Section (i): Brief account of Dexlansoprazole

37

Section (i): Brief account of Dexlansoprazole

Dexlansoprazole (DLP) is the R-enantiomer of lansoprazole (a racemic mixture of the R- and

S-enantiomers). It is chemically (R)-(+)2-([3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-

yl]methylsulfinyl)-1H-benzimidazole. Its empirical formula is: C16H14F3N3O2S, with a

molecular weight of 369.36. The structural formula is:

DLP is a white to nearly white crystalline powder which melts with decomposition at

140°C. DLP is freely soluble in dimethylformamide, methanol, dichloromethane, ethanol, and

ethyl acetate; and soluble in acetonitrile; slightly soluble in ether; and very slightly soluble in

water; and practically insoluble in hexane. DLP is stable when exposed to light. DLP is more

stable in neutral and alkaline conditions than acidic conditions [1]. DLP exhibits

polymorphism. It is available as both crystalline and amorphous forms. Amorphous forms are

generally more unstable than crystalline forms.

DLP is marketed with a brand name ‘DEXILANT” (earlier known as KAPIDEX) [2].

DEXILANT is the first proton pump inhibitor (PPI) with a Dual Delayed Release (DDR)

formulation designed to provide two separate releases of medication upon oral administration.

The capsules contain DLP in a mixture of two types of enteric-coated granules with different

pH-dependent dissolution profiles. DEXILANT is available in two dosage strengths: 30 mg

and 60 mg, per capsule.

DEXILANT is a proton pump inhibitor that is marketed by Takeda Pharmaceuticals,

Japan. DLP was approved by the U.S. Food and Drug Administration (FDA) on January 30,

2009 [3]. DLP DDR capsules approved for use of once-daily, oral treatment of heartburn

http://en.wikipedia.org/wiki/Proton_pump_inhibitor

http://en.wikipedia.org/wiki/Takeda_Pharmaceuticals

http://en.wikipedia.org/wiki/Food_and_Drug_Administration_(United_States)

38

associated with symptomatic non-erosive gastro esophageal reflux disease (GERD), the

healing of erosive esophagitis (EE) and the maintenance of healed EE.

DEXILANT is based on a dual release technology, with the first quick release

producing a plasma peak concentration about one hour after application, and the second

retarded release producing another peak about four hours later [4]. DEXILANT works by

turning off many of the millions of tiny pumps in stomach that produce acid. Clinical studies

have shown that DEXILANT provides up to 24 hours of relief from heartburn due to acid

reflux disease. Studies also showed DEXILANT heals damage (erosions) to the esophagus

and keeps it from coming back.

DLP drug substance and drug product is not official any pharmacopeia. Lansoprazole

drug substance and drug product which is a recimic mixture of R and S enantiomers is official

in USP.

Literature survey revealed, LC-MS method have been reported for the quantitative

determination of DLP in human plasma [5]. Few methods are reported for the quantification

of lansoprazole and its impurities pharmaceutical preparations, biological fluids and in

combination with other actives, these includes, colorimetry using dyes [6], UV spectrometry

[7-9], HPLC [10-13] and chemo metric approach using HPLC [14] and TLC [15]. UPLC-

MS/TOF[16] and HPLC[17] methods are reported for assay of lansoprazole oral suspension.

A chiral LC method [18] reported for enantiomeric separation of DLP. Few papers are

reported the synthesis, isolation, identification and characterization of the some of the

impurities in lansoprazole [19-21]. So far no method is reported for determination of all 11

impurities of DLP. The reported assay methods of lansoprazole are not capable of quantifying

DLP without interference from the other impurities.

DLP is unstable when exposed to acid, base, peroxide and thermal degradations. Three

unknown impurities (degradation products) present at a level below 0.05% in the initial

samples of DLP capsules increased to a level of 0.5% in 3 M accelerated stability studies, i.e;

40◦C/75%RH. The structures of these compounds are not reported in literature.

This prompted the author to develop stability indicating HPLC methods for the assay

and impurities. The three degradation impurities are enriched and isolated by using reverse-

phase preparative liquid chromatography and characterized.

39

This chapter describes development and validation of a stability indicating methods

for assay and impurities along with the identification, isolation and characterization of three

unknown degradation products formed in DLP capsules during the stability studies. Though

some of the impurities and degradation products were reported in the literature, identification,

isolation and characterization of these degradation products is not reported to the best of our

knowledge.

Based LC-MS and NMR data the impurity I is characterized as 2-{(1-H-

Benzoimidazol-2-ylsulfanyl)-[1-methyl-2-2,2,2-trifluoro-ethoxy)-4a,5,9b-triaza-indeno[2,1-

a]inden-10-yl]-methyl}-3-methyl-pyridin-4-ol, with molecular formula C30H23F3N6O2S and

molecular mass 588.16 Impurity-II is characterized as 10-{(1-H-Benzoimidazol-2-

ylsulfanyl)-[3-methyl-4-(2,2,2-trifluoro-ethoxy)-pyridin-2-yl]-methyl}-1-methyl-2-(2,2,2-

trifluoro-ethoxy)- 4a,5,9b-triaza-indeno[2,1-a]indene with molecular formula C32H24F6N6O2S

and molecular mass 670.16. Impurity-III is characterized as 1-Methyl-2-(2,2,2-trifluoro-

ethoxy)-4a,5,9b-triaza-indeno[2,1-a]indene with molecular formula C16H12F3N3O and

molecular mass 319.09.

40

Section (ii): Stability Indicating HPLC Assay method for Dexlansoprazole Capsules.

This section reports the various aspects relating to the development and validation of

stability indicating HPLC method for assay of Dexlansoprazole (DLP) capsules.

1. Experimental

1.1. Chemicals

Samples of DLP API are received from process R&D, Dr Reddy’s Laboratories,

Hyderabad, India. DLP Capsules of 30 and 60 mg & all excipients are received from

formulation R&D, Dr Reddy’s Laboratories, Hyderabad, India. HPLC grade acetonitrile,

methanol, triethylamine, sodium hydroxide and potassium dihydrogen phosphate are supplied

by Merck, Darmstadt, Germany. High purity water is prepared by using Millipore Milli-Q

plus purification system.

1.2. Determination of appropriate UV wavelength

The suitable wavelength for the determination of DLP in diluent is identified by

scanning over the range 200–400 nm with a Shimadzu UV-160 (Shimadzu, Japan) double

beam spectrophotometer.

1.3. Instrumentation and chromatographic conditions

The M/s Waters HPLC System with a photo diode array detector is used for the

method development and force degradation studies .The data is monitored and processed

using Waters Empower Networking software. The HPLC system used for method validation

is waters HPLC system with diode array detector and Agilent 1100 series LC system with

variable wavelength detector (VWD). The data is monitored and processed by using waters

Empower Networking Software. The chromatographic column used is an Xbridge C-18,

20mm x 4.6 mm column, with 5µ particle size with a Hypersil BDS C18, 10 mm x 4.6 mm

guard column. The chromatographic condition follows a gradient program consisting of

mixture of buffer and methanol in the ratio of 90:10 (v/v) as mobile phase A and of mixture of

methanol and acetonitril in the ratio of 50:50 (v/v) as mobile phase B. The buffer is prepared

as 0.05 M KH2PO4 with 0.8% v/v triethylamine and finally pH is adjusted to 8.0. The gradient

programme is : Time/%mobile phase A:% Mobile phase B is 0.0/75:25, 3.0/55:45, 4.0/55:45,

4.5/20:80, 5.5/20:80, 6.0/75:25, 8.0/75:25. The flow rate of the mobile phase is 1.2 ml min-1

.

41

The column temperature is maintained at 30ºC and the detection wavelength is 285 nm. The

injection volume is 20µl.

1.4. Diluent:

0.1N NaOH and methanol in the ratio 75:25(v/v) is used as diluent.

1.5. Preparation of standard drug solution:

The stock solution of DLP standard (equivalent to 0.68 mg ml-1

of DLP) is

prepared in diluent. The working standard solution (0.068mg ml-1

of DLP for both 30 mg and

60 mg sample analysis purpose) is obtained by dilution of the stock solution in diluent. The

Specimen chromatograms of diluent and standard is shown in fig.2.2.1.

Fig 2.2.1: Specimen chromatogram of diluent and DLP standard 0.068mg ml-1

.

Blank

Standard

42

1.6. Test Preparation for pharmaceutical formulations:

Contents of ten capsules of DLP are emptied and weighed the enteric coated pellets in

the capsules. Enteric coated pellets equivalent to 600 mg of DLP is weighed and transferred

into a clean dry 1000mL volumetric flask for 60mg and 500 mL volumetric flask for 30mg.

0.1N NaOH is added upto 20% of total volume and, swirled to avoid aggregation and

sonicated with frequent intermediate shaking to disperse the pellets. Then diluent is added to

make 60% total volume and sonicated for 20 min with intermediate shaking. The temperature

of the water in the sonicator bath is maintained between 20 ºC and 25ºC to avoid heating up

the solution during soniation. Then volume is made up to total volume and mixed well. The

concentration of this test stock solution is 0.6 mg of DLP per ml. The resulting solution is

centrifuged at 4000 rpm for 10 min. 5 ml of the centrifugate is then diluted to 50 ml in a

volumetric flask with diluent. The final concentration of this test preparation is 60 µg ml-1

of

DLP. Placebo sample is prepared in the same way by taking the placebo equivalent its weight

present in a test preparation .The Specimen chromatogram of placebo and test sample are

shown in fig.2.2.2.

Fig 2.2.2: Overlay chromatogram of placebo and DLP Capsules.

Placebo

Test

43

1.7. Specificity:

Regulatory guidances in ICH Q2A, Q2B, Q3B and FDA 21 CFR section 211, require the

development and validation of stability-indicating potency of assays. However, the current

guidance documents do not indicate detailed degradation conditions in stress testing. The

forced degradation conditions, stress agent concentration and time of stress, are found to

effect the % degradation. Preferably not more than 20% is recommended for active materials

to make the right assessment of stability indicating nature of the chromatographic methods.

The discovery of such stress conditions which can yield not more than 20% degradation is

based on experimental studies. Chromatographic runs of placebo solution and samples

subjected to force degradation are performed in order to provide an indication of the stability

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

base, neutral and oxidant media, moisture, heat 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 diluted with diluent to get the working concentrations equivalent

to test preparation. The samples are analyzed against a freshly prepared control sample (with

no degradation treatment) and evaluated for peak purity by using photo diode array detector.

Specific conditions are described below.

1.7.1. Placebo (excipients) interference:

Placebo solutions are prepared by taking the weight of placebo approximately

equivalent to its weight in the sample as described in the test preparation for DLP capsules

dosage form.

1.7.2. Effect of acid hydrolysis :

300 mg of DLP pellets powder is treated with 100 ml of 1N HCl for 5 minutes on

bench top with continuous shaking. The resulting solution is neutralized and then solution is

prepared as per the test preparation to obtain a stock solution of 0.6 mg ml-1

. Then the test

stock is diluted with diluent to get the test preparation having final concentration of drug at

about 60 µg ml-1

.

44

1.7.3. Effect of base hydrolysis

300 mg of DLP pellets powder is treated with 100 ml of 2N NaOH at 50°C using a

heating water bath for 24 hours. The resulting stress solution is neutralized, equilibrated to

room temparature and then solution is prepared as per the test preparation to obtain a stock

solution of 0.6 mg ml-1

. Then the test stock is diluted with diluent to get the test preparation

having final concentration of drug at about 60 µg ml-1

.

1.7.4. Effect of neutral hydrolysis

300 mg of DLP pellets powder is treated with 100 ml water at 50°C using a heating

water bath for 24 hours. The resulting stress solution is equilibrated to room temperature and

then treated same as per the test preparation to obtain a stock solution of 0.6 mg ml-1

. Then

the test stock is diluted with diluent to get the test preparation having final concentration of

drug at about 60 µg ml-1

.

1.7.5. Effect of oxidation

300 mg of DLP pellets powder is treated with 100 ml of 10%H2O2 for 15 minutes on

bench top with continuous shaking. The resulting solution is neutralized and then solution is

prepared as per the test preparation to obtain a stock solution of 0.6 mg ml-1

. Then the test

stock is diluted with diluent to get the test preparation having final concentration of drug at

about 60 µg ml-1

.

1.7.4. Effect of moisture and heat

To evaluate the effect of moisture and heat, DLP pellets powder is distributed as thin

layer over two glass plates. One plate is then exposed to 25ºC/90% relative humidity for 9

days. Similarly DLP pellets powder in another plate is exposed in an oven at 105ºC for 1

hour. Then, both the samples are subjected to sample preparation using diluents as described

in test preparation.

1.7.5. Effect of UV and visible light

To study the photochemical stability of the drug product, the DLP pellets powder is

exposed to 1200 K Lux hours of visible light and 200 Watt hours/ meter2 of UV light by using

photo stability chamber. After exposure the samples are subjected to sample preparation using

diluents as described in test preparation.

45

1.8. Method validation

1.8.1. Precision

Precision (intra-day precision) of the assay method is evaluated by carrying out six

independent assays of test sample of DLP capsules against qualified standard. The % RSD of

six assays obtained is calculated.

The intermediate precision (inter-day precision) of the method is also evaluated

using two different HPLC systems and different HPLC columns in different days in the same

laboratory.

1.8.2. Linearity

Linearity study for assay method is made using six different concentration levels in

the range of about 4- 150 µg ml-1

of DLP (corresponding to 6.6 to 250% of assay of nominal

sample concentration of 60 µg ml-1

.). The

data of peak area versus concentration is subjected

to least-square regression analysis.

1.8.3. Accuracy

A study of recovery of DLP from spiked placebo is conducted. Samples are prepared by

mixing placebo with DLP API equivalent to about 20%, 50%, 70%, 100%, 120%, and 150%

of the assay of nominal sample concentration. Sample solutions are prepared in triplicate for

each spike level as described in the test preparation. The % recovery is then calculated.

1.8.4. Robustness

To determine the robustness of the developed method, experimental conditions are

purposely altered one after the other to estimate their effect. Five replicate injections of

standard solution are injected under each parameter change. The effect of flow rate, pH,

column temperature and organic phase composition in mobile phase (methanol in mobile

phase A and acetonitrile and methanol in mobile phase B) on the tailing factor of DLP peak

and the %RSD for peak areas of replicate injections of standard is studied at flow rates of 1.0

ml min-1

and 1.4 ml min-1

, at pH of 7.8 and 8.2, at column temperatures of 25ºC and 35ºC and

organic phase compositions in mobile phase at + 10% respectively.

46

1.8.5. Solution stability and mobile phase stability

The solution stability of DLP in the assay method is carried out by leaving solutions of

both the test preparation and reference standard preparation in tightly capped volumetric

flasks at room temperature for 48 hours. The same sample solutions is assayed after every 24

hours during the study period.

The mobile phase stability is also carried out by assaying freshly prepared sample

solutions against freshly prepared reference standard solutions at 24 hours interval for 48

hours. Mobile phase prepared is kept constant during the study period. The % RSD of assay

of DLP is calculated for the study period during mobile phase stability and solution stability

experiments.

2. Results and discussion

2.1. Determination of suitable wavelength

The UV spectrum of DLP recorded in the range 200-400 nm is illustrated in fig.2.2.3.

The spectrum indicates that 285 nm gives a good sensitivity for the assay.

Fig 2.2.3: UV Spectra of DLP.

47

2.2. Optimization of chromatographic conditions

The HPLC procedure is optimized with a view to develop a stability indicating

assay method. Pure drug and stressed samples are injected and run in different solvent

systems. Selection of mobile phase pH is done based on stability of DLP. Drug is found to be

not stable and peak area continuously decreases in mobile phase with pH less than 7.0. Due to

problem in stability of standard and test solutions, after several experiments the diluent is

finalized as 0.1N NaoH and methanol in the ratio of 75 : 25 v/v. The pH of the diluents is

about 11. Normal silica based column stationary phases not workable at pH 8.0. Most of the

C18 column peak shape goes quickly as the silica starts dissolving at pH 8.0. Hence a choice

is made to work with hybrid silica based columns. After screening of columns which can

withstand for higher pH conditions, a choice of the column is made to waters Xbridge C18

column. A guard column is also chosen with C18 stationary phase in order to increase the life

of Xbridge column. DLP is prone to degradation upon stability, it generates number of

impurities during upon storage. As several late eluting non-polar degradants are possible to be

present in the sample, isocratic methods are found to be not feasible due to high run times in

order to elute all the degradants. As in the pharmaceutical industry, lot of potency analysis is

needed to check the quality of the formulated products, a study is conducted to get the

stability indicating method with shorter runtime. A number of experiments are done with

different lengths of the columns and different mobile phase compositions and with different

gradient programmes to separate all the degradants from DLP peak within short time.

Eventually, satisfactory peak shape and satisfactory separation is achieved using a 20 mm x

4.6 mm, Xbridge C18 column with 5 µm with mobile phase A consisting of mixture of buffer

and methanol in the ratio of 90:10 (v/v) as mobile phase A and of mixture of methanol and

acetonitril in the ratio of 50:50 (v/v) as mobile phase B with a gradient programme of :

Time/%mobile phase A:% Mobile phase B is 0.0/75:25, 3.0/55:45, 4.0/55:45, 4.5/20:80,

5.5/20:80, 6.0/75:25, 8.0/75:25. The optimum flow rate and column temperatures are found

to be 1.2 ml min-1

and 30ºC respectively.

48


2.3.1. Precision

Method repeatability (intra-day precision) is evaluated by assaying six samples,

prepared as described in the test preparation. The mean % assay and % RSD for assay values

are found to be 100.5 and 0.2 respectively. These are well with in the acceptance criteria i.e.

mean % assay between 97.0 -103.0 and RSD not more than 2.0 %. The intermediate precision

(inter day precision) is performed by assaying six samples on different HPLC systems and

different HPLC columns in different days as described in the sample preparation. The mean %

assay and % RSD for assay values are found to be 100.1 and 0.2 respectively. The result

shows good precision of the method (table 2.2.1).

Table 2.2.1: Results of precision of test method

Sample No. Assay of DLP as % of labeled amount

Intra-day precision Inter-day precision

1 100.7 99.8

2 100.4 99.9

3 100.7 100.2

4 100.3 100.2

5 100.7 100.3

6 100.4 100.4

Mean 100.5 100.1

RSD 0.2 % 0.2 %

2.3.2. LOQ and LOD

The LOQ and LOD are determined based on signal-to-noise ratios at analytical

responses of 10 and 3 times the background noise, respectively. The LOQ is found to be

0.14 μg ml -1

with a resultant %R.S.D. of 0.6 (n = 5). The LOD is found to be 0.028 μg ml.-1

2.3.3. Linearity

A linear calibration plot for assay of DLP is obtained over the calibration range 4-150

µg ml-1

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

fig.2.2.4 indicates that an excellent correlation exists between the peak area and concentration

of the analyte.

49

Fig.2.2.4: Linearity of detector response for DLP.

2.3.4. Accuracy

The percentage recovery of DLP in pharmaceutical dosage form of capsules is shown

in table 2.2.2 ranged from 98.2 to 100.7 and indicates high accuracy of the method.

Table 2.2.2: Recovery results of DLP in DLP capsules

Sample No.

level “mg”

added “mg” found

% Recovery

Mean % Recovery

1 20% 120.60 121.46 100.7

100.6 2 20% 120.60 121.23 100.5

3 20% 120.60 121.36 100.6

4 50% 301.50 296.04 98.2

98.4 5 50% 301.50 296.64 98.4

6 50% 301.50 297.48 98.7

7 70% 422.10 418.82 99.2

99.2 8 70% 422.10 418.66 99.2

9 70% 422.10 419.00 99.3

10 100% 603.00 604.23 100.2

100.1 11 100% 603.00 602.46 99.9

12 100% 603.00 604.14 100.2

13 120% 723.60 725.16 100.2

100.3 14 120% 723.60 725.38 100.2

15 120% 723.60 725.81 100.3

16 150% 904.50 907.72 100.4

100.4 17 150% 904.50 907.79 100.4

18 150% 904.50 907.77 100.4

50

2.3.5. Robustness

In all the deliberately varied chromatographic conditions studied (flow rate, column

temperature, ratio of acetonitrile and methanol composition in mobile phase), the tailing

factor and the % RSD for the DLP peak area for five replicate injections of standard is found

to be within the acceptable limit of not more than 2.0%, illustrating the robustness of the

method (table 2.2.3).

Table2.2.3: Results of Robustness study

Parameter Observed value

Variation Tailing factor %RSD

1.Flow rate 0.8 ml min-1 1.1 0.04

1.0 ml min-1 1.0 0.04

1.2 ml min-1 1.0 0.1

2.Column temperature 25ºC 1.2 0.1

30ºC 1.0 0.04

35ºC 1.1 0.1

3.Mobile phase A

(for methanol variation)

90% 1.2 0.1

100% 1.0 0.04

110% 1.1 0.0

4. Mobile phase B

(for acetonitrile variation)

90% 1.1 0.2

100% 1.0 0.04

110% 1.1 0.3

5. Mobile phase B

(for methanol variation)

90% 1.2 0.3

100% 1.0 0.04

110% 1.1 0.1

6. pH 7.8 1.1 0.1

8.0 1.0 0.04

8.2 1.1 0.1

51


The difference in % assay of DLP test and standard preparations upon storage on

bench top is found to be less than 1.2% up to 48 hours. Mobile phase stability experiments

showed that tailing factor and % RSD are less than 1.2 and 0.3% respectively up to 48 hours.

The solution stability and mobile phase stability experimental data confirms that standard and

test preparations and mobile phase used during assay determination are stable up to 48 hours.

2.3.7. Results of specificity studies

All the placebo and stressed samples prepared are injected into the HPLC system

with photodiode array detector as per the described chromatographic conditions.

Chromatograms of placebo solutions have shown no peaks at the retention time of DLP.

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

in capsules formulation dosage form.

Degradation is not found to be significant when exposed to light and humidity. In acid

hydrolysis, base hydrolysis, water hydrolysis, thermal stress and oxidative studies, significant

degradation is observed. All degradant peaks are well resolved from DLP peak in the

chromatograms of all stressed samples. The chromatograms of the stressed samples are

evaluated for peak purity of DLP using Waters Empower Networking software. For all forced

degradation samples, the purity angle (the weighted average of all spectral contrast angles

calculated by comparing all spectra in the integrated peak against the peak apex spectrum) is

found to be less than threshold angle (the sum of the purity noise angle and solvent angle, the

purity noise angles across the integrated peak) for DLP peak (table 2.2.4). This indicates that

there is no interference from degradants in quantitating the DLP in capsules dosage form.

Thus, this method is considered "Stability indicating”. The chromatogram and purity plots of

all stressed samples are shown in figs 2.2.5 to 2.2.12.

52

Table 2.2.4: Table of results of specificity

Stress condition

% Assay of

stressed

sample

Purity

Angle

Purity

Threshold

Kept on bench top for 5 minutes with 1N HCl

solution. 84.3 0.111 0.302

Stressed at 50°C in water bath for 24 hrs with

2N NaOH solution. 92.4 0.107 0.301

Kept on bench top for 15 minutes with 10%

H2O2 solution. 97.1 0.130 0.327

Stressed at 50°C in water bath for 24 hrs with

water. 91.5 0.116 0.299

Exposed to visible light for 1.2million lux

hours and UV light at 200 Watt hours/ meter2

99.7 0.117 0.318

Exposed to Dry heat at 105°C for about 1

hour. 84.7 0.081 0.292

Exposed to humidity at 25°C, 90% RH for 9

days. 99.8 0.110 0.318

Fig 2.2.5: Chromatogram and purity plot of acid stressed DLP capsules.

53

Fig 2.2.6: Chromatogram and purity plot of base stressed DLP capsules.

Fig 2.2.7: Chromatogram and purity plot of H2O2 stressed DLP Capsules.

54

Fig 2.2.8: Chromatogram and purity plot of water stressed DLP capsules.

Fig 2.2.9: Chromatogram and purity plot of light stressed DLP capsules.

55

Fig 2.2.10: Chromatogram and purity plot of Thermal stressed DLP capsules.

Fig 2.2.11: Chromatogram and purity plot of humidity stressed DLP capsules.

56

3. Conclusion:

A validated stability-indicating HPLC analytical method has been developed for the

determination of DLP in delayed release capsules dosage form. The results of stress testing

undertaken according to the International Conference on Harmonization (ICH) guidelines

reveal that the method is selective and stability-indicating. The proposed method is simple,

accurate, precise, specific and has the ability to separate the drug from degradation products

and excipients of capsules dosage form. The method is suitable for the routine analysis of

DLP in either bulk powder or in other pharmaceutical dosage forms. The HPLC procedure

can be applied to the analysis of samples obtained during accelerated stability experiments to

predict the expiry dates of DLP in bulk and in formulations.

57

Section (iii): Stability Indicating HPLC method for impurities Dexlansoprazole capsules.

This section reports the various aspects relating to the development and validation of

stability indicating HPLC method for impurities in DLP capsule dosage form.

1. Experimental

1.1. Chemicals

Dexlansoprazole capsules (dual delayed release) are formulated in Dr Reddy’s

laboratories Ltd, Hyderabad, India. The standards of DLP and its impurities namely

carboxylic acid, hydroxy benzimidazole, mercapto benzimidazole, N-oxide, nitrosulphoxide,

sulphone, impurity I, sulphide, impurity II, impurity III and impurity M+467 are supplied by

Dr. Reddy’s laboratories limited, Hyderabad, India. The HPLC grade acetonitrile, ethanol and

analytical grade KH2PO4, NaOH and ortho phosphoric acid are purchased from Merck,

Darmstadt, Germany. High purity water is prepared by using Milli Q Plus water purification

system (Millipore, Milford, MA, USA). The chemical names and structures of DLP and its

impurities are shown in the below.

S.No Name of the

impurity

Structure and IUPAC Name

1 Dexlansoprazole

(R)-2-(((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-

yl)methyl)sulfinyl)-1H-benzo[d]imidazole

2 Carboxylic acid

Impurity

1-(1H-benzo[d]imidazol-2-yl)-3-methyl-4-oxo-1,4-

dihydropyridine-2-carboxylic acid

3 Hydroxy-

benzimidazole

1H-benzo[d]imidazol-2-ol

58

4 Mercapto-

benzimidazole

1H-benzo[d]imidazole-2-thiol

5 N-oxide

(R)-2-(((1H-benzo[d]imidazol-2-yl)sulfinyl)methyl)-3-methyl-4-

(2,2,2-trifluoroethoxy)pyridine 1-oxide

6 Nirosulphoxide

2-(((3-methyl-4-nitropyridin-2-yl)methyl)sulfinyl)-1H-

benzo[d]imidazole

7 Sulphone

[[(1H-benzimidazole-2-yl)sulfinyl]methyl]-3-methyl-4-(2,2,2-

trifluoroethoxy)-pyridine 1-oxide

8 Cyclized

dessulphur- des

trifluoro ethyl

sulphide adduct

(Impurity I)

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

59

trifluoroethoxy)benzo[4',5']imidazo[2',1':2,3]imidazo[1,5-

a]pyridin-12-yl)methyl)-3-methylpyridin-4-ol.

9 Sulphide

2-(((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-

yl)methyl)thio)-1H-benzo[d]imidazole 10 Cyclized

dessulphur-

sulphide adduct

(Impurity II)

12-(((1H-benzo[d]imidazol-2-yl)thio)(3-methyl-4-(2,2,2-

trifluoroethoxy)pyridin-2-yl)methyl)-1-methyl-2-(2,2,2-


a]pyridine

11 Cyclized

dessulphur

(Impurity III)

1-methyl-2-(2,2,2-


a]pyridine.

60

12 Cyclized

dessulphur-

mercapto

benzimidazole

adduct

(Impurity

M+467)

12-((1H-benzo[d]imidazol-2-yl)thio)-1-methyl-2-(2,2,2-


a]pyridine

1.2. Determination of appropriate UV wavelength

The suitable wavelength for the determination of DLP and its impurities is identified by

taking the overlay spectra from 200–400 nm of all impurities and DLP from PDA detector.

1.3. Instrumentation and chromatographic conditions

The Waters HPLC System with a photo diode array detector is used for the

method development and force degradation studies .The data is monitored and processed

using Waters Empower Networking software. The HPLC system used for method validation

is waters HPLC system with diode array detector and Agilent 1100 series LC system with

variable wavelength detector (VWD). The chromatographic column used is an X-terra RP-18,

250mm x 4.6 mm column, with 3.5µ particle size with a 10 mm x 4.6 mm guard column. The

chromatographic condition follows a gradient program consisting of mixture of buffer and

acetonitrile in the ratio of 90:10 (v/v) as mobile phase A and of mixture of buffer and

acetonitrile in the ratio of 30:70 (v/v) as mobile phase B. The buffer is prepared as 0.01 M

KH2PO4 (6.8g in 1000ml) in water with 1.0% v/v triethylamine and finally pH is adjusted to

7.0. The gradient program is Time/% Mobile phase B: 0.0/10, 50/60, 67/80, 85/80, 90/10,

100/10. The flow rate of mobile phase is 0.8 ml min-1

. The column temperature is maintained

at 25ºC and the detection wavelength is 285 nm. The injection volume is 10µl. Sample cooler

temperature is used as 5°C.

61

1.4. Diluent:

0.1 N Sodium hydroxide and ethanol in the ratio of 3:5 (v/v) is used as a diluent.

1.5. Preparation of dexlansoprazole diluted standard solution:

An accurately weighed amount of about 54 mg of DLP working standard is

transferred into a 50 mL dried volumetric flask. 10 ml 0.1 N sodium hydroxide solution is

added, sonicated to dissolve the material completely and diluted to volume with diluent and

mixed well. This stock solution of DLP (equivalent to about 1.0 mg ml-1

of DLP) is then

diluted to get a working DLP diluted standard solution (about 2 µg ml-1

of DLP) by dilution

of the stock solution in with diluents along with 20% volume of 0.1N NaOH solution. The

specimen chromatogram of diluent and DLP diluted standard solution is shown in fig.2.3.1.

1.6. Test Preparation for dexlansaprazole pharmaceutical formulations:

Twenty capsules of DLP capsules (dual delayed release) are weighed and the contents

are emptied and the pellets are transferred into a clean dry poly bag. Pellets equivalent to

about 300 mg of DLP are transferred into a 500 ml volumetric flask. 100 ml of 0.1 N

sodium hydroxide solution is first added and sonicated for about 10 minutes to disperse the

pellets. Then 250 ml of diluent is added and, sonicated for 15 minutes with intermediate

shaking. The temperature of water in sonicator bath is maintained between 20°C to 25°C. The

volume is then made up with diluent and mixed well. A portion of the above solution is

centrifuged in a centrifuge tube with cap at 10000 RPM for 5 minutes. The resultant clear

supernatant solution is used for injection. Always freshly prepared solutions are used.

Placebo sample is prepared in the same way by taking the placebo equivalent its weight

present in a test preparation. The specimen chromatogram of placebo and test samples is

shown in fig.2.3.2 and 2.3.3.

62

Fig 2.3.1: Specimen chromatograms of diluent and DLP diluted standard.

Fig 2.3.2: Specimen chromatogram of placebo for DLP capsules.

Diluent

Placebo

Diluted standard

63

Fig 2.3.3: Specimen chromatogram of DLP capsules test sample spiked with known impurities.

64

1.7. Specificity:

Regulatory guidances ICH Q2A, Q2B, Q3B and FDA 21 CFR section 211, require the

development and validation of stability-indicating impurities method for all pharmaceutical

dosage forms. However, the current guidance documents do not indicate detailed degradation

conditions in stress testing. The forced degradation conditions, stress agent concentration and

time of stress, are found to effect the % degradation. Not more than 20% degradation is

recommended for active materials to make the right assessment of stability indicating nature

of the chromatographic methods. The optmisation of such stress conditions which can yield

not more than 20% degradation is based on experimental study. Chromatographic runs of

placebo and samples subjected to force degradation are performed in order to provide an

indication of the stability indicating properties and to establish the specificity of the method.

The stress conditions employed are acid, base, neutral and oxidant media, moisture, heat 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 diluted with diluent to

get the working concentrations equivalent to test preparation. The stressed samples are

subjected to assay analysis to assess the mass balance. The samples are analyzed against a

freshly prepared control sample (with no degradation treatment) and evaluated for peak purity

by using photo diode array detector. Specific conditions are described below.

1.7.1. Placebo (excipients) interference:

Placebo solutions are prepared in triplicate by taking the weight of placebo

approximately equivalent to its weight in the sample as described in the test preparation for

DLP capsules dosage form.

1.7.2. Effect of acid hydrolysis

About 300 mg of DLP pellets powder is treated with 25 ml of 1N HCl for 1 minute on

bench top with continuous shaking. The resulting solution is immediately neutralized and then

solution is prepared as per the test preparation to obtain a solution having final concentration

of drug at about 0.6 mg ml-1

.

1.7.3. Effect of base hydrolysis

About 300 mg of DLP pellets powder is treated with 25 ml 1N NaOH at 60°C using a

heating water bath for 24 hrs. The resulting stress solution is neutralized, equilibrated to room

65

temperature and then solution is prepared as per the test preparation to obtain a solution

having final concentration of drug at about 0.6 mg ml-1

.

1.7.4. Effect of neutral hydrolysis

About 300 mg of DLP pellets powder is treated with 25 ml water at 60°C using a

heating water bath for 9.5 hours. The resulting stress solution is neutralized, equilibrated to

room temperature and then treated same as per the test preparation to obtain a solution having

final concentration of drug at about 0.6 mg ml-1

.

1.7.5. Effect of oxidation

About 300 mg of DLP pellets powder is treated with 25 ml of 3%H2O2 for 30 minutes

on bench top with continuous shaking. The resulting solution is neutralized and then solution

is prepared as per the test preparation to obtain a solution having final concentration of drug at

about 0.6 mg ml-1

.

1.7.4. Effect of moisture and heat

To evaluate the effect of moisture and heat, DLP pellets powder is distributed as thin

layer over two glass plates. One plate is exposed to 25ºC/90% relative humidity for 7 days.

Similarly DLP pellets powder in another plate is exposed in an oven at 105ºC for 1 hour.

Then, both the samples are subjected to preparation using diluents as described in test

preparation.

1.7.5. Effect of UV and visible light

To study the photochemical stability of the drug product, DLP pellets powder is exposed

to 1200 K Lux hours of visible light and 200 Watt hours/ m2 of UV light by using photo

stability chamber. After exposure, the samples are subjected to preparation using diluents as

described in test preparation.

66


1.8.1. Relative response factors of all Known impurities

The relative retention times(RRTs) and relative response factors (RRFs) of all known

impurities are established against DLP. Different concentrations of DLP and its known

impurities are injected into the chromatographic conditions developed. The linearity graphs

are drawn for DLP and all its known impurities individually. The relative response factors are

then calculated by dividing the slope of impurity by slope of DLP. The relative retention

times(RRT’s) and relative response factors (RRF’s) of 11 known impurities are summarized

in table 2.3.1.

Table 2.3.1: RRT and RRF of known impurities of DLP.

S.No Name of the impurity RRT RRF

1 Carboxylic acid Impurity 0.14 1.04

2 Hydroxy benzimidazole 0.25 0.92

3 Mercapto benzimidazole 0.36 2.66

4 N-oxide 0.71 1.21

5 Nitrosulphoxide 0.74 1.26

6 Sulphone 1.06 0.92

7 Impurity I 1.41 1.11

8 Sulphide 1.43 1.11

9 Impurity II 1.65 0.88

10 Impurity III 1.68 0.69

11 Impurity M+467 1.85 1.96

1.8.2. Precision

Precision (intra-day precision) of the impurities method is evaluated by preparing six

different solutions of test sample of DLP capsules spiked with known impurities and injected

into the developed chromatographic conditions described above. % of impurities are

calculated against a qualified DLP standard. RSD is then calculated for % of impurities

individually obtained for six different preparations.

The intermediate precision (inter day precision) of the method is also evaluated using

different HPLC systems and different HPLC columns on different day in the same laboratory.

67

1.8.3. Limits of Detection (LOD) and Quantification (LOQ)

The LOD and LOQ for DLP and its 11 known impurities are determined at a signal-

to-noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with

known concentrations. Precision study is also carried out at the LOQ level by injecting six

individual preparations and % RSD is calculated.

1.8.4. Linearity

Linearity test solutions for DLP and all its known impurities are prepared by diluting

stock solutions to the required concentrations. The solutions are prepared at different

concentration levels from LOQ to equal to or more than 150% of the specification

concentration level for DLP and its all known impurities. The solutions are then injected into

the chromatographic conditions developed. The data of peak area versus concentration is

subjected to least-square regression analysis.

1.8.5. Accuracy

A study of recovery of DLP and its 11 known impurities from placebo is conducted.

Samples are prepared by mixing placebo with DLP as per the formulation composition and

then spiking all the known impurities at different spike levels starting from LOQ to 150% of

the specification level. Sample solutions are prepared in triplicate for each spike level as

described in the test preparation and injected into the chromatographic conditions developed.

The % recovery is then calculated against DLP diluted standard and by using relative

response factor and compared against the known amounts of impurities spiked.

1.8.6. Robustness


deliberately altered and the elution patterns, tailing factor and resolution between its

impurities are evaluated. The effect of flow rate is studied at 0.6 ml ml-1

and 1.0 ml min-1

. The

effect of the column temperature is studied at 20ºC and 30ºC instead of 25ºC. The effect of

pH is studied using mobile phase containing buffer with pH 7.0 ± 0.2. The effect of the

percent organic strength is studied by varying acetonitrile by −10 to +10% while other mobile

phase components were held constant.

68


The stability of DLP and its impurities in solution for the impurities method is

determined by leaving spiked sample solution in a tightly capped volumetric flask at room

temperature on bench top and refrigerator and by measuring the amounts of the impurities at

different intervals. The stability of mobile phase is also determined by analysing freshly

prepared solution of DLP and its impurities for 3 days by using the same mobile phase during

the study period.

2. Results and discussion

2.1. Determination of suitable wavelength

The UV spectrum of DLP and its 11 known impurities are extracted in PDA detector

from 200-400 nm and is illustrated in fig.2.3.4. The spectrum indicates that 285 nm gives a

good sensitivity for the all impurities of DLP.

Fig 2.3.4: UV Spectra of DLP and its impurities.

2.2. Optimization of chromatographic conditions

The HPLC procedure is optimized with a view to develop a stability indicating

impurities method. Pure drug and stressed samples are injected and run in different solvent

systems. Selection of mobile phase pH is done based on stability of DLP. Drug is found to be

not stable and impurities peak area continuously decreases in mobile phases with pH less than

7.0. Due to the problem in stability of standard, test and impurity solutions, after several

experiments the diluent is finalized as 0.1N NaOH and ethanol 75: 25 v/v. The pH of the

diluent is about 11. Normal silica based column stationary phases not workable beyond pH

8.0. Most of the C18 column peak shape goes quickly as the silica starts dissolving beyond

69

pH 8.0. Hence a choice is made to work with hybrid silica based columns. After screening of

columns which can withstand for higher pH conditions, a choice of the column is made to

waters X-terra RP18 column. A guard column is also chosen with C18 stationary phase in

order to increase the life of analytical column. DLP is prone to degradation upon stability, it

generates number of impurities upon storage. As several late eluting non-polar degradants are

found in the sample, isocratic method is found to be not feasible in order to elute all the

degradants. As in the pharmaceutical industry, lot of stability analysis is needed to check the

quality of the formulated products for shelf life determination, a study is conducted to get the

stability indicating method which can separate all known unknown impurities satisfactorily. A

number of experiments are done with different columns and different mobile phase

compositions and with different gradient programmes to separate all the degradants from each

other and from DLP peak. Eventually, satisfactory peak shape and satisfactory separation is

achieved using a 250 mm x 4.6 mm, X-terra RP18 column with 3.5 µm with mobile phase

consisting of mixture of buffer (pH 7.0) and acetonitrile in the ratio of 90:10 (v/v) as mobile

phase A and of mixture of buffer (pH 7.0) and acetonitrile in the ratio of 30:70 (v/v) as mobile

phase B with a gradient programme of Time/% Mobile phase B : 0.0/10, 50/60, 67/80, 85/80,

90/10, 100/10. The optimum flow rate and column temperatures are found to be 0.8 ml min-1

and 25ºC. As there are 11 known impurities which are differing in polarity very significantly,

with carboxylic acid impurity being most polar and impurity M+467 being most non polar, it

is learnt during the experiments that a run time of 100 min is necessary. Especially, the

separation between impurity I & sulphone and the separation between impurity II and III is

found to be critical. The column length reduction or making the faster gradient is not

successful in reducing the run time. Different pH conditions are also experimented without

success. Different buffers in mobile phase also explored but KH2PO4 with triethylamine as

ion pair is found to be showing the best separations especially between unknown and known

impurities when samples of stress solutions are evaluated. The wavelength of 285 nm is found

to be best suitable for all known and unknown impurities estimation, as all the impurities are

having good response at this wavelength. A test concentration of 0.6 mg ml-1

with an injection

volume of 10µl is found to provide adequate sensitivity to the method wherein the LOQ of

DLP and its known impurities is less than the reporting threshold as per ICH. Due to limited

stability of solution of test sample, selection of sample cooler temperature as 5°C is found

necessary.

70


2.3.1. Precision

The precision of test method (intra-day precision) is evaluated by analyzing six test

samples of DLP capsules by spiking test preparation with DLP impurities blend solution to

get about 0.2 to 0.3% of carboxylic acid, hydroxy benzimidazole, mercapto benzimidazole, N-

Oxide, nitrosulphoxide, impurity I, sulphide, impurity II, impurity III and impurity M+467

and about 0.4% of sulphone. Six different test preparations having placebo equivalent to test

and spiked with about 0.25% of DLP are prepared and injected into the chromatographic

condition developed. Relative standard deviations of % of DLP and its 11 known impurities

were evaluated. Inter-day Precision study is conducted on a different day with different

mobile phase, with different HPLC and different column. Six different test preparations of

sample are prepared similar to intra-day precision and the relative standard deviations of % of

DLP and its 11 known impurities are evaluated. The % RSD values are presented in table

2.3.2 and 2.3.3. % RSD values of less than 15% for DLP and its 11 known impurities shows

that method is precise and work satisfactorily on different day, with different column and

HPLC.

2.3.2. LOQ and LOD

The limit of detection, limit of quantification are determined by following signal to

noise ratio method. A precision study also conducted at LOQ level for DLP and its 11 known

impurities. The results shows that method is sensitive enough to quantify impurities well

below the ICH reporting threshold of 0.1%, as LOQ values are in the range of 0.02% to

0.05%. The data is summarized in table 2.3.4.

71

2.3.3. Linearity

A linear calibration plot for DLP and its 11 known impurities is drawn over the

calibration range LOQ to 150% of the specification levels. Correlation co-efficient for DLP

and its 11 known impurities is found to be greater than 0.997. The regression analysis results

are shown in table 2.3.4. The results indicates that an excellent correlation exists between the

peak area and concentration of the analyte for DLP and its 11 known impurities. The linearity

graphs are presented as figure 2.3.5 to 2.3.7.

2.3.4. Accuracy

The percentage recovery of DLP and its 11 known impurities in presence of placebo

matrix of DLP capsules from LOQ to 150% spike level are in the range of 90.9% to 109.2%.

The LC chromatogram of test preparation spiked with all 11 known impurities is shown in

Fig. 2.3.3. The % recovery values for DLP and impurities are presented in table 2.3.5. The

data shows that the method is having capability to estimate accurately all 11 known impurities

of DLP in DLP capsules.

72

Table 2.3.2: Results of Inter day precision of test method for DLP and its 11 known impurities.

Table 2.3.3: Results of Intra-day precision of test method for DLP and its 11 known impurities.

Sample

No.

DLP

Carboxylic

acid

Impurity

Hydroxy

benzimi

dazole

Mercapto

benzimid

azole

N-

Oxide

Nitro

sulph

oxide

Sulph

one

Impurity

I

Sulphide

Impurity

II

Impurity

III

Impurity

M+467

1 0.256 0.237 0.180 0.234 0.181 0.178 0.444 0.268 0.276 0.256 0.295 0.212

2 0.248 0.232 0.187 0.240 0.187 0.182 0.456 0.267 0.286 0.248 0.305 0.210

3 0.257 0.237 0.187 0.239 0.186 0.184 0.457 0.271 0.286 0.249 0.296 0.210

4 0.255 0.238 0.186 0.242 0.188 0.184 0.456 0.265 0.286 0.248 0.301 0.213

5 0.249 0.236 0.190 0.230 0.185 0.178 0.465 0.268 0.287 0.249 0.301 0.207

6 0.246 0.237 0.189 0.231 0.182 0.176 0.461 0.265 0.284 0.249 0.304 0.206

Avg 0.252 0.236 0.187 0.236 0.185 0.180 0.457 0.267 0.284 0.250 0.300 0.210

%RSD 1.9 0.9 1.9 2.1 1.5 1.9 1.5 0.9 1.4 1.2 1.4 1.3

Sample

No.

DLP

Carboxylic

acid

Impurity

Hydroxy

benzimi

dazole

Mercapto

benzimid

azole

N-

Oxide

Nitro

sulph

oxide

Sulph

one

Impurity

I

Sulphide

Impurity

II

Impurity

III

Impurity

M+467

1 0.252 0.227 0.200 0.197 0.205 0.171 0.437 0.235 0.254 0.244 0.225 0.191

2 0.246 0.229 0.205 0.195 0.197 0.176 0.440 0.241 0.249 0.232 0.239 0.188

3 0.251 0.226 0.214 0.201 0.193 0.166 0.437 0.231 0.261 0.228 0.242 0.191

4 0.235 0.231 0.193 0.194 0.190 0.171 0.427 0.252 0.252 0.221 0.238 0.183

5 0.245 0.239 0.195 0.190 0.184 0.171 0.441 0.226 0.274 0.226 0.241 0.204

6 0.248 0.234 0.207 0.192 0.181 0.169 0.418 0.229 0.251 0.230 0.237 0.188

Avg 0.250 0.231 0.202 0.195 0.192 0.171 0.433 0.236 0.257 0.230 0.237 0.191

%RSD 2.5 2.1 3.9 2.0 4.6 1.9 2.1 4.0 3.6 3.4 2.6 3.7

73

Table 2.3.4: LOD , LOQ data of DLP and its impurities

* RSD for 6 determinations.

Parameter

DLP

Carboxylic

acid

Impurity

Hydroxy

benzimida

zole

Mercapto

benzimid

azole

N-Oxide

Nitrosul

phoxide

Sulphone

Impurity

I

Sulphide

Impurity

II

Impurity

III

Impurity

M+467

LOD

In % 0.009 0.011 0.012 0.005 0.013 0.015 0.016 0.011 0.014 0.015 0.019 0.01

S/N ratio 2.82 3.117 2.93 2.95 3.01 3.04 3.01 3.464 3.01 2.815 2.370 3.26

LOQ

In% 0.026 0.0347 0.037 0.016 0.039 0.044 0.049 0.033 0.042 0.044 0.048 0.029

S/N ratio 9.94 10.594 10.15 10.32 9.93 10.22 9.58 10.827 9.51 9.363 9.698 10.48

Precision

at LOQ

(%RSD*)

3.0 2.6 2.5 7.7 3.5 2.1 3.1 7.7 5.5 2.4 1.6 1.9

74

Figure 2.3.5 : Linearity graphs of DLP and its impurities.

75

Figure 2.3.6 : Linearity graphs of DLP impurities.

76

Fig.2.3.7: Linearity graphs of DLP impurities.

77

Table 2.3.5: Recovery results of DLP impurities in pharmaceutical dosage forms

Values given in parenthesis represent standard deviation of triplicate results.

Spike level

DLP

Carboxylic

acid

Impurity

Hydroxy

benzimida

zole

Mercapto

benzimid

azole

N-Oxide

Nitrosul

phoxide

Sulphone

Impurity

I

Sulphide

Impurity

II

Impurity

III

Impurity

(M+467)

LOQ 101.4(2.0) 102.2(5.4) 103.9(6.6) 103.4(0.0) 97.5(1.6) 90.9(5.4) 107.6(0.7) 96.4(4.6) 94.6(3.9) 96.7(3.2) 96.6(3.5) 99.9(1.9)

50% 101.1(1.2) 102.7(4.3) 102.9(0.6) 91.4(0.0) 100(1.2) 91.3(0.6) 97.7(1.6) 103.8(0.6) 102.0(1.1) 102.6(3.3) 107.3(1.4) 102.7(1.0)

75% 99.6(0.7) 101.5(3.8) 105.3(0.7) 91.1(0.9) 97.6(0.7) 92.3(1.3) 98.1(0.5) 102.6(0.8) 96.5(0.7) 100.7(3.7) 105.6(2.4) 101.1(0.7)

100% 99.0(0.7) 98.1(2.0) 104.5(0.5) 91.1(0.8) 97.1(0.4) 94.0(1.0) 97.3(0.5) 105.4(1.2) 97.1(0.7) 102.2(2.1) 109.2(2.4) 101.2(0.1)

125% 94.5(0.5) 99.6(3.1) 104.2(0.6) 93.7(0.8) 98.1(0.5) 95.0(0.6) 98.3(0.7) 101.0(1.1) 99.2(0.8) 101.9(1.5) 106.5(4.1) 101.3(0.6)

150% 94.1(1.4) 100.0(1.6) 99.0(0.6) 92.0(0.6) 96.6(0.7) 93.6(0.7) 96.6(0.6) 97.8(2.3) 96.5(0.9) 108.0(0.7) 108.5(0.5) 102.5(0.7)

78

2.3.5. Robustness


deliberately altered and the elution pattern, separation between DLP and its impurities and

tailing factor for DLP and its impurities are recorded. In all the deliberately varied

chromatographic conditions (flow rate, column temperature, pH 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 2.3.6. The resolution between all critical pair components is found to

be greater than 2.0 and tailing factor for DLP and its impurities is found to be less than 1.4.


The stability of diluted standard solution is estimated against freshly prepared standard

and found that solutions are stable up to 2 days. DLP test preparation prepared as per test

method by spiking with 11 known impurities is injected at regular intervals up to 12 hours.

Although the difference in % of known individual impurity and % of total impurities are

found to be within the limits, the difference in % of unknown individual impurity is found to

be outside the limit of ± 0.04% after 6 hours. Therefore, a study to establish the stability of

DLP in test preparation on Refrigerator (between 2-8⁰C) is conducted for a period of about 15

hrs. DLP test preparation prepared as per test method by spiking with impurities is injected

using the sample cooler available in HPLC auto sampler by setting a temperature of 5⁰C. The

difference in % of individual impurities and % of total impurities are found to be within the

limits up to 15 hours. The results are summarized in table 2.3.7.

The stability of mobile phase-A & B on bench top is conducted for a period of about 3 days.

The difference in % of known impurities and % of total impurities from initial to 3 days is

found to be within the limits. The results are summarized in table 2.3.8. The elution pattern,

RRT’s of all 11 known impurities are found to be comparable between zero day and other

days during the study. From the above study it is established that the mobile phase is stable

for a period of 3 days on bench top.

79

Table 2.3.6: Results of robustness study

Condition

RRT of the impurity

Carbo

xylic

acid

imp

Hydroxy

benzimida

zole

Mercapto

benzimida

zole

N-

Oxide

Nitro

sul

phoxide

Sul

phone Imp I

Sul

phide

Imp

II

Imp

III M+467

0.6 ml/min 0.165 0.277 0.393 0.707 0.761 1.051 1.352 1.392 1.568 1.608 1.776

0.8 ml/min 0.142 0.241 0.345 0.692 0.731 1.054 1.407 1.424 1.642 1.663 1.859

0.85 ml/min 0.137 0.232 0.335 0.685 0.723 1.053 1.422 1.436 1.665 1.684 1.888

20°C 0.142 0.240 0.346 0.692 0.732 1.053 1.407 1.424 1.643 1.664 1.860

25°C 0.143 0.241 0.347 0.692 0.732 1.053 1.407 1.424 1.643 1.664 1.860

27°C 0.142 0.239 0.344 0.691 0.731 1.052 1.408 1.425 1.645 1.665 1.862

pH 6.8 0.143 0.246 0.357 0.695 0.743 1.086 1.392 1.415 1.612 1.650 1.842

pH 7.0 0.140 0.242 0.352 0.691 0.737 1.068 1.400 1.425 1.631 1.664 1.859

pH 7.2 0.140 0.243 0.351 0.688 0.732 1.032 1.404 1.433 1.652 1.677 1.875

M.P A ( acetonitrile)90% 0.139 0.244 0.354 0.691 0.737 1.059 1.400 1.426 1.635 1.665 1.860



M.P B ( acetonitrile)90% 0.131 0.228 0.331 0.686 0.722 1.056 1.420 1.434 1.654 1.675 1.872



80

2.3.7. Results of solution stability :

Duration

in hours

% of the impurity upon storage between 2-8ºC

Carbo

xylic

acid imp

Hydroxy

benzimid

azole

Mercapto

benzimida

zole

N-

Oxide

Nitrosul

phoxide

Sul

phone Imp I

Sul

phide Imp II Imp III

Imp

M+467

Total

impuriti

es

0 0.2522 0.2485 0.1809 0.1997 0.1682 0.4711 0.2882 0.3146 0.1622 0.2844 0.2044 2.7109

3 0.2356 0.2518 0.1853 0.2199 0.1668 0.4794 0.2678 0.3095 0.1314 0.2909 0.2049 2.7612

6 0.2350 0.2510 0.1840 0.2210 0.1681 0.4785 0.2712 0.3039 0.1252 0.2913 0.2145 2.7705

15 0.2360 0.2512 0.1820 0.2212 0.1673 0.4774 0.2716 0.3045 0.1224 0.2901 0.2146 2.7809

Maximum

Difference

from zero

hours

0.02 0.00 0.00 -0.02 0.00 -0.01 0.02 0.01 0.04 -0.01 -0.01 0.1

81

2.3.8. Results of mobile phase stability on bench top :

No of

days

% of impurity for mobile phase stability

Carboxylic

acid

Hydroxy

benzimida

zole

Mercapto

benzimida

zole N-oxide

Nitro

sulphoxide Sulphone

Impurity

1 Sulphide

Impurity

II

Impurity

III

Impurity

M+467 Total

0 0.2522 0.2485 0.1809 0.1997 0.1682 0.4711 0.2882 0.3146 0.1622 0.2854 0.2044 2.7755

1 0.2692 0.2484 0.171 0.1824 0.1827 0.4689 0.2564 0.275 0.1427 0.2776 0.1987 2.6729

2 0.2504 0.2435 0.1707 0.2002 0.1561 0.4433 0.286 0.3039 0.1309 0.2721 0.2035 2.6607

3 0.2272 0.2238 0.1673 0.1895 0.1564 0.4587 0.2606 0.2782 0.1545 0.2874 0.1883 2.5918

Max

diff.

from

initial 0.03 0.02 0.01 0.01 0.01 0.03 0.03 0.04 0.03 0.01 0.02 0.2

82

2.3.7. Results of specificity studies

All the placebo and stressed samples prepared are injected into the HPLC system

with photodiode array detector using the developed chromatographic conditions.

Chromatograms of placebo solutions have shown no peaks at the retention time of DLP and

its impurities. This indicates that the excipients used in the formulation do not interfere in

estimation of impurities in DLP capsules.

Degradation is not observed in light and humidity stress. Significant degradation is

observed in acid, base, water, peroxide and thermal degradation. All degradant peaks are well

resolved from each other and from DLP peak in the chromatograms of all stressed samples.

The chromatograms of the stressed samples are evaluated for peak purity of DLP using

Waters Empower Networking software. For all forced degradation samples, the purity angle

(the weighted average of all spectral contrast angles calculated by comparing all spectra in the

integrated peak against the peak apex spectrum) is found to be less than threshold angle (the

sum of the purity noise angle and solvent angle, the purity noise angles across the integrated

peak) for DLP peak. The details of known impurity formed in each stressed sample is

summarised in table 2.3.9. The stressed samples are subjected to mass balance study by

assaying the samples for DLP content apart from estimating the total impurities. The sum of

total % of impurities and assay is presented as mass balance, which is found to be in the range

of 98.3 to 100.4% for the stressed samples. The data indicates that there is no co-elution of

any degradants in the DLP peak and no impurity which is missing, as the mass balance is

close to 100%. Thus, this method is considered as specific and "Stability indicating”. The

chromatograms and purity plots of all stressed samples are shown in figs 2.3.8 to 2.3.15.

83

Table 2.3.9 : Summary of forced degradation studies Stress condition % Impurities observed %

Assay

Mass

balance

Carbo

xylic

acid

Imp

Hydro

xyl

benz

imida

zole

Mer

capto

benz

imida

zole

N-

Oxide

Nitro

sulpho

xide

Sul

phone

Imp I

Sul

phide

Imp II

Imp

III

Imp

M+467

TI

Control sample 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.08 0.00 0.00 0.00 0.27 99.8 100.1

Acid degradation 0.51 0.05 0.02 0.12 0.52 0.10 0.20 0.70 0.51 2.80 0.00 11.4 86.9 98.3

Base degradation 0.25 0.03 0.68 0.00 0.00 0.08 0.09 0.19 0.85 0.08 0.00 3.4 95.7 99.1

Oxidative

degradation 0.23 0.12 0.01 0.00 0.06 12.9 0.08 0.07 0.02 0.09 0.00 15.1 83.8 98.8

Hydrolytic

degradation 1.36 0.20 0.08 0.00 0.00 0.08 0.40 2.19 0.31 1.11 0.00 9.4 89.6 99.0

Visible light 0.00 0.03 0.02 0.00 0.00 0.05 0.00 0.08 0.00 0.00 0.00 0.25 99.9 100.2

UV light 0.00 0.01 0.02 0.00 0.00 0.07 0.00 0.08 0.00 0.00 0.00 0.26 99.8 100.1

Thermal

degradation 0.68 0.07 0.61 0.00 0.00 0.09 0.11 0.59 0.09 1.83 2.33 14.3 84.2 98.5

Humidity

degradation 0.00 0.01 0.02 0.00 0.00 0.05 0.00 0.06 0.00 0.00 0.00 0.27 99.7 100.4

TI: Total impurities; imp = impurity.

84

Fig 2.3.8: Chromatogram and purity plot of acid stressed DLP capsules.

85

Fig 2.3.9: Chromatogram and purity plot of base stressed DLP capsules.

86

Fig 2.3.10: Chromatogram and purity plot of H2O2 stressed DLP capsules.

87

Fig 2.3.11: Chromatogram and purity plot of water stressed DLP capsules.

88

Fig 2.3.12: Chromatogram and purity plot of visible light stressed DLP capsules.

89

Fig 2.3.13: Chromatogram and purity plot of UV light stressed DLP capsules.

90

Fig 2.3.14: Chromatogram and purity plot of heat stressed DLP capsules.

91

Fig 2.3.15: Chromatogram and purity plot of humidity stressed DLP capsules

92

3. Conclusion:

A precise, sensitive, specific, accurate, validated stability indicating LC method for the

determination of degradation products and its process- related impurities is described. The

behavior of DLP under various stress conditions is studied. All of the degradation products

and process impurities are well separated from each other and from DLP demonstrates the

stability- indicating nature of the method. The information presented in this study could be

very useful for quality monitoring of DLP pharmaceutical dosage forms and can be used to

check drug quality during stability studies.

94

1. Experimental :

1.1 chemicals and instrumentation :

DLP capsules (delayed release) are formulated in Dr Reddy’s laboratories Ltd,

Hyderabad, India. DLP drug substance and working standard is from Dr.Reddy’s laboratories

Ltd, Hyderabad, India. The HPLC grade acetonitrile, methanol, ethanol , ethyl acetate and

analytical grade KH2PO4, NaOH, HCl, triethyl amine and ortho phosphoric acid are

purchased from Merck, Darmstadt, Germany. High purity water was prepared by using Milli

Q Plus water purification system (Millipore, Milford, MA, USA).

The ESI MS spectrum is recorded using Applied 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 spectrums are recorded in

DMSO-d6 using Unity INOVA Varian 500 MHz spectrometer.

2. Isolation and purification of three unknown impurities :

2.1. Impurity-III (RRT 1.68):

2.1.1. Impurity enrichment :

About 500 mg of DLP API (amorphous) is dissolved in 10 ml of 1.0N HCl, kept on

bench top for 15 minutes with intermediate shaking. The solution is then neutralized with 10

ml of 1N NaOH. The solution is then subjected to liquid-liquid extraction using ethyl acetate.

Ethyl acetate fraction is evaporated with the help of rotavapour. The residue obtained is

further dissolved in one volume of ethyl acetate and three volumes of water and mixed well.

The ethyl acetate layer is then separated and the solvent is evaporated to get the crude residue.

The chromatogram of crude in analytical method is given in fig 2.4.1.

Figure. 2.4.1 : 1.68 RRT impurity crude.

95

2.1.2. Purification by flash chromatography :

A flash chromatographic method is developed to purify the impurity crude. Milli-Q

water is used as mobile phase A. Acetonitrile, water and TEA in the ratio of 800:200:5(v/v) is

used as mobile phase B. A Reveleris C18, 12 grams cartridge is used as column. A detector

wavelength of 285 nm, flow rate of 20 ml min-1

, injection volume of 10 ml is used. A gradient

programme of Time/%A:%B = 0.01/80:20, 2.00/80:20, 38.00/35:65, 10.00/25:75, 5.00/10:90,

5.00/5:95, 2.00/80:20, 2.00/80:20 with a run time of 65 min is employed for purification

purpose. About 1000 mg of extracted crude is dissolved in 5 ml of diluent and used for

purification using flash chromatography. 50: 50 (v/v) ratio of 0.1N NaOH and ethanol is used

as diluent. The sample preparations are purified further by injecting into flash

chromatographic conditions and the fractions are collected using automatic fraction collectors.

The purity of each fraction is verified using the analytical HPLC method. The high purity

fractions are then pooled. Acetonitrile present in the pooled fractions is evaporated using

rotavapour at room temperature. After this, the residual aqueous solution is subjected to

liquid-liquid extraction using ethyl acetate. The ethyl acetate layer is then separated and

evaporated using rotavapour at room temperature. The resultant residue is found to have a

purity of about 75% . The chromatogram of the purified fraction of flash is given in figure

2.4.2. The impurity is further purified using preparative HPLC.

Figure 2.4.2 : Chromatogram of flash purified fraction of 1.68 RRT impurity

96

2.1.3. Purification by preparative HPLC :

The impurity is purified further 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. A Sample

preparation of about 500 mg of flash crude dissolved in 10ml of diluent is used. Milli-Q water

is used as mobile phase A. Acetonitrile, water and TEA in the ratio of 800:200:5(v/v) was

used as mobile phase B. 250 mm x 20 mm, Inertsil C18, 10 µm column is used. A detector

wavelength of 285 nm, flow rate of 18 ml min-1

, injection volume of 1 ml is used. The

gradient programme is Time / %B : 0.01/10, 5/10, 41.5/50, 45/95, 58/95, 60/10, 65/10 with a

run time of 65 min. 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%. Chromatogram is shown in fig 2.4.3. The identity of the impurity is also confirmed by

ESI +ve mode ionization mass spectrum which showed a protonated molecular ion at m/z

320. The impurity isolated and purified is subjected for further characterization studies.

Figure 2.4.3 : Chromatogram of purified 1.68 RRT impurity.

97

2.2. Impurity-II(RRT 1.65) :

2.2.1. Enrichment of impurity : Unknown impurity which is eluting at RRT about 1.65,

which is found to be increasing in the stability studies is isolated and purified by using flash

chromatography and preparative HPLC. About 500 mg of DLP API is dissolved in 50 ml

methanol. 50 ml of 0.1N HCl is added to the above solution. The solution is kept on bench top

for 5 min with intermediate shaking. The solution is then neutralised with 50 ml of 0.1N

NaOH. The solution is centrifuged at 4000 RPM for 30 minutes. The residue is collected and

analysed by analytical HPLC method. The purity of the impurity is found to be about 27%.

2.2.2. Purification by Preparative HPLC :

500 mg of crude dissolved in 10ml of diluent (acetonitrile) is used as sample

preparation. The impurity is purified by injecting into preparative HPLC conditions and the

fractions are collected using automatic fraction collectors. Milli-Q water is used as mobile

phase A. Acetonitrile, water and TEA in the ratio of 800:200:5(v/v) is used as mobile phase

B. 500 mm x 20 mm, Zodiac C8, 7 µm column is used. A detector wavelength of 285 nm,

flow rate of 15 ml min-1

, injection volume of 4 ml is used. The gradient programme of Time /

%B : 0.01/35, 3/35, 38.0/20, 58/20, 60/35, 65/35 is used with a run time of 65 min.

The impurity fractions are collected for about 40 injections. The impurity fractions are

immediately extracted with ethyl acetate. All ethyl acetate layers are pooled. The solvent

present in the pooled fraction is evaporated using rotavapour under high vaccum at 25⁰C.

After evaporation of the total solvent, the solid is dissolved in few ml of acetonitrile and then

is subjected for lyophilization. Final chromatographic purity of the impurity is found to be

93.5% .Chromatogram is shown in Fig.2.4.4. The identity of the impurity is also confirmed by

ESI -ve ionization mass spectrum which showed a molecular ion at m/z = 669. The impurity

isolated and purified is subjected for further characterization studies.

98

Figure 2.4.4.: Chromatogram of purified 1.65 RRT impurity.

2.3. Impurity-I (RRT 1.40) :

2.3.1. Impurity enrichment :

Unknown impurity which is eluting at RRT about 1.40, which is found to be

increasing in the stability studies is isolated by using flash chromatography and preparative

HPLC. The impurity is enriched by degrading DLP API (amorphous) in oven at 105°c for 24

hours. 50:50(v/v) ratio of 0.1 N sodium hydroxide and ethanol is used as diluent. 500 mg of

crude sample dissolved in 5 ml of diluent is used as sample preparation.

The degraded sample is injected into the flash chromatographic system by following

the conditions given in section 2.1.2. Fractions are collected through the automatic fraction

collection system attached with flash chromatographic system. The chromatogram of

enriched sample is given as figure 2.4.5.

1.65 RRT impurity

99

Figure 2.4.5.: Chromatogram of degraded sample for 1.40 RRT impurity.

2.3.2. Purification by flash chromatography:

The impurity fractions are collected from about 10 injections. The purity of each

fraction is checked in the analytical HPLC method and purity of impurity is found to be

~40%. All the fractions of impurity are pooled. Acetonitrile present in the pooled fraction is

evaporated using rotavapour at room temperature. After evaporation of the total solvent,

impurity is extracted from aqueous layer using ethyl acetate, and ethyl acetate is evaporated

using rotavapour at room temperature. Final chromatographic purity of the impurity is found

to be 40%. The impurity is subjected to preparative HPLC for further purification.

2.3.3. Purification by Preparative HPLC :

500 mg of flash crude dissolved in 10ml of diluent is used as sample preparation. The

sample preparation is purified further by injecting into preparative HPLC and the fractions are

collected using automatic fraction collectors. Preparative HPLC conditions followed are same

as mentioned in section 2.1.3 for 1.68 RRT impurity. The impurity fractions are collected

from about 40 injections. The purity of each fraction is checked in the analytical HPLC

method and purity of impurity is found to be 96%.All the fractions of impurity are pooled.

Acetonitrile present in the pooled fraction is evaporated using rotavapour at room

temperature. After evaporation of the total solvent, aqueous layer was kept for lyophilization.

Final chromatographic purity of the impurity is found to be 92% .Chromatogram is shown in

fig.2.4.6.The identity of the impurity is also confirmed by ESI -ve ionization mass which

1.40 RRT

100

showed a molecular ion at m/z = 587. The impurity isolated and purified is subjected for

further characterization studies.

Figure 2.4.6.: Chromatogram of purified 1.40 RRT impurity.

3.0. Structural characterization of three unknown impurities :

3.1. Results and discussion:

3.1.1. Impurity –III (RRT 1.68):

The isolated and purified impurity (1.68 RRT) is subjected to LC-MS studies. Electro

spray mass spectrum in +ve mode of DLP 1.68 RRT impurity is presented in fig 2.4.7. The

compound mass spectrum shows that the mass number is 319, as it displayed a protonated

molecular ion at m/z=320. This indicates that the molecular weight of the impurity is 50 mass

units less than the DLP molecular weight.

The isolated and purified impurity (1.68 RRT) is subjected to high resolution mass

(HRMS) spectral studies. HRMS spectrum in +ve mode of 1.68 RRT impurity is presented in

fig 2.4.8. The HRMS spectrum showed that the impurity is having molecular formula of

C16H12N3OF3 with exact mass of 319.1008 daltons. The molecular formula shows the absence

of sulphur, one oxygen less and 2 protons less when compared to DLP. This clearly indicates

the possibility of cyclisation or cleavage of DLP.

The isolated and purified impurity (1.68 RRT) is subjected to NMR spectral studies.

1H NMR, 13C NMR, gDQ-COSY, gHSQC, gHMBC spectrums are recorded in DMSO-d6.

The Spectra are presented in fig 2.4.9 to 2.4.13. The 1H NMR spectrum of the impurity is

compared with that of DLP. The 1H NMR spectrum of DLP is presented in fig 2.4.20. The

structural formula of DLP is presented below.

1.40 RRT imp purified

101

The proton signals at 4.76, 4.83 ppm, which are due to the methylene group at C10

is absent in the impurity. The presence of extra aromatic proton signal at 7.98 ppm, and

the absence of benzimidazole-NH proton signal at 13.60 ppm in the impurity lend support

to the cyclisation between imidazole ring and pyridine ring. The extra aromatic signal at

7.98ppm shows correlation to a carbon signal at 93.1ppm in HSQC data. All other signals

in NMR data in the impurity matches with that of DLP.

In gHSQC spectrum, the presence of cross peak at 93.1 ppm showed the

correlation with signal at 7.98ppm supports to the cyclisation. Tracing the long range

connectivity of the aromatic signal at 132.0 ppm in HMBC data indicates the cyclised

structure given below.

102

Figure 2.4.7 : LC-MS (ESI+ve) Spectrum of 1.68 RRT impurity.

Fig2.4.8. HRMS Mass spectrum of 1.68 RRT impurity of DLP.

103

Fig.2.4.9. 1H NMR spectrum of 1.68 RRT impurity of DLP in DMSO-d6.

104

Fig.2.4.10. 13C NMR spectrum of 1.68 RRT impurity of DLP in DMSO-d6.

105

Fig.2.4.11. gDQ-COSY spectrum of 1.68 RRT impurity of DLP in DMSO-d6.

106

Fig.2.4.12. gHSQC spectrum of 1.68 RRT impurity of DLP in DMSO-d6.

107

Fig. 2.4.13. gHMBC spectrum of 1.68 RRT impurity of DLP in DMSO-d6.

108

3.1.2. Impurity II at 1.65 RRT:


spray mass spectrum in +ve mode of 1.65 RRT impurity is presented in fig 2.4.14. The


molecular ion ion at m/z=671. This indicates that the molecular weight of the impurity is 301

mass units extra than the DLP, which clearly indicates that the one more moiety is getting

added to DLP.


(HRMS) spectral studies. HRMS spectrum in +ve mode of DLP 1.65 RRT impurity is

presented in fig 2.4.15. The HRMS spectrum showed that the impurity is having molecular

formula of C32H23F6N6O2S with exact mass of 670.1688 daltons. The molecular formula

suggests that there is only one sulphur, number of fluorine are double (6) in the impurity and

number of nitrogens are double (6) and number of carbons are double (32) when compared to

DLP. This indicated that the there is possibility of adduct formation of between two modified

DLP compounds.


1H NMR, gDQ-COSY, gHSQC, gHMBC spectrums are recorded in DMSO-d6. The Spectra

are presented in fig 2.4.16 to 2.4.19. The 1H NMR spectrum of the impurity is compared with

that of DLP. The 1H NMR spectrum of DLP is presented in fig 2.4.20. The structural formula

of DLP is presented below.

109

The signals due to methylene protons at C10 and C17, which are in the region of 4.7

to 5.0ppm, are accounting for 4 protons in DLP. In the same region, the data of impurity

displays 5 signals at 4.32, 4.71, 4.80, 4.87 and 5.07ppm accounting for 5 protons. The absence

of doublets at 4.76 and 4.83 ppm observed in DLP which is due to C10 methylene group is

noteworthy.

The signals at 4.32, 4.71ppm and 4.87, 5.05ppm are found to be connected by

gDQ-COSY spectrum. These are assigned to the methylene signals connected to CF3 group.

This observation clearly indicates the two trifluoro ethoxy groups. 13

C signal for these

methylene carbons is found to be at 67.0ppm as indicated by g HSQC data.

The signal at 4.80ppm integrating for 1 proton, did not show any correlation in the

gDQ-COSY data. In gHSQC spectrum this signal is connected to the peak at 37.0ppm in 13

C.

Therefore, it is reasonable to infer that the C10 methylene of DLP has become a methine in

the impurity. This is indicative of site of addition of another moiety at C10.

When compared to DLP, the impurity displayed 6 additional protons in the aromatic region.

This indicates the presence of another aromatic ring system of DLP moiety in the impurity.

The aromatic ring system which got linked to modified DLP is found to be getting correlated

well with the structure of 1.68 RRT impurity. Based on the above discussion, the structure of

the impurity is proposed as given below.

110

Fig 2.4.14. LC-MS (ESI+ve) Mass spectrum of 1.65 RRT impurity of DLP.

111

Fig.2.4.15. HRMS Mass spectrum of 1.65 RRT impurity of DLP.

112

Fig.2.4.16. 1NMR spectrum of 1.65 RRT impurity of DLP in DMSO-d6.

113

Fig. 2.4.17. gDQ-COSY spectrum of 1.65 RRT impurity of DLP in DMSO-d6.

114

Fig.2.4.18. gHSQC spectrum of 1.65 RRT impurity of DLP in DMSO-d6.

115

Fig.2.4.19. gHMBC spectrum of 1.65 RRT impurity of DLP in DMSO-d6.

116

Fig.2.4.20. 1NMR spectrum of DLP drug substance in DMSO-d6.

117

3.1. 3. Impurity-I at 1.40 RRT:


spray mass spectrum in +ve mode of DLP 1.40 RRT impurity is presented in fig 2.4.21. The


molecular ion ion at m/z=589. This indicates that the molecular weight of the impurity is 219

mass units extra than the DLP molecular weight, which clearly indicates that the one more

moiety is getting added to DLP.


(HRMS) spectral studies. HRMS spectrum in +ve mode of DLP 1.40 RRT impurity is

presented in fig 2.4.22. The HRMS spectrum showed that the impurity is having molecular

formula of C30H23F3N6O2S with exact mass of 588.1613 daltons. The molecular formula

suggests that there is only one sulphur, 3 fluorine in the impurity and number of nitrogens are

double and number of carbons are almost double when compared to DLP. This indicated that

the there is possibility of adduct formation between two modified DLP compounds.


1H NMR, gHSQC, gHMBC spectrums are recorded in DMSO-d6. The Spectra are presented

in fig 2.4.23 to 2.4.25. The 1H NMR spectrum of the impurity is compared with that of DLP.

The 1H NMR spectrum of DLP is presented in fig 2.4.26. The structural formula of DLP is

presented below.

118

The signals due to methylene protons at C10 and C17, which are in the region of 4.7

to 5.0ppm, are accounting for 4 protons in DLP. In the same region, the data of impurity

displays signals at 4.55 and 4.75 ppm accounting for 3 protons. The absence of doublets at

4.76 and 4.83 ppm observed in DLP which is due to C10 methylene group is noteworthy. The

signal at 4.55 ppm integrating for 1 proton, indicates that C10 methylene of DLP has become

a methine in the impurity. This is indicative of site of addition of another moiety at C10.

The presence of cross peak at 67.0ppm in g HSQC showed the correlation with signal

at 4.75 ppm. These are assigned to the methylene signals connected to CF3 group. This

observation clearly indicates the presence of one trifluoro ethoxy group. When compared to

DLP, the impurity displayed 6 additional protons in the aromatic region. This indicates the

presence of another aromatic ring system of DLP moiety in the impurity. The aromatic ring

system which got linked to modified DLP is found to be getting correlated well with the

structure of 1.68 RRT impurity.

Few additional signals in 1HNMR data, namely one triplet at 0.9 ppm and one quartet at 2.4

ppm indicate that the compound contains traces of triethyl amine, which is due to the mobile

phase component during preparative purification.

Based on the above discussion, the structure of the impurity is proposed as given below.

119

Fig. 2.4.21. ESI +ve MS spectrum of 1.40 RRT impurity.

Fig. 2.4.22 : HRMS spectrum of 1.40 RRT impurity.

120

Fig.2.4.23 : 1H NMR spectrum of 1.40 RRT impurity of DLP in DMSO-d6.

121

Fig .2.4.24 : gHSQC spectrum of 1.40 RRT impurity of DLP in DMSO-d6.

122

Fig .2.4.25 : gHMBC spectrum of 1.40 RRT impurity of DLP in DMSO-d6.

123

Fig.2.4.26 : 1H NMR spectrum of DLP drug substance in DMSO-d6.

124

3.4. Conclusions:

The isolation, purification and characterization of the three unknown impurities

(RRT 1.40, 1.65 and 1.68) that are observed to be increasing during the accelerated

stability studies in DLP capsules are identified and all three are found to be structurally

interrelated as 1.68 RRT impurity structure is participated in forming adducts with

modified DLP to give rise to 1.40 and 1.65 RRT impurities. The chemical names are

2-(((1H-benzo[d]imidazol-2-yl)thio)(1-methyl-2-(2,2,2 -trifluoroethoxy)benzo[4',5']-

imidazo[2',1':2,3]imidazo[1,5-a]pyridin-12-yl)methyl)-3-methylpyridin-4-ol for 1.40 RRT

impurity,12-(((1H-benzo[d]imidazol-2-yl)thio)(3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-

2-yl)methyl)-1-methyl-2-(2,2,2-trifluoroethoxy) benzo[4',5']imidazo[2',1':2,3]imidazo

[1,5-a]pyridine for 1.65 RRT impurity and 1-methyl-2-(2,2,2-trifluoroethoxy)benzo[4',5']-

imidazo[2',1':2,3]imidazo[1,5-a]pyridine for 1.68 RRT impurity.

125

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Section (i): Brief account of Dexlansoprazole

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