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*Corresponding Author: Venkata Kishore, Department of Chemistry, AUEC (A), Visakhapatnam, India.
Citation: V Kishore, T Rao, A Deepthi, N Annapurna. (2021). Method Development of an Analytical Procedure for the Determination of Clofarabine in Pharmaceutical Formulations. J. Pharmaceutics and Pharmacology Research. 4(5); DOI: 10.31579/2693-7247/060
Copyright: © 2021 Venkata Kishore, This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received: 25 November 2021 | Accepted: 08 December 2021 | Published: 13 December 2021
Keywords: clofarabine; RP-HPLC; method development; validation; ICH guidelines
A novel, simple and economic high performance liquid chromatography (HPLC) method has been developed for the estimation of Clofarabine in bulk and tablet dosage form with greater precision and accuracy.The method was validated as per ICH guidelines. Validation studies demonstrated that the proposed HPLC method is simple, specific, rapid, reliable and reproducible. Hence the proposed method can be applied for the routine quality control analysis of Clofarabine in bulk and tablet dosage forms. All the components of the system are controlled using SCL-10Avp System Controller. Data acquisition was done using LC Solutions software.
For many years, the Southern Research Institute has had a programme, supported by the US National Cancer Institute, searching for new nucleoside anticancer drugs. In the early 1980s, two adenine-containing nucleosides, now known as fludarabine (Fludara; Berlex Oncology) and cladribine (Leustatin; Ortho Biotech) were in clinical trials. At the time, it was not clear whether either drug would gain approval by the FDA because some concerns were raised during preclinical and clinical development of these agents. Both drugs were susceptible to glycosidic bond cleavage with fludarabine subject to some phosphorylase cleavage and cladribine subject to both hydrolytic and enzymatic cleavage [1].
Clofarabine administered intraperitoneally had significant activity against a wide variety of human tumourxenografts implanted subcutaneously in athymic nude or severe combined immune deficiency mice [2]. Moderate to excellent sensitivity to tumour growth delays were seen in all eight human colon tumours, three out of four human renal tumours, all four non-small-cell lung tumours, and all three prostate tumours. This spectrum of widespread anticancer activity has been confirmed by other investigators in human tumourxenograft models in mice [3]. The anticancer activity of clofarabine was dose- and schedule-dependent, and greater antitumour activity was associated with more frequent administration [4].Clofarabine is a second generation purine nucleoside analog with antineoplastic activity. Clofarabine is phosphorylated intracellularly to the cytotoxic active 5'-triphosphate metabolite, which inhibits the enzymatic activities of ribonucleotide reductase and DNA polymerase, resulting in inhibition of DNA repair and synthesis of DNA and RNA [5-7].
Acute leukaemia is the most common paediatric cancer, with acute lymphoblastic leukaemia (ALL) and acute myelogenous leukemia (AML) being the two most common types. In the United States alone, ~2,000 children are diagnosed each year with ALL and 500 with AML [8]. Successful treatment of paediatric ALL and AML involves intensive, multi-cyclic therapy with multiple drugs that have various mechanisms of action and dosing regimens [9-10]. Such intense, cyclic treatment regimens with many different agents have reported a projected 5-year disease-free survival of 70% for paediatric patients with ALL and a complete response (CR) rate of 90% in certain forms of childhood acute leukaemia [11].
In this regard and view of the need for a suitable analytical HPLC method for routineanalysis of Clofarabinein formulations.Attempts were made to developsimple, precise and accurate analytical methods for estimation of Clofarabineand extend it for their determination in formulation.
The aim of the method is to develop an analytical procedure for the determination of Clofarabine in Pharmaceutical Formulations. The analytical procedure for determination of Assay in finished product of Clofarabine Injection, 1mg/mL is an In-House procedure.
The method shall be validated for the following parameters:
Precision
Specificity
Intermediate Precision
Accuracy
Robustness
Stability of Analyte in solution
System Suitability of overall validation study.
Instrumentation,Chromatographic Conditions & Method:
The Chromatographic system consisted of a Shimadzu Class VP Binary pump LC-10ATvp, SIL-10ADvp Auto sampler, CTO-10Avp Column Temperature Oven, SPD-10Avp UV-Visible Detector. All the components of the system are controlled using SCL-10Avp System Controller. Data acquisition was done using LC Solutions software.
The mobile phase consisted of 85:15 (v/v) of buffer solution and acetonitrileoperated on isocratic mode. The flow rate is 1.0 ml/min. Chromatographic Estimation of Clofarabinewas performed onInertsil ODS-2 (150 x 4.6) mm, 5µm column. The wavelength of detection is 263 nm. The injection volume is 25µL.
Chromatographic conditions
A High Performance liquid chromatography equipped with UV detector and an auto sampler or its equivalent
Column : Inertsil ODS-2 (150 x 4.6) mm, 5µm
Detection wavelength : 263 nm
Flow rate : 1.0 mL / min
Injection volume : 25µL
Run time : 10 min
Column temperature : 40°C
Sample cooling rack : 25°C
Standards
Clofarabine working standard.
Preparation of Buffer Solution
Dilute 1.0 mL of Glacial acetic acid in 1000 mL of water and mix.
Preparation of Mobile phase
Mix buffer solution and acetonitrile in 85:15 (v/v) portion, sonicate well.
Preparation of Diluents
Methanol 0.9% sodium chloride
Preparation of 0.9% Sodium Chloride Solution
Weigh and transfer accurately 0.9 g of sodium chloride into 100 mL volumetric flask dissolve and make up to the volume with water.
Preparation of Blank
Dilute 1.0 mL of methanol in 10 mL volumetric flask and make up to the mark with 0.9 % sodium chloride solution.
Preparation of Standard Solution
Weigh and transfer about 30 mg of Clofarabine standard in a 50 mL volumetric flask. Add about 10 mL of methanol and sonicate to dissolve. Make up to the volume with methanol and mix well. Transfer 5 mL of above solution in to 50 mL volumetric flask, dilute and make up to the volume with 0.9% sodium chloride solution.
Note:Standard solutions are stable up to 48 hrs at both room temperature and 2-8°C.
Preparation of Test Solution
Transfer 3.0 mL of sample solution into 50 mL volumetric flask. Add 5.0 mL methanol and 30 ml of 0.9% sodium chloride and mix well. Dilute up to the mark with 0.9% sodium chloride solution.
Note:Test solutions are stable up to 48 hrs at both room temperature and 2-8°C.
Procedure
Separately inject each solution into the chromatographic system in the following order.
Blank - Single injection
Standard solution - Five injections
Test solution - Two injections
Standard solution bracketing - Single injection.
Experimental:
Intermediate Precision
Intermediate precision expresses within-laboratories variations such as different days, different analysts, different columns, different equipment’s etc. Ruggedness incorporates the concept described under the terms “Intermediate Precision” as defined in USP <1225>.
Intermediate precision is established by doing same exercise as system and method precision by different analyst on different day using different column and different equipment. The same Lot/Batch of standard and sample were used within the laboratory. The results of Intermediate Precision are tabulated in below table. Comparison of Method Precision and Intermediate Precision result is summarized in the table below.
Acceptance Criteria:
The result meets the acceptance criteria and found comparable, indicates that the method is precise and rugged with respect to analyst to analyst, day to day, column to column and equipment to equipment for its intended use.
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. Robustness study is performed by analyzing the standard at different conditions. The results obtained with altered conditions are compared against results obtained under normal chromatographic conditions.
Variation in Flow Rate (± 0. 2 mL/min.)
The standard was carried out by varying the flow rate of mobile phase to 0.8 mL/min. and
1.2 mL/min. in place of actual flow rate 1.0 mL/min. The results are summarized in the below table. Typical chromatogram of Robustness for variation in flow rate (0.8 mL/min and 1.2 mL/ min) is exhibited below.
Variation in Column Oven Temperature (± 2°C)
The standard was carried out by varying the column oven temperature of 38°C and 42°C in place of actual column oven temperature 40°C. The results are summarized in the below table. Chromatogram of Robustness for variation in Column Oven Temperature (38°C and 42°C) is exhibited below.
Variation in Organic composition (142.5 and 157.5)
The standard was carried out by varying the Organic composition 142.5 mL and 157.5 mL in place of actual the 150mL. The results are summarized in the below table. Chromatogram of Robustness for variation in the Organic composition (142.5 mL and 157.5 mL) is exhibited below.
Acceptance Criteria:
The System suitability defined in test procedure should meet in each condition.
System suitability of overall validation study
The System suitability is an integral part of analytical procedure. The tests are based on the concept that the equipment, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. The system suitability results are tabulated in the below Table.
The analytical procedure for Assay is validated and found suitable for its intended use and it meets the acceptance criteria for:
Specificity:
No Interference should be observed at the retention time of peak in the chromatograms obtained from the diluent and the placebo solution.
Forced Degradation:
The method is specific and stability indicating for its intended use.
Linearity:
The analytical procedure is linear within the concentration range from 50 % to 150 % (30.24µg/mL to 90.72µg/mL) for Clofarabine peak
Intermediate Precision:
The method is precise and rugged with respect to analyst to analyst, day to day, column to column and equipment to equipment for its intended use.
Accuracy:
The analytical test procedure is accurate for its intended use.
Robustness:
The test method is robust enough as demonstrated by altering the Flow rate, Column oven temperature and Organic composition.
Stability of analyte in solution:
The Standard solution is stable up to 48 hours and sample solution is stable up to 48 hours at room temperature and 2-8°C for Clofarabine peak.
The data for each validation characteristic described in this report meets the acceptance criteria with respect to Specificity, Forced degradation, Stability of analyte in solution, Linearity, Precision, Intermediate Precision, Accuracy and Robustness.
The validation results reveal that the analytical procedure is suitable for determination of Assay in ClofarabineInjection, 1mg/mL. The method is stability indicating for determination of Assay of Clofarabine in Clofarabine Injection, 1mg/mL.
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