World Journal of Pharmaceutical Research et al. World ... · Tirukkovalluri et al. World Journal of...

Tirukkovalluri et al. World Journal of Pharmaceutical Research

www.wjpr.net Vol 6, Issue 7, 2017.

1158

DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING

RP–HPLC METHOD FOR ESTIMATION OF DAUNORUBICIN - A

CHEMOTHERAPIC DRUG IN BULK AND PHARMACEUTICAL

FORMULATIONS

Pallapati Suman1,2

, Tirukkovalluri Siva Rao*1, Kallam Venkata Siva Rama Krishna

Reddy2

1Department of Inorganic & Analytical Chemistry, Andhra University, Visakhapatnam-

530003, Andhra Pradesh, India.

2Laurus Labs Limited, Visakhapatnam- 531021, Andhra Pradesh, India.

ABSTRACT

Daunorubicin is a chemotherapy medicine used to treat cancer. A

simple, sensitive and specific stability indicating high performance

liquid chromatographic (HPLC) method for the determination of

Daunorubicin was developed and validated by using Kromasil C18

column (250 mm x 4.6 mm, 5µm) using a mixture of methanol and

Acetonitrile in the ratio 75:25 (v/v) as a mobile phase and at a flow rate

of 1.0mL/min. Quantification was achieved with an UV detector at 254

nm over the concentration range of 5–30µg/mL. The applied HPLC

method allowed the separation and quantification of Daunorubicin with

good linearity (r2 = 0.999) in the studied concentration range. Limit of

detection and limit of quantification were found to be 0.3µg/mL and

1.0µg/mL, respectively. The method was validated as per the

International Conference on Harmonization (ICH) guidelines. Daunorubicin stock solution

was subjected to different stress conditions. The degraded product peaks were well resolved

from the pure drug peak with significant difference in their retention time values. Stressed

samples were assayed using developed HPLC method. Statistical analysis of the data showed

that the method is precise, accurate, reproducible, and selective for the analysis of

Daunorubicin. The method was successfully applied to the estimation of Daunorubicin in

tablet dosage form.

World Journal of Pharmaceutical Research SJIF Impact Factor 7.523

Volume 6, Issue 7, 1158-1174. Research Article ISSN 2277–7105

Article Received on

30 April 2017,

Revised on 20 May 2017,

Accepted on 10 June 2017

DOI: 10.20959/wjpr20177-8791

*Corresponding Author

Tirukkovalluri Siva Rao

Department of Inorganic

& Analytical Chemistry,

Andhra University,

Visakhapatnam- 530003,

Andhra Pradesh, India.

[email protected]

Phone: +91-7702110459

mailto:[email protected]



1159

KEYWORDS: Daunorubicin, RP-HPLC, forced degradation, Daunotec©

- 20mg, Validation.

1. DRUG INTRODUCTION

Daunorubicin is a very toxic anthracycline aminoglycoside antineoplastic drug isolated from

Streptomyces peucetius.[1]

It is also known as daunomycin. Daunorubicin was approved for

medical use in the United States in 1979.[2-5] It is on the World Health Organization's List of

Essential Medicines.[1]

Specifically Daunorubicin is used for acute myeloid

leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia

(CML), and Kaposi's sarcoma. Daunorubicin is a cytotoxic agent, worked on preventing the

cancer cell from reproducing, which results in death of the cancer cell. The mechanism of

action of daunorubicin is as a Topoisomerase Inhibitor. It exhibits cytotoxic activity through

topoisomerase-mediated interaction with DNA, thereby inhibiting DNA replication and repair

and RNA and protein synthesis.[6-12]

Daunorubicin should only be administered in a

rapid intravenous infusion.[13-15] Common side effects with drug include hair loss,

vomiting, bone marrow suppression, and inflammation of the inside of the mouth.[1]

Other

severe side effects include heart disease and tissue death at the site of injection. Use

in pregnancy may harm the baby.Daunorubicin IUPAC name is (7S,9S)-9-acetyl-7-

[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-4-methoxy-

8,10-dihydro-7H-tetracene-5,12-dione,having molecular formula C27H29NO10 and molecular

weight 527.52 g/mol,structure is shown in figure-1.

Daunorubicin

Figure-1

There are various analytical methods have been developed reported for analysis

Daunorubicin. Among them Bio analytical methods[18-21]

, LC MS[22]

, HPLC[23, 24]

are



1160

analytical methods reported for analysis. Hence there is no stability indicating method was

reported with Daunorubicin, the present study was aimed for development and validation of

stability indicating RP-HPLC for Daunorubicin as well as formulation analysis.

2. MATERIALS AND METHODS

Chemicals

The reagents used in this work were methanol (HPLC grade- Lichrosolv), Acetonitrile

(HPLC grade- Lichrosolv), which were procured from Merck, India. The standard drug

Daunorubicin was obtained as gift sample from Chandra Bhagat Pharma Pvt Ltd, Mumbai.

The formulation injections of Daunorubicin (DAUNOTEC©

– 20mg) were purchased from

local pharmacy and the membrane filter was procured from Merck Milli Pour, India.

Equipment

Chromatography was performed using PEAK LC 7000 isocratic HPLC with PEAK 7000

delivery system, Rheodyne manual sample injector with switch (77251), Analytical column

Kromasil C18 column (250 X 4.6 mm, 5μ), Electronic balance-DENVER (SI234), manual

Rheodyne injector with a 20 μl loop was used for the injection of sample. PEAK LC software

was employed. UV 2301 Spectrophotometer was used to determine the wavelength of

maximum absorbance.

Chromatographic conditions

A number of HPLC systems were investigated to optimize the separation of Daunorubicin.

Separation was performed on a Kromasil C18 column (250 mm×4.6 mm i.d., 5 µm; at

ambient temperature. The mobile phase consisted of 750mL of HPLC grade methanol and

250mL of Acetonitrile of HPLC grade. The mobile phase was sonicated for 10 min and

filtered through a 0.45 μm membrane filter. The mobile phase flow rate was maintained at

1.0mL/min and eluents were monitored at 228nm. The samples were injected using a 20μL

fixed loop. All determinations were performed at ambient temperature for a run time of 10

min. The optimized chromatographic conditions are shown in Table 1.

Method development

Selection of mobile phase

Various mobile phases containing methanol, water, acetonitrile, and glacial acetic acid in

different ratios were tried with different flow rates. Good symmetrical peak was found with

the mobile phase comprising methanol and Acetonitrile in the ratio 75:25 (v/v).



1161

Preparation of standard stock solutions

The standard stock solutions of 100 μg/mL of the drug were prepared by dissolving 50 mg of

pure drug in the mobile phase in a 50 mL volumetric flask and the volume was made up to

the mark. Resulting solutions were further diluted with mobile phase to obtain a final

concentration of 100μg/mL and stored under refrigeration.

Preparation of calibration curve

Aliquots of standard stock solutions were put in a 10 mL volumetric flask and diluted up to

the mark with mobile phase. In such a way, the final concentrations of the drug were in the

range of 5–30μg/mL. Triplicate injections of 20 μL were made and analyzed by

chromatograph under the conditions as described above. Evaluation of the drug was

performed and peak areas were recorded. Calibration curves were constructed by plotting the

peak area on the y-axis against respective concentration of the drug on the x-axis. The

calibration curve was evaluated by its coefficient of determination.

Method validation

The developed method was validated by evaluating linearity, accuracy, precision, robustness,

ruggedness, detection limit, quantification limit and stability. Coefficients of variation and

relative errors of less than 2% were considered acceptable, except for the quantification limit,

for which these values were established at 2%, as recommended in the literature.

Linearity

A stock solution of Daunorubicin of 1000 μg/mL was prepared with mobile phase. From it,

various working standard solutions were prepared in the range of 5 to 100μg/mL and injected

into HPLC. It was shown that the selected drug had linearity in the range of 5–30 μg/mL. The

calibration plot (peak area of Daunorubicin versus concentration) was generated by replicate

analysis (n=9) at all concentration levels and the linear relationship was evaluated using the

least square method within Microsoft Excel® program.

Accuracy

The accuracy of the method was carried out using one set of different standard addition

methods at different concentration levels, 50%, 100% and 150%, and then comparing the

difference between the spiked value (theoretical value) and actual found value.



1162

Precision

The precision of the method was ascertained from the peak area obtained by actual

determination of six replicates of a fixed amount of the drug (15μg/mL). The precision of the

assay was also determined in terms of intra- and inter-day variation in the peak areas of a set

of drug solutions on three different days. The intra- and inter-day variation in the peak area of

the drug solution was calculated in terms of relative standard deviation (RSD).

Robustness

Robustness of the proposed method for Daunorubicin was carried out by the slight variation

in detector wavelength, pH and mobile phase ratio. The percentage change in each changed

condition was noted for Daunorubicin.

Ruggedness

The test solutions were prepared as per test method and injected under variable conditions.

Ruggedness of the method was studied by different analysts.

Detection limit and quantification limit

The limit of detection (LOD) and limit of quantification (LOQ) were established based on the

calibration curve parameters, according to the following formulas:

LOD=3.3SD/slope

LOQ=10SD/slope

or detection limit=3.3σ/s, quantification limit=10σ/s, where σ is the standard deviation of y-

intercept of regression line, and s is the slope of the calibration curve.

Forced degradation studies

The specificity of the method can be demonstrated through forced degradation studies

conducted on the sample using acid, alkaline, oxidative, thermal, photolytic, and ultra violet

(UV) degradations. The sample was exposed to these conditions, and the main peak was

studied for the peak purity, thus indicating that the method effectively separated the

degradation products from the pure active ingredient.

Hydrolytic degradation

Hydrolytic stress testing was performed to force the degradation of the drug substance to its

primary degradation products by exposure to neutral, acidic and basic conditions over time.

Functional groups likely to undergo hydrolysis are amides (lactams), esters (lactones),



1163

carbamates imides, imines, alcohols (epimerization for chiral center) and aryl amines.

Standard drug solution was prepared in aqueous, acidic (0.1N HCl) and basic (0.1N NaOH)

solutions. After the exposure time, the solution was neutralized and was diluted to get sample

concentration of 15μg/mL. The solution was analyzed in the optimized condition and the

degradation behavior was determined by comparing linear calibration curve results.

Oxidative degradation

Oxidative degradation of drug substances in pharmaceutical formulation is well documented.

Although exact mechanistic details about what promotes reaction between drug substance

and molecular oxygen in pharmaceutical formulations are not fully understood, such

reactions are generally thought to be in the category of auto-oxidation process. 3% Hydrogen

Peroxide solution was used for oxidative degradation study.

Photolytic degradation

For photolytic degradation, the drug was exposed to the direct sunlight and UV Light.

Sufficient amount of the drug was taken in a closed petri-dish and exposed to UV and

sunlight. After the exposing the drug to those stress condition the drug was taken out, diluted

appropriately and injected into HPLC to determine the amount of degradation of the drug.

Thermal degradation

To evaluate thermolytic pathways, evaluated temperature (80 °C) in the solid state and/or in

solution can be used. Many compounds began to degrade via different mechanisms above

80 °C, giving rise to degradation products. To solid-state stressing, the use of high and low

humidity atmosphere of the evaluated temperatures is appropriate. To evaluate stability under

the temperature the sample kept in a Petri dish and keep in oven at 80°C up to 24hours. After

expose of the samples and prepare sample solution and inject once. Evaluate the

degradedness in chromatogram and compare to Initial values.

3. RESULTS AND DISCUSSIONS

Chromatographic separation

A number of HPLC chromatographic systems were investigated to optimize the separation of

Daunorubicin. Retention time for Daunorubicin as function of stationary phase (Kromasil

C18column), the mobile phase and the other optimized chromatographic conditions are

shown in Table 1. Chromatogram of blank and Standard were given in figure 2 and 3.



1164

Table 1: Optimized chromatographic conditions.

S No Condition Results

1 Mobile phase Methanol: Acetonitrile 75:25 (v/v)

2 Pump mode Isocratic

3 PH 5.7

4 Diluents Mobile phase

5 Column Kromasil C18column (250 X 4.6 mm, 5μ)

6 Column Temp Ambient

7 Wavelength 228nm

8 Injection Volume 20μl

9 Flow rate 1.0ml/min

10 Run time 10min

11 Retention Time 4.5min

Figure 2: Blank chromatogram.

Figure 3: Standard chromatogram for Daunorubicin.



1165

Method Validation

Validation of a stability-indicating analytical method should demonstrate the capability of the

method for the quantitation of the active pharmaceutical ingredient and the determination of

possible degradation products without any interference. To obtain the best chromatographic

conditions, the mobile phase was optimized to provide sufficient selectivity and sensitivity in

a short separation time.

Specificity

Specificity is the ability to accurately and specifically measure the analyte of interest in the

presence of other components that may be expected to be present in the sample matrix. It is a

measure of the degree of interference from other active ingredients, excipients, impurities,

and degradation products. Specificity in a method ensures that a peak response is due to a

single component only. In the present study, the ability of the method to separate the drug

from its degradation products and the non-interference of the excipients indicate the

specificity of the method. Values of peak purity index were higher than 0.9999. These results

indicated that the proposed method is specific and stability-indicating, and can be applied for

stability studies and QC analysis of Daunorubicin in pharmaceutical products, with

advantages when compared to the previously published methods.

Calibration curve

The coefficient of determination (R2), slope and intercept for Daunorubicin were 0.999,

15819 and -3993, respectively. The retention time for Daunorubicin was 4.59min. HPLC

chromatogram of Daunorubicin at 228nm and calibration curve is shown in Fig. 4. Linearity

results were given in table 2.

Figure 4: Linearity Graph for Daunorubicin.



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Table 2: Linearity results.

Level Concentration

in µg/ml Peak Area

1 5 78420

2 10 150074

3 15 234125

4 20 311745

5 25 390140

6 30 472516

Precision

The precision, evaluated as the repeatability of the method, was studied by calculating the

RSD for six determinations of the 15µg/mL sample of Daunorubicin performed on the same

day and under the same experimental conditions. The obtained RSD value was 0.71%. The

RSD values for repeatability study was found to be <1%, which indicate that the proposed

method is repeatable. Among analysts precision was determined by analyzing 15µg/mL

sample of Daunorubicin performed on the same day and under the same experimental

conditions. The obtained RSD value was 0.48%. The RSD values for repeatability study was

found to be <1%, which indicate that the proposed method is re-producible.

Table 3: Precision results.

S No Peak Area at 15µg/ml

Intraday Precision Interday Precision

1 231641 232338

2 231471 230805

3 231457 233970

4 232285 232734

5 230818 231397

6 235405 232823

RSD = 0.71 RSD = 0.48

Accuracy

The accuracy was assessed by the standard addition method for three replicate determinations

of three different solutions containing 15, 20, and 25µg/mL of Daunorubicin. The recoveries

were obtained in a range of 98.04–100.67% for Daunorubicin using the proposed HPLC

method. (Table 4) The high values indicate that the proposed HPLC method is accurate.



1167

Table 4: Recovery Results.

Level Target

In µg/ml

Amount of

spiked (µg/ml)

Total in

µg/ml

Amount of

Recovered (µg/ml)

%

Recovery

50 %

10 5 15 15.07 100.44

10 5 15 14.80 98.63

10 5 15 14.71 98.04

100%

10 10 20 19.89 99.47

10 10 20 19.82 99.11

10 10 20 20.13 100.66

150%

10 15 25 24.96 99.85

10 15 25 24.64 98.56

10 15 25 25.17 100.67

Limit of detection and limit of quantification

The LOD and LOQ were determined from slopes of linear regression curves. The limit of

detection (LOD) and limit of quantification (LOQ) for Daunorubicin were found to be 0.13

and 1.00µg/mL, respectively. LOD and LOQ data show that Daunorubicin can be accurately

determined in the microgram quantity.

Robustness

Robustness of the method was studied by deliberate variations of the analytical parameters

such as detector wavelength, mobile phase ratio and different pH values. The results are

given in Tables 5. The % change was found to be less than 2% in all the changed condition.

Hence small variations in the method conditions don’t influence the results.

Table 5: Robustness results.

S. No Parameters

changes Change Peak Area % Change

1 Optimized …… 234125 ……

2 WL-change-1 232nm 235968 0.79

3 WL-change-2 222nm 230818 1.41

4 MP-change-1 Methanol: Acetonitrile

75:25 (v/v) 235086 0.41

5 MP-change-2 Methanol: Acetonitrile

75:25 (v/v) 238145 1.72

6 MP PH Change-1 5.8 232440 0.72

7 MP PH Change-2 5.7 236556 1.04

Forced Degradation studies

Forced degradation study of Daunorubicin was carried under different stress conditions. From

the neutral hydrolytic degradation study of Daunorubicin, it was found that two additional



1168

peaks were observed at a retention time of 3.94 and 5.28min. In this condition the % recovery

was found to be 97.92% and only 2.08% drug was degraded in this conditions. The results of

acidic hydrolysis showed degradation peaks at 0.76min, 5.37 and 5.89min along with the

drug peak. The peak area showed that 10.76% of degradation of the drug occurred when the

drug was kept in 0.1 M HCl up to 24 h. Daunorubicin upon alkaline degradation in 0.1 M

NaOH at 80 °C up to 24 h underwent degradation showing degradation peak at 5.37 and

5.89min in the chromatogram. The peak area of the drug showed that the percentage

degradation is 5.94% in the above condition. The degradation results of Daunorubicin were

given in table 6. The degradation chromatograms was given in figure 5-10 for Acidic, Base,

Light, Peroxide, Thermal and UV conditions respectively.

Table 6: Forced degradation results.

S No Condition No additional

peaks observed

Peak

Area % Obtained

%

degradation

1 Standard [15µg/ml] ...... 234125 100 0.0

2 Acidic 3 208929 89.24 10.76

3 Aqueous 2 229252 97.92 2.08

4 Base 2 220223 94.06 5.94

5 Light 2 222283 94.94 5.06

6 Peroxide 4 220530 94.19 5.81

7 Thermal 2 223909 95.64 4.36

8 UV 3 212189 90.63 9.37

Figure 5: Degradation chromatogram of Daunorubicin under Acidic condition.



1169

Figure 6: Degradation chromatogram of Daunorubicin under Base condition.

Figure 7: Degradation chromatogram of Daunorubicin under Light condition.



1170

Figure 8: Degradation chromatogram of Daunorubicin under Peroxide condition.

Figure 9: Degradation chromatogram of Daunorubicin under Thermal condition.



1171

Figure 10: Degradation chromatogram of Daunorubicin under UV condition.

Formulation analysis

The method was applied for the estimation of drug in marketed formulation. The prepared

formulation solution was analyzed in the method condition; the % assay was calculated by

comparing the peak area obtained in formulation analysis with the linear regression values.

The % assay was found to be 98.95. Hence the method was successfully applied for the

routine analysis of Daunorubicin in pharmaceutical formulations. Results were given in table

7 and formulation chromatogram was given in figure 11.

Table 7: Formulation analysis results.

S No Brand

Name Form Dosage

Amount

Prepared

Amount

Found

%

Assay

1 Daunotec©

Vial 20mg 15µg/ml 14.84µg/ml 98.95



1172

Figure 11: Formulation chromatogram of Daunorubicin.

4. CONCLUSION

This study is a typical example of the development of a stability-indicating assay following

ICH guidelines. The results of stress testing are undertaken according to the ICH guidelines

which reveal that the method is specific and stability-indicating. Based on the peak purity

results obtained from the analysis of forced degradation samples using the described method,

it can be concluded that there is no other co-eluting peak with the main peaks, and the method

is specific for the determination of Daunorubicin in the presence of degradation products.

A simple and rapid isocratic stability-indicating RP-HPLC method has been developed and

validated for the determination of Daunorubicin in tablet dosage form. The results of the

validation studies show that the RP-HPLC method is sensitive, accurate, specific, and

stability-indicating. It possesses significant linearity (r2 = 0.999), precision with a mean RSD

of <0.1%, high efficiency and resolution, and no interference from the excipients or

degradation products, as was demonstrated. The proposed method was successfully applied

and is suggested for the quantitative analysis of Daunorubicin in pharmaceutical formulations

for QC, where economy and time are essential and to assure therapeutic efficacy.

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1173

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