RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERTATION

1. / NAME OF THE CANDIDATE AND ADDRESS: / BODDU.SIRISHA
•Permanent Address:
D\O B.Patrudu,
Door no. 64-7-17/1, Ram nagar,
Sriharipuram,
Malkapuram, vizag-530011.
Andhra Pradesh.
Phone: 8123693928.
•Postal Address:
Door-no-16, Room-no-8,
Sri Vastham Residency,
Near S.G.R.Dental College Road,
Marthahalli,
Bangalore.
2. / NAME OF THE INSTITUTE: / Krupanidhi College of Pharmacy,
Chikkabellandur, Carmelaram Post,
Varthur Hobli,
Sarjapur road,
Bangalore- 560035.
Karnataka
3. / COURSE OF THE STUDY AND SUBJECT: / Master of Pharmacy
(Pharmaceutical Analysis)
4. / DATE OF ADMISSION TO THE COURSE: / 11th August 2010
5. / TITLE OF THE PROJECT:
“METHOD DEVELOPMENT AND VALIDATION FOR THE ANALYSIS OF IMPURITIES AND RELATED SUBSTANCES IN AN IMMUNOSUPPRESSANT USING HPLC TECHNIQUE”
6. / BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of the Study:
Impurity1: An impurity in the drug substance according to ICH Guidelines is any component present in the drug substance or drug product which is not the desired product, a product-related substance, or excipient including buffer components. It may be either process- or product-related.
Related substances2: Those impurities derived from the drug substance. Related substances include degradation products, synthetic impurities of drug substance, and manufacturing process impurities from the drug product.
It is necessary to incorporate stringent tests to control the impurities arising from different sources. This fact also evident from the requirements of the federal food, drug and cosmetic act and from a large number of pharmacopoeias that provide test for the control of specific impurities. A bulk drug after its scale up, it is necessary to analyse for the presence of any impurities or related substances in it according to ICH (International Conference of Harmonisation) Guidelines Q6B, Q3 A and B.
Guideline Q3A gives the impurities present in a drug substance; Guideline Q3B gives the impurities present in drug product. The Guideline addresses the chemistry and safety aspects of impurities, including the listing of impurities in specifications and defines the thresholds for reporting, identification and qualification.
Generally chromatographic methods (HPLC, HPTLC, and GC) are used because it fulfils requirement of various guidelines by effectively separating degraded product from the drug substance. The interest of work emphasizes on HPLC because, in the modern pharmaceutical industry HPLC (high performance liquid chromatography) is the basic requirement and integral analytical tool applied in all stages of drug discovery, development, and production.
HPLC Method development and validation are performed in order to meet the ICH requirements to prepare for regulatory submissions (e.g., NDA). “The object of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose.”
Immunosuppressive drugs3 or immunosuppressive agents are drugs that inhibit or prevent activity of the immune system. They are used in immunosuppressive therapy to:
·  Prevent the rejection of transplanted organs and tissues (e.g., bone marrow, heart, kidney, liver)
·  Treat autoimmune diseases or diseases that are most likely of autoimmune origin (e.g.,
rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, focal segmental glomerulosclerosis, Crohn's disease, Behcet's Disease, pemphigus, and ulcerative colitis.
An immunosuppressant is any substance that performs immunosuppression of the immune system. They may either be exogenous, as immunosuppressive drugs, or endogenous, as e.g. testosterone (or) a drug that lowers the body's normal immune response.
There are various immunosuppressant medicines that are used to cure eczema if it is severe that more conventional medicines cannot control it. They may be given by locally or parenterally.
The need of the proposed project is to identify the presence of impurities present in a drug substance (immunosuppressant) and to ensure regulatory complaints as per ICH Guidelines.
Main purpose of doing project is to develop asimple, rapid, sensitive and precise high performance liquid chromatographic (HPLC) methodfor the estimation of impurities and related substances in an Immunosuppressantin dosage form.
To Perform routine testing in aquality control laboratory withstandardized methods for activepharmaceutical ingredients (APIs)or final products requires analytical instrumentation with high reliability and ease-of-use, combinedwith optimal cost.
Very few methods were reported for the estimation of Immunosuppressants by using HPLC.

6.2 Review of Literature:
·  Development and validation of HPLC method for the determination of Cyclosporin A and its impurities in Neoral® capsules and its generic versions. Cyclosporin A (CyA) is a cornerstone immunosuppressant for the prophylaxis against allograft rejection after organ transplantation. In this paper, a simple and reliable HPLC method was developed and validated for the evaluation of four CyA degradation products and two related compounds. Isocratic elution at a flow rate of 1.0ml min-1 was employed on a Lichrospher RP-18 (4mm×250mm; 5µm) analytical column maintained at 75°C with a tetrahydrofuran:phosphoric acid (0.05M) (44:56,v/v) as mobile phase. The chromatograms were recorded using a Hewlett Packard 1100 chromatographic system. The developed method was validated in terms of selectivity, linearity, precision, accuracy, limit of detection and limit of quantitation4.
·  Normal phase and reverse phase HPLC-UV-MS analysis of process impurities for rapamycin analogue ABT-578: application to active pharmaceutical ingredient process development. Reverse phase (RP)-HPLC-UV-MS and normal phase (NP)-HPLC-UV-MS methods employing an LC/MSD trap with electrospray ionization (ESI) have been developed to track and map all significant impurities from the synthetic process. Trace-level tracking of key impurities occurring at various process points was achieved using complimentary methodologies, including a stability indicating reverse phase HPLC method. Process control strategies were established with these combinations of analytical technologies for impurities analyses to enable a rich understanding of the ABT-578 process (Rapamycin)5.
·  Isolation and characterization of process-related impurities and degradation products in larotaxel. The isolation and characterization of the process related impurities and degradation products of larotaxel drug substance were described. Forced degradation of larotaxel was carried out under acidic, basic, oxidation, light and thermal conditions to assess the nature of the impurities. The pure impurities were obtained by semipreparative Lcisolation and analyzed by NMR and MS6.
·  A HPLC validated assay of paclitaxel’s related impurities in pharmaceutical forms containing Cremophor® EL. A HPLC method has been developed for the determination of the paclitaxel’s related impurities in pharmaceutical forms. This method ensures the rapid determination of related impurities in the presence of polyoxyl castor oil—the main constituent of paclitaxel’s clinical formulation vehicle. The method is simple and does not require any preliminary treatment of the sample. The method was fully validated7.
·  A stability-indicating method for the determination of melphalan and related impurity content by gradient HPLC. A robust gradient high performance liquid chromatographic (HPLC) procedure is described for the simultaneous determination of melphalan content and related impurities in melphalan drug substance. The chromatographic conditions are capable of separating and quantifying all impurities found in routine production batches of melphalan at above 0.1% area:area. The method has been fully validated and is linear over the column loading range of 0-3mg of melphalan. The method has been applied to melphalan samples stored under stressed conditions, and shown to be stability-indicating8.
·  Characterization of impurities in semi-synthetic vinorelbine bitartrate by HPLC-MS with mass spectrometric shift technique. A simple and sensitive method of high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESI-MS) was developed to separate and identify impurities in semi-synthetic vinorelbine bitartrate sample. The analytical HPLC was carried out on a reversed-phase C8 column using 0.02M ammonium formicate buffer (pH 4.2) and methanol (46:54, v/v) as mobile phase at a flow rate of 0.8 ml/min at room temperature and a UV detection at 267 nm. Their structures were further confirmed by means of 1D and 2D NMR spectra9.
·  Validation of a capillary electrophoresis method for the analysis of ibandronate related impurities. A capillary zone electrophoretic (CZE) method has been developed for the determination of impurities ibandronate, which is a potent nitrogen-containing bisphosphonate. Successful separation of the drug from the impurities was achieved using 1mM tetradecyl-trimethyl-ammonium bromide (TTAB) and 5mM potassium chromate (pH 10.0) as background electrolyte with an indirect detection at 254 nm. The optimised method was validated for specificity, precision, linearity and accuracy. The limit of detection (LOD) was 2µg/mL and the limit of quantification (LOQ) was 7µg/ml for both phosphite and phosphate10.
·  Isolation and characterization of degradation impurities in docetaxel drug substance and its formulation. The majority of impurities were observed in a base degradation study and all five degradation products were characterized. These impurities were isolated, enriched and were subjected to mass and NMR spectral studies. Based on the spectral data, these were characterized as 10-deacetyl baccatin III, 7-epi-10-deacetyl baccatin III, 7-epi-10-oxo-10-deacetyl baccatin III, 7-epi docetaxel and 7-epi-10-oxo-docetaxel, respectively. The last two impurities were also detected in the stability study of docetaxel formulation11.
·  Gas chromatography–electron ionization mass spectrometry and liquid chromatography–electrospray tandem mass spectrometry for determination of impurities in the anti-cancer drug isophosphoramide mustard (IPM). Liquid chromatography– electrospray mass spectrometry (LC–ES–MS) and LC–ES–MS/MS methodologies have been developed and applied to the analysis of synthesized preparations of IPM. This impurity is present at levels in the range of 2–5% depending upon synthesis conditions. This work establishes that LC–ES–MS/MS is a viable tool for the detailed characterization of IPM and related products12.
·  LC–UV method development and validation for the investigational anticancer agent imexon and identification of its degradation products. A liquid chromatographic (LC) method with ultraviolet (UV) detection was developed, using a reverse phase column with phosphate buffer (pH 6; 50 mM) as mobile phase and UV detection at 230 nm. Moreover, the use of LC–mass spectrometry (MS) and on-line photodiode array (PDA) detection enabled us to propose structures for four degradation products. The developed LC–UV method was found suitable for the pharmaceutical quality control of imexon API and the drug in its pharmaceutical dosage form13.
·  Gradient RP-HPLC method for the determination of potential impurities in atazanavir sulfate (ATV). Chromatographic separation was achieved on Ascentis® Express C8, (150mm×4.6mm, 2.7µm) column thermostated at 30°C under gradient elution by a binary mixture of potassium dihydrogen phosphate (pH 3.5, 0.02 M) and acetonitrile at a flow rate of 1.0 ml/min. A photodiode array (PDA) detector set at 250nm was used for detection. The unknown process impurities and alkaline degradants are isolated by preparative LC and characterized by ESI-MS/MS, 1H NMR, and FT-IR spectral data. The developed method is validated with respect to sensitivity (LOD and LOQ), linearity, precision, accuracy and robustness and can be implemented for routine quality control analysis and stability testing of ATV14.
·  Qualification of a microfluidics-based electrophoretic method for impurity testing of monoclonal antibodies. In this method, they present a comprehensive evaluation of the Agilent Bioanalyzer, a microfluidics-based electrophoretic device that was used for impurity testing of a monoclonal antibody. They compared the system to SDS-PAGE, both operated under non-reducing conditions and found a significant improvement of accuracy for the Bioanalyzer. In addition, the latter exhibited a larger assay range and lower limit of quantitation (LOQ) based on a predefined total error limit of ±30%. Interestingly, after reducing the intact antibody into light and heavy chain, they resolved the outlier issue. Eventually, requalification of the micro-fabricated analytical device under reducing conditions revealed only 1 out of 32 quality control samples (QCs) exceeding the ±30% total error limits15.
·  Development of a stability-indicating UPLC method for determining olanzapine and its associated degradation products present in active pharmaceutical ingredients and pharmaceutical dosage forms. The repeatability and intermediate precision, expressed by the RSD, were less than 2.4%. The accuracy and validity of the method were further ascertained by performing recovery studies via a spike method. The accuracy of the method expressed as relative error was satisfactory. The performance of the method was validated according to the present ICH guidelines16.
·  Correlation of liquid chromatographic and biological assay for potency assessment of filgrastim and related impurities. The main goal of this research was to determine if a correlation between liquid chromatography assays and in vitro biological assay could be established for filgrastim (recombinant human granulocyte colony stimulating factor, rhG-CSF) samples containing various amounts of related impurities. For that purpose, relevant filgrastim related impurities were purified to homogeneity and characterized by liquid chromatography and mass spectrometry. Based on the results a conclusion was made that reversed phase high performance liquid chromatography could be used as an alternative for the in vitro biological assay for potency assessment of filgrastim samples17.
·  Rapid determination of the active leflunomide metabolite A77 1726 in human plasma by high-performance liquid chromatography. A simple method for the measurement of the active leflunomide metabolite A77 1726 in human plasma by HPLC is presented. Chromatographic separation of A77 1726 and the internal standard, phenylcinnamic acid, was achieved using a C18 column with UV detection at 305 nm. The reproducibility (%CV) for intra- and inter-day assays of spiked controls was 5%. The limit of quantification was 0.8µg/ml. The average absolute recovery was approximately 100%. This assay is suitable for the determination of A77 1726 in plasma of patients taking leflunomide, and is simpler to use than other HPLC methods reported previously18.
·  Isolation and characterization of process related impurities and degradation products of bicalutamide and development of RP-HPLC method for impurity profile study. The separation was accomplished on a Symmetry C18 (4.6mm×250 mm; particle size 5µm) column under isocratic mode. The mobile phase was 0.01M KH2PO4 (pH 3.0): acetonitrile (50:50 v/v) and a PDA detector set at 215 nm was used for detection. The unknown process impurities and alkaline degradation products were isolated and characterized by ESI-MS/MS, 1H NMR and FT-IR spectral data. Under alkaline conditions bicalutamide was degraded in to an acid and an amine. The kinetics of degradation was studied. Thus, the developed method can be used for process development as well as quality assurance of bicalutamide in bulk drug and pharmaceutical formulations19.
6.3 Objective of the Study:
From the literature review it is seen that not much work was carried out on method development and validation for the analysis of impurities and related substances in an immunosuppressant. So I am interested to work on this to develop a suitable method and validate it.