International Journal of Innovative Pharmaceutical …4Saraswathi College of Pharmaceutical...

RESEARCH ARTICLE D.Kalyan et.al / IJIPSR / 2 (7), 2014, 1495-1506

Department of Pharmaceutical Analysis ISSN (online) 2347-2154

Available online: www.ijipsr.com May Issue 1495

A RP-HPLC METHOD DEVELOPMENT & VALIDATION FOR

ESTIMATING DECITABINE WITH ITS STABILITY STUDIES s

1Donthineni Kalyan*,

2A.Swetha,

3Arabinda Patnaik,

4V.Om Prakash Chary

1,4

Saraswathi College of Pharmaceutical Sciences, Yethbarpally, Moinabad, R.R.dist. AP, INDIA

2,3Samskruti College of Pharmacy, Kondapur, Ghatkeswar, R.R Dist, A.P, INDIA

Corresponding Author:

Donthineni Kalyan

Saraswathi College of Pharmaceutical sciences,

Yethbarpally (V), Moinabad (M), R.R Dist, A.P, INDIA

Email: [email protected]

Phone: +91 9948177273

International Journal of Innovative

Pharmaceutical Sciences and Research www.ijipsr.com

Abstract

A simple, precise, accurate, economical and reproducible HPLC method for estimation of

Decitabine has been developed. Quantitative HPLC was performed with Hitachi L2130 with D2000

ELITE Software with UV-Visible Detector L-2400, PUMP, column c18, 5µm, 25cmx4.6mm i.d.

column was used in the study. The mobile phase of ACN: phosphate buffer (pH 4.2) in ratio 60: 40

were used in this study. The conditions optimized were: flow rate (1 ml/minute), wavelength (254

nm) and run time was 7 min. Retention time was found to be 2.85 min. The linearity was found to

be in the concentration range of 1-60g/ml. Results of analysis were validated statistically and by

recovery studies. The recovery studies 99.67 % was indicative of the accuracy of proposed method.

The precision was calculated as repeatability, inter and intraday variation (%RSD) for the drug. By

using the method, stability of the drug has been studied.

Keywords: Decitabine, method validation, ACN, precision, stability studies

mailto:[email protected]




INTRODUCTION

It is necessary to find the content of each drug either in bulk or single or combined dosage forms

for purity testing. It is also essential to know the concentration of the drug and it’s metabolites in

biological fluids after taking the dosage form for treatment. The scope of developing and

validating an analytical method is to ensure a suitable method for a particular analyte more

specific, accurate and precise [1-4]. The main objective for that is to improve the conditions and

parameters, which should be followed in the development and validation. According to the

literature survey it was found that few analytical methods such as (RP-HPLC, HPLC, UV-

Visible analysis and LC-MS) were reported for the estimation of Decitabine [5-8]. The objective

of the proposed method is to develop simple and accurate methods for the determination of

Decitabine by RP-HPLC method in pharmaceutical dosage forms & its stability indicative

studies. Decitabine is indicated for treatment of patients with myelodysplastic syndrome (MDS).

It is a chemical analogue of pyridine, a nucleoside present in DNA and RNA. Cells in the

presence of Decitabine incorporate it into DNA during replication and RNA during transcription.

The incorporation of Decitabine into DNA or RNA inhibits methyltransferase thereby causing

demethylation in that sequence. This adversely affects the way that cell regulatory proteins are

able to bind to the DNA/RNA substrate. Decitabine is believed to exert its antineoplastic effects

following its conversion to decitabine triphosphate, where the drug directly incorporates into

DNA and inhibits DNA methyltransferase, the enzyme that is responsible for methylating newly

synthesized DNA in mammalian cells. This results in hypomethylation of DNA and cellular

differentiation or apoptosis. Decitabine inhibits DNA methylation in vitro, which is achieved at

concentrations that do not cause major suppression of DNA synthesis. Decitabine-induced

hypomethylation in neoplastic cells may restore normal function to genes that are critical for the

control of cellular differentiation and proliferation. In rapidly dividing cells, the cytotoxicity of

decitabine may also be attributed to the formation of covalent adducts between DNA

methyltransferase and decitabine that has been incorporated into DNA. Non-proliferating cells

are relatively insensitive to decitabine. Decitabine is cell cycle specific and acts peripherally in

the S phase of the cell cycle. It does not inhibit the progression of cells from the G1 to S phase.




Fig 1: Structure of Decitabine.

MATERIALS AND METHOD

Instruments and Reagents

The chromatographic separation was performed on Hitachi L2130 with D2000 Elite Software

with Isocratic with UV-Visible Detector (L-2400),PUMP (LC-IOAT). Devilosil ODS C18, 5µm,

25cmx4.6mm i.d. column was used as a stationary phase. PH Analyzer (ELICO), Electronic

Balance (Shimadzu), Ultra Sonicator (ENERTECH) has been used in the work. Decitabine

Active Pharmaceutical Ingredient (API) was provided by Comprime Labs pvt ltd, Hydernagar,

Hyderabad. Ammonium acetate & water of HPLC grade were from Standard reagents,

Hyderabad. Dimethyl sulfoxide (S.D Fine chemicals, Mumbai).

Preparation of mobile phase

Acetonitrile and Acetate buffer were mixed in the ratio of 60:40 and filtered through membrane

filter and degassed in a sonicator for 10 minutes.

Preparation of buffer (0.01M)

1.3609 gm of Ammonium Acetate in sufficient water to produce 1000ml, pH adjusted to 4.2 with

Glacial acetic acid.

Preparation of Standard Drug Solutions

Standard stock solution of a concentration of 100 μg/ml of Decitabine was prepared by using

diluents (DMSO, Acetonitrile and buffer were mixed in the ratio of 1:1)




Preparation of Sample Solutions

Sample solution of a concentration of 100 μg/ml of Decitabine was prepared by using diluents.

The procedure for preparing the solution is as per the guideline.

Optimised Chromatographic conditions

The mobile phase of ACN: Acetate buffer= 6:4, diluents of DMSO were used in this study. The

conditions optimized were: flow rate (1 ml/minute), wavelength (254 nm, monitored by UV-VIS

detector) and run time was 20 min, column temperature was maintained at 250C. Retention time

was found to be 2.85 min. devilosil ODS C18, 5µm, 25cmx4.6mm i.d. column was used as

stationary phase in the study.

Fig 2: Chromatogram of Decitabine (100 ppm) in optimized conditions (RT 2.85 min)

Method Validation

As per the ICH guidelines [8-12], the method validation parameters checked were linearity,

accuracy, precision, limit of detection, limit of quantisation.

Preparation of Calibration Curves

Calibration curve was prepared by taking appropriate aliquots of standard [13]. Decitabine stock

solution in different 10 ml volumetric flask and diluted up to the mark with diluents to obtain the

final concentrations of 20, 40, 60, 80 μg/ml of Decitabine. Standard solutions (n=6) were

injected, the sample volume was 20 μl

1.47

1.71

2.85

5.75

0 1 2 3 4 5 6 7

Retention Time (min)

0

10

20

30

40

50

60

70

80

Intensity (mV)




Fig 3: Calibration curve for Decitabine

Forced degradation studies:

The protocol was strictly adhered to for forced degradation of Decitabine Active Pharmaceutical

Ingredient (API) [9]. The API (Decitabine) was subjected to stress conditions in various ways to

observe the rate and extent of degradation that is likely to occur in the course of storage and/or

after administration to body. This is one type of accelerated stability studies [14]. That helps us

determining the fate of the drug that is likely to happen after long time storage, within a very

short time as compare to the real time or long term stability testing. The various degradation

pathways studied are acid hydrolysis, basic hydrolysis and oxidative degradation.

Mother Sample

Before carry out the degradation studies the mother sample was prepared to find out the %

degradation. It is prepared as the same method followed in sample or standard preparation.

Acid Hydrolysis

An accurately weighed 25 mg. of pure drug was transferred to a clean & dry 25 ml volumetric

flask. To which 0.1N HCl was added & make up to the mark & kept for 24 hrs. from that 4 ml

was taken in to a 10 ml volumetric flask & make up to the mark with diluent, then injected for

HPLC analysis10

.




Fig.4 Chromatogram showing degradation of decitabine in 0.1 N HCl

Basic Hydrolysis


flask. To which 1 M NaOH was added & make up to the mark & kept for 24 hrs. from that 4 ml

was taken in to a 10 ml volumetric flask & make up to the mark with diluent, then injected for

HPLC analysis.

Fig. 5 Chromatogram showing degradation in 0.1 M NaOH

Oxidation with (3%) H2O2


flask. To which 3% H2O2 was added & make up to the mark & kept for 24 hrs. from that 4 ml

1.53

2.68

0 1 2 3 4 5 6 7


0

5

10

15

Intensity (mV)

1.53

2.69

0 1 2 3 4 5 6 7


0

5

10

15

20

25

Intensity (mV)




was taken in to a 10 ml volumetric flask & make up to the mark with diluent then injected for

HPLC analysis.

Fig. 6 Chromatogram showing degradation in 3% H2O2.

Thermal degradation


flask, make up to the mark with mobile phase & was maintained at 50 0C. for 24 hrs. then

injected into the HPLC system against a blank of mobile phase ( after all optimized conditions )

Fig. 7 Chromatogram showing thermal degradation

1.53

2.69

0 1 2 3 4 5 6 7


0

5

10

15

20

25

Intensity (mV)

1.47

1.71

2.85

5.75

0 1 2 3 4 5 6 7


0

10

20

30

40

50

60

70

80

Intensity (mV)




RESULTS & DISCUSSION

Results of degradation studies:

The results of the stress studies indicated the specificity of the method that has been developed.

Decitabine was degraded only in 3% H2O2 & temperature stress conditions. The result of forced

degradation studies are given in the following table.

Table - 1: Results of Force Degradation Studies of Decitabine Api.

Stress condition Time Assay of active

substance

Assay of degraded

products

Mass

Balance (%)

Acid Hydrolysis (0.1 M

HCl) 24 Hrs. 31.25 68.36 99.61

Basic Hydrolysis (0.I M

NaOH) 24 Hrs. 30.41 68.32 98.73

Thermal Degradation

(500C)

24 Hrs. 98.36 -------- 98.29

Oxidation(3% H2O2) 24 Hrs. 33.94 65.79 99.73

Method Validation [15]

Linearity and Range

The linearity of the method was determined at ten concentration levels ranging from 10-100

µg/ml for Decitabine. The calibration curve showed good linearity in the range of 10 – 100

µg/ml, for Decitabine (API) with correlation coefficient (r2) of 0.998 (Fig. 03). A typical

calibration curve has the regression equation of y = 4135x + 2102 for Decitabine.

Table- 2: Linearity of the Decitabine

CONC. in ppm AUC

0 0

20 782456

30 1286534

40 1678995

50 2092536

60 2442970




Accuracy

The accuracy of the method was determined by recovery experiments. The recovery study was

carried out by the standard addition method at three levels of 80, 100 and 120%. Each solution

was injected in triplicate and the percentage recovery was calculated. Recovery was within the

range of 100 ± 2% which indicates accuracy of the method.

Table- 3: Data of recovery studies

Precision

The precision of each method was ascertained separately from the peak areas & retention times

obtained by actual determination of six replicates of a fixed amount of drug. Decitabine. (API)

The percent relative standard deviations were calculated for Decitabine. The intra & inter day

variation of the method [16] was carried out & the high values of mean assay & low values of

standard deviation & % RSD (% RSD < 2%) within a day & day to day variations for

Decitabine.

Table-4: Data showing repeatability analysis

HPLC Injection

Replicates of Decitabine RT Area

Replicate – 1 2.85 1678995

Replicate – 2 2.86 1675346

Replicate – 3 2.86 1680314

Replicate – 4 2.85 1675315

Replicate – 5 2.87 1679463

Average 2.858 1677887

Level of Conc Conc. Injected AUC Conc. Found % Recovery

75 30 1255630 29.84417 99.48056

75 30 1245214 29.59972 98.66573

75 30 1286534 30.56944 101.8981

100 40 1678995 39.77998 99.44995

100 40 1679430 39.79019 99.47548

100 40 1678253 39.76257 99.40642

125 50 2092536 49.48524 98.97048

125 50 2092861 49.49287 98.98573

125 50 2103408 49.74039 99.48078

Avg 99.53481

SD 0.934993

% RSD 0.939362




Standard Deviation 0.0083666 2129.49

% RSD 0.292743186 0.126915

Limit of Detection and Limit of Quantification

The LOD was found to be 0.73 g/ml and LOQ was found to be 2.19 g/ml for Decitabine

which represents that sensitivity of the method is high.

Table-5: Data for Decetabine analysis

Conc. Of

Decetabine

(API) (µg/ml)

Observed Conc. Of Decetabine (µg/ml) by the proposed method

Intra-Day Inter-Day

Mean (n=6) % RSD Mean (n=6) % RSD

10 10.02 0.95 10.19 0.73

20 20.91 0.55 20.081 0.19

40 39.84 0.18 99.18 0.27

Method Robustness:

Influence of small changes in chromatographic conditions such as change in flow rate (

0.1ml/min), Temperature (20C), Wavelength of detection (2nm) & acetonitrile content in

mobile phase (2%) studied to determine the robustness of the method are also in favour of (%

RSD < 2%) the developed RP-HPLC method for the analysis of Decitabine ( API).

Table – 6: Result of method robustness test

Change in parameter % RSD

Flow (1.1 ml/min) 0.06

Flow (0.9 ml/min) 0.04

Temperature (270C) 0.08

Temperature (230C) 0.11

Wavelength of Detection (255 nm) 0.03

Wavelength of detection (253 nm) 0.02




SUMMARY & CONCLUSION

Table-7:- Summary Of Validation Parameters by Rp-Hplc Method

Validation parameters Decitabine

Specificity % interference <0.5 %

Range (µg/ml)

Linear range 0-15 μg/ml

Working range 0.03-15μg/ml

Target range 44,55,60.5 μg/ml

Target concentration 10 μg/ml

Accuracy (% Recovery) 98-102% 99.53481%

Precision (% RSD) Repeatability 0.865

Intraday(10,20,40 μg/ml) 0.95, 0.55, 0.18

Inter day(10,20,40 μg/ml) 0.73, 0.19, 0.27

LOD (µg/ml) 0.73

LOQ (µg/ml) 2.19

To develop a precise, linear, specific & suitable stability indicating RP-HPLC method for

analysis of Decitabine different chromatographic conditions were applied & the results observed

are presented. Isocratic elution is simple, requires only one pump & flat baseline separation for

easy and reproducible results. So, it was preferred for the current study over gradient elution. In

case of RP-HPLC various columns are available, but here waters C18, 5µm, 25cm x 4.6 mm i.d.

column was preferred because using this column peak shape, resolution and absorbance were

good. Mobile phase & diluents for preparation of various samples were finalized after studying

the solubility of API in different solvents of our disposal (methanol, DMSO, acetonitrile, water,

1M NaOH, IM HCl). The drug was found to be highly soluble in methanol. Drug was sparingly

soluble in acetonitrile. Using these solvents with appropriate composition newer methods can be

developed and validated. The result shows the developed method is yet another suitable method

for assay and stability studies which can help in the analysis of Decitabine in different

formulations. The precision of the method was demonstrated by intra-day and inter-day variation

studies. For intra-day studies the drug having concentration value 10%, 20 % & 40% of the

target concentration (n 3), were injected in triplicate into the HPLC system and for inter-day

studies the drug at above three concentrations were injected in triplicate into the HPLC system

for three days. Data were subjected to statistical treatment for the calculation of SD and %RSD.

The value of %RSD for Decitabine was found to be 0.95, 0.55, and 0.18 for intra-day studies.




The values for inter-day studies were 0.73, 0.19, and 0.27 respectively. This shows that values

are not more than 2%, indicates that the developed method is precise.

The proposed method is simple, sensitive and reproducible and hence can be used in routine for

determination of Decitabine in bulk as well as in pharmaceutical preparations. Statistical analysis

of the results has been carried out revealing high accuracy and good precision.

ACKNOWLEDGEMENT

The authors are thankful to the Director of Comprime Labs Pvt. Ltd. Hyderabad for providing

the necessary facilities to carry out this work.

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