HPLC Analytical Method Development and Validation for Estimation of Cytarabine and Daunorubicin in API and Pharmaceutical Formulation

 

T. Vijayalaxmi¹*, Vunjali Laxman Sai¹, Ramya Sri. S²

¹Department of Pharmaceutical Analysis, Samskruti College of Pharmacy,

Affiliated to JNTUH University, Hyderabad 501301, Telangana, India.

²Department of Pharmacy, University College of Technology,

Osmania University, Hyderabad, Telangana, 500007, India.

 

ABSTRACT:

A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of Cytarabine and Daunorubicin, in its pure form as well as in pharmaceutical dosage form. Chromatography was carried out on an Altima C18 (4.6mm x 150mm, 5µm) column using a mixture of ACN, Methanol and Phosphate buffer pH-4.6 (10:25:65 v/v) as the mobile phase at a flow rate of 1.0ml/min, the detection was carried out at 265nm. The retention time of the Cytarabine and Daunorubicin was 2.088, 6.068±0.02 min respectively. The method produces linear responses in the concentration range of 10-50mg/ml of Cytarabine and 20-100mg/ml of Daunorubicin. The method precision for the determination of assay was below 2.0%RSD. The method is useful in the quality control of bulk and pharmaceutical formulations.

 

 

 

INTRODUCTION:

High performance liquid chromatography (HPLC) is a technique used for analysis of drug substance, drug product and determination and quantification of known as well as unknown impurities at lower level, food and drug administration (FDA) also trust on the purity method of analysis by using HPLC, because of high accuracy and reproducibility of results. By using this technique we can separate drug related process impurities, degradation impurities as well as reactants¹.

 

 

According to the principle of separation of HPLC, as the particle size of column material decreases, the efficiency of the chromatographic separation, speed and resolution also increases. The HPLC is the most simple, economic, reliable and worldwide used technique in the pharmaceutical analysis².

 

Cytarabine, is cytosine arabinoside (ara-C), is a chemotherapeutic agent used to treat acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma, and chronic myelogenous leukemia (CML). It is administered via injection, under the skin, or into the cerebrospinal fluid. There is a pharmaceutical liposomal formulation for which there is tentative evidence of enhanced outcomes in lymphoma including the meninges³. Cancer is a group of diseases characterized by the disregulate proliferation of abnormal cells that invade and disrupt surrounding tissues. Being the second leading cause of death in the United States and in most parts of Europe, its social and economical impact is overwhelming⁴.

 

 

Fig 1: Chemical Structure of Cytarabine⁵

 

Cancer patients are facing a number of problems due to the disease itself or even with its treatment. Clinical anxiety and depression are apparent in one third of cancer patients⁶. Prolongation of survival, palliation of symptoms, preservation of quality of life are the main goals of chemotherapy. Even though chemotherapy can lead to nausea, vomiting, alopecia, fatigue, sexual dysfunction and reduction in quality of life⁷. The prevalence of cancer pain is estimated as 25% for those newly diagnosed, 33% for those undergoing active treatment and greater than 75% for those with advanced disease. Pain prevalence is also high in specific cancer type such as head and neck cancer (49%). With such high prevalence, cancer pain should be anticipated and responded as early as possible⁸.

 

Oral cancer occurs on sites in the oral cavity: tongue, lips, and floor of the mouth, soft palate, tonsils, salivary glands and oropharynx⁹.

 

Fig 2: Chemical Structure of Daunorubicin¹⁰

 

Leukaemia (blood cancer) is a non infectious and non genetic disease in humans. This may be caused by smoking, ionization, some dangerous chemicals but the exact cause of leukaemia is unknown. Leukaemia is a group of cancer that usally begins in the bone marrow and result in high number of abnormal white blood cells.¹¹

 

They can range from mouth ulcers, diarrhea, temporary hair loss, rashes, nausea, vomiting, and fatigue to low blood cell counts, increased risk of infections, graft versus host disease, tumor lysis syndrome, differentiation syndrome, and difficulty in conceiving¹². Due to these side effects, there is considerable research going on focusing on newer treatment options for it. Phytochemicals or plant-derived molecules are gaining popularity the world over for the treatment of various types of cancer.

 

Patient with leukemia often have bleeding due to decreased platelet number. This decrease is a result of malignant cell infiltration into the bone marrow as well as the effects of chemotherapy, furthermore because of  disseminated intravascular coagulopathy, immunological processes and secondary hypersplenism1. Bleeding that occurs can be a serious problem and even life-threatening¹³. In most patients with acquired imatinib resistance, the BCR-ABL kinase was still activated despite continuation of imatinib treatment. Point mutation of the kinase domain (KD) of BCR-ABL protein was found to be the commonest cause for the failure of inhibition and many different mutations were identified. On the other hand, KD mutation as the cause for primary resistance is much less common.¹⁴

 

MATERIALS AND METHODS:

Cytarabine from Sura labs, Daunorubicin from Sura labs, Water and Methanol for HPLC from LICHROSOLV (MERCK), Acetonitrile for HPLC from Merck.

 

HPLC Method Development:

Trails:

Preparation of standard solution:

Accurately weigh and transfer 10mg of Cytarabine and Daunorubicin working standard into a 10ml of clean dry volumetric flasks add about 7ml of Methanol and sonicate to dissolve and removal of air completely and make volume up to the mark with the same Methanol.

Further pipette 0.1ml of the above Cytarabine and Daunorubicin stock solutions into a 10ml volumetric flask and dilute up to the mark with Methanol.

 

Procedure:

Inject the samples by changing the chromatographic conditions and record the chromatograms, note the conditions of proper peak elution for performing validation parameters as per ICH guidelines.

 

Mobile Phase Optimization:

Initially the mobile phase tried was Methanol: Orthophosphoric acid and Phosphoric acid (pH 3): Acetonitrile and Methanol: ACN with varying proportions. Finally, the mobile phase was optimized to Buffer: Methanol: ACN in proportion 65:25:10v/v respectively. 

 

Optimization of Column:

The method was performed with various columns like C18 column, ODS and Zodiac column. Altima C18 (4.6×150mm, 5µ) was found to be ideal as it gave good peak shape and resolution at 1ml/min flow.

Validation methods procedures followed as per ICH guidelines^15-18.

 

RESULTS AND DISCUSSION:

Optimized Chromatogram (Standard):

Mobile phase :   Buffer: Methanol: ACN (65:25:10v/v/v)                                   

Column          :   Altima C18 (4.6×150mm, 5.0µm)

Flow rate        :   1ml/min

Wavelength    :   265nm

Column temp  :   38ºC

Injection Volume :  10µl

Run time        :  14minutes

 

 

Fig 3-: Optimized Chromatogram

 

Table 1: - Peak Results for Optimized Chromatogram

S. No	Peak name	Rt	Area	Height	USP Resolution	USP Tailing	USP plate count
1	Cytarabine	2.088	3425413	567933		1.0	5565.5
2	Daunorubicin	6.068	1629854	517733	2.5	1.1	5355.2

 

Optimized Chromatogram (Sample)

 

Figure 4-: Optimized Chromatogram (Sample)

 

Table 2-: Optimized Chromatogram (Sample)

S. No.	Name	Retention time(min)	Area (µV sec)	Height (µV)	USP resolution	USP tailing	USP plate count
1	Cytarabine	2.090	3468547	567933		1.0	5565.5
2	Daunorubicin	6.070	16289441	517733	2.5	1.1	5355.2

 

 

System Suitability:

Table 3-: Results of system suitability for Cytarabine

S. No.	Name	Rt	Area	Height	USP plate count	USP Tailing
1	Cytarabine	2.080	3569412	567917	5568.0	1.0
2	Cytarabine	2.080	3465125	517719	6359.2	1.1
3	Cytarabine	2.080	3598154	567933	5565.5	1.0
4	Cytarabine	2.081	3586491	517733	5355.2	1.1
5	Cytarabine	2.081	3582694	567917	6348.0	1.0
Mean			3560375
Std. Dev			54225.61
% RSD			1.523031

 

 

Table 3-: Results of method precession for Daunorubicin:

S. No.	Name	Rt	Area	Height	USP plate count	USP Tailing	USP Resolution
1	Daunorubicin	2.080	3582264	567917	5568.0	1.0	2.5
2	Daunorubicin	2.080	3586491	517719	5359.2	1.1	2.5
3	Daunorubicin	2.080	3598154	567933	5565.5	1.0	2.5
4	Daunorubicin	2.081	3564125	517733	5355.2	1.1	2.5
5	Daunorubicin	2.081	3569412	562173	5568.0	1.0	2.5
Mean			3580089
Std. Dev			13609.81
% RSD			0.380153

 

 

Assay (Standard):

Table 4-: Peak results for assay standard

S. No.	Name	Rt	Area	Height	USP Resolution	USP Tailing	USP plate count	Injection
1	Cytarabine	2.087	3425681	567917		1.0	5568.0	1
2	Daunorubicin	6.067	16235984	517719	2.5	1.1	5359.2	1
3	Cytarabine	2.088	3425413	567933		1.0	5565.5	2
4	Daunorubicin	6.068	16298543	517733	2.5	1.1	5355.2	2
5	Cytarabine	2.088	3465423	567933		1.0	5545.5	3
6	Daunorubicin	6.068	16265213	517733	2.5	1.1	5352.1	3

 

 

Assay (Sample):

Table 5-: Peak results for Assay sample

S. No.	Name	Rt	Area	Height	USP Resolution	USP Tailing	USP plate count	Injection
1	Cytarabine	2.089	3469821	567917		1.0	6568.0	1
2	Daunorubicin	6.069	16259845	517719	2.5	1.1	5359.2	1
3	Cytarabine	2.090	3468547	567933		1.0	5565.5	2
4	Daunorubicin	6.070	16287531	517733	2.5	1.1	5355.2	2
5	Cytarabine	2.090	3468143	567813		1.0	5391.1	3
6	Daunorubicin	6.070	16282431	517623	2.5	1.1	5564.0	3

 

 

% ASSAY =

 Sample area        Weight of standard     Dilution of sample     Purity     Weight of tablet

__________ ×   ________________ × _______________×_______×______________×100

Standard area      Dilution of standard    Weight of sample       100          Label claim

 

==16276602 / 16266580 × 10/60 × 60/0.0136 × 99.6/100 × 0.4102/300 × 100

= 100.1%

 

The % purity of Cytarabine and Daunorubicin in pharmaceutical dosage form was found to be 100.1%.

 

Linearity:

Chromatographic Data For Linearity Study:

Cytarabine:

 

Figure 5: Calibration Graph for Cytarabine

 

 

Daunorubicin

Concentration mg/ml	Average Peak Area
20	8040807
40	14318417
60	21087985
80	27913928
100	34584741

 

Figure 6: Calibration Graph for Daunorubicin

 

 

Repeatability:

Table 6-: Results of repeatability for Cytarabine:

S. No.	Name	Rt	Area	Height	USP Plate Count	USP Tailing
1	Cytarabine	2.084	3569412	567917	5568.0	1.0
2	Cytarabine	2.083	3465125	517719	5359.2	1.1
3	Cytarabine	2.082	3598154	567933	5565.5	1.0
4	Cytarabine	2.081	3586491	517733	5355.2	1.1
5	Cytarabine	2.080	3582694	567917	5568.0	1.0
Mean			3560375
Std. Dev			54225.61
% RSD			1.523031

 

Table 7-: Results of repeatability for Daunorubicin:

S. No.	Name	Rt	Area	Height	USP plate count	USP Tailing	USP Resolution
1	Daunorubicin	2.080	3582264	567917	5568.0	1.0	2.5
2	Daunorubicin	2.081	3586491	517719	5359.2	1.1	2.5
3	Daunorubicin	2.082	3598154	567933	5565.5	1.0	2.5
4	Daunorubicin	2.083	3564125	517733	5355.2	1.1	2.5
5	Daunorubicin	2.084	3569412	562173	5568.0	1.0	2.5
Mean			3580089
Std. Dev			13609.81
% RSD			0.380153

 

Accuracy:

Table 8-: The accuracy results for Cytarabine

%Concentration (At specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	1543793	15	15.2	101.9	100.9%
100%	3035883	30	30.4	101.4
150%	4451005	45	44.7	99.4

 

Table 9-: The accuracy results for Daunorubicin

%Concentration (At specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	1084420	30	30.07	100.2	99.6%
100%	2096069	60	59.6	99.4
150%	3112684	90	89.3	99.3

 

 

LIMIT OF DETECTION

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

 

LOD= 3.3 × σ / s

 

Where

σ = Standard deviation of the response

S = Slope of the calibration curve

σ = 58777.45

S= 98628

σ = 176374

S= 34187

 

RESULT:

Cytarabine:

=3.3 × 58777.45/98628

=1.9µg/ml

 

Daunorubicin:

=3.3 × 176374/34187

=17.0µg/ml

 

Limit of Quantitation:

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined.

 

LOQ=10×σ/S

Where

σ = Standard deviation of the response

S = Slope of the calibration curve

 

RESULT:

Cytarabine:

=10×58777.45/98628

= 5.9µg/ml

 

Daunorubicin:

=10 × 176374/34187

= 51.5µg/ml

 

Robustness

 

Table 10-: Results for Robustness

 

Cytarabine:

Parameter used for sample analysis	Peak Area	Retention Time	Theoretical plates	Tailing factor
Flow rate of 1.0 mL/min	3425413	2.088	5568.2	1.0
Flow rate of 0.9 mL/min	3425282	3.111	5922.2	1.2
Flow rate of 1.1 mL/min	3517879	1.880	5868.8	1.2
Less aqueous phase	3175485	3.101	5836.2	1.2
More aqueous phase	3365431	1.881	5282.6	1.1

 

Table 11-: Results for Robustness

Daunorubicin:

Parameter used for sample analysis	Peak Area	Retention Time	Theoretical plates	Tailing factor
Flow rate of 1.0 mL/min	2029854	6.068	5359.2	1.1
Flow rate of 0.9 mL/min	1738319	7.101	5999.1	1.2
Flow rate of 1.1 mL/min	1638304	5.007	5989.2	1.1
Less aqueous phase	1973724	7.108	5387.2	1.1
More aqueous phase	2102838	5.008	5938.1	1.1

 

CONCLUSION:

In the present investigation, a simple, sensitive, precise and accurate RP-HPLC method was developed for the quantitative estimation of Cytarabine and Daunorubicinin bulk drug and pharmaceutical dosage forms.

 

This method was simple, since diluted samples are directly used without any preliminary chemical derivatisation or purification steps.

 

Cytarabine was found to be soluble in organic solvents such as DMSO and dimethyl formamide (DMF), which should be purged with an inert gas. The solubility of Cytarabine in DMSO is approximately 0.2 mg/ml and approximately 0.1 mg/ml in DMF and is freely soluble in water and slightly soluble in alcohol and in chloroform. Daunorubicin was found to be soluble in water or methanol, DMSO, normal saline, Acetonitrile and tetrahydrofuran, slightly soluble in alcohol and practically insoluble in acetone.

 

 

ACN, Methanol and Phosphate buffer pH4.6 (10:25:65 v/v) was chosen as the mobile phase. The solvent system used in this method was economical.

 

The %RSD values were within 2 and the method was found to be precise.

 

The results expressed inTablesfor RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectrophotometric methods.

 

This method can be used for the routine determination of Cytarabine and Daunorubicin in bulk drug and in pharmaceutical dosage forms.

 

ACKNOWLEDGEMENT:

Thе Authors arе thankful to the Management and Principal, Department of Pharmacy, Samskruti College of Pharmacy, Hyderabad, for extending support to carry out the research work. Finally, the authors express their gratitude to the Sura Pharma Labs, Dilsukhnagar, Hyderabad, for providing research equipment and facilities.

 

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Received on 23.10.2022       Modified on 21.11.2022

Accepted on 25.12.2022   ©AandV Publications All Right Reserved