Available online on 15.06.2022 at ijmspr.com

International Journal of Medical Sciences and Pharma Research 

Open Access to Medical and Research

Copyright  © 2022 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Stability Indicating RP-HPLC Method for the Estimation of Clofarabine in Parenteral Formulation

Deepak Kumar Sehrawat¹*, Neetesh Kumar Jain², Apoorva Tiwari¹, Prerna Chaturvedi³

¹ Department of Quality Assurance, Faculty of Pharmacy, Oriental University Indore-India

² Department of Pharmacology, Faculty of Pharmacy, Oriental University Indore-India

³ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Oriental University Indore-India

Email: dksehrawat123@gmail.com

1. INTRODUCTION:

 Pharmaceutical products formulated with more than one drug, typically referred to as combination products. These combination products can present daunting challenges to the analytical chemist responsible for the development and validation of analytical methods. The development and validation of analytical methods [Spectrophotometric, High performance liquid chromatography (HPLC) & High performance thin layer chromatography (HPTLC)] for drug products containing more than one active ingredient. The official test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency, and performance of drug products.

The number of drugs introduced into the market is increasing every year. These drugs may be either new entities or partial structural modification of the existing ones. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market), development of patient resistance and introduction of better drugs by competitors. Under these conditions, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop newer analytical methods for such drugs.

2. DRUG PROFILE: 

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. This nucleoside analog also disrupts mitochondrial function and membrane integrity, resulting in the release of pre-apoptotic factors, including cytochrome C and apoptotic-inducing factors, which activate apoptosis.

3. MATERIALS AND METHODS:

3.1 Reagents and Materials used: 

3.1.1 Selection of Chromatographic condition:

• Column: Inertsil C₁₈ (150mm×4.6mm) 5µm
• Mobile Phase: Buffer : Acetonitrile (90:10)V/V
• Flow Rate: 1.0 ml/min
• Column Temperature: 40^◦C
•  Detection Wavelength: 263 nm
•  Run time: 10 min
•  Injection volume: 5 μl

3.1.2 Selection of mobile phase:

• Buffer: 1ml Glacial acetic acid à 1000 ml water
• Mobile phase: Prepare a mixture of 90 volume of Buffer and 10 volumes of Acetonitrile. Filter through 0.45 µm filter and degas before use. 

(A)  Clofarabine standard preparation: 

(B)  Preparation of working standard solution:

(C)  Preparation of Sample standard stock solution of Clofarabine   

4. RESULT AND DISCUSSION

4.1 Identification of Drugs:

 4.1.1Melting Point Determination

Melting point of the APIs were determined by using melting point apparatus. The observed melting points of APIs were compared with the reported melting point.

Table 1: Melting point determination

4.1.2 Infrared Spectroscopy

IR spectrum of Clofarabine were taken by KBr pellet method on FTIR and characteristic peaks were compared with IR spectrum of Reference standard given in Indian Pharmacopoeia.

Figure 1 Reference IR Spectra of Clofarabine

Figure 2 IR Spectra Sample of Clofarabine

Table 2 Interpretation of IR Spectra of Clofarabine

4.1.3 Solubility Determination

The solubility of Clofarabinewere checked in various solvents like distilled water, methanol, and Dimethyl Sulphoxideetc. The results are shown in table 

Table 3 Solubility determination of Clofarabine

4.2 METHOD DEVELOPMENT

4.2.1 Selection of wavelength

Scan the standard solution and test solution on UV/Visible spectrophotometer, over the spectral range 200 to 400 nm. Use diluent as blank. The UV spectrum of the test solution should exhibit maxima at the same wavelength (±2 nm) as that of a standard solution.Clofarabine show reasonably good response at 263 nm.

A) Standard preparation 

Fig. 3 Uv Spectrum of Clofarabine Standard solution showing selection of wavelength detection

B) Assay preparation

Fig. 4 UV Spectrum of Clofarabine Assay preparation showing selection of wavelength detection

4.2.2 Selection of Mobile Phase

To optimize the RP-HPLC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for Clofarabine was obtained with a mobile phase Buffer (1 ml Glacial acetic acid in 100 ml of water) : Acetonitrile (85:15) at a flow rate of 1.0 mL/min

(A) Development trials: 

Trial: 1   

Table: 4 Trial in mobile phase Water: Methanol (60:40)%v/v

Fig. 5 Trial in mobile phase Water: Methanol (60:40)%v/v

Trial: 2     Table: 5 Trial in mobile phase Buffer: Acetonitrile (65:35)%v/v

Fig. 6 Trial in mobile phase Buffer: Acetonitrile (65:35)%v/v

Trial 3           Table: 6 Trial in mobile phase Buffer: Acetonitrile (70:30)%v/v

Fig. 7 Trial in mobile phase Buffer: Acetonitrile (70:30)%v/v

Trial:4         Table: 7 Trial in mobile phase Buffer: Acetonitrile (85:15)%v/v

Fig. 8 Trial in mobile phase Buffer: Acetonitrile (85:15)%v/v

Trial:5      Table: 8 Trial in mobile phase Buffer: Acetonitrile (90:10)%v/v

Fig. 9 Trial in mobile phase Buffer: Acetonitrile (90:10)%v/v

Observation:

After considering the varying combinations of various mobile phases, Buffer: Acetonitrile (90:10)%v/v was finalized as it was showing good peak shapes and a significant amount of resolution.

4.3 FORCE DEGRADATION:

4.3.1 Acid Degradation:                         

Fig. 10 Blank Chromatogram of Acidic Degradation

Fig. 11 Standard Chromatogram of Clofarabine(15 µg/ml) for Acid  Degradation

Fig. 12 Test Chromatogram ofClofarabine(15 µg/ml) for Acid Degradation

Table 9 Acid Degradation

4.3.2 Basic Degradation:

[Fig. 13 Blank Chromatogram of Basic Degradation]

[Fig. 14 Standard Chromatogram of Clofarabine(15 µg/ml) for Basic Degradation]

[Fig. 15 Test Chromatogram of Clofarabine(15 µg/ml) for Basic Degradation]

Table 10: Basic Degradation

4.3.3 Oxidative Degradation: 

[Fig. 16 Blank Chromatogram of Oxidative Degradation]

[Fig. 17 Standard Chromatogram of Clofarabine (15 µg/ml) for Oxidative Degradation]

Fig. 18 Test Chromatogram of Clofarabine (15 µg/ml) for Oxidative Degradation

Table 11: Oxidative Degradation

4.3.4 Thermal Degradation:

[Fig. 19 Blank Chromatogram of Clofarabine for Heat Degradation]

[Fig. 20 StandardChromatogram of Clofarabine (15 µg/ml) for Heat Degradation]

[Fig. 21 Test solution Chromatogram of Clofarabine (15 µg/ml) for Heat Degradation]

Table 12: Thermal Degradation

4.3.5 Photolytic Degradation:

[Fig. 22 Blank Chromatogram of Clofarabine for Photolytic Degradation]

[Fig. 23 Standard solution Chromatogram of Clofarabine (15 µg/ml) for Photolytic Degradation]

[Fig. 24 Test solution Chromatogram of Clofarabine (15 µg/ml) for Photolytic Degradation]

Table 13: Photolytic Degradation

[Table 14 Data of Force degradation study of Standard Solution]

[Table 15 Data of Force degradation study of Test Solution]

5. CONCULSION:

The method was used for estimation of Clofarabine in parenteral formulation. For the sample preparation Mobile phase was used as a solvent. 10 ml of parenteral solution , accurately weighed (equivalent to 10 mg) and transferred in to 10 ml volumetric flask, added about 5 ml of Mobile phase in to it, sonicated for 30 minutes with intermittent shaking, cooled to attain room temperature and added up to 100ml of  Mobile phase and mixed well. It was filtered through 0.45 µ syringe filter. Further 1.5 ml of the above filtrate was diluted to 10 ml with Mobile phase to get 15 µg/ml concentration of Clofarabine sample respectively.

REFERENCES:

1. Tripathi KD., Essentials of Medical Pharmacology; 6th Edn; Jaypee Brothers Medical Publishers (P) Ltd, New Delhi,2008-09, pp 819

2. Malcolm R Alison, "Cancer" Imperial College School of Medicine, London, UK

3. Buddhini Samarasinghe, "The Hallmarks of Cancer tissue Invasion and Metastasis" Oct 30 2013

4. "Clolar Mechanism of Action" Sep 2015 http://www.clolar.com/Hcp-mechanism-of-action

5. International Conference on Harmonization, Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products, International Conference on Harmonisation, IFPMA, Geneva, 1995.

6. FDA, Guideline for Submitting Documentation for the Stability of Human Drugs and Biologics. Food and Drug Administration, Rockville, MD, 1987.

7. Dong W M. Modern HPLC for Practicing Scientists; A Wiley- Interscience publication, USA, 2006, pp 1-9. https://doi.org/10.1002/0471973106

8. Kazakevich Y and LoBrutto R. HPLC for pharmaceutical Scientists;A John Wiley and sons, 2007, pp 1-6. https://doi.org/10.1002/9780470087954.ch1

9. Snyder LR, Kirkland JJ and Glajch LJ. Introduction to Modern Liquid Chromatography; 2nd Edn; A Wiley- Inter science publication, NY, USA, 1997, pp 5-42.

10. Skoog D.A, West D.M and Holler D.F; Fundamentals of Analytical Chemistry; 8th Edn, Saunders College Publishing, 2004, pp 973-992.

11. Sharma B.K, Instrumental Methods of Chemical Analysis; 25th Edn; GOEL Publication House, Meerut, pp 286-385.

12. Michael E S., and Ira S K. Analytical Method Development and Validation; Marcel Dekker, Inc., New York, 1997, pp 25-29.

13. John Lacey, Patrick Flanigan, "Genzyme Seeks U.S Approval for Clolar to treat adult AML" Cambridge, 2008.

14. Clofarabine "Drug profile" , Oct 2015, http://us.chemicalbook.com/ChemicalProductProperty_US_CB2277491.aspx

15. Clofarabine "Drug profile" , Oct 2015, http://www.chemicalland21.com/specialtychem/nd/CLOFARABINE.htm

16. Clofarabine "Drug profile" , Oct 2015, http://www.drugs.com/cdi/clofarabine.html

17. Clofarabine "Drug profile" , Oct 2015, http://www.scbt.com/datasheet-278864-clofarabine.html

18. Pokharana M, Vaishnav R, Goyal A, Shrivastava A, Stability testing guidelines of pharmaceutical products, Journal of Drug Delivery and Therapeutics 2018; 8(2):169-175

19. Wang Juan, Zhang Rong, ZHAO Yan-yan, Lu Lai-chun, "High- performance liquid chromatography Clofarabine Injection Content." Med Pap Pharm Pap 2009

20. Hsieh Y, Duncan CJ, Lee S, Liu M. Comparison of fast liquid chromatography/tandem mass spectrometric methods for simultaneous determination of cladribine and clofarabine in mouse plasma. J Pharm Biomed Anal. 2007 Jun 28; 44(2):492-7. Epub 2007 Feb 13. https://doi.org/10.1016/j.jpba.2007.02.007

21. Tu X, Lu Y,,Zhong D, Zhang Y, Chen X, "A sensitive LC-MS/MS method for quantifying Clofarabine triphosphate concentrations in human peripheral blood mononuclear cells." J Chromatogr B Analyt Technol Biomed Life Sci. 2014; 202-207 https://doi.org/10.1016/j.jchromb.2014.01.021

22. Venkata N, Jalandhar D, Gnana G, Rajendar B, Manoj P, Dhananiay D, Venkata N "Development of supercritical fluid (carbon dioxide) based ultra performance convergence chromatographic stability indicating assay method for the determination of clofarabine in injection" Mylan Laboratories Ltd, 2013; 7008-7013 https://doi.org/10.1039/c3ay41561a