development of a validated rp-hplc method for separation and ...

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DEVELOPMENT OF A VALIDATED RP-HPLC METHOD FOR SEPARATION AND DETERMINATION OF PROCESS-RELATED IMPURITIES OF OMEPRAZOLE IN BULK DRUGS CRISTINA IUGA1*, MARIUS BOJIŢĂ1, SORIN E. LEUCUŢA2 University of Medicine and Pharmacy „Iuliu Haţieganu”, Faculty of Pharmacy, 400349, str. Louis Pasteur 6, Cluj Napoca, Romania 1 Department of Drugs Analysis 2 Department of Pharmaceutical Technology and Biopharmacy *corresponding author: [email protected] Abstract A gradient reversed phase liquid chromatographic (RP-LC) method has been developed and subsequently validated for the determination of omeprazole and its process-related impurities (noted as: impurity A, B, C, D, G, H).. Separation was achieved with a Zorbax Extend C18 column and acetonitrile: water: triethylamine1% (pH adjusted to 9.5) as eluent, at a flow rate of 0.8 mL/min. UV detection was performed at 280 nm. The described method was linear over a range of 40.6203µg/mL for omeprazol, 0.9556-14.334µg/mL for impurity A, 1.1568-17.352µg/mL for impurity B, 1.0772-16.158µg/mL for impurity C, 1.289-19.344 µg/mL for impurity D and 0.796811.952µg/mL for impurity H. The accuracy of the method has been demonstrated at 5 concentration levels in the range of 60–140% of the specification limit and the recovery of impurities was found to be in the range of 90–109%. The method is simple, rapid, selective, accurate and useful for indicating the stability of omeprazole from dosage forms. The method can be useful in the quality control of bulk manufacturing and pharmaceutical formulations. Rezumat A fost elaborată şi validată o metodă RP-HPLC de separare şi determinare a omeprazolului şi impurităţilor sale de sinteză (notate astfel: impuritatea A, B, C, D, G, H). Separarea s-a realizat pe o coloană ZorbaxExtend C18 cu o fază mobilă formată din acetonitril: apa: trietilamină 1% (pH=9,5), cu un debit de 0,8 mL/min. Detecţia a fost UV la 280nm. Metoda descrisă a fost lineară pe domeniul de concentraţii de 40,6-203µg/mL pentru omeprazol, 0,9556-14,334µg/mL pentru impuritatea A, 1,1568-17,352µg/mL pentru impuritatea B, 1,0772-16,158µg/mL pentru impuritatea C, 1,289-19,344 µg/mL pentru impuritatea D and 0,7968-11,952µg/mL pentru impuritatea H. Acurateţea metodei a fost demonstrată pentru 5 nivele de concentraţie în domeniul de 60–140% făţă de limitele specificate, gradul de regăsire pentru impurităţi a fost cuprins în domeniul 90–109%. Metoda este simplă, rapidă, selectivă, are acurateţe şi precizie, şi poate fi folosită şi pentru evaluarea stabilităţii omeprazolului în forme farmaceutice. Este o metodă utilă de control al purităţii omeprazolului din materia primă şi din formele farmaceutice. Keywords: omeprazole; process-related impurities; RP-HPLC; validation

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Introduction Omeprazole (OPZ), 5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2pyridinyl)methyl]sulphinyl]-1H-benzimidazole is a substituted benzimidazole compound and a prototype anti-secretory agent, being the first “proton pump inhibitor” widely used for the prophylaxis and treatment of gastro-duodenal ulcers and for the treatment of symptomatic gastro-oesophageal reflux. It acts by interacting with H+/K+ ATPase in the secretory membranes of the parietal cells and is very effective in the treatment of Zollinger–Ellison syndrome. It is a lipophilic, weak base with pKa1 = 4.2 and pKa2 = 9 and can be degraded unless it is protected against acid conditions. OPZ contains a tricoordinated sulphur atom in a pyramidal structure and therefore can exist in two different optically active forms, (S)- and (R)-omeprazole. OPZ was first approved as a racemic mixture, but the (S) isomer was recently introduced on the market [1]. Few methods for the determination of the impurities either in bulk drugs or pharmaceuticals have been reported. In the last few years, it can be observed an increased interest for identification and quantification of impurities in bulk drugs using new methodologies [2, 3]. There are only a few analytical methods available in literature for the determination of omeprazole and its related impurities (Fig. 1) in bulk drugs and pharmaceuticals [1,4,5]. Thus, there is a need for the development of analytical methods, which will be useful for monitoring the levels of impurities in the finished products with omeprazole during process development. Experimental Materials Samples of omeprazole (batch nr. 90807/BA22347) and its related impurities - impurity A (batch nr. 17998/ BA20692), impurity B (batch nr. Rx935/BA20697), impurity C (batch nr. 310790/BA20693), impurity D (batch nr. 210997/BA22424), impurity G (batch nr. 321_E/BA20694) and impurity H (batch nr. AOP-324-1/BA22425) - were obtained from Union Quimica Farmaceutica, Barcelona, Spain. HPLC grade acetonitrile, triethylamine and orthophosforic acid 85% were obtained from Merck, Darmstadt, Germany. LC grade water was deionized with Milli-Q and then filtered using Milli-Q Academic, Millipore water purification system (Milford, MA, USA). All other reagents were of analytical grade purity. Equipment The LC system, HPLC Agilent Series 1100, consisted of binary gradient pump, autosampler, column oven and a UV detector. The output signal was monitored and integrated using HP Chemstation software.

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Figure 1 Chemical structures of omeprazole and its process-related impurities

Solutions Mobile phase. A. Water: triethylamine 1% pH adjusted to 9.5 with 85%, w/v H3PO4 and acetonitrile in ratio (90:10v/v). It was filtered through a 0.45 µm nylon membrane filter prior to use and degassed for 15 min. B. Acetonitrile Dilution Solution. A mixture of sodium tetraborat 0.01M and acetonitrile in ratio (3:1 v/v)

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Impurities standard solutions. 0.5mg/mL impurities stock standard solutions were prepared in methanol. These solutions were kept at +4°C, protected from light. In these conditions the solutions were stable for two months. Working solutions were prepared by dilution to a concentration of 1 µg/mL and injected into the system. Omeprazole standard solution. 1.015mg/mL omeprazole stock standard solution was prepared in methanol and was kept at +4°C, protected from light. In these conditions it was stable for two months. Omeprazole working solutions were prepared by dilution using previously prepared impurities solutions at concentrations between 40-200µg/mL and injected into the system. Sample solution. 2mg/mL solution of omeprazole sample was prepared in methanol and diluted to a concentration of 0.200µg/mL and injected into the system. Forced degradation samples for the specificity study. Omeprazole was kept in a climatic chamber at 40±5°C, 75±5% humidity and was exposed to artificial light for 6 months to study the formation of the degradation products. Every two weeks, an omeprazole sample was withdrawn from the climatic chamber to perform the analyse. Chromatographic conditions. A Zorbax Extend C18 analytical column (150mm×4.6 mm, 5µm packing) (Agilent) was used for analysis at 25°C. The mobile phase was pumped through the column at a flow rate of 0.8 mL/min. Gradient program was: 0min(18%B), 7.2min(18%B), 10.5min(50%B), 12min(50%B), 16min(18%B) and 18min(18%B). The sample injection volume was 20µL. The UV detector was set to a wavelength of 280 nm for the detection. Results and discussion Method development In order to develop a suitable and robust LC method for the determination of omeprazole and its process-related impurities, different mobile phases and columns were employed to achieve the best separation and resolution. Finally, the mobile phase consisting of water:triethylamine 1% pH adjusted to 9.5 with 85% H3PO4 and acetonitrile at a flow rate of 0.8 mL/min, using a ZorbaxExtend C18 (150mm×4.6mm, 5µm) column was found to be appropriate, allowing good separation of omeprazole and its process-related impurities. Because, the impurity D and the impurity G were not separated in the described chromatographic conditions, we choose to use only impurity D along with others impurities in future analysis. The reason

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for this choice is because it is a higher probability to find impurity D than impurity G as degradation product in time. Omeprazole and its process-related impurities show significant UV absorbance at the wavelength of 280nm. Hence, this wavelength has been chosen for detection. Method validation Specificity. In the above conditions, impurity A eluted first followed by impurities D, B, omeprazole, H and C. The chromatogram showing the separation of omeprazole and all its process-related impurities is shown in Fig. 2.

Figure 2 The chromatogram of omeprazole and its impurities (standard solutions)

Linearity. Calibration curves were performed in the following concentration ranges: for omeprazol (40.6-203 µg/mL), impurity A (0.955614.334 µg/mL), impurity B (1.1568-17.352 µg/mL), impurity C (1.077216.158 µg/mL), impurity D (1.289-19.344 µg/mL) and impurity H (0.796811.952 µg/mL). The parameters of linearity curves are shown in table I.

Omeprazole Impurity A Impurity B Impurity C Impurity D Impurity H

Table I Parameters of linearity curves of omeprazole and its impurities Slope Intercept r Limit of detection (LOD ) (b) (a) (µg/mL) 39.210 -29.414 0.9997 0.70 30.397 -12.095 0.9998 0.40 57.505 -14.112 0.9992 0.20 36.713 -18.166 0.9998 0.50 39.920 -1.554 0.9999 0.03 46.287 -10.037 0.9989 0.20

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Accuracy. The accuracy of the analytical method was evaluated by analysing standard solutions of omeprazole and its related impurities at five concentration levels. The obtained values are reported in Table II.

Compound

Omeprazole

Impurity A

Impurity B

Impurity C

Impurity D

Impurity H

Theoretical concentration (µg/mL) 40.60 81.20 121.80 162.40 203.00 0.96 1.91 3.82 7.64 14.33 1.15 2.13 4.62 9.25 17.35 1.07 2.15 4.30 8.61 16.15 1.28 2.52 5.15 10.31 19.34 0.79 1.59 3.18 6.37 11.95

Table II Accuracy of omeprazole and its impurities Calculated Recovery % Accuracy concentration ±SD % µg/mL ±SD 41.28 ± 0.03 101.67 ± 0.06 1.67 83.69 ± 0.56 103.07 ± 0.69 3.07 122.35 ± 0.53 100.45 ± 0.43 0.45 165.96 ± 0.61 102.19 ± 0.38 2.19 206.50 ± 0.58 101.72 ± 0.28 1.72 1.0 ± 0.03 109.18 ± 3.02 9.18 1.89 ± 0.15 99.13 ± 7.86 -0.87 3.90 ± 0.17 102.09 ± 4.37 2.09 7.11 ± 0.03 93.02 ± 0.38 -6.98 14.27 ± 0.05 99.58 ± 0.36 -0.42 1.15 ± 0.01 100.29 ± 1.00 0.29 2.13 ± 0.02 100.16 ± 0.98 0.16 4.52 ± 0.01 97.84 ± 0.22 -2.16 9.20 ± 0.05 99.46 ± 0.56 -0.54 17.33 ± 0.02 99.90 ± 0.09 -0.10 1.05 ± 0.03 98.13 ± 2.80 -1.87 2.15 ± 0.04 100.16 ± 1.76 0.16 4.33 ± 0.02 100.62 ± 0.36 0.62 8.51 ± 0.08 98.80 ± 0.93 -1.20 16.22 ± 0.02 100.45 ± 0.09 0.45 1.17 ± 0.02 91.41 ± 1.35 -8.59 2.46 ± 0.02 97.62 ± 0.69 -2.38 5.12 ± 0.02 99.42 ± 0.34 -0.58 10.32 ± 0.03 100.06 ± 0.31 0.06 19.30 ± 0.11 99.79 ± 0.58 -0.21 0.86 ± 0.07 108.86 ± 8.77 8.86 1.62 ± 0.04 101.68 ± 2.38 1.68 2.93 ± 0.03 92.03 ± 0.79 -7.97 6.27 ± 0.02 98.38 ± 0.33 -1.62 12.24 ± 0.69 102.45 ± 5.77 2.45

Precision. The precision of the analytical method was evaluated by analysing standard solutions of omeprazole and its related impurities at 100% level. The obtained values for intra-day precision are reported in Table III and for inter-day precision are reported in Table IV.

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FARMACIA, 2009, Vol. 57, 5 Table III Intra-day precision of the RP-HPLC method, evaluated by analysing standard solutions of omeprazole and its impurities Compound Theoretical Calculated CV % concentration concentration (µg/mL) µg/mL ±SD 0.12 Omeprazole 121.80 121.74 ± 0.105 3.82 0.18 Impurity A 4.07 ± 0.007 0.29 Impurity B 4.62 4.53 ± 0.008 0.26 Impurity C 4.30 3.74 ± 0.010 0.13 Impurity D 5.15 5.07 ± 0.005 0.34 Impurity H 3.18 2.70 ± 0.009 Table IV Inter-day precision of the RP-HPLC method, evaluated by analysing standard solutions of omeprazole and its impurities Compound Theoretical Calculated CV % concentration concentration (µg/mL) µg/mL ±SD 0.39 Omeprazole 121.80 121.905 ± 0.479 3.82 1.20 Impurity A 4.131 ± 0.049 0.35 Impurity B 4.62 4.539 ± 0.016 2.66 Impurity C 4.30 3.828 ± 0.101 0.55 Impurity D 5.15 5.109 ± 0.028 1.42 Impurity H 3.18 2.715 ± 0.038

Limit of detection (LOD). Limits of detection for omeprazole and its impurities were calculated based on the signal to noise ratio (S/N=10σ), and were between 0.7µg/mL and 0.03 µg/mL, being presented in table I. Limit of quantification (LOQ). Limits of quantitation for omeprazole and its impurities were calculated based on the signal to noise ratio (S/N=10σ), and were the following: omeprazole (2.331µg/mL), impurity A (3.331µg/mL), impurity B (0.666µg/mL), impurity C (1.665µg/mL), impurity D (0.099µg/mL), impurity H (0.666µg/mL). Conclusion A gradient reversed phase HPLC method has been developed and validated for the determination of omeprazole and its process-related impurities from bulk drug. This chromatographic assay fulfilled all the requirements for being a reliable and feasible method, including accuracy, linearity, recovery and precision. It is a highly specific and precise analytical procedure. Therefore, this HPLC method can be used as a routine sample analysis.

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Acknowledgements The authors would like to acknowledge the help of Union Quimica Farmaceutica, Barcelona, for providing process-related impurities of omeprazole. References 1. 2. 3. 4. 5.

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