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 Sci- entific-Qualigens), Ammonium bicarbonate (Sigma Aldrich), Formic acid (Alfa Aesar), Trifluroacetic acid (Alfa Aesar) and Water used was Milli-Q grade.

Acid degradation studies were carried out as per the guidelines of ICH. 200 mg of standard drug was refluxed with 0.1 N HCl solution at 60˚C for 8 h to study acidic degradation. Normality of acidic solution increased gradually from 0.1 N to 2 N to achieve sufficient degradation. For analytical study, the resultant acid degrada- tion 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.

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.

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 bi- carbonate and acetonitrile as a mobile phase and X-Select column (150 × 4.6 mm, 5µ). In the developed me- thod 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-

Acid forced degradation products of Lansoprazole.

Base forced degradation products of Lansoprazole.

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 spec- trums were shown in Figures 2-6.

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 the mass spectrum it was showing:

1) Monoisotopic mass with even electron ions (m/z: 468.0966 (M + H));

2) Elements observed: C: 0-23 H: 0-17 N: 0-5 O: 0-1 S: 0-1;

3) Molecular formula: C23 H17 N5O S (M + H).

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.

The numbering scheme for the NMR assignments and HMBC correlation for critical singlet proton at 7.914 ppm are shown in Figure 8. The ¹H NMR, ¹³C NMR spectral data of degradation product was compared with those of Lansoprazole. In ¹H NMR, one set of ?CH₂ (Methylene) group protons in Lansoprazole are missing in degradation product. As well, in ¹³C NMR one set of ?CH₂ (Methylene) group carbon in Lansoprazole missing in degradation product. NMR chemical shifts and 2 D NMR (HSQC, HMBC Correlations) of base degradation pro- duct 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). ¹H NMR spectrum, ¹³C NMR spectrum, purity chromatogram of degradation product of Lansoprazole and COSY correlation spectrum were shown in Figures 9-12.

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 (¹H-¹H) 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.

Lable-2 (7.793, d) Lable-3 (6.945, d)

Lable-19 (8.533, d) Lable-18 (7.501, t)

Lable-16 (7.680, d) Lable-17 (7.394, t)

Lable-25 (7.742, d) Lable-26 (7.296, t)

Lable-28 (7.547, d) Lable-27 (7.254, t)

From the above information obtained from the HRMS, ¹H NMR, ¹³C NMR and 2D NMR spectral data it is clearly matching the assumed structure of base degradation product.