Development and validation of a stability indicating method for the enantioselective estimation of omeprazole enantiomers in the enteric-coated formulations by high-performance liquid chromatography.

Omeprazole is widely prescribed in the form of enteric-coated formulations, due to the rapid degradation of the drug in the acidic condition of the stomach. In the current article, we are reporting the development and complete validation of a stability indicating chiral high-performance liquid chromatography (HPLC) method for the enantioselective analysis of omeprazole in the enteric-coated formulations. A precise and sensitive enantiomeric separation of omeprazole was obtained on Chiralcel OD-H analytical column (250mm × 4.6 mm, 5μm particle size) using normal phase chromatography. The analysis was performed under UV detection at 301nm wavelength. During method development, the addition of methanol to the mobile phase helped in getting the sharp peaks. The developed method showed linear response over a wide concentration range of 0.39-800μg/ml and the regression coefficients value (r(2)) was obtained more than 0.999 for (S)- and (R)-omeprazole. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for (R)-omeprazole were found to be 0.39 and 0.78 μg/ml, respectively for 5 μl injection volume. The percentage recovery of (R)-omeprazole ranged from 93.5 to 104 in spiked formulation samples and omeprazole sample solution and mobile phase were found to be stable for at least 24 h at room temperature. The proposed method was found to be suitable and accurate for the quantitative determination of undesired enantiomer in the enteric-coated omeprazole formulations.

Omeprazole is one of the most important Proton Pump Inhibitors (PPIs). PPIs are a group of drugs whose main action is pronounced and long-lasting reduction of gastric acid production. Omeprazole has a stereogenic center at the sulphur atom, and it exists as the two optically active forms, (S)-(-) and (R)-(+) omeprazole [Figure 1].[1] Omeprazole was first approved as a racemic mixture, but the (S)-enantiomer was later introduced to the market. The major difference is that (S)-omeprazole is metabolized more slowly and reproducibly than the (R)-omeprazole and racemic omeprazole, because of the stereoselective metabolism by human cytochrome P450 enzyme.[2] Therefore lower doses of (S)-omeprazole can be used to produce equivalent acid suppression than omeprazole doses. Omeprazole is unstable in acidic environment.[3] In aqueous media, the degradation rate proceeds with a half-life of less than 10 min at pH values lower than 4.3.[4] Omeprazole is thus formulated as enteric-coated granules encapsulated in a gelatin shell or as enteric-coated tablets.[56]

Figure 1

Enantiomers of omeprazole

One study reported the comparison of these two techniques, high-performance liquid chromatography (HPLC) and capillary electrophoresis for the enantioselective analysis of omeprazole.[7] The major objective of this present work is to develop a stability indicating method and perform the systematic validation as per the International Conference of Harmonization (ICH) guidelines.[8] The same method was used to estimate the (R)-omeprazole in the enteric-coated pharmaceutical formulations.

Chemicals and drugs

HPLC grade solvents were used as mobile phase, which is manufactured by Merck and procured from commercial source. Bulk drug samples and enteric-coated capsule formulation of omeprazole, (R) and (S)-omeprazole were obtained from local market.

Chromatographic conditions

The chiral analysis was performed on a Shimadzu LC-2010 HPLC system consist of a quaternary pump, a column oven, a photo diode array detector and an auto injector. Enantiomeric separation achieved at 40°C column oven temperature using Chiralcel OD-H column (250 × 4.6 mm, 5 μm particle size, Daicel make). The mobile phase consisted of 85% of n-hexane, 8% of methanol and 7% a mixture of isopropylalcohol and ethanol (85:15, v/v). Mobile phase was chosen as the diluent to achieve clean blank chromatogram without any interference. Flow rate was 0.75 ml/min and injection volume was 5 μl. Data analysis performed at a wavelength of 301 nm.

Sample preparation

The standard stock solutions of (R)- and (S)-omeprazole were prepared by dissolving appropriate amount of the standard samples in mobile phase. A stock solution concentration was fixed at 800 μg/ml. For formulation sample, 8 capsules (10 mg of (S)-omeprazole label claim) were opened and the enteric-coated granules were finely ground using agate mortar and pestle. The ground material, which was equivalent to 80 mg of (S)-omeprazole was extracted in to methanol in a 50-ml volumetric flask by ultrasonication. The resultant mixture was filtered through a 0.45-μm membrane filter. This solution corresponds to analyte concentration of 1600 μg/ml, and further dilutions were prepared in diluent.

Method validation

Specificity

Specificity of this method was indicated by the absence of any endogenous interference at retention times of enantiomeric peaks. The absence of interfering peak was evaluated by injecting a blank sample consisting of diluent and placebo.

Stability indicating method

The drug was subjected to forced degradation under acidic (1M hydrochloric acid, 5 ml), basic (1M sodium hydroxide, 5 ml), and oxidative (3% hydrogen peroxide, 5 ml) stress conditions.

Precision

The precision of the method was checked by an analyzing nine replicate samples of (S)-omeprazole (at analyte concentration, i.e., 800.00 μg/ml) spiked with 0.1% (0.8 μg/ml) of (R)-omeprazole on different days and RSD of area under the peaks was calculated.

Linearity of omeprazole enantiomers

Linearity corresponds to the capacity of the method to supply results directly proportional to the concentration of the substance being determined within a certain interval of concentration.[910] Detector response linearity was assessed by preparing 12 calibration sample solutions covering from 0.39 to 800 μg/ml (0.39, 0.78, 1.56, 3.13, 6.25, 12.50, 25.00, 50.0, 100.00, 200.00, 400.00, and 800.00 μg/ml), Regression curve was obtained by plotting peak area versus concentration, using the least squares method. Duplicate injections given for each concentration level.

Sensibility (LLOD and LLOQ)

LLOD and LLOQ were achieved by giving six injections of lowest three concentration levels, prepared for linearity study. The signal to noise ratio and RSD of the area is considered to evaluate LLOD and LLOQ.

Recovery study of (R)-omeprazole in formulation

The standard addition and recovery experiments were conducted to determine accuracy of the present method. The study was carried out in triplicate by spiking placebo with three concentrations (0.12, 0.15 and 0.18%) of standard (R)-omeprazole and assaying for the chromatographic method. The recovery for (R)-omeprazole was calculated from the slope and Y-intercept of the calibration curve, drawn in the concentration range of 0.39-800 μg/ml.

Ruggedness

To determine the ruggedness, the recovery experiments carried out for (R)-omeprazole in formulation samples were again carried out in laboratory B using a different instrument.

Robustness

For the HPLC method, the robustness was determined by the analysis of the samples under a variety of conditions making small changes in the percentage of methanol in mobile phase (7 and 9%, v/v), in the flow rate (0.7 and 0.8 ml/min), in the column temperature (35 and 45°C), and changing the wavelength (299 and 303 nm). The change in chromatographic resolution between enantiomers was evaluated for the study.

Solution stability

The sample was analyzed for 24h at room temperature, i.e., at 25°C. Resolution and composition of omeprazole enantiomers were observed during the study period.

Results and Discussion

Method development and optimization

Among the chiral columns evaluated, Chiralcel OJ-R and Chiralcel OJ-H did not show potential results for enantiomeric separation of omeprazole. The Chiralcel OD-H column was found to be suitable for better resolution of omeprazole enantiomers. The solutes can bind to the carbamate groups on the chiral stationary phase forming transient diastereomers through hydrogen bonding using the C=O and NH groups and also through dipole–dipole interaction using the C=O moiety. Omeprazole has NH functional group and this could well be contributing to the interactions with the carbamate groups on CSP, resulting in separation.[11]

During method development the methanol was chosen as a polar organic modifier of the mobile phase, because the methanol has proven good organic modifier for resolution of omeprazole enantiomers.[1213] The percentage of methanol between 0 and 10% had strong effect on separation and sharpness of the enantiomeric peaks corresponding to omeprazole. The increase in certain percentage of methanol content in mobile phase drastic increased the resolution, and number of theoretical plates of the two enantiomers, but decreased the resolution with the higher percentage of methanol [Table 1]. In order to obtain sharp peaks without compromising on the resolution, the 8 % (v/v) of methanol content chosen in mobile phase.

Table 1

Optimization of methanol content in mobile phase

In the final method, the typical retention times of (S)-omeprazole and (R)-omeprazole were about 14 and 15 min, respectively [Figure 2]. A chromatogram of spiked (R)-omeprazole at 0.1% of concentration level in (S)-omeprazole formulation sample is shown in Figure 3.

Figure 2

Enantiomeric resolution of omeprazole on Chiralcel OD-H column. Mobile phase consisted of 85% of n-hexane, 8% of methanol and 7% a mixture of isopropylalcohol and ethanol (85:15, v/v), flow rate: 0.75 ml/min, UV-301 nm, column temperature: 40°C

Figure 3

Typical chiral HPLC chromatogram of (S)-omeprazole formulation sample (0.8 mg/ml) spiked with (R)-omeprazole (0.1%)

Validation

The blank chromatogram was clean without any interference from diluent and placebo of the capsules.

The drug shows 30%, 15% and 21% degradation under acidic, basic and oxidative conditions, respectively. The purity factor is within the threshold limit for forced degradation and formulation samples, hence the developed method was found to be stability indicating and results are free from any interference.

Precision and sensibility

The determined precision, intraday precision, LLOD and LLOQ values are reported in Table 2.

Table 2

Validation parameters and results of LC method validation

Recovery

recovery experiments were conduced to determine the accuracy of the present method for the quantification of (R)-omeprazole in formulation samples. (R)-omeprazole was spiked to the extracted (S)-omeprazole sample (800 μg/ml) in triplicate at 0.12, 0.15 and 0.18% of target analyte concentration. Recovery was calculated from the slope and Y-intercept of the calibration curve obtained in linearity study. The same recovery experiments were also conducted using a different system in laboratory B at the same concentration levels tested in laboratory A and results were well in agreement. This confirms the ruggedness of the method. The results are summarized in Table 3.

Table 3

Recovery result of (R)-omeprazole in enteric-coated formulation

Linearity

The calibration curve constructed for omeprazole was linear over the wide concentration range of 0.39-800 μg/ml. The each solution was injected in duplicate and RSD of area under the peak was < 2% across the study. The regression was found to be linear over the concentration range and correlation coefficient was greater than 0.999 for both enantiomers.

The chromatographic resolution of the (S)- and (R)-omeprazole enantiomers peaks was remain more than 1.5 under all modified conditions, which demonstrate the sufficient robustness of the method.

No significant change was observed in resolution and composition of omeprazole enantiomers during the solution stability study for 24h at room temperature, i.e., at 25°C.

Application

The accuracy data proved that the developed method can be used for the quantitative determination of undesired enantiomer of omeprazole in the enteric-coated pharmaceutical formulations.

Conclusions

This is the first report to describe the stability indicating chiral HPLC method for enantioselective analysis of omeprazole enantiomers. In this study we found the importance of methanol as polar organic modifier in normal phase chiral chromatography, which improved the peak shape of omeprazole enantiomers. The method was completely validated and shown satisfactory data for all the method validation parameters tested. The method was sensitive (LLOQ=0.78 μg/ml) and linear over the thousand fold concentration range. This method can be use for routine analysis in quality control laboratories.