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-2yl]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
140C. 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

37

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]. UPLCMS/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;
40C/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 reversephase preparative liquid chromatography and characterized.

38

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-HBenzoimidazol-2-ylsulfanyl)-[1-methyl-2-2,2,2-trifluoro-ethoxy)-4a,5,9b-triaza-indeno[2,1a]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-2ylsulfanyl)-[3-methyl-4-(2,2,2-trifluoro-ethoxy)-pyridin-2-yl]-methyl}-1-methyl-2-(2,2,2trifluoro-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-trifluoroethoxy)-4a,5,9b-triaza-indeno[2,1-a]indene with molecular formula C16H12F3N3O
molecular mass 319.09.

39

and

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 Reddys Laboratories,
Hyderabad, India. DLP Capsules of 30 and 60 mg & all excipients are received from
formulation R&D, Dr Reddys 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 200400 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 5 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.
40

The column temperature is maintained at 30C and the detection wavelength is 285 nm. The
injection volume is 20l.

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.

Blank

Standard

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

41

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 C and 25C 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 g 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.

Placebo

Test

Fig 2.2.2: Overlay chromatogram of placebo and DLP Capsules.

42

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 g ml-1.

43

1.7.3. Effect of base hydrolysis

300 mg of DLP pellets powder is treated with 100 ml of 2N NaOH at 50C 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 g ml-1.
1.7.4. Effect of neutral hydrolysis
300 mg of DLP pellets powder is treated with 100 ml water at 50C 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 g 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 g 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 25C/90% relative humidity for 9
days. Similarly DLP pellets powder in another plate is exposed in an oven at 105C 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.

44

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 g ml-1 of DLP (corresponding to 6.6 to 250% of assay of nominal
sample concentration of 60 g 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 25C and 35C and
organic phase compositions in mobile phase at + 10% respectively.

45

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.

46

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 m 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 30C respectively.

47

2.3. Method validation

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

100.7

99.8

100.4

99.9

100.7

100.2

100.3

100.2

100.7

100.3

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 g ml -1 with a resultant %R.S.D. of 0.6 (n = 5). The LOD is found to be 0.028 g ml.-1
2.3.3. Linearity
A linear calibration plot for assay of DLP is obtained over the calibration range 4-150
-1

g ml 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.
48

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.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

level
20%
20%
20%
50%
50%
50%
70%
70%
70%
100%
100%
100%
120%
120%
120%
150%
150%
150%

mg
added
120.60
120.60
120.60
301.50
301.50
301.50
422.10
422.10
422.10
603.00
603.00
603.00
723.60
723.60
723.60
904.50
904.50
904.50

mg found
121.46
121.23
121.36
296.04
296.64
297.48
418.82
418.66
419.00
604.23
602.46
604.14
725.16
725.38
725.81
907.72
907.79
907.77

49

%
Recovery
100.7
100.5
100.6
98.2
98.4
98.7
99.2
99.2
99.3
100.2
99.9
100.2
100.2
100.2
100.3
100.4
100.4
100.4

Mean % Recovery
100.6

98.4

99.2

100.1

100.3

100.4

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
Observed value
Parameter
1.Flow rate

2.Column temperature

3.Mobile phase A
(for methanol variation)
4. Mobile phase B
(for acetonitrile variation)
5. Mobile phase B
(for methanol variation)
6. pH

Variation

Tailing factor

%RSD

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

25C

1.2

0.1

30C

1.0

0.04

35C

1.1

0.1

90%

1.2

0.1

100%

1.0

0.04

110%

1.1

0.0

90%

1.1

0.2

100%

1.0

0.04

110%

1.1

0.3

90%

1.2

0.3

100%

1.0

0.04

110%

1.1

0.1

7.8

1.1

0.1

8.0

1.0

0.04

8.2

1.1

0.1

50

2.3.6. Solution stability and mobile phase stability

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.

51

Table 2.2.4: Table of results of specificity

Stress condition
Kept on bench top for 5 minutes with 1N HCl
solution.
Stressed at 50C in water bath for 24 hrs with
2N NaOH solution.
Kept on bench top for 15 minutes with 10%
H2O2 solution.
Stressed at 50C in water bath for 24 hrs with
water.
Exposed to visible light for 1.2million lux
hours and UV light at 200 Watt hours/ meter2
Exposed to Dry heat at 105C for about 1
hour.
Exposed to humidity at 25C, 90% RH for 9
days.

% Assay of
stressed
sample

Purity
Angle

Purity
Threshold

84.3

0.111

0.302

92.4

0.107

0.301

97.1

0.130

0.327

91.5

0.116

0.299

99.7

0.117

0.318

84.7

0.081

0.292

99.8

0.110

0.318

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

52

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.
53

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.
54

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.

55

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.

56

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 Reddys
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. Reddys 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
1
Dexlansoprazole

Structure and IUPAC Name

(R)-2-(((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2yl)methyl)sulfinyl)-1H-benzo[d]imidazole
2

Carboxylic acid
Impurity

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

Hydroxybenzimidazole
1H-benzo[d]imidazol-2-ol
57

Mercaptobenzimidazole

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

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)-1Hbenzo[d]imidazole
7

Sulphone

[[(1H-benzimidazole-2-yl)sulfinyl]methyl]-3-methyl-4-(2,2,2trifluoroethoxy)-pyridine 1-oxide
8

Cyclized
dessulphur- des
trifluoro ethyl
sulphide adduct
(Impurity I)

2-(((1H-benzo[d]imidazol-2-yl)thio)(1-methyl-2-(2,2,258

trifluoroethoxy)benzo[4',5']imidazo[2',1':2,3]imidazo[1,5a]pyridin-12-yl)methyl)-3-methylpyridin-4-ol.
9

Sulphide

2-(((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2yl)methyl)thio)-1H-benzo[d]imidazole
10

Cyclized
dessulphursulphide adduct
(Impurity II)

12-(((1H-benzo[d]imidazol-2-yl)thio)(3-methyl-4-(2,2,2trifluoroethoxy)pyridin-2-yl)methyl)-1-methyl-2-(2,2,2trifluoroethoxy)benzo[4',5']imidazo[2',1':2,3]imidazo[1,5a]pyridine
11

Cyclized
dessulphur
(Impurity III)

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

59

12

Cyclized
dessulphurmercapto
benzimidazole
adduct
(Impurity
M+467)

12-((1H-benzo[d]imidazol-2-yl)thio)-1-methyl-2-(2,2,2trifluoroethoxy)benzo[4',5']imidazo[2',1':2,3]imidazo[1,5a]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 200400 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 3.5 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 25C and the detection wavelength is 285 nm. The injection volume is 10l. Sample cooler
temperature is used as 5C.
60

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 g 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 20C to 25C. 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.

61

Diluent

Diluted standard

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

Placebo

Fig 2.3.2: Specimen chromatogram of placebo for DLP capsules.

62

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

63

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 60C using a
heating water bath for 24 hrs. The resulting stress solution is neutralized, equilibrated to room
64

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 60C 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 25C/90% relative humidity for 7 days.
Similarly DLP pellets powder in another plate is exposed in an oven at 105C 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.

65

1.8. Method validation

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(RRTs) and relative response factors (RRFs) 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

Carboxylic acid Impurity

0.14

1.04

Hydroxy benzimidazole

0.25

0.92

Mercapto benzimidazole

0.36

2.66

N-oxide

0.71

1.21

Nitrosulphoxide

0.74

1.26

Sulphone

1.06

0.92

Impurity I

1.41

1.11

Sulphide

1.43

1.11

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.
66

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 signalto-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
To determine the robustness of the developed method, experimental conditions are
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 20C and 30C instead of 25C. 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.

67

1.8.7. Solution stability and mobile phase stability

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
68

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 m 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 25C. 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 10l 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 5C is found
necessary.
69

2.3. Method validation

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, NOxide, 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.

70

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.

71

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

Sample
No.

1
2
3
4
5
6
Avg
%RSD

DLP

Carboxylic
acid
Impurity

0.256
0.248
0.257
0.255
0.249
0.246
0.252
1.9

0.237
0.232
0.237
0.238
0.236
0.237
0.236
0.9

Hydroxy Mercapto
benzimi benzimid
dazole
azole
0.180
0.187
0.187
0.186
0.190
0.189
0.187
1.9

0.234
0.240
0.239
0.242
0.230
0.231
0.236
2.1

NOxide

Nitro
sulph
oxide

Sulph
one

0.181
0.187
0.186
0.188
0.185
0.182
0.185
1.5

0.178
0.182
0.184
0.184
0.178
0.176
0.180
1.9

0.444
0.456
0.457
0.456
0.465
0.461
0.457
1.5

Impurity
I

Sulphide

0.268
0.267
0.271
0.265
0.268
0.265
0.267
0.9

0.276
0.286
0.286
0.286
0.287
0.284
0.284
1.4

Impurity
Impurity
Impurity
II
M+467
III
0.256
0.248
0.249
0.248
0.249
0.249
0.250
1.2

0.295
0.305
0.296
0.301
0.301
0.304
0.300
1.4

0.212
0.210
0.210
0.213
0.207
0.206
0.210
1.3

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

Sample
No.

1
2
3
4
5
6
Avg
%RSD

DLP

Carboxylic
acid
Impurity

0.252
0.246
0.251
0.235
0.245
0.248
0.250
2.5

0.227
0.229
0.226
0.231
0.239
0.234
0.231
2.1

Hydroxy Mercapto
benzimi benzimid
dazole
azole
0.200
0.205
0.214
0.193
0.195
0.207
0.202
3.9

0.197
0.195
0.201
0.194
0.190
0.192
0.195
2.0

NOxide

Nitro
sulph
oxide

Sulph
one

0.205
0.197
0.193
0.190
0.184
0.181
0.192
4.6

0.171
0.176
0.166
0.171
0.171
0.169
0.171
1.9

0.437
0.440
0.437
0.427
0.441
0.418
0.433
2.1

72

Impurity
I

Sulphide

0.235
0.241
0.231
0.252
0.226
0.229
0.236
4.0

0.254
0.249
0.261
0.252
0.274
0.251
0.257
3.6

Impurity
Impurity
Impurity
II
M+467
III
0.244
0.232
0.228
0.221
0.226
0.230
0.230
3.4

0.225
0.239
0.242
0.238
0.241
0.237
0.237
2.6

0.191
0.188
0.191
0.183
0.204
0.188
0.191
3.7

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

Parameter

DLP

Carboxylic
acid
Impurity

LOD
0.009
0.011
In %
S/N ratio
2.82
3.117
LOQ
0.026
0.0347
In%
S/N ratio
9.94
10.594
Precision
at LOQ
3.0
2.6
(%RSD*)
* RSD for 6 determinations.

Hydroxy
benzimida
zole

Mercapto
benzimid
azole

N-Oxide

Nitrosul
phoxide

Sulphone

Impurity
I

Sulphide

Impurity
II

0.012

0.005

0.013

0.015

0.016

0.011

0.014

2.93

2.95

3.01

3.04

3.01

3.464

0.037

0.016

0.039

0.044

0.049

10.15

10.32

9.93

10.22

2.5

7.7

3.5

2.1

73

Impurity
III

Impurity
M+467

0.015

0.019

0.01

3.01

2.815

2.370

3.26

0.033

0.042

0.044

0.048

0.029

9.58

10.827

9.51

9.363

9.698

10.48

3.1

7.7

5.5

2.4

1.6

1.9

Figure 2.3.5 : Linearity graphs of DLP and its impurities.

74

Figure 2.3.6 : Linearity graphs of DLP impurities.

75

Fig.2.3.7: Linearity graphs of DLP impurities.

76

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

Hydroxy
benzimida
zole

Mercapto
benzimid
azole

N-Oxide

Nitrosul
phoxide

Sulphone

Impurity
I

Sulphide

Impurity
II

Spike level

DLP

Carboxylic
acid
Impurity

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)

Values given in parenthesis represent standard deviation of triplicate results.

77

Impurity
III

Impurity
(M+467)

2.3.5. Robustness
To determine the robustness of the developed method, experimental conditions are
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.

2.3.6. Solution stability and mobile phase stability

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 2-8C) 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 5C. 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,
RRTs 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.

78

Table 2.3.6: Results of robustness study

RRT of the impurity

Condition
0.6 ml/min
0.8 ml/min
0.85 ml/min
20C
25C
27C
pH 6.8
pH 7.0
pH 7.2
M.P A ( acetonitrile)90%
M.P A ( acetonitrile)100%
M.P A ( acetonitrile)110%
M.P B ( acetonitrile)90%
M.P B ( acetonitrile)100%
M.P B ( acetonitrile)110%

Carbo
xylic
acid
imp
0.165
0.142
0.137
0.142
0.143
0.142
0.143
0.140
0.140
0.139
0.140
0.143
0.131
0.134
0.136

Hydroxy
benzimida
zole
0.277
0.241
0.232
0.240
0.241
0.239
0.246
0.242
0.243
0.244
0.242
0.245
0.228
0.231
0.242

Mercapto
benzimida
zole
0.393
0.345
0.335
0.346
0.347
0.344
0.357
0.352
0.351
0.354
0.352
0.355
0.331
0.337
0.342

NOxide
0.707
0.692
0.685
0.692
0.692
0.691
0.695
0.691
0.688
0.691
0.691
0.694
0.686
0.687
0.686

79

Nitro
sul
phoxide
0.761
0.731
0.723
0.732
0.732
0.731
0.743
0.737
0.732
0.737
0.737
0.739
0.722
0.726
0.728

Sul
phone
1.051
1.054
1.053
1.053
1.053
1.052
1.086
1.068
1.032
1.059
1.068
1.059
1.056
1.059
1.057

Imp I
1.352
1.407
1.422
1.407
1.407
1.408
1.392
1.400
1.404
1.400
1.400
1.394
1.420
1.415
1.412

Sul
phide
1.392
1.424
1.436
1.424
1.424
1.425
1.415
1.425
1.433
1.426
1.425
1.421
1.434
1.431
1.432

Imp
II
1.568
1.642
1.665
1.643
1.643
1.645
1.612
1.631
1.652
1.635
1.631
1.626
1.654
1.649
1.651

Imp
III
M+467
1.608 1.776
1.663 1.859
1.684 1.888
1.664 1.860
1.664 1.860
1.665 1.862
1.650 1.842
1.664 1.859
1.677 1.875
1.665 1.860
1.664 1.859
1.656 1.848
1.675 1.872
1.672 1.869
1.676 1.874

2.3.7. Results of solution stability :

% of the impurity upon storage between 2-8C

Duration
in hours

0
3
6
15
Maximum
Difference
from zero
hours

Carbo
xylic
acid imp

Hydroxy
benzimid
azole

Mercapto
benzimida
zole

NOxide

Nitrosul
phoxide

Sul
phone

Imp I

Sul
phide

Imp II

Imp III

Imp
M+467

Total
impuriti
es

0.2522

0.2485

0.1809

0.1997

0.1682

0.4711

0.2882

0.3146

0.1622

0.2844

0.2044

2.7109

0.2356

0.2518

0.1853

0.2199

0.1668

0.4794

0.2678

0.3095

0.1314

0.2909

0.2049

2.7612

0.2350

0.2510

0.1840

0.2210

0.1681

0.4785

0.2712

0.3039

0.1252

0.2913

0.2145

2.7705

0.2360

0.2512

0.1820

0.2212

0.1673

0.4774

0.2716

0.3045

0.1224

0.2901

0.2146

2.7809

0.02

0.00

0.00

-0.02

0.00

-0.01

0.02

0.01

0.04

-0.01

-0.01

0.1

80

2.3.8. Results of mobile phase stability on bench top :

% of impurity for mobile phase stability
No of
days
0
1
2
3
Max
diff.
from
initial

Carboxylic
acid

Hydroxy
benzimida
zole

Mercapto
benzimida
zole

N-oxide

Nitro
sulphoxide

Sulphone

Impurity
1

Sulphide

Impurity
II

Impurity
III

Impurity
M+467

0.2522
0.2692
0.2504
0.2272

0.2485
0.2484
0.2435
0.2238

0.1809
0.171
0.1707
0.1673

0.1997
0.1824
0.2002
0.1895

0.1682
0.1827
0.1561
0.1564

0.4711
0.4689
0.4433
0.4587

0.2882
0.2564
0.286
0.2606

0.3146
0.275
0.3039
0.2782

0.1622
0.1427
0.1309
0.1545

0.2854
0.2776
0.2721
0.2874

0.2044
0.1987
0.2035
0.1883

2.7755
2.6729
2.6607
2.5918

0.03

0.02

0.01

0.01

0.01

0.03

0.03

0.04

0.03

0.01

0.02

0.2

81

Total

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.

82

Table 2.3.9 : Summary of forced degradation studies

Stress condition

Control sample
Acid degradation
Base degradation
Oxidative
degradation
Hydrolytic
degradation
Visible light
UV light

% Impurities observed

Carbo
xylic
acid
Imp

Hydro
xyl
benz
imida
zole

Mer
capto
benz
imida
zole

NOxide

Nitro
sulpho
xide

Sul
phone

Imp I

Sul
phide

Imp II

Imp
III

0.00

0.00

0.00

0.00

0.00

0.08

0.00

0.08

0.00

0.51

0.05

0.02

0.12

0.52

0.10

0.20

0.70

0.25

0.03

0.68

0.00

0.00

0.08

0.09

0.23

0.12

0.01

0.00

0.06

12.9

1.36

0.20

0.08

0.00

0.00

0.00

0.03

0.02

0.00

0.00

0.01

0.02

Thermal
0.68
0.07
degradation
Humidity
0.00
0.01
degradation
TI: Total impurities; imp = impurity.

%
Assay

Mass
balance

Imp
M+467

TI

0.00

0.00

0.27

99.8

100.1

0.51

2.80

0.00

11.4

86.9

98.3

0.19

0.85

0.08

0.00

3.4

95.7

99.1

0.08

0.07

0.02

0.09

0.00

15.1

83.8

98.8

0.08

0.40

2.19

0.31

1.11

0.00

9.4

89.6

99.0

0.00

0.05

0.00

0.08

0.00

0.00

0.00

0.25

99.9

100.2

0.00

0.00

0.07

0.00

0.08

0.00

0.00

0.00

0.26

99.8

100.1

0.61

0.00

0.00

0.09

0.11

0.59

0.09

1.83

2.33

14.3

84.2

98.5

0.02

0.00

0.00

0.05

0.00

0.06

0.00

0.00

0.00

0.27

99.7

100.4

83

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

84

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

85

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

86

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

87

Fig 2.3.12: Chromatogram and purity plot of visible light stressed DLP capsules.
88

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

89

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

90

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

91

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.

92

Section (iv): Isolation, identification and characterization of three unknown

impurities of dexlansoprazole in dexlansoprazole capsules.
2.4. Introduction :
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-2yl]methylsulfinyl)-1H-benzimidazole. Its empirical formula is: C16H14F3N3O2S, with a
molecular weight of 369.36. The structural formula is:

DLP is unstable when exposed to acid, base, peroxide and thermal degradations.
During formulation development, it is observed that three late eluting unknown impurities are
increasing upon stability. These degradants at RRT 1.40, 1.65 and 1.68 are found to be
increasing to a level of 0.3% in 3 M, 40oC/75%RH stability studies. A mass spectral study is
conducted to understand the mass of the unknown degradants. Literature survey done based
on mass numbers (588, 670 and 319) did not yield any structural information of these
unknown degradants.
This section describes the isolation and characterization of these three unknown
degradation products formed in DLP capsules during the stability studies. Though some of the
impurities and degradation products are reported in the literature, isolation and
characterization of these degradation products is not reported to the best of our knowledge.

93

1. Experimental :
1.1 chemicals and instrumentation :
DLP capsules (delayed release) are formulated in Dr Reddys laboratories Ltd,
Hyderabad, India. DLP drug substance and working standard is from Dr.Reddys 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.

94

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

95

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 m 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.

96

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 m 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 25C.
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.

97

1.65 RRT impurity

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 105c 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.
enriched sample is given as figure 2.4.5.

98

The chromatogram of

1.40 RRT

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

99

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

1.40 RRT imp purified

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.
1

H 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.
100

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.

101

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.

102

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

103

Fig.2.4.10.

13

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

104

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

105

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

106

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

107

3.1.2. Impurity II at 1.65 RRT:

The isolated and purified impurity (1.65 RRT) is subjected to LC-MS studies. Electro
spray mass spectrum in +ve mode of 1.65 RRT impurity is presented in fig 2.4.14. The
compound mass spectrum shows that the mass number is 670, as it displayed a protonated
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.
The isolated and purified impurity (1.65 RRT) is subjected to high resolution mass
(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.
The isolated and purified impurity (1.65 RRT) is subjected to NMR spectral studies.
1

H 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.

108

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 13C.
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.

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116

3.1. 3. Impurity-I at 1.40 RRT:

The isolated and purified impurity (1.40 RRT) is subjected to LC-MS studies. Electro
spray mass spectrum in +ve mode of DLP 1.40 RRT impurity is presented in fig 2.4.21. The
compound mass spectrum shows that the mass number is 588, as it displayed a protonated
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.
The isolated and purified impurity (1.40 RRT) is subjected to high resolution mass
(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.
The isolated and purified impurity (1.40 RRT) is subjected to NMR spectral studies.
1

H 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.

117

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.

118

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

Fig. 2.4.22 : HRMS spectrum of 1.40 RRT impurity.

119

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

120

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

121

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

122

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

123

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)pyridin2-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.

124

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