A Single Gradient Stability-Indicating Reversed-Phase LC Method for the Estimation of Impurities in Omeprazole and Domperidone Capsules

A gradient reversed-phase liquid chromatographic (RP-LC) method was developed for the quantitative estimation of impurities in the pharmaceutical dosage form of Omeprazole and Domperidone capsules. The developed method is a stability-indicating test method for the estimation of impurities generated during the formulation and storage of Omeprazole and Domperidone capsules. The chromatographic separation was achieved on a column packed with octadecyl silane, having a column length of 250 mm and diameter of 4.6 mm with a particle size of 5 μm, and by following a gradient program using a combination of a monobasic potassium phosphate buffer (0.05M) and acetonitrile. Since the spectral properties were similar, both compounds’ individual impurities were estimated at 285 nm. Forced degradation studies were performed on Omeprazole pellets (enteric coated) and Domperidone pellets (SR coated) encapsulated in size ‘1’ hard gelatin capsules. Omeprazole and Domperidone were degraded using acid hydrolysis (0.1 N hydrochloric acid), base (0.1 N sodium hydroxide), oxidation (50% hydrogen peroxide), heat (105 °C), and UV light (254 nm). The established method was validated and found to be linear, accurate, precise, specific, robust, and rugged.


Introduction
The combination of Omeprazole (OZ) and Domperidone (DP) is used for duodenal ulcers, gastric ulcers, reflux, or ulcerative esophagitis, etc. OZ is a class of medications called proton pump inhibitors [1,2]. It suppresses gastric acid secretion through the specific inhibition of the H + /K + -ATPase enzyme system at the secretory surface of the gastric parietal cell [1]. By acting specifically on the proton pump, OZ blocks the final step in acid production, thus reducing the gastric acidity. DP is a dopamine receptor antagonist, which works as an upper gastrointestinal prokinetic and increases the tone of the lower esophageal sphincter and enhances gastric emptying [3,4]. It does not produce dopamine antagonist effects in the CNS, probably because it fails to cross the blood brain barrier. It facilitates gastrointestinal smooth muscle activity by inhibiting dopamine at the D1 receptors. The chemical structures of OZ and DP are shown in Fig. 1.
So far, various reported RP-LC (HPLC) methods [5][6][7] include the estimation of impurities in OZ and OZ capsules, but none of the methods described a single method for the estimation of impurities in the combination product of OZ and DP capsules. The present paper describes the quantitative estimation of impurities in the combination product. This paper also deals with the forced degradation study under various stress conditions such as acid hydrolysis, base hydrolysis, oxidation, heat, and UV and also method validation [8] for the accurate quantification of known impurities in pharmaceutical formulations. The chemical structures of impurities are shown in

Equipment
The Waters RP-LC (HPLC) system with a diode array detector was used for the method development and forced degradation studies. The output signal was monitored and processed using Millenium software.
The LC (HPLC) system used for method validation was the Agilent HPLC. The output signal was monitored and processed using HP ChemStation software (Agilent) on a Pentium computer.

Chromatographic conditions
The chromatographic column used was the Inertsil ODS 3V 250 x 4.6mm with 5 μm particles. The buffer used was 0.05M monobasic potassium phosphate pH-adjusted to 7.2 using a 0.1N sodium hydroxide solution. The buffer and acetonitrile in the ratio of 75:25 was used as mobile phase A, and the buffer and acetonitrile in the ratio of 45:55 were used as mobile phase B. The flow rate of the mobile phase was 1.

Preparation of standard solution
A standard stock solution of OZ and DP (0.5mgmL −1 and 0.75mgmL −1 respectively) was prepared by dissolving an appropriate amount of substance in diluent. The stock solution was further diluted with diluent to obtain a standard solution of 3 µgmL −1 and 4.5 µgmL −1 respectively for the determination of impurities. The typical chromatograms of the standard and diluent are shown in Fig. 3 and Fig. 4.

Preparation of Test solution
The test solution was prepared by taking the pellet powder equivalent to 1.0mgmL −1 and 1.5mgmL −1 of OZ and DP respectively. The typical chromatograms of the placebo and OZ and DP capsule tests are shown in Fig. 5 and Fig. 6.

Preparation of System suitability solution
The system suitability solution was prepared with OZ sulphone (3 ppm) with 1.0 mgmL −1 solution of OZ. The resolution between the OZ and OZ sulphone impurity was measured.

Response factor
The measurement of the response factor for each impurity's determination is important when the calculations are being made on a relative percent basis. Hence, authentic samples of the related substances and R were dissolved in the diluent and injected, and responses were calculated. RRF values are mentioned in Table 1.

Tab. 1.
Relative response factor results

Precision
The precision of the test method was evaluated by analyzing six samples of OZ and DP capsules by spiking the test preparations with the OZ and DP impurities blend solution at 0.3% concentration of each impurity with respect to the test concentration. The relative standard deviation was calculated for the response of each impurity.

Intermediate Precision
The intermediate precision study was conducted by a different analyst on a different day, column, and on a different LC (HPLC) system. Six samples of OZ and DP capsules were prepared by spiking the test preparations with the OZ and DP impurities blend solution at 0.3% concentration of each impurity with respect to the test concentration. The RSD was calculated for the response of each impurity.

Limit of detection (LOD) and limit of quantification (LOQ)
The LOD and LOQ of OZ and DP impurities were estimated based on the signal-to-noise ratio to get a signal-to-noise ratio of 3:1 and 10:1, respectively, for each impurity by injecting a series of dilute solutions with known concentration. Precision and accuracy studies were also carried out at the LOQ level by injecting six individual preparations of impurities and calculating the % RSD of the area.

Linearity
Linearity test solutions for the related substance method were prepared from the impurities stock solution at six concentration levels from the LOQ to 150% of the specification (% of LOQ, 0.15%, 0.225%, 0.3%, and 0.45%). Plotting the peak areas of impurities versus their corresponding concentrations drew the calibration curve. We then calculated the slope and Y-intercept and correlation coefficient of the calibration curve for each impurity.

Accuracy
A recovery study of OZ and DP impurities from spiked samples of the test preparation was conducted. Samples were prepared in triplicate by spiking the test preparations with 50%, 75%, 100%, 125%, and 150% of the target concentration (0.3%) of OZ and DP impurities. The % recovery of each individual impurity was calculated by the external standard method.

Specificity
Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities. The specificity of the developed LC (HPLC) method for OZ and DP was carried out in the presence of its related potential impurities.
Forced degradation studies were performed on OZ and DP pellet powders to provide an indication of the stability-indicating property and specificity of the proposed method. Samples were degraded intentionally by stressing samples under UV light (254 nm), heat (60 °C), acid (0.1 N HCl), base (0.1 N NaOH), and oxidation (3% H 2 O 2 ) to evaluate the ability of the proposed method to separate OZ, DP, and their known impurities from their degradation products as well as the placebo. For the heat and light studies, the time period for stress was 24 hours, whereas acid, base hydrolysis, and oxidation was 30 minutes. The peak purity test was carried out by using a photodiode array detector for OZ and DP peaks, and the peaks were found to be pure.

Robustness
To determine the robustness of the developed method, experimental conditions were intentionally altered and the resolution between the OZ sulphone and OZ peaks was evaluated. The flow rate of the mobile phase was 1.2mLmin −1 . To study the effect of flow rate on the resolution, it was changed by 0.2 units from 1.0 mLmin −1 to 1.4 mLmin −1 , while the other mobile phase components were held constant. The effect of the percent organic strength on the resolution was studied by varying the acetonitrile percentage from -10 to +10%, while the other mobile phase components were held constant. To study the effect of a buffer pH on the resolution, 0.2 units changed it from 7.0 to 7.4, while the other mobile phase components were held constant.

Solution stability of test preparation
The benchtop solution stability of the test preparation and standard preparation of OZ and DP was carried out up to 48 hours. The standard preparation was found to be stable up to 48 hours, whereas the test preparation was stable only up to 24 hours.

Optimization of chromatographic conditions
OZ Desmethoxy, OZ sulphone, OZ sulphide, OZ N-oxide, and OZ c-789 impurity, DP Impurity A, B, C, D, and F in DP were the potential impurities. The main target of the chromatographic method is to get the separation of all potential impurities in a single chromatographic condition. The separation of all impurities was tried using different mobile phases containing acetate buffers like ammonium acetate and phosphate buffers like potassium dihydrogen phosphate, along with various ratios of organic modifiers like acetonitrile and methanol using a different gradient program. The impurities and degradants pertaining to the individual active moiety are estimated at the specific wavelength of 285 nm.
The chromatographic separation was achieved by the following gradient program using the 0.05M monobasic potassium phosphate buffer, pH adjusted to 7. System suitability was established as OZ and OZ sulphone peaks were eluting closely. The resolution between OZ and OZ sulphone peaks was found to be more than 2.0. The relative retention time and relative response factors were evaluated for impurities. The developed LC method was found to be specific for OZ and DP and their impurities.

Results of Forced degradation experiments
Degradation was observed in OZ and DP under stress conditions like UV light, heat, and also acid, base peroxide hydrolysis. Peak purity has been verified for all of the impurities and for both main peaks; the peak purity matches for all impurities and main compounds were found to be more than 990. Peak purity shows that impurity peaks as well as main peaks are homogeneous under all the stress conditions. By the above-mentioned fact we can confirm that the method is a stability-indicating method. The summary of the forced degradation studies and peak purity details are given in Table 2. The chromatograms and purity plots of the stressed samples are shown in Fig. 7 to Fig. 11.

Precision
The % RSD of the response of all impurities during precision and intermediate precision was found to be less than 10%. The results are shown in Table 3, which indicate good precision of the method

Limit of detection (LOD) and limit of quantification (LOQ)
The limit of detection (LOD) and limit of quantification (LOQ) for all the impurities were established by the signal-to-noise method, and precision and accuracy is verified at the limit of quantification level. The limit of quantification of the OZ Desmethoxy impurity, OZ sulphone, OZ sulphide, OZ N-oxide, and OZ c-789 are 0.015%, 0.015%, 0.015%, 0.015%, and 0.016% respectively.
The % recoveries of DP Imp A, B, C, D, and F at the LOQ level were found to be 98.7%, 108.6%, 95.5%, 100.4%, and 98.3% respectively.
The precision of OZ Desmethoxy impurity, OZ sulphone, OZ sulphide, OZ N-oxide, and OZ c-789 and of DP Imp A, B, C, D, and F at the LOQ level was indicated by the RSD of the response, which was below 15%. The concentration of impurities at the LOQ is summarized in Table 3.

Linearity
The linear calibration plot for the impurities method was obtained over the calibration ranges tested, i.e. LOQ to 150% of target concentration (0.3%) of individual impurities for all impurities. The correlation coefficient was measured from each impurity's linear calibration plot and found to be greater than 0.997. The results showed that a good correlation existed between the peak area and concentration of impurities. The results are shown in Fig. 12 to Fig. 21

Accuracy
The recovery studies were performed from 50 % to 150% of the target concentration (0.3%). The % of mean recovery and % RSD of individual impurities of OZ and DP from the formulation samples were found to be satisfactory.
The mean recovery and RSD of the OZ Desmethoxy impurity was 99.42% and 2.46%. The mean recovery and RSD of OZ sulphone was 103.44 % and 5.12%. The mean recovery and RSD of OZ sulphide was 107.64% and 2.8%. The mean recovery and RSD of OZ Noxide was 96.15% and 1.81%. The mean recovery and RSD of OZ c-789 was 97.07% and 1.58%.
The mean recovery and RSD of DP Impurity A was 109.98% and 1.87%. The mean recovery and RSD of DP Impurity B was 87.30% and 1.88%. The mean recovery and RSD of DP Impurity C was 99.59% and 2.16%. The mean recovery and RSD of DP Impurity D was 102.73% and 2.4%. The mean recovery and RSD of DP Impurity F was 101.78% and 3.74%. The summary of the % recovery and RSD of individual impurities is mentioned in Table 5.

Tab. 5.
Results of Impurities Recovery study on formulation sample

Robustness
In all the deliberately varied chromatographic conditions (flow rate, buffer pH, and percentage of organic strength) the resolution between OZ and OZ sulphone peaks was greater than 4.0, which illustrates the robustness of the method.

Test Solution stability
After 24 hours on the benchtop, no significant change was observed in the % of impurities of DP, whereas a slight variation was observed in the % of impurities of OZ. Hence, the test solutions were freshly prepared and injected into the chromatographic system during method validation.