Detection , Isolation and Characterization of Principal Synthetic Route Indicative Impurity in Lansoprazole

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.


Introduction
Lansoprazole (1) (Figure 1), a substituted benzimidazole, 2-[[[3-methyl-4-(2,2,2trifluoroethoxy)-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 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 Prevacid 1 ® (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 spectrophotometry 2 .Several HPLC methods involving assay and a LC-MS/MS method for the determination of impurities and degradation products of lansoprazole have been published [3][4][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 lansoprazole 6 .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 sample 7 .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.

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.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 o C. 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 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 1 H 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. 1

Figure 2 .
Figure 2. Typical chromatograms of lansoprazole (a) Spiked with all impurities (b) Under acid hydrolytic conditions and (c) Isolated impurity 1.
acetonitrile, HPLC grade triethylamine (TEA) and HPLC grade ortho phosphoric acid were purchased from Merck (Darmstadt, Germany).

1 H
and 13 C 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.
H and 13 C NMR, MS and IR assignments for des-(trifluoroethoxy) lansoprazole.Based on the above spectral data the molecular formula for impurity (5) could be C 14 H 13 N 3 OS and the corresponding structure was characterized as 2-((3-methylpyridin-2yl)methylsulfinyl)-1H-benzo[d]imidazole.