CHAPTER III STABILITY INDICATING ASSAY AND IMPURITIES METHODS FOR FIXED DOSE COMBINATION PRODUCT OF OMEPRAZOLE AND DOMPERIDONE

Transcription

1 CHAPTER III STABILITY INDICATING ASSAY AND IMPURITIES METHODS FOR FIXED DOSE COMBINATION PRODUCT OF OMEPRAZOLE AND DOMPERIDONE 91

2 Section (i): Brief account of Omeprazole and Domperidone Omeprazole (OZ),5-methoxy-2-[{(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl}sulfinyl]-1H-benzimidazole is a Proton pump inhibitor. It suppresses gastric acid secretion by specific inhibition of H + /K + - ATPase enzyme system at the secretary surface of the gastric parietal cell [1, 2]. By acting specifically on the proton pump, OZ blocks the final step in acid production, thus reducing the gastric acidity. Omeprazole is used to treat symptoms of gastroesophageal reflux disease (GERD) and other conditions caused by excess stomach acid. It is also used to promote healing of erosive esophagitis (damage to your esophagus caused by stomach acid). Domperidone(DP),5-chloro-1-[1-{3-(2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)propyl}-4- piperidinyl]-1,3-dihydro-2h-benzimidazol-2-one 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 on CNS, probably because it fails to cross the blood brain barrier. It facilitates gastrointestinal smooth muscle activity by inhibiting dopamine at the D1 receptors. It acts as an antiemetic and a prokinetic agent through its effects on the chemoreceptor trigger zone and motor function of the stomach and small intestine. The combination of Omeprazole (OZ) and Domperidone (DP) is used for treatment of duodenal ulcers, gastric ulcers, reflux or ulcerative oesophagitis, etc. It is available as fixed dose combination product which contains 10 mg of Omeprazole and 30 mg of Domperidone in Sustain release form for oral administration. Omeprazole is white to off white crystalline powder with a molecular weight of It is acid labile decomposes rapidly at ph less than 5 and sensitive to heat, moisture and organic solvents. Domperidone is white or almost powder with molecular weight of The chemical structures of OZ and DP are as follows: Omeprazole : 92

3 5-methoxy-2-[{(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl}sulfinyl]-1H-benzimidazole Domperidone : 5-chloro-1-[1-{3-(2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)propyl}-4-piperidinyl]-1,3-dihydro-2H-benzimidazol- 2-one Omeprazole drug substance is official in USP/EP/BP/IP, Domperidone drug substance is official in EP/BP/IP where as drug product of Omeprazole is official in UPS/BP/IP and Domeridone is official in BP/IP [5-8]. But combination product is not official in any of the pharmacopeia. Literature survey reveals that methods have been reported for estimation of impurities in OZ and DP drug substance and individual drug products [9-11]. But none of the reported articles described the single method for estimation of impurities in fixed 93

4 dosage combination product of OZ+DP. Instead of following two individual methods, author has seen an opportunity to develop a single analytical method for estimating impurities for this combination product. Although several methods are reported for assay of individual drugs and combination of two drugs [12-25], the analytical time for each sample is reported about 10 to 15 min. The author felt that developing single method on UPLC can achieve short time analysis which is useful for industry with respect to time and cost. This prompted the author to develop stability indicating single methods for the assay and impurities. This chapter describes development and validation of stability indicating methods for assay and impurities for OZ+DZ capsules, the method validation performed as per International Conference on Harmonization (ICH) guidelines [26]. Section (ii): Stability Indicating method for Assay of Omeprazole and Domperidone by UPLC. This section describes the various aspects related to method development and vali- dation of stability indicating UPLC method for assay of Omeprazole (OZ) + Domperidone (DP) in Capsules. 1. Experimental 1.1. Chemicals Active pharmaceutical ingredient of OZ and DP are procured from bulk manufacturers of Dr Reddy s Laboratories Ltd., Hyderabad. OZ+DP capsules and placebo are procured from formulation R&D, Dr. Reddy s 94

5 Laboratories. HPLC grade acetonitrile and methanol are purchased from Merck, Germany. Analytical reagent potassium dihydrogen orthophosphate, triethylamine, ortho phosphoric acid and sodium hydroxide are purchased from Merck, Germany. High purity water is prepared by using Millipore Milli Q plus purification system Determination of appropriate UV wavelength The suitable wavelength for the determination of OZ and DP in diluent is identified by scanning over the range nm with a double beam UV spectrophotometer Instrumentation and chromatographic conditions The waters UPLC system with a diode array detector is used for method development and forced degradation studies. The output signal is monitored and processed using Empower software. Cintex digital water bath is used for hydrolysis studies. Photo stability studies are carried out in Sanyo photo stability chamber. Thermal stability studies are performed in a Cintex dry air oven. The ph of buffer is measured using Thermo Orion ph meter. The chromatographic column Aquity Shield-RP18, 100 x 2.1mm, 1.7µm is used for developing a method. ph 6.4 buffer prepared with 0.01M potassium dihydrogen ortho phosphate and 0.1% triethylamine, ph adjusted to 6.4 ± 0.05 using orthophosphoric acid. Mobile phase A consists a phosphate buffer and acetonitrile in the ratio of 50:50 (v/v). Mobile phase B consists a phosphate buffer and acetonitrile in the ratio of 90:10 (v/v). The flow rate of mobile phase is 0.30 ml/min, column is maintained at 30 C and detection wavelength is 280 nm. The injection volume is 5 µl and the data acquisition time is 4 min. The gradient program is as follows: Time (min)/%b; 0/35, 1/40, 2/40, 2.5/35 and 4/ Diluent 0.1N methanolic sodium hydroxide is used as diluent Preparation of standard solution A standard stock solution is prepared in diluent containing 400 µg/ml of OZ and 600 µg/ml of DP. 1 ml of stock is diluted to 50 ml using diluent to obtain 20 µg/ml of OZ and 30 µg/ml of DP. A specimen overlay chromatogram of diluent and standard is shown in Fig

6 Fig Overlay chromatogram of diluent and OZ+DP standard 1.6. Preparation of Test solution Five capsule contents are transferred (each capsule containing 20 mg of OZ delayed release pellets and 30 mg of DP sustain release pellets) into 250 ml dried volumetric flask. 50 ml of 0.1N sodium hydroxide is added and sonicated for about 10 minutes in a sonicator (maintain the sonicator temperature between 10 C- 15 C) with intermediate shaking, 100 ml of 0.1 N methanolic sodium hydroxide is added and sonicated for 20 minutes and made up to volume with 0.1 N methanolic sodium hydroxide. A part of solution is centrifuged to get clear solution. 5 ml clear centrifuged solution is transferred to 100 ml volumetric flask and made up to volume with diluent to obtain sample solution concentration 20 µg/ml of OZ and 30 µg/ml of DP respectively. Placebo samples are prepared in the same way by taking the placebo equivalent its weight present in the test preparation. A specimen overlay chromatogram of placebo and sample is shown in Fig

7 Fig Overlay chromatogram of placebo and sample 1.7. Specificity As per ICH guidelines, stability indicating methods are required for evaluating quality of finished dosage forms. The current ICH guidelines do not talk about degradation conditions for stress study. The forced degradation conditions, stress agent concentration and time of stress, are found to be effective based on % degradation. Preferably not more than 30% of degradation is recommended for active material to make the right assessment of stability indicating nature of the chromatographic methods. The optimization of such stress conditions which can yield not more than 30% degradation is based on experimental conditions. Chromatographic run times are decided for placebo and samples subjected to force degradation in order to provide an indication of the stability indicating properties and specificity of the method. The stress conditions employed are acid, base, neutral and oxidant media, water, heat and light. After the degradation treatments are completed, the samples are allowed to equilibrate to room temperature, neutralized with acid or base (as necessary), and made up with diluent. The samples are analyzed against a freshly prepared control sample (with no degradation treatment) and evaluated for peak purity by using photo diode array detector. Specific conditions are described below: Placebo (excipients) interference 97

8 Placebo sample solutions are prepared in duplicate by taking the weight of placebo approximately equivalent to its weight in the test preparation as described in Effect of acid hydrolysis OZ+DP pellet powder equivalent to 100 mg of OZ and 150 mg of DP is transferred into 100 ml round bottom flask, treated with 10 ml of 0.1N HCl for 30 minutes at 60 C. The sample is allowed to equilibrate to room temperature, neutralized with base and resulting solution is prepared as per test procedure to obtain final concentration of OZ and DP of 20 µg/ml and 30 µg/ml respectively Effect of base hydrolysis OZ+DP pellet powder equivalent to 100 mg of OZ and 150 mg of DP is transferred into 100 ml round bottom flask, treated with 10 ml of 0.1N NaOH for 60 minutes at 60 C. The sample is allowed to equilibrate to room temperature, neutralized with acid and resulting solution is prepared as per test procedure to obtain final concentration of OZ and DP of 20 µg/ml and 30 µg/ml respectively Effect of neutral hydrolysis OZ+DP pellet powder equivalent to 100 mg of OZ and 150 mg of DP is transferred into 100 ml round bottom flask, treated with 10 ml of water for 60 minutes at 60 C. The sample is allowed to equilibrate to room temperature; resulting solution is prepared as per test procedure to obtain final concentration of OZ and DP of 20 µg/ml and 30 µg/ml respectively Effect of oxidation OZ+DP pellet powder equivalent to 100 mg of OZ and 150 mg of DP is transferred into 100 ml round bottom flask, treated with 10 ml of 3% H 2 O 2 for 30 minutes at 60 C. The sample is allowed to equilibrate to room temperature; resulting solution is prepared as per test procedure to obtain final concentration of OZ and DP of 20 µg/ml and 30 µg/ml respectively Effect of humidity and heat 98

9 To evaluate the effect of humidity and heat, the contents of capsules is crushed to a powder and distributed uniformly in petri dish and then exposed to 25ºC/90% RH (Relative Humidity) for 7 days. A similar sample is kept in an oven at 105 C for 60 minutes. The exposed samples are prepared for analysis as described in the test preparation Effect of UV and visible light To study the photochemical stability, the content of capsules is crushed to a powder and distributed uniformly in petri dish and exposed to 1200 K Lux of visible light and 200 W h/ m 2 of UV light in photo stability chamber. The exposed samples are prepared for analysis as described in the test preparation Method validation Precision Precision (intra-day precision) of the assay method is established by carrying out six independent assays of test sample of OZ+DP capsules against reference standard. The % of RSD of six assays obtained is calculated. The intermediate precision (inter-day precision) of the method is also established by using two different UPLC systems and on two different UPLC columns in different days in the same laboratory Linearity Linearity test for assay is established using six different concentration levels in the range of about 5-30 µg/ml for OZ (corresponding to 25% to 150% of assay concentration) and µg/ml for DP (corresponding to 25% to 150% of assay concentration). The peak area versus concentration data is evaluated by least-square regression analysis Accuracy A study of recovery of OZ and DP from spiked placebo is conducted. Samples are prepared by mixing placebo with OZ and DP equivalent to about 50%, 75%, 100%, 125% and 150% of the assay of highest test concentration. Samples are prepared in triplicate for each spike level as described in the test preparation. The % recovery is calculated Robustness 99

10 To determine the robustness of the test method, experimental conditions are purposely altered one after the other to establish their effect. Five replicate injections of standard solution is injected into chromatographic system with each parameter change. The effect of flow rate, column temperature and organic phase composition (acetonitrile) in mobile phase is studied by verifying tailing factor for OZ and DP peaks and %RSD for peak areas of replicate injections of OZ and DP standard. The flow rates of 0.25 ml/min and 0.35 ml/min, column temperatures of 25ºC and 35ºC and organic phase compositions (acetonitrile) in mobile phase A at + 10% along with the method conditions of 0.3 ml/min, 30ºC and 100% organic phase composition in mobile phase A is studied Solution stability and mobile phase stability The solution stability of sample and reference standard is established by allowing solutions on bench top at controlled room temperature for 24 hours interval up to the study period. The solutions are stored in volumetric flasks by tightly capping. The assay is determined for both test and reference standard solutions by using freshly prepared reference standard at each interval. The mobile phase stability is also established by determining the assay of freshly prepared sample solutions against freshly prepared reference standard solutions at 24 hour interval for 48 hours. Mobile phase prepared is kept constant during the study period. The tailing factor and % RSD of peak area of standard for replicate injections is evaluate for the study period. 2. Results and discussion 2.1 Determination of suitable wavelength The UV spectrum of OZ and DP recorded in the range nm is illustrated in Fig The spectrum indicates that 280 nm gives a good sensitivity for the assay of OZ and DP. 100

11 Fig Overlay UV Spectra of OZ and DP 2.2 Optimization of chromatographic conditions Assay method plays a major role in dosage form, to quantify the amount of analyte. The main target of the chromatographic method is to get the separation of all potential degradants and impurities of OZ and DP without interfering with the main analyte peaks with shorter analysis time. Since OZ and DP have ionizable functional groups, the reverse phase technique is selected along with UPLC system for simultaneous determination of OZ and DP. Aquity Shield-RP18 (encapped) 100 x 2.1 mm, 1.7 µm column is selected due to its high efficiency and suitability for polar moieties compared with other commercially available octadecyl silanized silica packed columns. Lower particle size column is used to achieve the better resolution. The selection of Buffer and its ph and organic modifiers in mobile is critical to achieve desired objective. The pka values for OZ and DP are about 4.13 and 7.9. Based on pka values buffer is selected as 0.01M potassium dihydrogen ortho phosphate and 0.1% triethylamine, and ph adjusted to 6.4 ± 0.05 using orthophosphoric acid. Acetonitrile is selected as organic solvent for better peak shapes and resolution. Initial experiments are conducted using isocratic method with mobile phase consists buffer and acetonitrile in the ratio of 70:30 (v/v) at 0.35 ml/min flow rate. Acid stress sample is analyzed, all degradant peaks are separated well from the OZ and DP peaks, but the run time is found to be longer i.e about 15 min. To shorten the run time, acetonitrile concentration is increased in mobile phase, but OZ is co-eluted with DP. Gradient method is chosen to get better separation of all degradant peaks from main active peaks. Various 101

12 experiments are conducted with different gradient programmes using different ratios of buffer and acetonitrile in mobile phase. The chromatographic separation is achieved on Aquity Shield-RP18, 100 x 2.1 mm, 1.7 µm column by following gradient program Time (min)/%b; 0/35, 1/40, 2/40, 2.5/35 and 4/35 using mobile phase A and B. ph 6.4 buffer is prepared using 0.01M potassium dihydrogen ortho phosphate and 0.1% triethylamine, and ph adjusted to 6.4 ± 0.05 using orthophosphoric acid. Mobile phase A consists a ph 6.4 phosphate buffer and acetonitrile in the ratio of 50:50 (v/v). Mobile phase B consists a ph 6.4 phosphate buffer and acetonitrile in the ratio of 10:90 (v/v). The flow rate of the mobile phase is 0.35 ml/min, column is maintained at 30 C, detection wavelength is 280 nm, injection volume is 5 µl Method validation Precision Method repeatability (intra-day precision) is evaluated by assaying six samples, prepared as described in the sample preparation. The mean % assay values of OZ and DP are found to be and respectively, % RSD for assay values of OZ and DP are found to 0.3 and 0.5 respectively. These values are within the acceptable limits of between 97.0% % and %RSD not more than 2.0. The intermediate precision (inter day precision) is performed by assaying six samples on different UPLC systems and different UPLC columns in different days as described in the sample preparation. The mean % assay values of OZ and DP are found to be and respectively, % RSD for assay values of OZ and DP are found to 0.6 and 0.8 respectively. The precision and intermediate precision results are summarized in Table Table Precision and intermediate precision results % Assay ( Precision ) % Assay( Intermediate precession) S.NO. 1. Omeprazole Domperidone Omeprazole Domperidone

13 Average % RSD

14 LOQ and LOD The LOQ and LOD are determined based on signal-to-noise noise ratios at analytical responses of 10 and 3 times the background noise, respectively. The LOQ is found to be 0.48 µg/ml with a resultant % RSD of 0.5 (n = 6) for OZ and 0.78 µg/mll with a resultant % RSD of 0.5 (n=6) for DP. The LOD is found to be 0.16 µg/ml for OZ and 0.26µg/mL for DP Linearity A linear calibration plot for assay of OZ and DP is obtained over the calibration range of 5-30 µg/ml of OZ (corresponding to 25% to 150% of assay of highest sample concentration) and µg/ml of DP (corresponding to 25% to 150% of highest test concentration and the correlation co-efficient is found to be for both OZ and DP. The graphical plot shown in Fig and indicates that a very good correlation exists between the peak area and concentration of the analyte. Fig Linearity of detector response graph of OZ Fig Linearity of detector response graph of DP Accuracy 101

15 The percentage recovery of OZ and DP from the drug product is found to be 98.9 to for OZ and 99.8 to for DP which indicates the high accuracy of the test method. The results are summarized in Table Table Recovery results of OZ and DP in capsules Spike level (%) Average µg/ml added Average µg/ml found Mean % recovery % RSD OZ DP OZ DP OZ DP OZ DP 50% % % % % Robustness In all the deliberately varied chromatographic conditions studied (flow rate, column temperature and ratio of acetonitrile in Mobile phase A), the tailing factor and the % RSD for the OZ and DP peak areas for five replicate injections of standard is found to be within the acceptable limits of NMT 2 for tailing factor and NMT 2% for RSD, the results are summarized in Table Table Results of Robustness study Observed value Variation Tailing factor % RSD for five 102

16 injections of standard Flow rate Column temperature Mobile phase A Composition (acetonitrile) OZ DP OZ DP 0.25 ml/min ml/min ml/min ºC ºC ºC % % % Solution stability and mobile phase stability The difference in % assay of test and standard preparations upon storage on bench top is found to be less than 1.0% up to 48 hours. Mobile phase stability experiments showed that tailing factor and % RSD are less than 1.1 and 0.6 respectively up to 48 hours. The solution stability and mobile phase stability experimental data confirmed that sample solutions and mobile phase used during assay determination are stable up to 48 hours Results of specificity studies Placebo and stressed samples prepared are injected into the UPLC system with photo diode array detector as per the described chromatographic conditions. Chromatograms of placebo solutions have shown no peaks at the retention time of OZ and DP peaks. This indicates that the excipients used in the formulation do not interfere in estimation of OZ and DP in capsules. All degradant peaks are well resolved from OZ and DP peaks in the chromatograms of all stressed samples. The chromatograms of the stressed samples are evaluated for peak purity for OZ and DP peaks using Waters Empower Networking software. For all forced degradation samples, the purity angle (the weighted 103

17 average of all spectral contrast angles calculated by comparing all spectra in the integrated peak against the peak apex spectrum) is found to be less than threshold angle (the sum of the purity noise angle and solvent angle, the purity noise angles across the integrated peak for OZ and DP peaks (Table 3.2.4). This indicates that there is no interference from degradants in quantitating the OZ and DP. Thus, this method is considered "Stability indicating. The typical chromatogram and purity plots of all stressed samples are shown in Fig to

18 Table Specificity study results Stress conditions Omeprazole Domperidone % Purity Purity % Purity Purity Degradation angle threshold Degradation angle threshold Treated with 0.1 N HCI solution for 30 minutes at 60 C Treated with 0.1 N NaOH solution for 1 hour at 60 C Treated with 3% H 2 O 2 solution for 30 minutes at 60 C Exposed to photo stress (1200 K Lux of visible light followed by 200 W h/ m 2 of UV light) Exposed to Heat for 1 hour at 105 C Treated with purified water for 1 hour at 60 C Exposed to humidity at 25 C, 90% RH for 7 days

19 Fig Chromatogram and purity plot of acid stress sample of OZ+DP 114

20 Fig Chromatogram and purity plot of base stress sample of OZ+DP 115

21 Fig Chromatogram and purity plot of H 2 O 2 stress sample of OZ+DP 116

22 Fig Chromatogram and purity plot of water stress sample of OZ+DP 117

23 Fig Chromatogram and purity plot of photo stress sample of OZ+DP 118

24 Fig Chromatogram and purity plot of heat stress sample of OZ+DP 119

25 Fig Chromatogram and purity plot of humidity stress sample of OZ+DP 3. Conclusion 120

26 The Stability indicating assay method by UPLC is developed for estimation of OZ and DP in capsules. The method is validated as per ICH guidelines and found to be specific, precise, linear, accurate, rugged, and robust. This chromatographic method with run time of 4 minutes allows the analysis of a large number of samples in a short period of time. The developed method is stability indicating and can be used for quantifying OZ and DP in capsule dosage form and in their individual pharmaceutical dosage forms. Section (iii): Stability Indicating HPLC method for determination of impurities in Omeprazole + Domperidone. This section describes the various aspects of method development and validation of stability indicating HPLC method for determination of impurities in Omeprazole (OZ) + Domperidone (DP) capsules. 1. Experimental 1.1. Chemicals Active Pharmaceutical ingredient samples of OZ and DP as well as five OZ impurities and five DP impurities are procured from Bulk manufacturer of Dr. Reddy s Laboratories Limited, Hyderabad, India and British Pharmacopeia. OZ+DP capsules and placebo are received from formulation R&D, Dr Reddy s Laboratories. HPLC grade acetonitrile is purchased from Merck, Germany. Analytical reagents monobasic potassium phosphate and sodium hydroxide are purchased from Merck, Germany. High pure water is prepared by using Millipore Milli Q plus purification system. Chemical name, structures of OZ, DP and its impurities are shown in Table and Table Chemical name and structure of OZ and its five impurities Name Structure IUPAC Name 121

27 Omeprazole 5-methoxy-2-[{(4- methoxy-3,5-dimethyl-2- pyridinyl)methyl}sulfinyl]- 1H-benzimidazole Omeprazole Impurity C 2-Mercapto-5-methoxy benzimidazole Omeprazole N Oxide Omeprazole Desmethoxy 5-methoxy-2-[[(4- methoxy-3,5-dimethyl pyridine-2yl)-1-oxide methyl]-sulphinyl]-1hbenzimidazole 5-Methoxy-2-[[(3,5- dimethyl-2-pyridine-2- yl)methyl]sulfinyl]-1hbenzimidazole Omeprazole Sulphone 5-methoxy-2-[(4-methoxy- 3,5-dimethylpyridin-2- yl)methylsulphonyl]-1hbenzimidazole 122

28 Omeprazole Sulphide 5-methoxy-2-[[(4- methoxy-3,5- dimethylpyridin-2- yl)methyl]sulphanyl]-1hbenzimidazole Table Chemical name and structure of DP and its five impurities Name Structure IUPAC Name Domperidone 5-chloro-1-[1-{3-(2,3-dihydro- 2-oxo-1H-benzimidazol-1- yl)propyl}-4-piperidinyl]-1,3- dihydro-2h-benzimidazol-2- one Domperidone Impurity A 5-chloro-1-(piperidin-4-yl)-1,3- dihydro-2h-benzimidazol-2- one Domperidone Impurity B 4-(5-chloro-2-oxo-2,3-dihydro- 1H-benzimidazol-1-yl)-1- formylpiperidine 123

29 Domperidone Impurity C cis-4-(5-chloro-2-oxo-2,3- dihydro-1h-benzimidazol-1- yl)-1-[3-(2-oxo-2,3-dihydro- 1H-benzimidazol-1- yl)propyl]piperidine 1-oxide Domperidone Impurity D 5-chloro-3-[3-(2-oxo-2,3- dihydro-1h-benzimidazol-1- yl)propyl]-1-[1-[3-(2-oxo-2,3- dihydro-1h-benzimidazol-1- yl)propyl]piperidin-4-yl]-1,3- dihydro-2h-benzimidazol-2- one Domperidone Impurity F 1,3-bis[3-[4-(5-chloro-2-oxo- 2,3-dihydro-1H-benzimidazol- 1-yl)piperidin-1-yl]propyl]-1,3- dihydro-2h-benzimidazol-2- one 1.2. Determination of appropriate UV wavelength The suitable wavelength for the determination of OZ, DP and its impurities is identified by scanning overlay spectra from nm for all impurities, OZ and DP using PDA detector Instrumentation and chromatographic conditions 124

30 The Waters HPLC system with a diode array detector is used for method development and forced degradation studies. The out put signal is monitored and processed using Millenium software. The Agilent HPLC system is used for method validation. The output signal is monitored and processed using HP chemstation software (Agilent). Cintex digital water bath is used for hydrolysis studies. UV exposure study is carried out in a Kompakt UV cabinet. Thermal stability studies are performed in a Cintex dry air oven. The ph of buffer is measured using Thermo Orion ph meter. The chromatographic column Inertsil ODS 3V 250 x 4.6 mm with 5 µm is used for development of method. 0.05M monobasic potassium phosphate buffer is prepared and ph is adjusted to 7.2 using 0.1N sodium hydroxide solution. Mobile phase A consist of phosphate buffer and acetonitrile in the ratio of 75:25 (v/v), Mobile phase B consist of phosphate buffer and acetonitrile in the ratio of 45:55 (v/v). The flow rate of the mobile phase is 1.2 ml/min, analytical column is maintained at 25 C and the detection wavelength is 285 nm. The injection volume is 20 µl. Gradient programme is as follow: Time Flow % A % B Diluent 0.1N sodium hydroxide is used as diluent Preparation of Standard solution A standard stock solution is prepared in diluent by dissolving appropriate quantities of OZ and DP (500 µg/ml of OZ and 750 µg/ml of DP). Stock solution is further diluted with diluent to obtain a standard solution of 3 µg/ml and 4.5 µg/ml respectively for impurities determination. The typical chromatograms of diluent and standard are shown in Fig and

31 Fig Typical chromatogram of diluent Fig Typical chromatogram of OZ +DP standard Preparation of Test solution The contents of ten OM+DP capsules are transferred to petri dish and crushed to a powder. Accurately about100 mg pellet powder is transferred to100 ml volumetric flask, 75 ml diluents is added and sonicated for 15 min and made up to volume to give a solution containing 1 mg/ml of OM and 1.50 mg/ml of DP. Placebo sample is prepared in the same way by taking the placebo equivalent weight present in a test preparation Preparation of Impurity stock solution Impurity stock solutions are prepared individually by weighing accurately about 10 mg each of OZ impurities and 25 mg each of DP impurities into two separate 100 ml volumetric flasks. 25 ml of diluent is added 126

32 and dissolved with aid of sonication and made upto volume with diluent to obtain stock solutions of 100 µg/ml each of OZ impurities and 150 µg/ml each of DP impurities Preparation of Test solution with spiking of Impurities The content of ten OM+DP capsules are transferred to pet dish and crushed to a powder. Accurately about 100 mg pellet powder is transferred to 100 ml volumetric flask, 3 ml of OZ and 4.5 ml of DP impurity stock solutions are added and dissolved in 60 ml of diluent by aid of sonication for 15 minutes and made up to volume with diluent. The typical chromatogram of sample spiked with impurities and placebo are shown in Fig and Fig Typical chromatogram of sample spiked with impurities. 127

33 Fig Typical chromatogram of placebo 1.8. Specificity As per ICH guidelines, require development and validation of stability indicating methods for all pharmaceutical dosage forms. The current ICH guidelines do not describe degradation conditions for stress study. The forced degradation conditions, stress agent concentration and time of stress, are found to be effective based on % degradation. Preferably between 20% to 30 % of degradation is recommended for active material to make the right assessment of stability indicating nature of the chromatographic methods. The optimization of such stress conditions which can yield desired % degradation is based on experimental conditions. Chromatographic run times are decided for placebo and samples subjected to force degradation in order to provide an indication of the stability indicating properties and specificity of the method. The stress conditions employed are acid, base, neutral,peroxide, heat, humidity and light. After the degradation treatments are completed, the samples are allowed to equilibrate to room temperature, neutralized with acid or base (as necessary), and made up with diluent to 1000 µg/ml of OZ and 1500 µg/ml of DP. Peak purity test is carried out for the OZ and DP peaks by using PDA detector in stress samples. Specific conditions are described below: Placebo (excipients) interference Samples are prepared in duplicate by taking the weight of placebo approximately equivalent to its weight in the test sample as described in the Test preparation. 128

34 Effect of acid hydrolysis OZ+DP pellet powder equivalent to 100 mg of OZ and 150 mg of DP is transferred into 100 ml round bottom flask, treated with 5 ml of 1N HCl for 30 minutes at 60 C. The sample is allowed to equilibrate to room temperature, neutralized with base and resulting solution is prepared as per test procedure to obtain final concentration of OZ and DP of 1.0 mg/ml and 1.5 mg/ml respectively and solution is filtered through 0.45 µm nylon 66 membrane filter Effect of base hydrolysis OZ+DP pellet powder equivalent to 100 mg of OZ and 150 mg is transferred of DP into 100 ml round bottom flask, treated with 5 ml of 1N NaOH for 30 minutes at 60 C. The sample is allowed to equilibrate to room temperature, neutralized with aicd and resulting solution is prepared as per test procedure to obtain final concentration of OZ and DP of 1.0 mg/ml and 1.5 mg/ml respectively and solution is filtered through 0.45 µm nylon 66 membrane filter Effect of oxidation OZ+DP pellet powder equivalent to 100 mg of OZ and 150 mg of DP is transferred into 100 ml round bottom flask, treated with 5 ml of 50% H 2 O 2 for 30 minutes at 60 C. The sample is allowed to equilibrate to room temperature, resulting solution is prepared as per test procedure to obtain final concentration of OZ and DP of 1.0 mg/ml and 1.5 mg/ml respectively and solution is filtered through 0.45 µm nylon 66 membrane filter Effect of humidity and heat To evaluate the effect of moheat, the contents of capsules is crushed to a powder and distributed uniformly in petri dish and kept in an oven at 105 C for 2 days. Similarly sample is exposed separately to 25ºC/90% RH (Relative Humidity) for about 7 days. The stressed samples are used for analysis by preparing the solution as described in the test preparation Effect of UV and visible light 129

35 To study the photochemical stability, the content of capsules is crushed to a powder and distributed uniformly in petri dish and exposed to 1200 K Lux of visible light and 200 W h/ m 2 of UV light in photo stability chamber. The stressed samples are used for analysis by preparing the solution as described in the test preparation. 1.9 Method validation Relative retention times and relative response factors: Relative retention times (RRT) are established for all the known impurities of OZ against OZ and all the known impurities of DP against DP by injecting all the impurity standards. Relative response factors (RRFs) are established for all the known impurities of OZ against OZ and all the known impurities of DP against DP. RRFs are established as the ratio of slope of impurities to slope of OZ and DP respectively. Slope value obtained with linearity calibration plots. Established RRT and RRF values of OZ and DP impurities are summarized in Table Table RRT and RRF values of impurities of OZ and DP S.No. Name RRT( about) RRF 1 Omeprazole N Oxide Omeprazole C Omeprazole Sulphone Omeprazole des methoxy Omeprazole Sulphide Domperidone Impurity A Domperidone Impurity B Domperidone Impurity C Domperidone Impurity D Domperidone Impurity F Precision The precision of test method is evaluated by analyzing six individual test preparations spiking with OZ and DP impurities at 0.3% concentration of each impurity with respect to test concentration. The Relative standard deviation is calculated for the response of each impurity. The intermediate precision of test method is evaluated on different HPLC system by different analyst on different column in different days Limits of Detection (LOD) and Quantification (LOQ) 130

36 The LOD and LOQ for all known impurities of OZ and DP are estimated based on the signal to noise ratio of 3:1 and 10:1 respectively by injecting a series of diluted solutions with known concentration. Precision and accuracy studies are conducted at the LOQ level by injecting six individual preparations for precision and triplicate preparations for accuracy and % RSD calculated for each impurity of OZ and DP Linearity Linearity solutions for the impurities are prepared by diluting impurity stock solution to get the solutions of impurities having different concentrations. The solutions are prepared at different concentration levels from LOQ to 0.45% for OZ and DP impurities. The peak area versus concentration data is treated by least-squares linear regression analysis Accuracy Recovery study is conducted to determine accuracy of impurities method for the quantification of all impurities in OZ+DP capsules. The study is carried out in triplicate at LOQ, 50%, 75%, 100%, 125% and 150% of the target concentration (0.3% for all OZ and DP impurities) of each impurity. The % recovery is calculated for OZ and DP impurities Robustness To determine the robustness of the test method, experimental conditions are purposely altered one after the other to establish their effect, Relative retention times of all impurities are measured and tailing factor for OZ, DP are measured. The effect of flow rate is studied at 1.2 ± 0.2 ml/min. The effect of column temperature is studied at 25ºC ± 5 ºC (at 20 ºC and 30ºC). The effect of percent organic strength is studied by varying acetonitrile percentage from -10% to +10% while the other mobile phase components are held constant. To study the effect of buffer ph on the separation of peaks, 0.2 units changed from 7.0 to 7.4, while the other mobile phase components are held constant Solution stability and mobile phase stability The solution stability of OZ, DP standards and test preparation spiked with impurities is established by allowing solutions on bench top at controlled room temperature for 24 hours. The solutions are stored in volumetric flasks by tightly capping. The amounts of OZ, DP and its impurities in the above solutions are 131

37 measured. The stability of mobile phase is also determined by analyzing freshly prepared solution of OZ, DP and its impurities at 24 hours intervals for 48 hours using same lot of mobile phase. 2. Results and discussion 2.1. Determination of suitable wavelength Based on the UV spectra (Fig 3.3.5), OZ having absorption maxima at about 301, where as DP is having absorption maxima at about 285 nm. To get the optimum absorbance for OZ, DP and its impurities, 285 nm is selected for quantification all the impurities of OZ and DP. Fig The Overlay UV spectra of OZ, DP and its impurities 132

38 2.2. Optimization of chromatographic conditions OZ Desmethoxy, OZ sulphone, OZ sulphide, OZ N-oxide and OZ c-789 impurity in OZ pellets, DP Impurity-A, B, C, D and F in DP pellets are potential impurities. The main target of the chromatographic method is to get the separation of all potential impurities in Single chromatographic condition. The separation of all impurities are tried using different buffers like acetate and phosphate buffers based on pk values of OZ and DP along with various ratios of organic modifiers like acetonitrile and methanol. Gradient program is used with different compositions of buffer and organic solvents to have separation between polar and non polar impurities. Different C18 stationary phases are screened with different gradient programs to see the separation of all impurities from each other and from OZ and DP peaks. Based on UV spectra of OZ and DP, wavelength of 285 nm is selected for determination of impurities and degradants pertaining to individual active moiety. All impurities and degradants separation from each other and from OZ and DP is achieved by following Gradient program using Mobile phase A and B compositions which contains different concentration of buffer and acetonitrile. Mobile phase A consists of 0.05M monobasic potassium phosphate buffer, ph adjusted to 7.2 with 0.1N sodium hydroxide solution and acetonitrile in the ratio of 75:25 (v/v) and Mobile Phase B consists of buffer and acetonitrile in the ratio of 45:55 (v/v). A Gradient program is necessary to elute all the impurities and degradation products with good resolution. The gradient program is as follows (Time/%B) : 0/0, 20/0, 30/75, 40/75, 50/0 and 55/0 is used for impurities detection of both compounds. The injection volume is 20 µl. System suitability is established as OZ and OZ sulphone peaks are eluting closely. The resolution between OZ and OZ sulphone peaks found to be more than 2.0. The relative retention time and Relative response factors are evaluated for impurities. The developed LC method is found to be specific for OZ and DP and their impurities Method validation Precision The % RSD of replicate test preparations spiked with impurities (Intra-day and inter-day precision study) is found to be less than 3.85, conforming good precision of the method. All values are well within the acceptance criteria i.e. % RSD not more than 15.0 %. The data is presented in Table to

39 Table Precision results of OZ impurities TEST OZ Impurity C OZ impurity N Oxide (%) OZ Impuritydemethoxy (%) OZ impuritysulphone (%) OZ impurity- Sulphide (%) (%) Mean %RSD Table Precision results of DP impurities TEST DP Impurity A DP Impurity B DP Impurity C DP Impurity D DP Impurity F (%) (%) (%) (%) (%) Mean %RSD

40 Table Intermediate precision results of OZ impurities TEST OZ Impurity C OZ impurity N Oxide (%) OZ Impuritydemethoxy (%) OZ impuritysulphone (%) OZ impurity- Sulphide (%) (%) Mean %RSD Table Intermediate precision results of DP impurities TEST DP Impurity A DP Impurity B DP Impurity C DP Impurity D DP Impurity F (%) (%) (%) (%) (%) Mean %RSD LOQ and LOD 135

41 The determined limit of detection (LOD), limit of quantification (LOQ) and precision at LOQ values for OZ and DP impurities are reported in Table Table LOD, LOQ and precision at LOQ for OZ and DP impurities LOD LOQ Name of the Impurity Concentration in % S/N ratio Concentration in % S/N ratio % RSD LOQ OZ- Impurity C OZ - N-Oxide OZ impurities OZ Desmethoxy OZ - Sulphone OZ sulphide DP Impurity A DP Impurity B DP impurities DP Impurity C DP Impurity D DP Impurity F

42 Linearity A linear calibration plot for OZ and DP impurities is obtained over the calibration range LOQ (~0.02) to 0.45 µg/ml and the correlation co-efficient is found to be greater than The result shown in Fig and indicates that an excellent correlation exists between the peak area and concentration of the analyte for all the impurities. area 5 0 Linearity of Detector response of Omeprazole Impurity C y = 0.481x R = concentration 5 Linearity of Detector response of Omeprazole Impurity N Oxide y = 0.498x R = area concentration 5 Linearity of Detector response of Omeprazole Desmethoxy Impurity y = 0.498x R = area area area concentration 2 0 Linearity of Detector response of Omeprazole Sulphone y = 0.327x impurity R = concentration 5 0 Linearity of Detector response of Omeprazole Sulphide Impurity y = 0.491x R = concentration Fig Linearity graphs of OZ impurities. 131

43 area Linearity of Detector response of Domperidone Impurity A y = 0.529x R = concentration area Linearity of Detector response of Domperidone Impurity B y = 0.223x R = concentration in ppm Linearity of Detector response of Domperidone Impurity C y = 0.382x R = area 1 area concentration Linearity of Detector response of Domperidone Impurity D y = 0.528x R = concentration area Linearity of Detector response of Domperidone Impurity F y = 0.424x R = concentration Fig Linearity graphs of DP impurities. 132

44 Accuracy The percentage recoveries of all impurities in OZ+DP samples are found to be between 85.0 to for OZ and DP impurities. The % recovery values for OZ and DP impurities are presented in Table and Table Recovery results of OZ impurities in OZ+DP capsules Amount spiked a %Recovery b OZ- Impurity C OZ-N Oxide OZ-Desmethoxy OZ-sulphone OZ-Sulphide 50% 97.8± ± ± ± ±1.7 75% 100.6± ± ± ± ± % 97.5± ± ± ± ± % 96.9± ± ± ± ± % 96.9± ± ± ± ±1.1 a All impurities of OZ individually spiked at 0.3 % level of test concentration (1.0 mg/ml of OZ and 1.5mg/mL of DP), b Mean ± % RSD for three determinations Table Recovery results of DP impurities in OZ+DP capsules Amount spiked a %Recovery b DP-Impurity A DP-Impurity B DP-Impurity C DP-Impurity D DP-Impurity F 50% 106.3± ± ± ± ±1.3 75% 107.4± ± ± ± ± % 108.5± ± ± ± ± % 109.8± ± ± ± ± % 111.6± ± ± ± ±0.9 a All impurities of DP individually spiked at 0.30 % level of test concentration (1.0 mg/ml of OZ and 1.5mg/mL of DP), b Mean ± % RSD for three determinations Robustness 133

45 To determine the robustness of the developed method, experimental conditions are deliberately altered and the elution pattern, separation between OZ and its impurities, DP and its impurities and tailing factor for OZ and DP and its impurities are recorded. In all the deliberate varied chromatographic conditions (flow rate, column temperature, ph of the mobile phase and composition of organic solvent), all analytes are adequately resolved and elution orders remained unchanged. RRT of all the known impurities for all deliberately varied conditions along with original conditions are summarized in Table The variability in the estimation of OZ and DP impurities is within ± 10% Solution stability and mobile phase stability The stability of diluted standard solutions is estimated against freshly prepared standard and found that solutions are stable up to 2 days, as the difference in assay is found to be less than 3.0% up to 2 day. The results from test solution stability confirmed that the solution is stable for 10 hours on bench top for impurities quantification analysis. The results are summarized in Table The variability in the estimation of OZ and DP impurities is within ± 0.04% during mobile phase stability experiments. The results from mobile phase stability experiments confirmed that mobile phases are stable up to 24 hours for impurities quantification analysis. The results are summarized in Table

46 14

47 Table Results of Robustness study Impurity Name As per the method conditions RRT s of OZ and DP impurities Acetonitrile composition in Flow rate Column temperature ph of the buffer Mobile phase B C 30 C 7.0 (v/v) 7.4 (v/v) 90% 110% ml/min ml/min OZ- Impurity C OZ - N-Oxide OZ Desmethoxy OZ - Sulphone OZ sulphide DP Impurity A DP Impurity B DP Impurity C DP Impurity D DP Impurity F

48 Table Results of Test solution stability on bench top OZ- Impurity C OZ-N Oxide OZ- Desmethoxy % of impurities OZsulphone OZ- Sulphide DP- Impurity A DP- Impurity B DP- Impurity C DP- Impurity D Initial After 12h Difference from Initial DP- Impurity F Table Results of mobile phase stability Initial After 24h Difference from Initial OZ- Impurity C OZ-N Oxide % of OZ impurities % of DP Impurities OZ- Desmethoxy OZsulphone OZ- Sulphide DP-Impurity A DP- Impurity B DP- Impurity C DP- Impurity D DP- Impurity F

49 Results of specificity studies Placebo and stressed sample solutions are injected into the HPLC system with photodiode array detector as per the described chromatographic conditions. Chromatograms of placebo solutions have shown no peaks at the retention time of OZ and DP and its impurities. This indicates that the excipients used in the formulation do not interfere in estimation of impurities in OZ+DP capsules. Degradation is not observed in light and humidity exposure. Significant degradation observed in acid, alkaline and oxidative studies. All degradant peaks are well resolved from OZ and DP peak in the chromatograms of all stressed samples. The chromatograms of the stressed samples are evaluated for peak purity of OZ and DP using chemistration software. For all forced degradation samples, purity match for OZ and DP peak are found to be more than 990. This indicates that there is no interference from degradants in quantitating the impurities in OZ+DP capsules. The details of % of degradation and peak purity of OZ and DP peaks are summarized in Table The data indicates that there is no co elution of any degradants in the OZ and DP peaks and no impurity which is missing. Thus, this method is considered Stability indicating. The chromatogram and purity plots of stressed samples are shown in Fig to Table Summary of forced degradation study Unstressed sample Stress Condition UV and visible stress study Peak purity match of Omeprazole Drug product Peak purity match of Domperidone % of degradation