detection, isolation and characterization of principal ...ich, draft revised guidance on impurities...

ISSN: 0973-4945; CODEN ECJHAO

E-Journal of Chemistry

http://www.e-journals.net 2010, 7(3), 844-848

Detection, Isolation and Characterization of Principal

Synthetic Route Indicative Impurity in Lansoprazole

K.S.V SRINIVAS*, K MUKKANTI,

R BUCHI REDDY§ and P SRINIVASULU

*Analytical Development Laboratory,

Inogent Laboratories Private Limited, No. 28A, IDA,

Nacharam, Hyderabad, 500 076, India. §Research & Development Laboratory,

Inogent Laboratories Private Limited, No. 28A, IDA,

Nacharam, Hyderabad, 500 076, India.

Centre for Environment,

JNT University, Kukatpally, Hyderabad, 500 072, India.

[email protected]

Received 1 April 2009; Accepted 1 June 2009

Abstract: An unknown impurity in lansoprazole (2-[[[3-methyl-4-(2, 2,

2-trifluoroethoxy)-2-pyridyl] methyl] sulfinyl] benzimidazole) was

detected by HPLC and was identified as des-(trifluoroethoxy)

lansoprazole, an principal synthetic route indicative impurity of

lansoprazole. Lansoprazole was subjected to different ICH prescribed

stress conditions like hydrolysis, oxidation, photolysis and thermal

degradation conditions to enrich the impurity. The impurity was enriched

by using acid catalytic degradation, isolated by using preparative HPLC

and characterized (FTIR, MS and NMR). Limit of Detection (LOD) and

Limit of Quantification (LOQ) are found to be 0.014% and 0.035%

respectively.

Keywords: Impurity, Isolation, Characterisation, Preparative HPLC, Degradation.

Introduction

Lansoprazole (1) (Figure 1), a substituted benzimidazole, 2-[[[3-methyl-4-(2,2,2-

trifluoroethoxy)-2-pyridyl] methyl] sulfinyl] benzimidazole, a compound that inhibits gastric

acid secretion. Lansoprazole is in a class of drugs called proton pump inhibitors (PPI)

which block the production of acid by the stomach. Proton pump inhibitors are used for the

Detection, Isolation and Characterization 845

treatment of conditions such as ulcers, gastroesophageal reflux disease (GERD) and

Zollinger-Ellison Syndrome that are caused by stomach acid. Lansoprazole, like other

proton-pump inhibitors, blocks the enzyme in the wall of the stomach that produces acid. By

blocking the enzyme, the production of acid is decreased, and this allows the stomach and

esophagus to heal. The recemic mixture of the lansoprazole was introduced as a therapeutic

agent under the trade name of Prevacid1® (TAP Pharmaceutical Products Inc.)

Here, we report the structural characterization of the unknown impurity (5) (Figure 1).

Figure 1. Structures of lansoprazole and impurities.

The different analytical techniques reported so far for the determination of this drug

along with corresponding impurities by UV-Visible spectrophotometry2. Several HPLC

methods involving assay and a LC–MS/MS method for the determination of impurities and

degradation products of lansoprazole have been published3-5

.

The lansoprazole sample containing 0.09% of unknown impurity at 0.52 RRT was

observed when the sample was analysed by using chromatographic conditions published in

the USP monograph for lansoprazole6. As per regulatory guidelines, the pharmaceutical

studies using a sample of the isolated impurities can be considered for safety assessment. It

is therefore, essential to isolate and characterize unidentified impurities present in the drug

sample7. Forced degradation studies were conducted to enrich this impurity and were formed

under acid hydrolytic conditions along with other known impurities. The acid degraded

sample was taken for the isolation of unknown impurity by using preparative HPLC and the

isolated impurity was charecterised by using LC-MS, NMR and IR .To the best of our

knowledge, the impurity detected at 0.52 RRT was published for first time.

Chromatogram

Ab

sorb

ance

, A

U

Time, min

Time, min

Ab

sorb

ance

, A

U

Time, min

Ab

sorb

ance

, A

U

846 K.S.V SRINIVAS et al.

Figure 2. Typical chromatograms of lansoprazole (a) Spiked with all impurities (b) Under

acid hydrolytic conditions and (c) Isolated impurity 1.

(a)

(b)

(c)

Detection, Isolation and Characterization 847

Experimental

HPLC grade acetonitrile, HPLC grade triethylamine (TEA) and HPLC grade ortho

phosphoric acid were purchased from Merck (Darmstadt, Germany).

Instrumentation

The HPLC system was equipped with quaternary gradient pumps with auto sampler and auto

injector (Model Alliance 2695, Make Waters USA ) connected with photo diode array

detector (PDA Model 2996, Make Waters USA) controlled with Empower

software(Make Waters , USA).

The preparative HPLC system was equipped Prep LC controller with binary gradient

pumps with Waters fraction collector (model 2767, Make Waters USA) and with photo

diode array detector (PDA Model 2996, Make Waters USA) controlled with Empower 2

software (Make Waters, USA).

Positive ion ESI-MS was performed on 6310 ion-trap mass spectrometer (Agilent, USA)

equipped with an ESI source. Sample solutions were introduced into the ion source at a flow

rate of 4 Lm−1

, capillary voltage 3.8 kV, drying gas temperature 250 ◦C, drying gas flow rate

5 Lm−1

and nebulizer pressure 14 psi nitrogen was used as both nebulizing gas and drying gas.

1H and

13C NMR spectra were acquired Varian with autosampler version 2.2 C at 305 K.

The hydrogen and carbon chemical shifts are referred to the internal tetramethylsilane

(TMS). The coupling constants are expressed in Hertz.

The IR spectra were recorded in the solid state as KBr dispersion medium using Perkin-

Elmer Spectrum One FT IR spectrophotometer.

Isolation of the destrifuoro ethoxy impurity

Acid catalytic conditions

Sample was dissolved in 0.1N HCl and stirred for 30 minutes at 60 oC. The degraded sample

was subjected for preparative HPLC and isolated the impurity. The impurity (5) was

enriched to 17%.

Chromatographic conditions for preparative HPLC

The chromatographic separation was achieved on an Gemini C18 (Phenomenex, USA),

150 mm × 30 .0 mm, 5 µ semi preparative column using a mobile phase containing mixture

of water and acetonitrile (70 :30, v/v). The flow rate of the mobile phase was 20.0 mL min-1. The

column temperature was maintained at 25 ◦C and the wavelength was monitored at 285 nm. The

injection volume was 1000 µL. The test concentration for the analysis was 50 mg mL-1

. The

standard and the test dilutions were prepared in the mobile phase.

Isolation of the impurity The collected fractions from preparative HPLC were subjected to distillation for 60 min. to

remove the solvent and then crystallized by using lyophilizer.

Results and Discussion

The solid crystallized compound was analyzed by HPLC and the HPLC purity was found to

be 98.1%. The impurity was then characterized by using LC-MS, NMR and IR.

The +ve ES-MS spectrum of the impurity showed peaks at m/z 272.2 and 543.2

corresponding to the adduct ions (M+H)+ and (2M+H)+. Further the −ve ES-MS spectrum

showed peaks at m/z 270 corresponding to (M−H)−. The adduct ions confirm the molecular

ion of the impurity (5) to be m/z 271. The DEPT spectra displayed one negative signal due to

848 K.S.V SRINIVAS et al.

one methylene group and five positive peaks due to the presence of one methyl group and

the rest are due to the methine groups (all in aromatic). IR spectrum displayed characteristic

absorptions at 3061(Aromatic C-H stretching), 2967(Aliphatic C-H stretching), 1560

(Aromatic C=C, C=N stretching), 1433 (Aliphatic C-H bonding), 1267 (C-N

stretching),1022 (S=O stretching) and 748 cm-1

(Aromatic C-H stretching). Presence of

signals at 2.37 ppm (3H) and 4.89 ppm (2H) in 1H NMR spectrum was attributed to methyl

and methylene groups respectively. Other signal at 7.29, 7.47, 7.66 and 8.26 ppm attributed

to aromatic protons (Table.1). Limit of detection (LOD) and Limit of quantification (LOQ)

of the impurity was found to be 0.014% and 0.035% respectively.

Table 1. 1H and

13C NMR, MS and IR assignments for des-(trifluoroethoxy) lansoprazole.

Position 1H ppm/J 13

C MS IR

1 3H 2.37/s 15.19

2 -- -- 139.3

3 1H 7.47/m 116.0

4 1H 7.66/m 123.7

5 1H 8.26/d 132.3

6 -- -- 151.8

7 2H 4.89/s 60.2

8 -- -- 153.0

9 -- -- 123.2

10 -- -- 123.2

11 1H 7.66/m 143.7

12 1H 7.29/m 147.2

13 1H 7.29/m 147.2

14 1H 7.66/m 143.7

+ve ES-MS :

272.2 (M+H)+

543.2 (2M+H)+

-ve ES-MS:

270 (M-H)-

3061: Aromatic C-H

stretching

2967: Aliphatic C-H

stretching

1560: Aromatic C=C, C=N

stretching

1433 : Aliphatic C-H

bonding

1267 : C-N stretching

1022 : S=O stretching

748 : Aromatic C-H

stretching

Based on the above spectral data the molecular formula for impurity (5) could be

C14H13N3OS and the corresponding structure was characterized as 2-((3-methylpyridin-2-

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

References

1. Prevacid (Lansoprazole), Drug description, Pharmacology, Pharmacokinetics, Studies

Metabolism, http://www.rxlist.com/prevacid-drug.htm#cp. Accessed on March 2010.

2. Karljikovic-Rajic K, Novovic D, Marinkovic V and Agbaba D, J Pharm Biomed

Anal., 2003, 32,1019-1027.

3. El-Sherif Z A, Osman Mohamed A, El-Bardicy M G and El-Tarras M F, Chem Pharm

Bull., 2006, 54, 814-818.

4. DellaGreca M, Iesce M R, Previtera L, Rubino M, Temussi F and Brigante M,

Chemosphere, 2006, 63(7), 1087-1093.

5. Selenka J, Duff S, He J, Li K, Sunthankar P and Ding X, Aapspharmsci Abstracts,

AM_2007, AAPS2007-003647.PDF.

6. The United States Pharmacopoeia 30, Monographs: Monographs: Lansoprazole, 1229, 2007.

7. ICH, Draft Revised Guidance on Impurities in New Drug Substances Q3A (R2),

International Conference on Harmonisation, IFPMA, Geneva, 2006.

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