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Development of International Pharmacopoeia monographs for selected protease inhibitors

Development of International Pharmacopoeia monographs for selected protease inhibitors. KATHOLIEKE UNIVERSITEIT LEUVEN GROUP BIOMEDICAL SCIENCES FACULTY OF PHARMACEUTICAL SCIENCES. J.HOOGMARTENS Yekkala Raja Laboratory for Pharmaceutical Analysis. OUTLINE. Introduction.

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Development of International Pharmacopoeia monographs for selected protease inhibitors

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  1. Development of International Pharmacopoeia monographs for selected protease inhibitors KATHOLIEKE UNIVERSITEIT LEUVEN GROUP BIOMEDICAL SCIENCES FACULTY OF PHARMACEUTICAL SCIENCES J.HOOGMARTENS Yekkala Raja Laboratory for Pharmaceutical Analysis

  2. OUTLINE Introduction Aim of the study WHO strategy Development of monographs Results - LC method improvement Indinavir sulfate • Selectivity of C18 columns towards indinavir sulfate components • Validation Saquinavir (mesilate) Nelfinavir mesilate Conclusions

  3. INTRODUCTION Current status of protease inhibitors (PIs) • Relatively new class of drugs • Extensively used in human (chronic) therapy eg: highly active anti retroviral therapy (HAART) • Obtained by synthetic procedures • Impurities are often closely related substances - difficult to remove

  4. INTRODUCTION Current status of protease inhibitors (PIs) • Change of quality - transfer of production to other countries with cheaper production capabilities and less stringent GMP and environment regulations - use of different synthetic route • Impurities in pharmaceuticals can have significant effect on their quality and safety

  5. INTRODUCTION Current status of protease inhibitors (PIs) • Very few papers have been published on LC methods for assay and purity control • Some monographs are published in official compendia such as Indian Pharmacopoeia (IP) and United States Pharmacopeia (USP) • Most often these monographs prescribe to use expensive reference standards • Simple and better analytical methods needed for assay and purity control in International Pharmacopoeia

  6. OUTLINE Introduction Aim of the study WHO strategy Development of monographs Results - LC method improvement Indinavir sulfate • Selectivity of C18 columns towards indinavir sulfate components • Validation Saquinavir (mesilate) Nelfinavir mesilate Conclusions

  7. AIM OF THE STUDY • HIV infected patients have no cure but their quality of life can be improved with antiretroviral (HAART) therapy • The World Health Organization (WHO) is interested in having analytical methods and specifications for the International Pharmacopoeia (Int. Ph.) • Monographs are made available worldwide to evaluate generic drugs • Analytical methods and specifications provided by the laboratories collaborating in this project were used as starting point

  8. Saquinavir (mesilate) Indinavir sulfate Nelfinavir mesilate Ritonavir AIM OF THE STUDY To develop complete monographs for selected PIs

  9. OUTLINE Introduction Aim of the study WHO strategy Development of monographs Results - LC method improvement Indinavir sulfate • Selectivity of C18 columns towards indinavir sulfate components • Validation Saquinavir (mesilate) Nelfinavir mesilate Conclusions

  10. WHO STRATEGY Quality control methods at different levels (step-wise strategy) 1. Basic tests (an indication of identity) - Colour reactions - Precipitation reactions Simple and readily applicable Useful when fully equipped laboratory and/or analytical expertise are not available and when rapid control is necessary Limited role but helps in primary testing and for detection of counterfeit drugs

  11. WHO STRATEGY 2. Screening tests (confirmation of identity) - Thin layer chromatography (TLC*) These tests help to detect counterfeit drugs and gross contamination However, screening tests can not replace full analysis * WHO collaborating Center for Chemical Reference Substances, Centrallaboratoriet, Kungens Kurva, Sweden

  12. WHO STRATEGY 3. Full analysis (identification, purity testing and assay) Identification tests - Infrared spectroscopy (IR) - UV spectroscopy Purity testing - Specific optical rotation - Limit tests for minerals Heavy metals and Sulfated ash - Water content / Loss on drying - Residual solvents (ICH guidelines) - Related substances Assay - Titration - UV spectroscopy

  13. OUTLINE Introduction Aim of the study WHO strategy Development of monographs Results - LC method improvement Indinavir sulfate • Selectivity of C18 columns towards indinavir sulfate components • Validation Saquinavir (mesilate) Nelfinavir mesilate Conclusions

  14. DEVELOPMENT OF MONOGRAPHS The ICH guidelines for residual solvents are applicable and monographs do not repeat this - A method is prescribed where needed - Limits are reported only when higher than these of ICH A recently adopted approach in the Int. Ph. is implemented to provide alternative tests - This approach helps many laboratories in developing countries who do not have access to the more sophisticated techniques

  15. DEVELOPMENT OF MONOGRAPHS When more sophisticated techniques like LC are prescribed for assay, easier to perform techniques like UV spectroscopic method or potentiometric titrations are also developed as alternative For related substances, special attention was given to evaluate the existing LC methods The best ones were used as a starting point for further method development To avoid use of expensive reference compounds in routine analysis, the system suitability tests (SSTs) were developed by degradation of sample

  16. OUTLINE Introduction Aim of the study WHO strategy Development of monographs Results - LC method improvement Indinavir sulfate • Selectivity of C18 columns towards indinavir sulfate components • Validation Saquinavir (mesilate) Nelfinavir mesilate Conclusions

  17. INDINAVIR SULFATE 1. Cipla 2. Indian Pharmacopoeia and Ranbaxy 3. USP 4. Farmanguinhos-Fiocruz

  18. C4 (25 cm x 4.6 mm I.D.) 5 µm column, at room temperature Stationary phase: Kromasil C4 Mobile phase: - 35 vol Acetonitrile 0.1 M ammonium phosphate buffer pH 4.8 (containing 0.2 g of sod.1 hept. sulfonate) - 65 vol Detection: 220 nm Flow rate: 1.0 ml/min INDINAVIR SULFATE 1. Cipla

  19. A B 25 70 25 25 50 5 INDINAVIR SULFATE 1. Cipla adapted Stationary phase: Hypersil C18 Mobile phase: gradient: faster and more sensitive Acetonitrile 0.1 M ammonium phosphate buffer pH 4.8 (containing 0.2 g of sod.1 hept. sulfonate) Water Detection: 220 nm Flow rate: 1.0 ml/min

  20. INDINAVIR SULFATE Cipla Typical chromatogram of indinavir impure sample (1 mg/ml) using the optimized Cipla method

  21. INDINAVIR SULFATE 2. Indian Pharmacopoeia and Ranbaxy Stationary phase: C8 (20 cm x 4.6 mm I.D.) 5 µm column, at 40 °C Hypersil C8 (25 cm x 4.6 mm I.D.) Mobile phase: acetonitrile - 40 vol 0.05 M sodium citrate buffer pH 5.0 - 60 vol Detection: 260 nm Base line noise due to the high absorbance of citrate buffer at lower wavelength Flow rate: 1.0 ml/min

  22. INDINAVIR SULFATE 3. USP Stationary phase: C18 (25 cm x 4.6 mm I.D.) 5 µm column, at room temperature Hypersil BDS C8 Hypersil BDS C18 Mobile phases: • potassium phosphate buffer 0.54 g K2HPO4 + KH2PO4 in 2 L of water B) acetonitrile optimized Gradient: 0-40 min: 20% B, 40-45 min: 20% to 70% B many ghost peaks were observed Detection: 220 nm could not be continued Flow rate: 1.0 ml/min

  23. INDINAVIR SULFATE 4. Farmanguinhos-Fiocruz Stationary phase: Hypersil C18 BDS (250 x 4.6 mm I.D.,) 5 µm column, at room temperature A B 30 70 Mobile phase: Acetonitrile Sodium phosphate buffer pH 7.5 5 5 (1.0 g/100 ml) Water 65 25 Detection: 220 nm Gradient: faster elution better sensitivity Flow rate: 1.0 ml/min

  24. INDINAVIR SULFATE Farmanguinhos-Fiocruz Typical chromatogram of indinavir impure sample (1 mg/ml) using the optimized Farmanguinhos-Fiocruz method

  25. INDINAVIR SULFATE The chromatograms shown were obtained under the same integration and detection conditions Adapted Cipla method Adapted Farmanguinhos method - detects more impurities - less tailing for principal peak Further optimization - less complex mobile phase Sample solution: 1.0 mg/ml to 2.0 mg/ml Column temperature: 30 °C, 35 °C, 40 °C and 45 °C

  26. INDINAVIR SULFATE Typical chromatograms of 2.0 mg/ml (A) IDV commercial sample and (B) IDV spiked sample solution

  27. INDINAVIR SULFATE Proposed system suitability test indinavir SSTPK 3.5 Typical chromatogram of SST solution prepared by heating 2 ml of 2 mg/ml of indinavir sulfate and 2 ml of sulfuric acid (190 g/l) in boiling water for 10 minutes

  28. OUTLINE Introduction Aim of the study WHO strategy Development of monographs Results - LC method improvement Indinavir sulfate • Selectivity of C18 columns towards indinavir sulfate components • Validation Saquinavir (mesilate) Nelfinavir mesilate Conclusions

  29. INDINAVIR SULFATE Selectivity of C18 columns towards indinavir sulfate Hypersil BDS C18 (25 cm x 4.6 mm I.D.) 5 µm The manufacturer claims: - both base-deactivated and end-capped Separation was examined on a set of 16 columns - at least either base-deactivated or end-capped The columns were chosen based on a column ranking system developed in our laboratory http://pharm.kuleuven.be/pharmchem/columnclassification The ranking system is based on four chromatrographic parameters

  30. INDINAVIR SULFATE The list of C18 columns (25 cm x 4.6 mm I.D.), 5 µm examined and their characteristics provided by the manufacturers

  31. INDINAVIR SULFATE Quality of the separation was evaluated by both SST and CRF Chromatographic response function (CRF) 0 < CRF < 1 co-elution of two or more peaks CRF = 0 complete baseline separation CRF = 1

  32. F < 2 2 <F < 6 F > 6 INDINAVIR SULFATE Quality of the separation was evaluated by both SST and CRF No column has CRF = 0 2 columns have CRF = 0 3 columns have CRF = 0

  33. INDINAVIR SULFATE Supelcosil DB; F = 0.667; CRF = 0.86 Hypersil BDS; F = 0.000; CRF = 1.00 Apex ODS II; F = 26.256; CRF = 0.23 Validated; F = 2.303; CRF = 0.58 Chromatograms for purity control obtained on different columns for a spiked IDV sample

  34. INDINAVIR SULFATE Relation between end-capping and/or base-deactivation, SST (≥ 3.5) and sufficient quality of separation (CRF = 0.80) • The probability of obtaining a good separation for indinavir sulfate and its impurities (CRF >0.80) was higher on the end-capped columns than on the base-deactivated columns examined It is observed that SST criteria do not always give the required information

  35. OUTLINE Introduction Aim of the study WHO strategy Development of monographs Results - LC method improvement Indinavir sulfate • Selectivity of C18 columns towards indinavir sulfate components • Validation Saquinavir (mesilate) Nelfinavir mesilate Conclusions

  36. INDINAVIR SULFATE Robustness • The influence of 4 chromatographic paramaters on the separation was investigated using Hypersil BDS C18 column Chromatographic parameter settings applied: • Central composite design was applied using Modde 5.0 statistical graphic software (Umetrics, Umeå, Sweden)

  37. INDINAVIR SULFATE Regression coefficient plots UNK3-EPO regression coefficient bar error bar Individual and interaction parameter effects on the resolution for the UNK3-EPO

  38. INDINAVIR SULFATE Response surface plot UNK3-EPO Response surface plot of UNK3-EPO as a function of acetonitrile in the mobile phase and temperature of the column

  39. INDINAVIR SULFATE Quantitative aspects R2: coefficient of determination Sy,x: standard error of estimate nc: number of experimental concentrations studied ni: number of injections for each concentration y: peak area; x: concentration injected (µg/ml)

  40. INDINAVIR SULFATE Precision data for IDV and some of its impurities

  41. INDINAVIR SULFATE Analysis of commercial samples Purity control of IDV samples, expressed as IDV (100 % = 2.0 mg/ml)

  42. OUTLINE Introduction Aim of the study WHO strategy Development of monographs Results - LC method improvement Indinavir sulfate • Selectivity of C18 columns towards indinavir sulfate components • Validation Saquinavir (mesilate) Nelfinavir mesilate Conclusions

  43. (C) (B) (A) SAQUINAVIR (MESILATE) Proposed LC conditions Typical chromatogram of 0.5 mg/ml of (A) SQV, (B) SQVM sample 1 and (C) SQVM sample 2 using the proposed experimental conditions

  44. SAQUINAVIR (MESILATE) 14 2.0 A typical chromatogram of 0.5 mg/ml solution of SQVM capsules (Invirase) using the proposed experimental conditions

  45. SAQUINAVIR (MESILATE) Proposed system suitability test RRT 0.45 Saquinavir 14 RRT 1.9 RRT 1.8 2.0 Typical chromatogram of SST solutions prepared by heating 2 ml of 0.5 mg/ml of SQV and 5 ml of sulfuric acid (475 g/l) in boiling water for 30 minutes

  46. SAQUINAVIR (MESILATE) ACE; F = 0.436; CRF = 1.00 Hypersil BDS; F = 0.000; CRF = 1.00 Apex ODS II; F = 26.256; CRF = 0.00 Chromatograms for purity control obtained on different columns for a spiked SQV sample

  47. OUTLINE Introduction Aim of the study WHO strategy Development of monographs Results - LC method improvement Indinavir sulfate • Selectivity of C18 columns towards indinavir sulfate components • Validation Saquinavir (mesilate) Nelfinavir mesilate Conclusions

  48. NELFINAVIR MESILATE Proposed LC conditions Typical chromatograms of (A) a 20 µg/ml of reference impurity mixture and (B to H) 2 mg/ml of NFVM commercial samples using the proposed experimental conditions

  49. NELFINAVIR MESILATE Typical chromatograms of commercial samples using the proposed method (A to C) NFVM tablets and (D) NFVM oral powder

  50. RRT 1.8 RRT 1.9 RRT 0.2 Nelfinavir NELFINAVIR MESILATE System suitability test 15 4.0 Typical chromatogram of SST solutions prepared by heating 2 ml of 2.0 mg/ml solution of NFVM and 5 ml of sulfuric acid (475 g/l) in boiling water for 30 minutes.

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