pingili RR, Jambula MR, Ganta MR, Sajja E, Sundaram V, Boluggdu VB, Identification

and synthesis of potential impurities of rabeprazole sodium, Pharmazie. 2005, 60, 814–

818.

14. Reddy GM, Mukkanti K, Bhaskar BV, Reddy PP, Synthesis of metabolites and related

substances of rabeprazole, an anti-ulcerative drug, Synth. Commun. 2009, 39, 278–290.

15. Reddy PR, Himabindu V, Jayadeepkumar L, Reddy GM, Kumar JV, Reddy, An

improved process for the production of rabeprazole sodium substantially free from

impurities, Org. Process Res.Develop. 2009, 13, 896–899.

16. Reguri Buchireddy, Khagga Mukkanti, Polisetty Srinivasulu, Koduri SV Srinivas, A Validated, Stability-

Indicating, LC Method for Rabeprazole Sodium, Chromatographia. 2008, 68, 275-280

17. Koduri SV Srinivas, Reguri Buchireddy, Khagga Mukkanti, Polisetty Srinivasulu, A Validated RP-

LC Method for Advanced Intermediate of Rabeprazole Sodium, Chromatographia. 2009, 69, 381-

384

18. Shan Ren, Mi-Jin Park, Hongkee Sah, Beom-Jin Lee, Effect of pharmaceutical excipients on aqueous

stability of rabeprazole sodium, Int. J. Pharm. 2008, 350, 197-204

19. Nafisur Rahman, Zehra Bano, Syed Najmul Hejaz Azmi, Quantitative Analysis of Rabeprazole Sodium in

Commercial Dosage Forms by Spectrophotometry, Chem. Pharm. Bull, 2008, 56, 995-1001

20. Sreenivasa Rao P, Uttam Kumar Ray, Badarinadh Gupta P, Srinivasa Rao DVN, Aminul Islam, Pradeep Rajput,

Mukkanti K, Identification, isolation and characterization of new impurity in rabeprazole sodium, J.

Pharm. Biomed. Anal. 2010, 52, 620–624

21. Vasu Dev R, Sai Uday Kiran G, Venkata Subbaiah B, Suresh Babu B, Moses Babu J, Dubey PK, Vyas K,

Identification of degradation products in stressed tablets of Rabeprazole sodium by HPLC-hyphenated

techniques, Magn Reson Chem. 2009, 47, 443-448.

22. ICH. Harmonized Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology Q2(R1).

2005.