Chapter-7 Determination of Assay and Related Substances of Omeprazole in Pharmaceutical Dosage Form Overview:

Transcription

1 Chapter-7 Determination of Assay and Related Substances of Omeprazole in Pharmaceutical Dosage Form Overview: Method development and validation of rapid stability indicating method for determination of omeprazole (OME) and its related substances in solid oral dosage form is presented in this chapter. 208

2 Determination of Assay and Related Substances of Omeprazole in Pharmaceutical Dosage Form 7.1 LITERATURE REVIEW Impurity profiling of active pharmaceutical ingredients (API) in both bulk material and finalized formulations is one of the most challenging tasks of pharmaceutical analytical chemists under industrial environment [1]. The presence of unwanted or in certain cases unknown chemicals, even in small amounts, may influence not only the therapeutic efficacy but also the safety of the pharmaceutical products [2]. For these reasons, all major international pharmacopoeias have established maximum allowed limits for related compounds for both bulk and formulated APIs. As per the requirements of various regulatory authorities, the impurity profile study of drug substances and drug products has to be carried out using a suitable analytical method in the final product [3, 4]. Literature search revealed that, papers on degradation of omeprazole [5], determination by UV spectrophotometry method [6], omeprazole (OME) in human plasma and urine by LC-MS-MS [7], colorimetric method [8], determination of S-omeprazole, R-omeprazole and racemic omeprazole [9] are available, but as such there is no validated method available, which reports more known and unknown impurities precisely and significantly for omeprazole (OME), as such and in drug product. It s validated analytical performance in terms of major parameters such as selectivity, accuracy, precision and sensitivity is adequate for the routine quality control of the purity of omeprazole containing pharmaceutical formulations. The important part of method is with help of single injection, quantification of omeprazole and its degradable impurities and process impurities. Common method for assay and related substances, as well as analytical methods, are validated to ensure they are suitable for their intended use and give accurate and reliable data. 209

3 Chapter THE SCOPE AND OBJECTIVES OF PRESENT STUDY There is no stability-indicating HPLC method reported in the literature that can adequately separate all impurities and accurately quantify omeprazole API and dosage forms. It is, therefore, felt necessary to develop a new rapid, stability-indicating method for the related substance determination and quantitative determination of omeprazole. An attempt is made to determine whether HPLC can reduce analysis times without compromising the resolution and sensitivity. The objectives of the present work are as follow: To developed a rapid, stability indicating RP-HPLC method for determination of omeprazole and its related substances in solid oral dosage form. Forced degradation study. To separates omeprazole from its all impurities (Benzamithazone, N-Oxide, Sulphone, Des-Methoxy and Sulphide) known impurities/ degradation products and any unknown degradation product generated during forced degradation study. Perform analytical method validation for the proposed method as per ICH guideline. 7.3 OMEPRAZOLE Omeprazole is highly effective inhibitor of gastric acid secretion used in the therapy of stomach ulcers and zollinger-ellison syndrome. The drug inhibits the H(+)-K(+)-ATPase (H(+)-K(+)- exchanging ATPase) in the proton pump of gastric parietal cells [10, 11]. The chemical IUPAC name of omeprazole is 6-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl]-1Hbenzimidazole. Its empirical formula is C 17 H 19 N 3 O 3 S, and its structural formula is shown in Figure 7.1 [12]; omeprazole Mg is a white to off-white free-flowing crystalline powder with a molecular weight of The route of synthesis of omeprazole Mg resulted five known impurities, Benzamidazole, N-Oxide, Sulphone, Des-Methoxy and Sulphide. The presented method is capable for quantification of all known and unknown impurities of omeprazole Mg accurately. 210

4 Determination of assay and related substances of omeprazole in pharmaceutical dosage form Figure 7.1 3D and 2D Structure of omeprazole Indications For the treatment of acid-reflux disorders (GERD), peptic ulcer disease, H. pylori eradication, and prevention of gastroinetestinal bleeds with NSAID use. Pharmacodynamics Omeprazole is a compound that inhibits gastric acid secretion and is indicated in the treatment of gastroesophageal reflux disease (GERD), the healing of erosive esophagitis, and H. pylori eradication to reduce the risk of duodenal ulcer recurrence. Omeprazole belongs to a new class of antisecretory compounds, the substituted benzimidazoles, that do not exhibit anticholinergic or H2 histamine antagonistic properties, but that suppress gastric acid secretion by specific inhibition of the H + /K + ATPase at the secretory surface of the gastric parietal cell. As a result, it inhibits acid secretion into the gastric lumen. This effect is dose-related and leads to inhibition of both basal and stimulated acid secretion irrespective of the stimulus. Mechanism of action Omeprazole is a proton pump inhibitor that suppresses gastric acid secretion by specific inhibition of the H + /K + -ATPase in the gastric parietal cell. By acting specifically on the proton pump, omeprazole blocks the final step in acid production, thus reducing gastric acidity. 211

5 Chapter-7 Absorption Absorption is rapid, absolute bioavailability (compared to intravenous administration) is about 30-40% at doses of mg. Protein binding 95% Metabolism Hepatic Route of elimination Urinary excretion is a primary route of excretion of omeprazole metabolites. Half life hour Toxicity Symptoms of overdose include confusion, drowsiness, blurred vision, tachycardia, nausea, diaphoresis, flushing, headache, and dry mouth. 7.4 EXPERIMENTAL Materials and reagents Omeprazole tablets, placebo, omeprazole working standard (90.2% w/w), benzamidazole working standard (99.7% w/w), N-oxide working standard (98.4% w/w), sulphone working standard (98.7% w/w), des-methoxy working standard (95.1% w/w) and sulphide working standard (99.8% w/w) are provided by Cadila Pharmaceutical Ltd. Dholka, Ahmedabad, India. HPLC grade methanol and acetonitrile are obtained from J.T. Baker (NJ., USA). GR grade glycine and sodium hydroxide are obtained from Merck Ltd (Mumbai, India). Nylon membrane 212

6 Determination of assay and related substances of omeprazole in pharmaceutical dosage form filters (0.45 µ), nylon syringe filters and PVDF syringe filters are purchased from Pall Life Science Limited (India). High purity water is generated with Milli-Q Plus water purification system (Millipore, Milford, MA, USA) Equipments Cintex digital water bath (Mumbai, India) is used for specificity study. Photo stability studies are carried out in a photo-stability chamber (SUNTEST XLS+, ATLAS, Germany). Thermal stability studies are performed in a dry air oven (Cintex, Mumbai, India) Preparation of mobile phase Mobile phase-a: 0.04 M glycine (adjusted ph 8.8 with diluted sodium hydroxide solution) buffer solution is used as a MP-A. Mobile Phase-B: Mixture of acetonitrile and methanol in ratio of (83:17) is used as MP-B. Table 7.1 Gradient Program Time in min Mobile-A Mobile-B Diluent preparation Dissolve 4.47 gm of Na 2 HPO 4 and 0.73 gm of NaH 2 PO 4 in water. Make the volume up to 1000 ml with water. Dilute 250mL of this solution to 1000 ml with water (ph 8.0, adjusted with dilute NaOH solution). Mix buffer (ph 8.0) and acetonitrile at a ratio of 90:

7 Chapter Standard solution (RS) An accurately weight and transfer about 5 mg of each impurity and 5 mg of OME standard into 100mL volumetric flask dissolve and dilute with diluent and mix. Dilute 5.0mL of this solution to 50mL with diluent Resolution solution An accurately weight and transfer about 1 mg of each impurity and 50 mg OME standard into 100mL volumetric flask, dissolve and dilute up to the mark with diluent, mix well Standard solution (Assay) An accurately weight and transfer about 25 mg of OME working standard in to 50mL volumetric flask add 10mL of dimethylformamide to dissolve it, dilute up to the mark with diluent (500 µg/ml), mix well Sample solution preparation Transfer 5 tablets in to 200mL volumetric flask add about 10mL of dimethylformamide and sonicate for five minutes and add about 150mL of diluent and sonicate for 20 minutes, dilute to volume with diluent. Filter this solution with 0.45µ nylon filter Placebo solution preparation Transfer 5 placebo tablets in to 200 ml volumetric flask add about 10mL of dimethylformamide and sonicate for five minutes and add about 150mL of diluent and sonicate for 20 minutes, dilute to volume with diluent. Filter this solution with 0.45µ nylon filter Chromatographic conditions The liquid chromatography consisted of an Agilent and waters system, equipped with automatic sample injector and PDA detector. For data collection and calculation chemstation Software and Waters Empower TM -2 software is used. The chromatographic condition is optimized using a Zorbax XDB C8 (150 x 4.6mm, 5µ) column. 0.04M glycine buffer (ph adjusted 8.8) as a MP-A 214

8 Determination of assay and related substances of omeprazole in pharmaceutical dosage form and mixture of acetonitrile: methanol (83:17) as a MP-B. The mobile phase is filtered through a 0.45µ Nylon membrane filter and degassed under vacuum prior to use. The flow rate is 1.2 ml/min with gradient program [Table 7.1]. The monitoring wavelength is 302 nm and the injection volume is 10 µl with maintaining column oven temperature at 25ºC and sample cooler temperature is 10 C. 7.5 METHOD VALIDATION The method described herein has been validated for simultaneous determination of assay and its related substances by RP-HPLC Specificity Forced degradation studies are performed to demonstrate selectivity and stability-indicating capability of the proposed method. The sample are exposed to acid hydrolysis [0.1 N HCl (5 ml), 25 C, 15 min], alkaline hydrolysis [0.1N NaOH (5 ml), 60 C, 3h], oxidative [1 % H 2 O 2 (5 ml), 60 C, 3h], water hydrolysis [at 60 C for 4h], thermal [104 C, 4h], and photolytic degradation [1.2 million Lux hours]. All exposed samples are than analysed by the proposed method System suitability Inject diluent, one injection of resolution solution, six replicate of standard solution (RS), five replicate injection of standard solution (Assay) and check the system suitability as follows. 1) In resolution solution, the resolution between OME and des-methoxy should not less than ) The % RSD of area due to all impurities peaks and OME peaks in six replicate injections of standard solution (RS) should not more than ) The % RSD of the area due to OME in five replicate injection of standard preparation (Assay) should not more than

9 Chapter Precision The precision of the system is determined using the sample preparation procedure described above for six real samples of formulation and analysis using the same proposed method. Intermediate precision is studied by other scientist, using different columns, different HPLC, and is performed on different day Accuracy To confirm the accuracy of the proposed method, recovery experiments are carried out by the standard addition technique for assay and related substances. The accuracy of the assay method for OME is evaluated in triplicate (n=3) at the three concentrations of 250, 500 and 750 µg/ml (50, 100 and 150 %) of drug product, and the recovery is calculated for each added (externally spiked) concentration. The mean of percentage recoveries (n=9) and the relative standard deviation are calculated. For all impurities, the recovery is determined in triplicate (n=3) for LOQ, 0.5, 1.0 and 1.5 µg/ml (LOQ, 50, 100, and 150 %) and the recovery of the impurities are calculated. The mean of percentage recoveries (n=12) and the relative standard deviation are also calculated for related substances Linearity For related substances test, linearity is demonstrated from 0.1 to 1.5µg/mL of standard concentration of impurities using a minimum of ten calibration levels (0.10, 0.15, 0.20, 0.25, 0.30, 0.50, 0.75, 1.0, 1.25 and 1.50µg/mL) for benzamidazole, N-oxide, suphone, des-methoxy, and sulphide impurities. For assay test, linearity is demonstrated from 50% to 150% of standard concentration using a minimum of five calibration levels (250, 375, 500, 625 and 750µg/mL) for OME. The method of linear regression is used for data evaluation. The peak areas of the standard compounds are plotted against the respective OME, benzamidazole, N-oxide, sulphone, desmethoxy and sulphide concentrations. Linearity is described by the linearity equation, correlation coefficient and Y-intercept bias is also determined. 216

10 Determination of assay and related substances of omeprazole in pharmaceutical dosage form Limit of detection (LOD) and limit of quantification (LOQ) Limit of detection and quantification concentrations of omeprazole determined based on standard deviation of response and slope method. The linearity study is performed in the range of 10% to 150.0% of the limit concentration of OME, benzamedazole, N-Oxide, sulphone, des-methoxy and sulphide, considering 0.2% limit for all known impurities and 0.10% for any unknown impurities. Duplicate injection of linearity solutions are injected performed. Linearity graph of concentration in µg/ml (X-axis) versus peak area response (Y-axis) is plotted. LOD and LOQ concentrations of omeprazole and its impurities are determined on the basis of equation given below. Limit of Detection = (3.3 X σ) / S Limit of Quantification = (10 X σ) / S Where, σ = Residual standard deviation of regression line S = Slope of calibration curve Injected six replicate injections of these LOD and LOQ concentrations and ensured the peak is detected and responses are measured Robustness The robustness is a measure of the capacity of a method to remain unaffected by small but deliberate changes, change in wavelength (± 2.0nm), change in column oven temperature (± 5 C), changes in flow rate (± 0.1mL/min), and change in buffer ph (± 0.2). All important characteristic including resolution between des-methoxy and OME, tailing factor, theoretical plate number and the retention behaviour of interested compound are evaluated Solution stability The stability of the sample solution (for related substances) is established by storage of the sample solution at 10 C temperature for 25h. The sample solution is re-analyzed after 7h, 16h, 217

11 Chapter-7 21h, 25h and the results of the analysis are compared with the results of the fresh sample. The stability of standard solution and sample solution for assay are established by the storage of them at ambient temperature for 54h. The standard and sample solution are re-injected after 7h, 16h, 21h, 25h, 32h, 44h and 54h, % difference is calculated for it Filter compatibility Filter compatibility is performed for 0.45 μ nylon syringe filter and 0.45 μ PVDF syringe filter. To confirm the filter compatibility in proposed analytical method, filtration recovery experiment is carried out by sample filtration technique. Sample is filtered through both syringe filters and percentage assay and impurities are calculated and compared against centrifuged sample. 7.6 RESULTS AND DISCUSSION Method Development and Optimization The main objective of the RP-HPLC method development is to rapid assay and related substances determination of omeprazole in pharmaceutical formulation. Developed method should be able to determine assay (AS) and related substances (RS) in single run and should be accurate, reproducible, robust, stability indicating, filter compatible, linear, free of interference from blank/ placebo/ impurities / degradation products and straightforward enough for routine use in quality control laboratory. The spiked solution of omeprazole (500 µg/ml), benzamidazole (10 µg/ml), N-oxide (10 µg/ml), sulphone (10 µg/ml), des-methoxy (10 µg/ml) and sulphide (10 µg/ml) is subjected to separation by RP-HPLC. Initially the separation of all compounds is studied using water as a mobile phase-a (MP-A) and acetonitrile (ACN) as a mobile phase-b (MP-B) on a HPLC column Hypersil BDS (C8, 150 x 4.6mm, 5µ) using a Waters (HPLC) system with the linear gradient program. The flow rate of 1.0 ml/min is selected with regards to the backpressure and analysis 218

12 Determination of assay and related substances of omeprazole in pharmaceutical dosage form time as well. Various types of MP-A and MP-B are studied to optimize the method; some of them are summarized in Table 7.2 with the associated observations. Table 7.2 Summary of method optimization Experimental condition Water (MP-A) and ACN (MP-B), linear gradient; Hypersil BDS-C8 ( mm, 5µ); 25 C 0.01 M KH 2 PO 4, ph-7.0 (MP-A) and ACN (MP-B), linear gradient; Hypersil BDS-C8 ( mm, 5µ); 25 C 0.04 M Glycine ph-9.0 (MP-A) and ACN (MP-B), linear gradient; Zorbax Eclipse XDB C8 (4.6 x 150 mm) 5 µm; 25 C 0.04 M Glycine ph-8.8(mp-a) and ACN : methanol, (83:17)(MP-B), linear gradient; Zorbax Eclipse XDB-C8 (150 x 4.6 mm, 5µ); 25 C Observation No resolution found between omeprazole and impurities Peak shape is not good Peak separation observed but peak shape is not good Satisfactory peak separation and sharp peak shape ACN Acetonitrile Based on above experimental study, the optimized HPLC parameters are; flow rate 1.2 ml/min; column oven temperature 25 C; 0.04 M glycine buffer ph-8.8 as MP-A and, a mixture of ACN, and MeOH in the ratio of 83:17 v/v as MP-B. In order to achieve symmetrical peak shape of all substances and more resolution between OME and des-methoxy, different stationary phases are explored. Finally the desired separation with symmetrical peaks is obtained using Zorbax Eclipse XDB C8 (150 x 4.6 mm, 5 µ) column. Column oven temperature is also studied and found that 25 C is more appropriate with respect to separation and peak shape. Based on compounds UV response, 302nm is found to be more appropriate for determination of OME and its impurities from single run. OME, benzamidazole, N-oxide, sulphone, des-methoxy and sulphide are well resolved from each other and there is no chromatographic interference observed due to blank and placebo in a reasonable time of 25.0 minutes, which are presented in Figure

13 Chapter-7 Figure 7.2 Expanded overlaid specimen chromatograms of blank, placebo and sample Analytical Parameters and Validation After satisfactory development of the method it is subjected to method validation as per ICH guideline [13]. The method is validated to demonstrate that it is suitable for its intended purpose by the standard procedure to evaluate adequate validation characteristics (specificity, system suitability, precision, accuracy, linearity, LOD, LOQ, robustness, solution stability and filter compatibility) Specificity Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities. Forced degradation studies are performed to demonstrate selectivity and stability indicating capability of the proposed RP-HPLC method. Figure 7.2 is show that there is no any interferences at the RT (retention time) of OME due to blank, placebo and impurities. Stress studies are performed at concentration of 500 µg/ml of OME to provide the stability indicating property and specificity of the proposed method. The sample is exposed to acid hydrolysis, alkaline hydrolysis, oxidative, water hydrolysis, thermal, and photolytic degradation to evaluate the ability of the proposed method to separate OME from its degradation products. Minor degradation products are observed when OME is 220

14 Determination of assay and related substances of omeprazole in pharmaceutical dosage form subjected to base, heat, photolytic and hydrolytic conditions. Significant degradation is observed when the drug product is subjected to acid hydrolysis, and oxidation. The purity of the peaks obtained from the stressed sample is verified using the PDA detector. The results of forced degradation study are summarized in Table 7.3. Specimen chromatogram of specificity study presented in Figure 7.3 to Table 7.3 Summary of forced degradation results Degradation condition Mass balance # Purity Observation Control sample 99.8 Pass NA Acidic hydrolysis Pass Significant degradation observed Alkaline hydrolysis 98.8 Pass No significant degradation observed Oxidation 97.5 Pass Significant degradation observed Water hydrolysis 98.4 Pass No significant degradation observed Thermal (solid) 98.3 Pass No significant degradation observed Photolytic 99.1 Pass No significant degradation observed NA= Not applicable; # = % assay + % known impurities + area % unknown impurities Figure 7.3 Specimen chromatogram of diluent 221

15 Chapter-7 Figure 7.4 Specimen chromatogram of placebo Figure 7.5 Specimen chromatogram of OME Figure 7.6 Specimen chromatogram of benzamedazole 222

16 Determination of assay and related substances of omeprazole in pharmaceutical dosage form Figure 7.7 Specimen chromatogram of N-oxide Figure 7.8 Specimen chromatogram of sulphone Figure 7.9 Specimen chromatogram of des-methoxy 223

17 Chapter-7 Figure 7.10 Specimen chromatogram of sulphide Figure 7.11 Specimen chromatogram of impurities spiked in OME sample System suitability The % RSD of area counts of five replicate injections is below 1.0 % in OME standard solution (Assay). The % RSD of all impurities area counts for six replicate injections are below 1.0% in standard solution (RS). The resolution between des-methoxy and OME is more than 3.0 in resolution solution. The all parameters complied with the acceptance criteria and system suitability is established. The system suitability result for system precision, method precision and intermediate precision are summarized in Table

18 Determination of assay and related substances of omeprazole in pharmaceutical dosage form Table 7.4 System suitability results (system precision, method precision and intermediate precision) Test Parameters % RSD Resolution between OME and Des-Methoxy System precision Omeprazole (assay) Benzamedazole 0.4 N-Oxide 0.5 Sulphone 0.3 Des-Methoxy 0.2 Sulphide 0.2 Omeprazole (RS) 0.9 Precision Omeprazole (assay) Intermediate precision RS Related substance Benzamedazole 0.3 N-Oxide 0.4 Sulphone 0.2 Des-Methoxy 0.1 Sulphide 0.4 Omeprazole (RS) 0.7 Omeprazole (assay) Benzamedazole 0.1 N-Oxide 0.4 Sulphone 0.3 Des-Methoxy 0.5 Sulphide 0.2 Omeprazole (RS) Precision In this study, % RSD of sample is observed, by injecting six sets of sample solution. %RSD for assay and related substances results of six samples are calculated. The result should not deviate more than 1.0% for assay value and for related substances it s not deviate more than 0.01% for all impurities. It is well within the acceptances criteria for all the sets and method found repeatable and precise for intended purpose. Method precision and intermediate precision results are summarized in Table

19 Chapter-7 Table 7.5 Precision (n=6) and intermediate precision (n=6) results Substance Precision Intermediate precision Mean % % RSD Mean % % RSD % Difference OME (1000µg/mL) Benzamedazole N-Oxide Sulphone Des-Methoxy Sulphide Unknown impurity Total impurities Accuracy Accuracy of a method is defined as the closeness of the measured value to the true value for the sample. Accuracy has been performed for RS in the range of LOQ to 150.0% (LOQ, 50.0%, 100.0%, and 150.0%) of target concentration of OME considering limit 0.1% (limit of individual unknown impurity) and 0.2% of (limit of all known impurities). Accuracy has been performed for assay in the range of 50 to 150 % of target concentration (500 µg/ml). The % recovery for the range 50.0% to 150.0% of target concentration has found within the acceptance criteria with acceptable % RSD of NMT 2.0 at each level. The recovery at each level is within 98.0% to 102.0%. The recovery at each level of each impurity is within 80% to 120% of target concentration. This indicates that the method is accurate for the analysis of OME assay and related substances method. Accuracy study results are summarized in Table 7.6. Overlaid specimen chromatograms of accuracy study (related substance) are presented in Figure

20 Determination of assay and related substances of omeprazole in pharmaceutical dosage form Figure 7.12 Overlaid specimen chromatograms of accuracy study Table 7.6 Accuracy results Substance LOQ (n=3) At 50% (n=3) At 100% (n=3) At 150% (n=3) % % Rec. $ RSD % % Rec. $ RSD % % Rec. $ RSD % % Rec. $ RSD OME (Assay) NA NA #Benzamedazole #N-Oxide #Sulphone #Des-Methoxy #Sulphide Unknown impurity $... Recovery, #... For Related Substances, NA Not applicable Linearity Concentration levels are 50, 75, 100, 125 and 150% of the claimed analyte concentration of assay, corresponding to the range of about µg/ml. For related substances each impurities concentration levels are LOQ, 15%, 20%, 25%, 30%, 50%, 75%, 100%, 125% and 227

21 Chapter-7 150% of the claimed analyte concentration of impurities, corresponding to the range of about LOQ to1.5 mcg/ml. The calibration curve obtained by plotting the peak area versus the concentration is linear in the mentioned concentration range of 50% to 150% for assay and LOQ to 150% for related substances. For assay correlation coefficient of linearity curve is less than and Y-intercept bias is within ± 2.0% of 100% linearity response. For RS correlation coefficient of linearity curve is less than and Y-intercept bias is within ± 5.0% of 100% linearity response. Linearity regression statistic results are summarized in the Table 7.7. Linear curve of OME (assay), OME (RS) and all known impurities are presented in Figure 7.13 to Obtained results are indicated that the method is linear up to the specified range of concentrations. Overlaid specimen chromatograms of linearity (RS) study are presented in Figure Table 7.7 Regression statistics Compound Linearity (µg/ml) Correlation coefficient (r 2 ) Linearity (Equation) Y- intercept bias at 100% Omeprazole (Assay) 250 to Y= 21034x Benzamidazole 0.1 to y= 47630x N-Oxide 0.1 to y= 13718x Sulphone 0.1 to y= 14419x Des-Methoxy 0.1 to y= 18966x Sulphide 0.1 to y= 17722x Omeprazole (RS) 0.1 to y= 59502x

22 Area Area Area Determination of assay and related substances of omeprazole in pharmaceutical dosage form Linearity of Omeprazole (Assay) y = 21034x R² = Conc. In ppm Figure 7.13 Linearity curve of omeprazole (assay) Linearity of Benzamedazone y = 47630x R² = Conc. in ppm Figure 7.14 Linearity curve of benzamedazole Linearity of N-Oxide y = 13718x R² = Conc. in ppm Figure 7.15 Linearity curve of N-Oxide 229

23 Area Area Area Chapter Linearity of Sulphone y = 14419x R² = Conc. In ppm Figure 7.16 Linearity curve of sulphone Linearity of Des-Methoxy y = 18966x R² = Conc. In ppm Figure 7.17 Linearity curve of Des-Methoxy Linearity of Sulphide y = 17722x R² = Conc. In ppm Figure 7.18 Linearity curve of sulphide 230

24 Area Determination of assay and related substances of omeprazole in pharmaceutical dosage form Omeprazole (Related Substance) y = 59502x R² = Conc. In ppm Figure 7.19 Linearity curve of omeprazole (RS) Figure 7.20 Overlaid specimen chromatograms of linearity study (RS) Limit of detection (LOD) and limit of quantification (LOQ) The detection limit of individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantified as an exact value and quantification limit is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. Limit of detection and quantification concentrations of omeprazole determined based on standard deviation of response and slope method. 231

25 Chapter-7 Duplicate injection of linearity solutions are injected into chromatography system. Linearity graph of concentration in µg/ml (X-axis) versus peak area response (Y-axis) is plotted. The correlation coefficient, slope of regression line and RSD of regression line are calculated. Injected six replicate injections of these LOD and LOQ concentrations and ensured the peak is detected and responses are measured. Chromatogram of LOD and LOQ are presented in Figure 7.21 and Figure 7.22 respectively. Results for LOD, LOQ and LOQ precision are summarized in Table 7.8. Figure 7.21 Specimen chromatogram of LOD Figure 7.22 Specimen chromatogram of LOQ 232

26 Determination of assay and related substances of omeprazole in pharmaceutical dosage form Table 7.8 Results of LOD, LOQ and LOQ precision (n=6) Name of compound LOD (µg/ml) LOQ (µg/ml) LOQ precision (% RSD) OME Benzamedazole N-Oxide Sulphone Des-Methoxy Sulphide Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. No significant effect is observed on system suitability parameters such as RSD, and resolution, when small but deliberate changes are made to chromatographic conditions. The results are summarized in Table 7.9, along with the system suitability parameters of normal conditions. Thus, the method is found to be robust with respect to variability in applied conditions. Table 7.9 Robustness study results Condition Name of component Resolution between Des- Methoxy and OME Normal methodology % RSD of five injections Omeprazole (Assay) Benzamidazone 0.34 N-Oxide 0.42 Sulphone 0.23 Des-Methoxy 0.11 Sulphide 0.40 Omeprazole (RS)

27 At 300 nm Wavelength At 304 nm Wavelength Column temperature 35 C Column temperature 25 C Omeprazole (Assay) Benzamidazone 0.40 N-Oxide 0.33 Sulphone 0.32 Des-Methoxy 0.57 Sulphide 0.29 Omeprazole (RS) 0.43 Omeprazole (Assay) Benzamidazone 0.32 N-Oxide 0.19 Sulphone 0.11 Des-Methoxy 0.95 Sulphide 0.05 Omeprazole (RS) 0.31 Omeprazole (Assay) Benzamidazone 0.50 N-Oxide 0.31 Sulphone 0.03 Des-Methoxy 1.50 Sulphide 0.04 Omeprazole (RS) 0.56 Omeprazole (Assay) Benzamidazone 0.38 N-Oxide 0.11 Sulphone 0.08 Des-Methoxy 0.23 Sulphide 0.04 Omeprazole (RS) 0.78 At 1.3 ml/min flow Omeprazole (Assay) Benzamidazone 0.28 N-Oxide 0.16 Sulphone 0.37 Des-Methoxy 0.11 Chapter-7 234

28 Determination of assay and related substances of omeprazole in pharmaceutical dosage form Sulphide 0.10 Omeprazole (RS) 1.21 At 1.1 ml/min flow Omeprazole (Assay) Benzamidazone 1.58 N-Oxide 0.14 Sulphone 0.37 Des-Methoxy 0.37 Sulphide 0.21 Omeprazole (RS) 0.23 Buffer ph 8.6 Omeprazole (Assay) Benzamidazone 0.47 N-Oxide 0.21 Sulphone 0.16 Des-Methoxy 0.59 Sulphide 0.16 Omeprazole (RS) 0.79 Buffer ph 9.0 Omeprazole (Assay) Benzamidazone 0.90 N-Oxide 0.36 Sulphone 0.41 Des-Methoxy 0.58 Sulphide 0.31 Omeprazole (RS) Solution stability The solution stability is checked for sample preparation from initial to 25h for related substances and initial to 54h for assay. For related substances % difference is not more than 0.1. For assay % area difference is not more than 2.0. The results obtained are well within the acceptance criteria up to 25h at 10 C temperature. Solution stability result of sample, standard for assay and related substance summarized in Table 7.10 and

29 Chapter-7 Table 7.10 Solution stability of sample preparation for related substances Time in Hours Benzamidazole Sulphone N-Oxide Uk- (RRT- 0.36) Uk- (RRT- 0.85) Total impurity % Difference Initial NA 7 h h h h NA Not Applicable; Uk un known; RRT related retention time Table 7.11 Solution stability of standard and sample preparation for assay Time in Hours Area of standard solution % Difference Area of sample solution % Difference Initial NA NA 7 h h h h h h h NA Not Applicable Filter compatibility The filter compatibility is studied for two different filters 0.45 µm PVDF filter and 0.45 µm nylon filters. A single set of sample solution is prepared and some of the portion of this solution is centrifuged, filtered 10mL of sample solution through 0.45μm nylon filter and 0.45µm PVDF filter. Centrifuged sample solution and filtered solutions are injected in the chromatography system. For assay, the % difference in assay value is less than 1.0, from centrifuge solution 236

30 Determination of assay and related substances of omeprazole in pharmaceutical dosage form value. For RS, the % difference in the value is less than 0.05, from centrifuge solution value. Filter compatibility study results for assay and RS are summarized in Table 7.12 and 7.13, respectively. Based on obtained results PVDF and Nylon both the filters are compatible for the developed method. Table 7.12 Filter compatibility study result for Assay Component Name Centrifuge sample Sample filter with Nylon filter Sample filter with PVDF filter Omeprazole 100.0% 99.8% 99.5% % difference Not applicable 0.2% 0.5% Table 7.13 Filter compatibility study result for related substance Name of Impurity Centrifuged sample Sample filter with Nylon %Difference against centrifuge Sample filter with PVDF %Difference against centrifuge Un Single Max 0.04% 0.03% 0.01% 0.01% 0.01% Benzamidazole 0.16% 0.15% 0.01% 0.16% 0.00% N-Oxide 0.14% 0.14% 0.00% 0.15% 0.01% Sulphone 0.15% 0.15% 0.00% 0.14% 0.01% Des-methoxy 0.15% 0.14% 0.01% 0.15% 0.00% Sulphide 0.16% 0.15% 0.01% 0.15% 0.01% Total impurities 0.80% 0.76% 0.04% 0.76% 0.04% Un unknown; Max Maximum 7.7 CALCULATION FORMULA Assay (% w/w) Calculated the quantity, in mg, of OME in the portion of solid oral pharmaceutical formulation using the following formula: 237

31 Chapter-7 Where, C std = Concentration of standard solution in mg/ml C s = Concentration of sample solution in mg/ml R s = Compound peak response obtained from the sample preparation R std = Compound peak response (mean peak area) obtained from the standard preparation Impurity (% w/w) Calculate % impurity present in the finished product formulation using the following formula: Where, C std = Concentration of standard solution in mg/ml C s = Concentration of sample solution in mg/ml R s = Compound peak response obtained from the sample preparation R std = Compound peak response (mean peak area) obtained from the standard preparation Relative standard deviation (% RSD) It is expressed by the following formula and calculated using Microsoft excel program in a computer. Where, SD= Standard deviation of measurements = Mean value of measurements 238

32 Determination of assay and related substances of omeprazole in pharmaceutical dosage form Accuracy (% Recovery) It is calculated using the following equation: 7.8 CONCLUSION A precise and accurate method is successfully developed and validated for simultaneous determination of omeprazole and its related substances in omeprazole dosage form, to meet global regulatory requirements. The methodology is evaluated for specificity, linearity, precision, accuracy and range in order to establish the suitability of the analytical method. Stability of analytical solution, filter compatibility, LOD and LOQ is also studied. The total run time is 25.0 min, within which the drug and their degradation products are eventually separated. This method can be successfully applied for the routine analysis as well as stability study. 239

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