Chapter VII

175

7.1 Introduction

Drug profile of Pantoprazole

Structure

NH

N

S

N

O O

OF

FO

CAS Registry Number 102625-70-7

Chemical Name 6-(difluoromethoxy)-2-[(3, 4-dimethoxypyridin -2-

yl) methylsulfinyl]-1H-benzo[d]imidazole

Molecular Formulae C16H15F2N3O4S

Molecular Weight 383.37

Appearance Yellowish to off-white powder

Solubility Freely soluble in water

pKa 3.9

Therapeutic Category Proton pump inhibitor

Brand Name Protonix, Protonix IV

Pantoprazole, 5-(difluoromethoxy)-2-[[(3, 4-dimethoxy-2-pyridinyl)

methyl] sulfinyl]-1H-benzimidazole is an oral pharmaceutically active

compound having promising anti-ulcer activity [1] and belongs to the class of

2-[[(2-pyridyl) methyl] sulfinyl]- 1H-benzimidazoles. [(Pyridylmethyl)

sulfinyl] benzimidazoles (PSBs) have proved to be highly active inhibitors of

the gastric (H+, K+)-ATPase both in vitro and in vivo with high and long

Chapter VII

176

lasting antisecretory activity [2, 3]. In general these classes of compounds were

used for the prevention and treatment of gastric acid related diseases [4]. In the

literature few methods found for the preparation of Pantoprazole [5]. Some

spectrophotometric methods for the determination of Pantoprazole sodium

sesquihydrate with the combination of other drugs were described earlier [6–

10]. However, very little information was available for the determination of its

forced degradation impurities. In the synthesis of PPS, 2-(chloromethyl)-3, 4-

dimethoxy pyridine hydrochloride (CDP) is a key raw material and dimethyl

sulphate (DMS) is an important reagent. Identification and determination of

these two impurities in PPS is essential because CDP is toxic and DMS is

genotoxic [11] in nature. As per the regulatory guidelines [12], a threshold of

toxicological concern (TTC) value of 1.5 µg day-1

intake of a toxic impurity is

permitted. The permitted quantity in ppm is the ratio of TTC in microgram day-

1 and dose in gram day

-1. Since 40 mg of PPS is administered per day [13] in

the form of tablets (20, 40 mg with the trade name Pantin), the estimated

permissible quantity of these impurities is 37.5 ppm per day. In the literature

one GCMS method was found for the identification and determination of these

two impurities in PPS [14] and also the reported methods [15-17] for the

determination of Pantoprazole were found. Further, a low cost RP-LC method

was also developed for CDP alone, since DMS does not have UV absorbance.

Pantoprazole is a proton pump inhibitor drug used for short-term

treatment of erosion and ulceration of the esophagus caused by gastro

esophageal reflux disease. Initial treatment is generally of eight weeks duration,

after which another eight week course of treatment may be considered if

necessary. It can be used as a maintenance therapy for long term use after

initial response is obtained. This medication may affect the results of certain

lab tests, such as drug screenings (Pantoprazole can cause a false positive for

THC, the psychoactive component of cannabis). The active ingredient in

Protonix (Pantoprazole sodium) delayed-release tablets is a substituted

benzimidazole, sodium 5-(difluoromethoxy)-2-[[(3, 4-dimethoxy-2-pyridinyl)

Chapter VII

177

methyl] sulfinyl]-1 H -benzimidazole sesquihydrate, a compound that inhibits

gastric acid secretion. Safety and efficacy of pharmaceuticals are two

fundamental issues of importance in drug therapy. Instability of

pharmaceuticals can cause a change in physical, chemical, pharmacological and

toxicological properties of the active pharmaceutical ingredients (API), thereby

affecting its safety and efficacy. Hence, the pharmacists should take cognizance

of various factors such as drug stability, possible degradation products,

mechanisms and routes of degradation and potential interactions with

excipients utilized in the formulation to ensure the delivery of their therapeutic

values to patients. In order to assess the stability of a drug product, one needs

an appropriate analytical methodology, so called the stability indicating

methods which allow accurate and precise quantitation of the drug, its

degradation products and interaction products, if any. In recent times, the

development of stability-indicating assays has increased enormously [18–20],

using the approach of stress testing as outlined in the International Conference

on Harmonization (ICH) guideline Q1AR2 [21] and even this approach is

being extended to drug combinations [22–24]. This ICH guideline requires that

stress testing on API and drug products should be carried out to establish their

inherent stability characteristics which should include the effect of temperature,

humidity, light, oxidizing agents as well as susceptibility across a wide range of

pH. However, there are no detailed regulatory guidelines that direct how stress

testing is to be done and hence stress testing has evolved into an ‘‘artful

science’’ that is highly dependent on the experience of the pharmaceutical

industries or the individuals directing the studies [25]. The knowledge gained

from stress testing can be useful for a) the development of stable formulation

and appropriate packaging design, b) controlling of manufacturing and

processing parameters, c) identification and isolation of toxic degradents during

API synthesis, d) recommendation of appropriate storage conditions and shelf-

life determination and e) designing and interpreting environmental studies, as

the degradation of the drug in the environment will often be similar to

Chapter VII

178

degradation observed during stress-testing studies. It is also recommended that

analysis of stability samples should be done through the use of a validated

stability-indicating testing method.

The inhibition of the gastric proton pump or H+/K+-ATPase, suppresses

gastric acid secretion and hence hyperacidity can be controlled by Pantoprazole

[26]. Domperidone, 5-chloro-1-[1-[3- (2-oxo-2, 3-dihydro-1H-benzimidazol-1-

yl) propyl]-piperidin-4-yl]-1, 3-dihydro-2 H benzimidazol- 2-one acts by

selectively antagonizing the peripheral dopaminergic D2 receptors in the

gastrointestinal wall, thereby enhancing gastrointestinal peristalsis and motility

and increasing lower esophageal sphincter tone. This increased gastrointestinal

motility can facilitates the movement of acid contents further down in the

intestine preventing reflux esophagitis and thereby controlling nausea and

vomiting [27]. Thus, the pharmacology of Pantoprazole and Domperidone

corroborates their use in combined dosage form to treat various gastro

intestinal disorders in particular for hyperacidity frequently associated with

gastro intestinal dysmotility. Combination drug products of Pantoprazole and

Domperidone are hence widely marketed and successfully used in the treatment

of gastro esophageal reflux disease and non ulcer dyspepsia. Several HPLC

methods have been cited in the literature for the estimation of proton pump

hibitors Pantoprazole [28–31] and Domperidone [32–35] individually and to

our knowledge no analytical method for the simultaneous determination of the

two drugs in dosage forms has been published. Recently one HPLC method

found for the routine quality control analysis of Pantoprazole and Domperidone

simultaneously from tablets and capsule dosage forms [36]. The method gave

acceptable results for fresh quality control samples, but gave overestimation

during analysis of stability samples and aged products, as it lacks assay

specificity in presence of their degradation products. Further, no stability-

indicating method has been reported in literature for simultaneous

determination of Pantoprazole and Domperidone in presence of their

degradents. Different analytical methods are reported in the literature for the

Chapter VII

179

assay of lansoprazole, Omeprazole and Pantoprazole in dosage forms and in

biological fluids including spectrophotometry [37-44]. Fluorimetry [44], TLC

[44, 45], HPTLC [46-48], HPLC [49-55], capillary electrophoresis [56] and

polarography [57].

Hence in this article we have focused on the development of HPLC

method for the analysis of Pantoprazole and simultaneously transfer this

method to LCMS for the identification of degradation products formed on

stress condition. The proton pump inhibitors are acid sensitive and gets

degraded in very short span of time hence it is very necessary to characterise

and identify the degradation products. The reported methods only give the

information about potential impurity and process related impurity and none of

the method is reported for the characterisation of degradation products. The

developed method can be used for qualitative as well as quantitative analysis of

Pantoprazole and also for the characterisation of degradation products. The

structures of Pantoprazole and degradation products formed on oxidation are as

shown in figure 7.5.1.

7.2. Experimental:

7.2.1 Material and Reagents

The HPLC grade solvents acetonitrile and methanol, AR grade sodium

hydroxide, ortho phosphoric acid, and ammonium formate buffer were

procured from Qualigens fine chemicals, Mumbai, India. Hydrochloric acid

and hydrogen peroxide were purchased from Merck (Darmstadt, Germany).

Milli-Q water was used throughout the experiment.

7.2.2 Equipments

The analysis of Pantoprazole has been done on HPLC system (LC2010,

Shimadzu Corporation, Kyoto, Japan) consisted of low-pressure gradient

quaternary pump, auto sampler, column oven and photo diode array detector

(SPD M20A). LCsolution software was used for data acquisition. The

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180

separation of impurities from drug was achieved using YMC C18 column (150

mm X 4.6 mm, 5 µm).

Mass spectroscopic analysis was performed using LCMS-2010 equipped

with electrospray ionization interface (Shimadzu Corporation, Kyoto, Japan).

The data were collected and processed using LCMSsolution software.

7.2.3 Chromatographic conditions

Chromatographic separation was achieved using YMC C18 column (150

mm × 4.6 mm) with acetonitrile-ammonium formate (10 mM) 35:65 (v/v) as a

mobile phase at flow rate of 1.2 mL min-1

. Mobile phase was filtered through

0.45 µm filter and degassed for 10 min. Column oven temperature was

maintained at 30 °C and quantitation was achieved at 288 nm on the basis of

peak area. Injection volume was 10 µL. Standard and test solutions were

prepared with mobile phase.

An LCMS-2010 single quadrupole mass spectrometer was interfaced

with electrospray ionization (ESI) probe. The temperatures were maintained at

250, 250 and 200 °C for the probe, CDL and block respectively. The voltages

were set at 4.5 kV, -30 V, 25 V, 150 V and 1.6 kV for the probe, CDL, Q-array

1, 2, 3 bias, Q-array radio frequency and detector respectively. The flow rate of

nebulizer gas and dried gas were set at 1.5 L min-1

.

7.2.4 Sample Preparation

Stock solution (500 µg mL-1

) was prepared by dissolving 100 mg Pantoprazole

in minimum amount of methanol, kept for sonication up to 15 min and diluted

to 100 mL using volumetric flask. Then standard solutions were prepared by

dilution of stock solutions using mobile phase within the range 1-100 µg mL-1

.

Triplicate 10 µL injection of each solution were chromatographed. Average

peak areas were plotted against concentration to obtain the calibration plot.

Chapter VII

181

7.2.5 Validation of the Method

The developed chromatographic method was validated for linearity, range,

accuracy, precision, selectivity, sensitivity, ruggedness, robustness and system

suitability [58–60].

7.2.6 Linearity

Linearity test solutions for developed method were prepared from stock

solutions at six concentrations levels of 1, 5, 10, 25, 50 and 100 µg mL-1

.

Standard curve was obtained by plotting peak area against concentrations for

evaluation of linearity by linear regression analysis using least square method.

An excellent correlation existed between the peak area and concentration of

Pantoprazole.

7.2.7 Limit of detection (LOD) and limit of quantitation (LOQ)

The LOD and LOQ for Pantoprazole can be estimated by either signal to noise

ratio of 3:1 and 10:1 respectively by injecting a series of dilute solutions with

known concentrations or from linear regression plot using intercept and slop

values. LOD and LOQ can be calculated by using formulae’s 3.3(σ/slope) and

10(σ/slope) respectively. The precision study also carried out at LOQ level by

injecting six individual injections of sample solution.

7.2.8 Specificity

Specificity is the ability of method to measure analyte response in the presence

of its potential impurities. Specificity of developed HPLC method was carried

out in presence of its degradation products formed on hydrolysis, oxidation,

heat and photolysis. Stress studies were performed for Pantoprazole bulk drug

to provide an indication of stability indicating property and specificity of

proposed method. Peak purity test was carried out for Pantoprazole peak by

using photo diode array (PDA) detector in stress samples.

Chapter VII

182

7.2.9 Robustness

To determine the robustness of the developed method, experimental conditions

were purposely altered and resolution of Pantoprazole from its degradation

products was evaluated. The flow rate of the mobile phase was 1.2 mL min-1

.

To study the effect of flow rate on the resolution, it was changed by 0.2 units

from 1 to 1.4 mL min-1

while the other mobile phase composition was kept

constant. The effect of the percent organic strength on resolution was studied

by varying acetonitrile from ±2 % while other mobile phase components were

held constants. The effect of temperature on the resolution was studied at 25 °C

and 35 °C while the other mobile phase components were held constant.

7.2.10 Solution Stability and Mobile Phase Stability

Solutions of the sample prepared from stock solution and diluted by mobile

phase were kept in tightly capped volumetric flask for 48 hours at room

temperature and analysed by preparing the fresh mobile phase. Mobile phase

stability was checked by analysing the freshly prepared sample solutions at an

interval of 8, 24 and 48 hours by keeping the same mobile phase throughout

analysis.

7.2.11 Accuracy

Accuracy of developed method was evaluated in triplicate at three

concentration levels i.e. 25, 50 and 75 µg mL-1

in bulk drug sample. The

percentage recoveries were calculated from slope and y intercept on the

calibration curve.

7.3 Results and Discussion

7.3.1 Optimisation of Chromatographic Condition

Literature survey prevails that previously attempts have been made to

identify process related toxic impurities by GCMS and LCMS for the

Pantoprazole and also stability study for simultaneous determination of

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183

Domperidone and Pantoprazole. But there was not a single article found in

literature for identification of degradation products formed in oxidative

degradation. After revealing literature search we decided to develop such

method which will provide qualitative and quantitative analysis as well as

identification of degradation products collectively. For the development of

HPLC method we tried variety of buffers and columns. At the time of

optimisation of the chromatographic conditions we tried the phosphate,

ammonium formate, ammonium acetate, ammonium bicarbonate to achieve

better separation and symmetrical peak shape. For the proposed method we

selected the ammonium formate buffer and acetonitrile since phosphate is non

volatile buffer and we cannot go for LCMS with same buffer. In column

selection we tried various columns like YMC C18, Waters X-Bridge, Waters

Sunfire, Phenomenex Luna, cyano, Zorbax phenyl. Since YMC C18 column

gives better results and cost of the column is less hence we preferred for the

same. The results of system suitability are as shown in table 7.6.1. At the time

of study of effect of pH on retention and stability we observe that the

compound is sensitive to acidic pH which made us to work without ion pairing

reagents and acids. By using the phosphate buffer and waters X-bride, Sunfire

column, separation of impurities from drug was poor and we got better

resolution using ammonium formate and acetonitrile. Instead of acetonitrile we

tried the methanol as organic solvent to reduce the analysis cost but we got

broad peak shapes and tailing. Separation of all impurities formed in stress

condition was achieved using C18 YMC column (150 mm X 4.6 mm, 5µ) with

mobile phase ammonium formate and acetonitrile (65:35, v/v). The flow rate

was 1.2 ml min-1

and quantitation was achieved at 288 nm on the basis of peak

area. The chromatogram of Pantoprazole using ammonium formate buffer and

acetonitrile is as shown in figure 7.5.2. The UV spectrum and peak purity

profile is as shown in figure 7.5.3.

Chapter VII

184

7.3.2 Forced degradation study

Proton pump inhibitors are very sensitive to acid and undergo

immediately degradation on forced degradation in stress condition.

Degradation of Pantoprazole was performed with various stress conditions like

0.1 N HCl, 0.1 N NaOH, 3 % H2O2, heating at 60 °C and light at 254 nm in UV

chamber. Pantoprazole dissolved in mobile phase was kept in various stress

conditions by adding 0.1 N HCl, 0.1 N NaOH and 3 % H2O2 up to 8 hours for

monitoring the degradation. In case of photolysis Pantoprazole dissolved in

mobile phase was kept in UV chamber at 254 nm up to 8 hrs. Thermal

degradation was carried out by keeping the solid Pantoprazole in oven at 60 °C

up 8 hrs. Pantoprazole was found to be stable in base hydrolysis and thermally

but degradation occurs in acid hydrolysis and oxidation. The chromatogram of

Pantoprazole and total ion chromatograph (TIC) of mass on base hydrolysis is

as shown in figure 7.5.4. There is no interference of any impurity to drug as in

photo diode array (PDA) detector as well as mass TIC which confirms the

specificity of method. In acid hydrolysis Pantoprazole gradually undergoes

degradation and converts into seven impurities as shown in figure 7.5.5. On

oxidation with 3 % H2O2 Pantoprazole undergoes degradation and found to be

converts in to two impurities. The impurities formed in stress conditions were

successfully separated and identified by mass spectroscopy to elucidate

probable structure of the degradation product. The non interference of forced

degradation product with Pantoprazole confirms specificity of developed

method. The detail results of forced degradation study are as shown in table

7.6.2.

7.3.3 Limit of detection (LOD) and limit of quantification (LOQ)

In accordance with International Conference on Harmonisation (ICH)

recommendations, the approach based on the standard deviations (SD) of the

response and the slope of the calibration plot was used for determinations of

Chapter VII

185

limit of detection and limit of quantification. The calculated values of LOD and

LOQ are 0.18 µg mL-1

and 0.49 µg mL-1

respectively.

7.3.4 Linearity

The linearity calibration plot was obtained on six points over the

calibration ranges tested i.e.1-50 µg mL-1

. The values of correlation coefficient

and slope were 0.9998 and 23403 respectively.

7.3.5 Accuracy

To obtain the accuracy of the method, recovery experiments were

carried out at three concentration levels i.e. 10, 25 and 50 µg mL-1

. The

percentage recovery of Pantoprazole in bulk drug samples was ranged from

99.0 to 100.3. From this result it was confirmed that the method is remarkably

accurate. The recovery results are summarised in table 7.6.3.

7.3.6 Precision

The relative standard deviation (RSD) of Pantoprazole during intra-day

study was found to be 0.65 % and inter day study was within 0.82 %. The

results confirm the repeatability of the method.

7.3.7 Robustness

In all the deliberate varied chromatographic conditions (flow rate,

percentage organic strength, column temperature), well resolution was

observed between Pantoprazole and its degradation product, illustrating the

robustness of method.

7.3.8 Identification of Degradation Product

Mass spectroscopy is the best analytical tool for fast characterisation of

new chemical entity, impurities in stability study and even in API bulk drug

samples along with the UV, IR and NMR tools. For the analysis of the

degradation product of Pantoprazole we preferred mass spectroscopy as one

Chapter VII

186

can analyse the sample within vary short period and with sample matrix.

Developed method was compatible with MS and same method was transferred

to LCMS for the determination of molecular weight of impurities. LCMS

method was successfully applied for identification of degradation product

formed on stress condition in a single run. The degraded samples of

Pantoprazole in various stress condition were diluted in mobile phase and

directly injected to LCMS for scanning of M/Z value in the range 50-800

Daltons. Pantoprazole ionises in the positive mode and shows the M+1 mass

peak at 384 in ESI ionisation source. Pantoprazole is unstable in acid

hydrolysis and gradually undergoes degradation to form seven impurities. In

oxidative degradation two polar impurities were formed and eluted earlier than

drug at retention time 2.5 minute and 3.0 minute respectively at LCMS as

shown in figure 7.5.6. The typical mass spectra is as shown in figure 7.5.7 and

the two impurities formed on oxidation have the same mass 400 (M+1) as

shown in figure 7.5.8. The impurities of same mass indicate the formation of

structural isomers and more 16 Dalton mass indicates the introduction of

oxygen atom to the structure of Pantoprazole. There is major possibility of

addition of oxygen atom at sulphoxide group to form sulphone and other

possibility is formation of n-oxide of Pantoprazole.

7.4 Conclusion

Developed and validated stability indicating liquid chromatographic

method has been carried out for analysis of Pantoprazole in presence of its

degradation products. Method is validated according to ICH guidelines and

further work of identification of degradation product can also be applicable in

impurity profile. The method is simple, rapid, accurate and there is no

interference of any impurity to Pantoprazole hence specific.

Chapter VII

187

7.5 Figures

7.5.1 Figure: Structures of Pantoprazole and degradation products formed on

oxidation

NH

N

S

N

O O

OF

FO

N

NH

N

S

OF

FO

O

OO

NH

N

S

N

O O

OF

FO

O

Pantoprazole

Pantoprazole sulphone

Pantoprazole N-Oxide

Chapter VII

188

7.5.2 Figure: Typical HPLC chromatograph of Pantoprazole

Chapter VII

189

7.5.3 Figure: UV spectra and peak purity profile of Pantoprazole.

Chapter VII

190

7.5.4 Figure: LCMS chromatograph and TIC of Pantoprazole

Chapter VII

191

7.5.5 Figure: HPLC chromatograph of degradation products formed on acid

hydrolysis

Chapter VII

192

7.5.6 Figure: LCMS chromatograph and TIC of Oxidation of Pantoprazole

Chapter VII

193

7.5.7 Mass spectra of Pantoprazole

7.5.8 Mass spectra of impurity formed on oxidation of Pantoprazole

Chapter VII

194

7.6 Tables

Table 7.6.1: System- Suitability Report

Compound

(n=3)

tR RS N T

Pantoprazole 3.4 ± 0.1 3.3 13504 1.2

n = number of determinations.

tR = Retention time in minutes.

Rs= USP Resolution.

T = USP tailing factor.

N = number of theoretical plates.

Chapter VII

195

Table 7.6.2: Summary of forced degradation study of Pantoprazole

Stress condition Time Assay

(%)

Mass balance

(Assay +Imp) %

Remarks

Acid hydrolysis

(0.1 N HCl)

1 h 65.2 99.98 Degrades into

seven

impurity

Base hydrolysis

(0.1 N NaOH)

8 h 99.98 99.98 No

Degradation

Oxidation

(3 % H2O2)

8 h 90 99.95 Degrades into

two impurity

Thermal

(60 °C)

48

days

99.99 99.99 No

degradation

UV (254 nm) 48 h 99.99 99.99 No

degradation

Chapter VII

196

Table 6.6.3: Recovery results of Pantoprazole

Added (µg) (n=3) Recovered (µg) % Recovery % R.S.D.

10.2 10.1 99.0 0.5

25.1 25.2 100.3 0.9

50.2 50.0 99.6 0.7

Where,

n- Number of determinations

Chapter VII

197

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