DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP HPLC METHOD FOR ANALYSIS OF ACYCLOVIR IN API AND PHARMACEUTICAL DOSAGE FORM

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING

RP-HPLC METHOD FOR ANALYSIS OF ACYCLOVIR IN API AND

PHARMACEUTICAL DOSAGE FORM

Atul Musmade1, Hemant Jain2* and Rohit Prajapati3

1_{Department Quality Assurance Techniques, STES’s, Sinhgad College of Pharmacy,}

Vadgaon (Bk.), Pune- 411041, Maharashtra, India.

2_{SPTM, SVKM’s NMIMS, Shirpur, Dhule-425 405, Maharashtra, India.}

ABSTRACT

A rapid stability indicating RP-HPLC method was developed and

validated for determination of Acyclovir in bulk and tablet dosage

form. Sample was analysed on a Kromasil ODS C18 column (250 × 4.6

mm, 5µ). The mobile phase consist of MeCN: Ammonium acetate

buffer (pH 4.5) in the ratio of 50:50 at a flow rate 1 ml min-1 with UV

detection wavelength at 253 nm. The retention time of Acyclovir was

2.47 minutes. The calibration curve was linear over the concentration

range of 10-60 µg/ml (r2=0.998) Acyclovir was found to degrade in

alkaline and oxidative stress conditions. However it was stable in acid

and dry heat conditions. The validation studies were carried out

according to ICH guidlines, The developed method was found to be

linear, precise, accurate and robust.

KEYWORDS: Acyclovir, ICH, Validation, RP-HPLC.

INTRODUCTION

Acyclovir (ACV) [(9,2-ethoxyhydroxy) methyl guanine] is a nucleoside analogue with

potent antiviral activity for herpes simplex viruses (HSV), varicella zoster virus (VZV),

Epstein- Barr virus and cytomegalovirus (CMV).[1,2] It is official in Indian Pharmacopoeia.[3]

Determination of acyclovir in bulk drugs and formulations by spectphotometric method, [4]

and chromatography method.[5-7] were reported. Acyclovir determination in biological

samples like serum,[8] skin layers,[9] plasma,[10] saliva and urine.[11] was subjected to extensive

study by RP-HPLC method. But none of the literature described validated stability indicating

Volume 4, Issue 8, 1043-1052. Research Article ISSN 2277– 7105

Article Received on 10 May 2015,

Revised on 05 June 2015, Accepted on 02 July 2015

*Correspondence for

Author

Dr. Hemant Kumar Jain

Department Quality Assurance Techniques, STES’s, Sinhgad College

of Pharmacy,

RP-HPLC method. A new method for the RP-HPLC analysis of acyclovir is described in this

paper. The method is substantially simpler, faster and more sensitive.

EXPERIMENTAL Instuments

A HPLC (Perkin Elmer Lambda 25) equipped with Perkin Elmer Binary LC Pump 200B/

250, solvent degasser, Series 200 UV/VIS detector and Kromasil ODS C18 column was used

with Total Chrome Navigator Software (version 6.3.1).

Chemical and Reagent

Pharmaceutical grade acyclovir was kindly provided by Lupin Ltd. Boiser, Mumbai and was

used as received. Proprietary drug Sitavig-50 mg. was purchased from local store. Analytical

reagent grade Ammonium acetate (Qualigens Fine Chemicals), MeCN (Merck Specialities

Private Limited), AcOH (Merck Specialities Private Limited) and distilled water filtered

through a 0.45 µm Millipore PVDF (polyvinyl difluoride) filter were used.

Standard solution

Accurately weighed quantity of acyclovir 10 mg was transferred to 100 ml volumetric flask

and made up to volume with diluent. Samples were dilute to concentration of 10, 20, 30,

40,50 and 60 µg ml-1 and used for validation study.

Assay of tablet formulation

Sitavig-50 mg were procured from local market. Tablet of Acyclovir equivalent to 49.7 mg

Of Acyclovir was accurately weighted and tranfered in to 100 ml volumetric flask. About 50

ml of diluent was added to dissolve the components present in tablet. The contents were

sonicated for 15 minutes with intermittent shaking and diluted up to the mark with the diluent

This solution was filtered through 0.45µm nylon filter, discarding first five ml of filtrate. The

sample solution was prepared in triplicate and 20µl volume of each sample solution was

injected in to the sample injector of RP-HPLC under the optimized chromatographic

condition (Table:1 ).

PROCEDURES

Method development and validation

A literature survey indicated that number of HPLC methods were developed and validated for

guideline Q1A (R2).[12] Thus method was developed which can used for quantification of

acyclovir and its degradation product and validated for linearity, accuracy, precision,

repeatability and robustness as per ICH guideline Q2 (R1).[13] Method was developed on a

reverse-phase Kromasil ODS C18 column (250 × 4.6 mm, 5µ) at ambient temperature with a

mobile phase consisting of MeCN:20 mM ammonium acetate buffer (pH 4.5) (50:50) at a

flow rate 1 ml min-1 with 20 µl injection volume. The detector wavelength was set at 253 nm

as determined by Perkin Elmer Lambda 25 UV/VIS spectrometer with distilled water as

solvent [Fig.2].

Force degradation studies

The force degradation studies were carried out under the conditions of hydrolysis, oxidation

and dry heat as described in ICH guideline Q1A (R2).[12] The approach suggested by Bakshi

and Singh.[15] was adopted for these studies. The drug solution prepared in stressor (e.g. HCl,

NaOH, H2O2) at concentration of 1 mg ml-1 was used for force degradation study. Acidic and

alkaline hydrolysis was carried out by heating acyclovir in 0.1 M HCl and 0.1 M NaOH at

60°C for 4 h and then samples were neutralised. The oxidative study was carried out in 3 %

v/v H2O2 at 60°C for 3 h. 0.1 ml neutralised sample of hydrolysis and oxidative study were

diluted with 10 ml diluent and injected into HPLC. For thermal degradation study, drug was

sealed in glass vial and heated at 80°C for 24 h then 10 µg ml-1 solution in diluent was

injected.

Method Development

The method utilising Methanol: Water as mobile phase yielded broad peak, whereas with

MeCN: Water tailing was observed with water as diluent. Procedure utilising Ammonium

acetate buffer (pH 4.5): Water as mobile phase with water as diluents also yielded tailing

where as with MeCN: Ammonium acetate buffer (pH 4.5) with MeCN: Water as diluent

sharp peak was obtained.

During method development, a number of variations were tested like pH, buffer

concentration, MeCN concentration and flow rate to give a symmetric peak. With a mobile

phase MeCN: 20mM ammonium acetate buffer (pH 4.5) (50:50) at flow rate 1 ml min-1 and

MeCN: Water (60:40), symmetric peak was obtained [Fig. 4].

Validation Linearity

A linear calibration graph (y = 103887x + 24776; where y and x are peak area and

concentration, respectively) was obtained over six concentrations 10, 20, 30, 40, 50, 60 µg

ml-1 in triplicates with 20 µl injection volume. Correlation coefficient was found to be 0.9989

[Fig. 3].

Accuracy

Accuracy was evaluated at three different concentrations 4, 5 and 6 µg ml-1 equivalents to 80,

100 and 120% of the active ingredient. To a pre quantified sample solution known amount of

acyclovir was spiked then acyclovir amount was estimated by measuring the peak area and

fitting these values to the straight line equation of linearity curve. The recoveries at three

different concentrations were found to be within the range of 98 to 102% as per ICH

guidelines Q2 (R1).[17] Mean % recovery (mean ± SD) was found to be 99.83 ± 0.27 [Table

3].

Limit of detection (LOD) and Limit of Quantification (LOQ)

Four sets of known concentration (10-40µg/ml)were prepared . calibration curve plotted for

each set. LOD and LOQ were calculated using the formula as

Where,

= the standard deviation of the response.

S = Average of the slope of the calibration curves.

The results are discussed in Table:4

Precision

Intra-day precision of the method was determined by repeat analysis (three identical

injections) at three concentration levels. Inter-day precision was established by performing

the analysis next day on a freshly prepared solution. The low RSD values of Table 3 indicate

Repeatability

For repeatability study, 30 µg ml-1 acyclovir solution was analyzed six times on the same day.

% RSD was calculated for the resultant peak area and retention time as shown in Table 5.

Robustness

The robustness was assessed by altering the experimental conditions such as, by changing the

flow rate from 0.8 to 1.2 ml/min, the mobile phase composition with MeCN-ammonium

acetate buffer (pH 4.5) (55:45, 45:55) and analysed in triplicate.

In all varied Chromatographic conditions, there was no significant change in

chromatographic parameters. There was no effect of mobile phase composition on retention

time as seen in Table 6.

Degradation study Acid hydrolysis

Initially 0.1 M HCl was used for the degradation of acyclovir but it was found that the drug

was stable and no degradation was observed With 1M HCl no degradation was observed

which indicates that drug is stable to acid.

Alkali hydrolysis

Fig. 6 shows that drug degraded in 0.1 M NaOH at 60°C. At the end of 4 hrs it was degraded

to an extent of 9 % with retention time of degradation product at 4.490 minutes and that of

drug at 2.457 minutes.

Oxidative degradation

Drug was also degraded in 3 % H2O2 as seen in Fig. 7. At the end of 3 hrs 27 % degradation

was observed. Retention time of degradation product was 2.465 minutes and retention time of

drug was 2.783 minutes.

Thermal degradation

Shows no degradation at 80°C for 24 hrs. It indicated that the drug is stable to heat.

RESULT AND DISSCUSSION

The calibration curve was plotted of Acyclovir peak area v/s concentration. The generated

regression equation was y = 103887x +24776(R20.998). The R2 value as 0.999 indicates that

10-60µg/ml. The proposed method was found to be precise as % R.S.D. values for intraday as

well as interday precision was satisfactory. The drug at each of the 80%, 100% and 120%

levels 100.1, 99.95, 99.52 showed good recoveries . Hence it can be said that this method was

accurate. The LOD and LOQ were calculated as 0.015 µg/ml and 0.047 µg/ml, respectively.

The result of analysis of final formulation by the developed method consistent with the lable

claim, highly reproducible and reliable. The method can be used for the routine analysis of

the Acyclovir in tablet dosage form.

Figure 1: Chemical structure of Acyclovir.

Figure: 2: λmax of Acyclovir by Perkin Elmer Lambda 25UV/VIS Spectrometer

Figure 4: Chromatogram of Acyclovir (20 µg ml-1).

Figure 5: Chromatogram of Acyclovir in 0.1 M HCl at 60°C

Figure 6: Chromatogram of Acyclovir in 0.1 M NaOH at 60°C

Figure 8: Chromatogram of Acyclovir at 80°C for 24 hrs.

Table 1: Result of assay of Acyclovir

Table 2: Force degradation conditions. Force degradation

condition

Concentration of stressor

Exposure

condition Duration

Hydrolysis Acid 0.1 M HCl 60°C 4 h

Base 0.1 M NaOH 60 °C 4 h

Oxidation 3 % v/v H2O2 60°C 3 h

Thermal - 80°C 24 h

Table 3: LOD and LOQ of Acyclovir.

Table 2: Recovery study.

Table 3: Precision study Sample Solution Concentration (µg/ml) Sample solution area Mean sample Solution area % Drug Found

50 5175

5177 100.2%

50 5177

50 5179

Drug LOD LOQ

Acyclovir 0.015 µg/ml 0.047 µg/ml

Spiked conc. (µg ml-1)

Calculated spiked conc. ± SD (µg ml-1), % RSD

% Recovery

4 4.00 ± 0.008, 0.21 100.01

5 5.00 ± 0.035, 0.70 99.95

6 5.95 ± 0.475, 0.79 99.52

Conc. (µg ml-1)

Intra-day precision Inter-day precision Measured conc. ± SD (µg ml-1),

% RSD

Measured conc. ± SD (µg ml-1),% RSD

Table 4: Repeatability study

a

Based on peak area

b

Based on retention time

Table 5: Robustness study

Conc. Conditions changed Mean Peak area % RSDa Mean RT % RSDb

50 µg ml-1

Mobile phase composition

45:55 5178084.79 0.60 2.646 0.69

55:45 5176384.03 0.74 2.462 0.33

Flow rate

0.8 ml min-1 5167450.17 0.69 3.272 0.22 1.2 ml min-1 5186230.70 0.40 1.974 0.39

a

Based on peak area

b

Based on retention time

CONCLUSION

This study is typical example of development of stability indicating method, established

following the recommendations of ICH guidelines Q2 (R1).[17] It is one of the rare studies

where degradation product and drug were resolved in a single isocratic run.The developed

method is simple, accurate, precise and rugged. This method can be applied to the quality

control of acyclovir raw material and long term stability control of pharmaceutical product.

ACKNOWLEDGEMENT

The authors are grateful to Lupin Ltd. Boiser, Mumbai, India for providing API of Acyclovir

as gift sample. Authors also thanks to Sinhgad College of Pharmacy, Pune, India for

providing necessary facilities to complete this project.

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