Hydrolytic Degradation Study of Lansoprazole , Identification , Isolation and Characterisation of Base Degradation Product

Lansoprazole degradation is accelerated in both acidic and basic medium in water. The present investigation deals with the hydrolytic degradation of Lansoprazole. Acidic medium degradation show all known impurities and degradation products whereas basic degradation studies show new impurity which has higher molecular weight than Lansoprazole. New impurity was identified, isolated using mass based auto purification system and characterised by 1H NMR, 13C NMR, HMBC, HSQC, NOE, COSY and HRMS experiments. Isolated impurity was showing molecular weight of 467.10, molecular formula of C23H16F3N5OS and its name is 7-(3-Methyl-4-(2,2,2-trifluoroethoxy) pyridin-2-yl)-7H-benzo[4,5]imidazo[2,1-b]benzo[4,5]imidazo[2,1-d][1,3,5]thiadiazine.

In general, active pharmaceutical ingredients (APIs) are formulated with excipients as tablets, syrup and capsules.Since the active ingredient interacts with the excipients and the formulated product is stored at different conditions, the study of API stability is critical in the drug development process.Many factors can affect the stability of a pharmaceutical product, some of them include the stability of the active ingredient, the manufacturing process, and the environmental conditions (such as oxidation, reduction, hydrolysis, and racemization that might occur [7] [8].The study of stability under stressed conditions is important since it can cause many degradation reactions. Prevacid I.V. for injection contains 30 mg of the active ingredient of Lansoprazole, 60 mg mannitol, 10 mg meglumine and 3.45 mg sodium hydroxide and is supplied as a sterile, lyophilized powder for I.V. (intravenous) use.Drug is a combination of sodium hydroxide and Lansoprazole, so it is important to study the degradation in basic medium and characterisation of degradation products.
The different analytical techniques were reported so far for the determination of this drug along with corresponding impurities by UV-Visible spectrometry [9]- [12].Several HPLC assay and LC-MS/MS method for determination of impurities and degradation products of Lansoprazole have been published [13]- [22].Some research work has been published on characterisation of process related impurities of Lansoprazole [23] [24].Literature survey indicates that no work is done on the identification, isolation and characterisation of major unknown degradation product formed in the base degradation study.All chemical structures of Lansoprazole and its impurities were shown in Figure 1.

Materials
The investigated sample of Lansoprazole was procured from NOSCH Labs Private Limited, India.Solvents and buffers used for analysis were HPLC grade Acetonitrile and Methanol (Rankem), Ammonium acetate (Fisher Scientific-Qualigens), Ammonium bicarbonate (Sigma Aldrich), Formic acid (Alfa Aesar), Trifluroacetic acid (Alfa Aesar) and Water used was Milli-Q grade.

Base Degradation Procedure
Base degradation studies were carried out as per the guidelines of ICH.200 mg of standard drug was refluxed with 0.1 N NaOH solution at 60˚C for 8 h to study basic degradation.Normality of base solution increased gradually from 0.1 N to 2 N to achieve sufficient degradation.For analytical study, the resultant base degradation sample was dissolved in methanol and diluted with mobile phase and 10 µl injected into the system and the chromatograms were recorded to assess the stability of the sample.

Identification of New Base Degradation Product
After acid and base degradation the crude sample was injected into the analytical column for the separation of degradation products from the drug peak.To obtain the baseline separation different mobile phases have been used like ammonium acetate, trifluro acetic acid, formic acid, ammonium bicarbonate.Various columns were screened to check the separation and symmetrical peak shape like YMC ODS, YMC Triart, Sunfire, X-terra, X-bride, Agilent zorbax CN, X-Select.Finally desired separation was achieved using the 10 mM ammonium bicarbonate and acetonitrile as a mobile phase and X-Select column (150 × 4.6 mm, 5µ).In the developed method observed peaks, retention times, molecular weights and area percentages were shown in below tables (Table 1 and Table 2).
From Table 1 and Table 2, it was clearly observed that in acid degradation study observed all peaks are re-

Isolation of Base Degradation Product
Base degradation study clearly indicates that the peak observed at 12.63 min retention time and having mass m/z 468.11 (M + H) was new entity in base degradation and remaining peaks were matching with the already reported impurities.To separate this novel impurity formed in base degradation condition, we scaled up the analytical method to preparative scale.Using the same mobile phase and column having dimensions (150 × 19 mm, 5µ) and a flow rate of 19 ml•min −1 degradation product has been isolated.In mass based preparative system splitter has been used after the column through which flow has been splitted in the ratio of 1000:1.The one part of the flow is passed through mass detector by diluting with make pump using 1 ml•min −1 flow rate.For the proper ionisation of the desired impurity 0.1% formic acid has been used as it is more volatile and enhances the ionisation.The delay time of the fraction collector has been investigated by using dye at same flow rate and the same parameters have been given as a input to get the good recovery.The crude sample was neutralised with HCl solution, diluted with mobile phase and injected into the preparative column in three consecutive injections.
The fractions have been collected on the basis of mass threshold parameters of total ion chromatogram.After completion of purification collected all fractions of mass 468.11 (M + H) pooled together and lyophilized to get free solid.
From information provided by HRMS report it is matching with expected structure of base degradation product.HRMS report of base degradation product was shown in Figure 7.

Nuclear Magnetic Resonance ( 1 H & 13 C-NMR, HMBC, HSQC, COSY, NOE)
The numbering scheme for the NMR assignments and HMBC correlation for critical singlet proton at 7.914 ppm are shown in Figure 8.The 1 H NMR, 13 C NMR spectral data of degradation product was compared with those of Lansoprazole.In From HMBC, Proton of chemical shift at 7.914 singlet (Label 11) giving correlations to the carbons of labels 8, 10, 13, 22 and 29.The above correlation clearly indicates presence of benzimidazoles substituted [1,3,5] Thiadiazine.
From COSY ( 1 H-1 H) study, assumed structure of base degradation product contains four aromatic doublets and four aromatic triplets in two benzimidazole rings and two doublets in pyridine ring.From cosy spectrum it clearly shows correlations with adjacent protons.

Conclusion
The fast, simple and sensitive method has been optimised for the separation of degradation product of Lanso-

Figure 1 .
Figure 1.Chemical structures of Lansoprazole and its related impurities.

Figure 7 .
Figure 7. Mass elemental composition report of base degradation product.
1 H NMR, one set of -CH 2 (Methylene) group protons in Lansoprazole are missing in degradation product.As well, in13 C NMR one set of -CH 2 (Methylene) group carbon in Lansoprazole missing in degradation product.NMR chemical shifts and 2 D NMR (HSQC, HMBC Correlations) of base degradation product are shown in Table 3.In NOE study, while irradiating proton chemical shift at 7.914 (Label 11) enhancing signals which are spatially closed signals of methyl protons (Label 9) and aromatic proton (Label 28).It clearly indicates presence of benzimidazoles substituted [1,3,5] Thiadiazine.(HMBC, HSQC, NOE and all other NMR data enclosed in supplementary data). 1 H NMR spectrum, 13 C NMR spectrum, purity chromatogram of degradation product of Lansoprazole and COSY correlation spectrum were shown in Figures 9-12.

Figure 8 .
Figure 8. Numbering scheme and HMBC correlation structures of base degradation product.

Figure 9 . 1 H
Figure 9. 1 H NMR spectrum of degradation product in DMSO.

Figure 10 .
Figure 10. 13 C NMR spectrum of base degradation product.

Table 1 .
Degradation products formed in acid forced degradation.

Table 2 .
Degradation products formed in base forced degradation.
ported in literature.In base degradation study well resolved unknown major peak of m/z 468.11 (M + H) was not reported in literature.So we intended to isolate m/z 468.11 by using mass mediated auto purification system.Standard chromatogram of Lansoprazole, acid degradation, base degradation chromatograms and mass spectrums were shown in Figures2-6.